WO2013067173A1 - Systematic control of protein interaction using a modular er/k linker - Google Patents

Abstract

Disclosed herein is a genetically encoded biosensor for use in measuring protein-protein interactions such as affinity of intra- or intermolecular interactions. The disclosed biosensor utilizes an unusual polypeptide linker, an ER/K motif. The ER/K motif is a polypeptide composed of 70% or more of the amino acids glutamic acid, arginine and/or lysine. The ER/K motif, by virtue of its alternating charge is unlikely to interact with protein domains at its ends and is stable under extremes of pH, salt concentrations, and temperature. ER/K motif linkers can be used, because of unusual structural properties to measure the frequency of protein-protein interactions and identify compounds that alter such interactions.

Description

SYSTEMATIC CONTROL OF PROTEIN INTERACTION USING A

MODULAR ER/K LINKER

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to USSN 61/554,671, filed November 2, 201 1 , herein incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT [0002] This invention was made with U.S. Government support under Grant No. GM33289 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

BACKGROUND [0003] Interactions between proteins are essential for cellular function. Association of proteins facilitates their localization to, and therefore function in, specific subcellular compartments (1). Proteins often associate to form a macromolecular complex that can perform functions that the individual components cannot (2). Brief bimolecular interactions amongst a series of proteins are characteristic of signaling cascades that are essential to propagate and amplify an extracellular signal across the cell and elicit an appropriate response (3). Interactions between proteins are dictated by their binding affinity and the local cellular concentrations of the proteins involved. In addition to bimolecular interactions between proteins, many proteins have intramolecular interactions between distinct domains that regulate their function (4). Such intramolecular interactions within a protein in turn are regulated by the free solution binding affinity of the two domains and the effective concentration experienced by the two domains within the protein.

[0004] Regulation of intermolecular and intramolecular protein interactions is important to gain mechanistic insight into cellular processes and is an essential component of pharmacological intervention in disease states. However, proteins have complex structures with predictions of ~ 1000 structural families (5) and ~ 10,000 distinct types of interactions between proteins (6). Detailed insight into the intermolecular or intramolecular interaction interface gained from techniques such as x-ray crystallography, N R and computational modeling can be used to perturb the interaction by altering the sequence of one or both proteins/domains using site-directed mutagenesis (7, 8). This approach, however, is limited by the effects of the mutation on the structure and folding of the protein domains, which is complicated by the allosteric nature of protein structure (9). Further, site directed mutagenesis often results in a loss of protein-protein interaction and is therefore usually an all-or-nothing approach to regulation.

[0005] Given the importance of protein-protein interactions, a need in the art exists to have an alternate approach that can systematically modulate protein-protein interactions without perturbing the structure of the proteins or protein domains involved.

[0006] The instant disclosure addresses this need and others.

BRIEF SUMMARY OF THE INVENTION

[0007] Modern biological research is concerned with how, when, and where proteins interact with other proteins. Protein-protein interactions occur at almost every level of cell function; in the structure of sub-cellular organelles, transport across membranes, the packaging of chromatin, muscle contraction, signal transduction, and the regulation of gene expression. Abnormal protein-protein interactions have been implicated in a number of diseases including Alzheimer's disease.

[0008] The frequency of protein interactions is a function of the local concentration of two proteins and their affinity with one another. When two proteins are tethered together, the link between them influences their effective concentrations and therefore the frequency of their interactions. The instant invention provides methods and compositions to systematically vary the effective concentration of proteins and thereby measure protein-protein and/or domain interactions and this can be further applied to the identification of compounds that inhibit these interactions. [0009] Accordingly, in some embodiments, a biosensor is provided, the biosensor encompassing at least two test proteins, a polypeptide linker, and a reporter, wherein the at least two test proteins are covalently linked to, and separated by, the polypeptide linker, and wherein the polypeptide linker possesses an ER/K domain and wherein the interaction of the at least two test proteins results in a detectable signal. In some embodiments, the biosensor is genetically encoded. In some embodiments, the detectable signal is a fluorescent signal.

[0010] In some embodiments, the amino acid sequence of the polypeptide linker is such that a three-fold increase in the length of the linker results in a 30 fold decrease in the effective concentration of the test proteins. In some embodiments, the linker is at least 30 amino acids in length.

[0011] In some embodiments, a biosensor is provided, the biosensor encompassing a test protein A and a test protein B, a Forster's resonance energy transfer (FRET) acceptor, a FRET donor, and a polypeptide linker, wherein the test protein A and the test protein B are covalently attached, and separated by, the polypeptide linker, and wherein the polypeptide linker possesses an ER/K domain.

[0012] In some embodiments, the amino acid sequence of the polypeptide linker is such that a three-fold increase in the length of the linker results in a 30 fold decrease in the effective concentration of the test proteins.

[0013] In some embodiments, a method for measuring the equilibrium dissociation constant of a protein - protein or protein-ligand interaction, the method encompassing detecting the signal from a biosensor at two or more different concentrations of the biosensor wherein the biosensor comprises a test protein A, a test protein B, a polypeptide linker, and a reporter, wherein the test protein A and the test protein B are covalently attached, and separated by, the polypeptide linker, and wherein the polypeptide linker possesses an ER/K domain. [0014] In some embodiments, the amino acid sequence of the polypeptide linker is such that a three-fold increase in the length of the linker results in a 30 fold decrease in the effective concentration of the test proteins. In some embodiments, the linker is at least 30 amino acids in length.

[0015] In some embodiments, a method for measuring the equilibrium dissociation constant of a protein - protein or protein-ligand interaction, the method encompassing detecting the signal from a biosensor in the presence of two or more concentrations of a competitive binding ligand, wherein the biosensor comprises a test protein A, a test protein B, a polypeptide linker, and a reporter, wherein the test protein A and the test protein B are covalently attached, and separated by, the polypeptide linker, wherein the polypeptide linker possesses an ER/K domain, and wherein the competitive binding ligand competes with the test polypeptide B for binding to the test polypeptide A. [0016] In some embodiments, an assay for identifying compounds which alter a protein- protein interaction is provided, the assay encompassing detecting a signal from a biosensor in the absence and in the presence of a test compound, wherein the biosensor comprises a test protein A, a test protein B, a polypeptide linker, and a reporter, wherein the test protein A and the test protein B are covalently attached, and separated by, the polypeptide linker, and wherein the polypeptide linker possesses an ER/K domain.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 depicts Systematic Protein Affinity Strength Modulation ("SPASM") sensor design, (a) Schematic of protein domains (His₆ is at the N-terminus) in the SPASM sensor. A 12 amino acid Gly-Ser-Gly (GSG)₄ linker is placed between different domains to ensure rotational freedom, (b) Structural model of the SPASM sensor in the open (left) and closed (right) conformation, (c) Sequences of two ER/K a-helices. Note that the 20 nm helix used in this study is the first 130 amino acids of the 30 nm helix sequence, (d) (Left) Coomassie gel staining of purified SPASM sensors with 30 nm (lanes 1, 2) and 20 nm (lanes 3,4) ER/K a- helices. Lane 2 and 4 were treated with Tev-protease, which cleaves at its recognition site engineered between the CAM and ER/K a-helix. (Right) Laser fluorescence gel scan (457 nm eCFP excitation with 520BP40 emission). This condition captures both eCFP and mCitrine fluorescent bands (e) Absorption spectrum of purified SPASM sensor showing peaks at 433 nm (eCFP) and 514 nm (mCit). Note that mCit has ~ 2 fold higher extinction coefficient compared to eCFP.

[0018] Figure 2 depicts FRET as a readout of protein interaction, (a) Emission spectra of control SPASM sensors with calmodulin (CAM) linked to a 12 amino acid Gly-Ser-Gly peptide (GSG)₄ by 10, 20 and 30 nm ER/K a-helices. Spectra are taken at 4 mM Ca²⁺.

Excitation of eCFP (430 nm) results in eCFP emission (475 nm) without significant mCit emission (525 nm). (b) Sample emission spectrum of SPASM sensor with CAM linked by a 20 nm ER/K a-helix to a CI 5 W peptide(25) that interacts with 1 μΜ affinity to Ca²⁺ activated CAM. Excitation of eCFP (430 nm) results in eCFP emission (475 nm) with no detectable FRET for mCit in the absence of Ca (chelation by ImM EGTA) or strong FRET (large mCit peak at 525 nm) in the presence of 4 mM Ca²⁺.

[0019] Figure 3 depicts FRET ratio varies linearly with fraction of interacting protein in the bound state, (a) Emission spectra of SPASM sensors digested with Tev protease to cleave a site between CAM and the ER/K a-helix, resulting in a bimolecular interaction between eCFP-tagged CAM and mCit-tagged C15W peptide (~ 1 μΜ affinity). Sample spectra corresponding to 160 nM and 16 μΜ interaction affinities are shown, (b) Measured FRET Ratio (mCit/eCFP) as a function of sensor concentration (top abscissa) and equilibrium fraction bound (bottom abscissa). Dotted line represents fit to equation (3). Linear regression fit of FRET ratio to equilibrium fraction bound has R of 0.996.

[0020] Figure 4 depicts quantifying the equilibrium dissociation constant using FRET; determination of the effective concentration of interacting partners in a SPASM sensor, (a) FRET ratio (mCit/eCFP) as a function of protein concentration. Blue dots - intact SPASM sensor with C15W peptide in the presence of 4 mM Ca2+. Red dots - SPASM sensor with CI 5W peptide digested with Tev-protease to cleave SPASM sensor into eCFP-tagged CAM and mCit-tagged C15W peptide. Black line - Bimolecular interaction fit to equations (2) and (4) to calculate Kd of bimolecular interaction, (b) Competitive binding of Vasoactive Intestinal Peptide (VIP) (50 nM affinity) to Tev-cleaved SPASM sensor with CAM and Trp3 peptide (0.3 nM Kd). Inverse FRET ratio (eCFP/mCit) increases as eCFP-CAM/mCit-peptide interaction is disrupted by VIP. (c) Competitive binding of free unlabeled CAM to intact SPASM sensor linked to C15W peptide by 10 nm (red), 20 nm (green) and 30 nm (blue) ER/K a-helices. Inverse FRET ratio (eCFP/mCit) increases as the open conformation of the sensor is favored by increased unlabeled CAM concentration. (Insets (a)-(c) low

concentration data shown on log-axis for clarity), (d) Effective concentration of CAM- peptide interaction within the SPASM sensor measured by competitive binding to free unlabeled CAM. Black squares - Peptides that bind to CAM with indicated affinities in the presence of Ca2+. Blue diamond - ApoCAM binding to peptide with 0.7 μΜ affinity. All data shown are mean +SD of at least four different experiments.

[0021] Figure 5 depicts measuring off-rate (k_ope„) using FRET. Change in FRET ratio (mCit/eCFP) following the addition of 100 μΜ unlabeled CAM at time = 0 s to 10 nM of intact SPASM sensor with 10, 20 and 30 nm ER/K a-helix. SPASM sensor switches from the closed to the open conformation at the rate of k_open and is held in the open conformation by binding of unlabeled CAM to sensor peptide. There is no significant change in k_ope„ relative to the bi-molecular interaction (Tev cleaved sensor). Note that zero time on the abscissa corresponds to the time of addition of excess CAM. The time lag in the data set represents the time from mixing of unlabeled CAM with sensor to the beginning of data collection. The rate constants are mean±STD of three separate data sets.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

[0022] Many cellular processes involve proteins with multiple domains. This modular nature of proteins provides many advantages, providing increased stability and new cooperative functions. The amino acid linkers that join domains can play an important role in the structure and function of multi-domain proteins. There are numerous examples of proteins whose catalytic activity requires proper linker composition. In general, altering the physical characteristics of linkers connecting domains has been shown to affect protein stability, folding rates and domain-domain orientation {see George and Heringa (2003) Prot. Eng. 15:871-879).

[0023] Many studies of natural linker peptides in various protein families have come to the conclusion that linkers lack regular secondary structure, they display varying degrees of flexibility to match their particular biological purpose and are rich in Ala, Pro and charged residues. For example, in a study by Argos (1990) J Mol. Biol. 21 1 :943-958 it was concluded that preferred linker amino acids are mostly hydrophilic, often polar and usually small.

[0024] The ER7K motif, described in more detail below, represents an amino acid sequence composed of repeating sequences of negatively charged amino acid residues followed by positively charged amino acid residues and found to be present in a wide variety of proteins; at least 123 known distinct proteins in 137 organisms ranging from archaea to humans. The ER/K motif has been shown to form isolated stable a-helices in the proteins caldesmon, myosin X, myosin VI, GPC60 and MEKKK.

[0025] Despite the importance of understanding protein assembly in biological processes, there are few convenient methods for studying protein-protein interactions. Gene fusion offers one avenue for exploring protein-protein and/or protein domain interactions. The construction of a fusion protein involves the linking of two proteins or domains of proteins by a peptide linker. Unfortunately, linkers can perturb protein and/or domain function and thereby limit the usefulness of gene fusion in understanding protein-protein and/or protein domain interactions.

[0026] It has been reported that the ER/K a-helix behaves as an ideal worm like chain, acting as a relatively rigid spacer between protein domains. Thus, it would be anticipated that if an ER/K motif were used as a linker, separating two proteins or protein domains, that as the length of the ER/K motif linker (length of the a-helix) was increased, this would facilitate the interaction between the separated proteins or domains. That is, increasing the length of the ER/K motif would in turn facilitate the interaction between separated proteins or domains.

[0027] Contrary to this expectation, the present disclosure shows that increasing the length of the ER K motif (a-helix) results in the reduction in the frequency in the interaction of the separated proteins or domains and in turn a decrease in the effective concentration of the proteins or protein domains attached at the ends of the ER/K motif. By virtue of this unexpected property, the ER/K motif can be used to "dial-in" the frequency of interaction of two proteins or protein domains attached to its ends by, for example, altering the length of the ER/K motif and thereby, offers a way by which fusion proteins can be used to model protein- protein interactions, protein domain interactions and to identify compounds that interfere with such interactions.

[0028] In addition, these interactions can be studied in the absence of detailed knowledge of the structures of the protein or protein (polypeptide) domains. Because the ER/K motif represents a polypeptide sequence, a fusion polypeptide encompassing the ER/K motif can be genetically encoded and expressed in vitro or in vivo and thereby allowing for the detection, localization, and quantification of protein-protein and/or polypeptide domain interactions in the setting of an intact living experimental or clinical subject.

[0029] In order for the present invention to be more readily understood, certain terms and phrases are defined below as well as throughout the specification.

Definitions

[0030] Generally, the nomenclature used herein and many of the fluorescence, luminescence, computer, detection, chemistry, and laboratory procedures described herein are commonly employed in the art. Standard techniques are generally used for chemical synthesis, fluorescence or luminescence monitoring and detection, optics, molecular biology, and computer software and integration. Chemical reactions, cell assays, and enzymatic reactions are typically performed according to the manufacturer's specifications where appropriate. See, generally, Lakowicz, J. R. Topics in Fluorescence Spectroscopy, (3 volumes) New York: Plenum Press (1991), and Lakowicz, J. R. Emerging applications of florescence spectroscopy to cellular imaging: lifetime imaging, metal- ligand probes, multi photon excitation and light quenching, Scanning Microsc. Suppl. Vol. 10 (1996) pages 213-24, for fluorescence techniques; Sambrook et al, Molecular Cloning: A Laboratory Manual, 2^ed (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., for molecular biology methods; Cells: A Laboratory Manual, 1 st edition (1998) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., for cell biology methods; and Optics Guide 5 (Melles Griot, Irvine Calif), and Optical Waveguide Theory, Snyder & Love (published by Chapman & Hall) for general optical methods, all of which are incorporated herein by reference.

[0031] General methods for performing a variety of fluorescent or luminescent assays on luminescent materials are known in the art and are described in, e.g. , Lakowicz, J. R., Topics in Fluorescence Spectroscopy, volumes 1 to 3, New York: Plenum Press (1991 ); Herman, B., Resonance Energy Transfer Microscopy, in Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San

Diego: Academic Press (1989), pp. 219-243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361 ; and Bernard Valeur, "Molecular Fluorescence: Principles and Applications" Wiley VCH, 2002. Guidance in the selection and use of specific resonance acceptor moieties is available at, for example, Berlman, I. B., Energy transfer parameters of aromatic compounds, Academic Press, New York and London (1973), which contains tables of spectral overlap integrals for the selection of resonance energy transfer pairs. Additional information sources include the Molecular Probes Catalog (2003); Invitrogen New Products Catalog (2006); Full Invitrogen Catalog (2005); Invitrogen Drug Discovery Solutions Catalog (2004); Tsien et al., 1990 Handbook of Biological Confocal Microscopy, pp. 169-178.

[0032] It is noted here that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

[0033] The term "polymer" means any compound that is made up of two or more monomeric units covalently bonded to each other, where the monomeric units may be the same or different, such that the polymer may be a homopolymer or a heteropolymer. Representative polymers include peptides, polysaccharides, nucleic acids and the like, where the polymers may be naturally occurring or synthetic. [0034] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer. Methods for obtaining (e.g. , producing, isolating, purifying, synthesizing, and recombinantly manufacturing) polypeptides are well known to one of ordinary skill in the art.

[0035] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g. , an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g. , homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g. , norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. [0036] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0037] As to "conservatively modified variants" of amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention. [0038] The following eight groups each contain amino acids that are conservative

substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g. , Creighton, Proteins (1984)).

[0039] A charged amino acid refers to an amino acid residue which contains a charge at neutral pH. Charged amino acids include the negatively charged amino acids Aspartic acid (D) and Glutamic acid (E) as well as positively charged amino acids Histidine (H), Lysine (K), and Arginine (R).

[0040] As referred to herein, a linked fusion polypeptide is a polypeptide made up of two smaller polypeptide constituents, each constituent being derived from a single chain protein or a single chain of a multi-chain protein, where the constituents are combined in a non- naturally occurring arrangement using a peptide linker. Linked fusion polypeptides mimic some or all of the functional aspects or biological activities of the protein(s) from which their polypeptide constituents are derived. The constituent at the amino terminal portion of the linked fusion polypeptide is referred to herein as the first polypeptide. The constituent at the carboxy terminal portion of the linked fusion polypeptide is referred to herein as the second polypeptide. By "non-naturally occurring arrangement" is meant an arrangement which occurs only through in vitro manipulation of either the polypeptide constituents themselves or the nucleic acids which encode them.

[0041] A first polypeptide and a second polypeptide are joined through a linker of defined tertiary structure, particularly of defined alpha helical structure, to form a fusion protein. As used herein, the terms "fusion protein" or "fusion polypeptide" or grammatical equivalents herein are meant to denote a protein composed of a plurality of protein components, which are typically unjoined in their native state but are joined by their respective amino and carboxyl termini through a linker of defined tertiary structure to form a single continuous polypeptide. Plurality in this context means at least two, and preferred embodiments generally utilize a first and a second polypeptide joined through a linker. [0042] The term "peptide linker" or "linker" refers to polypeptide typically ranging from about 2 to about 350 amino acids in length, which is designed to facilitate the functional connection of two polypeptides into a linked fusion polypeptide. The term functional connection denotes a connection that facilitates proper folding of the polypeptides into a three dimensional structure that allows the linked fusion polypeptide to mimic some or all of the functional aspects or biological activities of the protein(s) from which its polypeptide constituents are derived. Linkers of the invention are typically able to fold into a

thermodynamically stable structure, for example an a-helix.

[0043] The presence of a helix in a sequence can be empirically determined, e.g. by circular dichroism (CD) spectra, where a polypeptide retains CD spectra characteristic of an a helix, and where the characteristic spectra persists in the presence of up to 2 M urea. Methods relating to spectral analysis of tertiary structures in polypeptides may be found, inter alia, in Turner et al. J Phys Chem B. 2007 Feb. 22; 1 1 1(7): 1834; Shepherd et al. J Am Chem Soc.

2005 Mar. 9; 127(9):2974-83; Thulstrup et al. Biopolymers. 2005 May; 78(l ):46-52; Jeong et al. Mol Cells. 2004 Feb. 29; 17(l):62-6; Maiti et al. J Am Chem Soc. 2004 Mar. 3;

126(8):2399-408; Maeda et al. J Pept Sci. 2003 February; 9(2): 106-13; Verzola et al.

Electrophoresis. 2003 March; 24(5): 794-800; Wallimann et al. J Am Chem Soc. 2003 125(5):1203-20; Lawrence et al. Biophys Chem. 2002 Dec. 10; 101-102:375-85.

[0044] The presence of a helix structure can also be predicted based on the amino acid sequence, e.g. as described by Phoenix et al. Curr Protein Pept Sci. 2002 April; 3(2):201-21 ; Miinoz et al. Curr Opin Biotechnol. 1995 August; 6(4):382-6; Godzik et al. J Comput Aided Mol Des. 1993 August; 7(4):397-438; Viswanadhan et al. Biochemistry. 1991 Nov. 19;

30(46)1 1 164-72; Gamier et al. Biochem Soc Symp. 1990; 57: 1 1-24.

[0045] a helix boundary residues (the first and last helical residues) are called Neap and Ccap at the N- and C-terminus, respectively (Richardson & Richardson, 1988), which yields the following representation: -N"-N'-Ncap-Nl-N2-N3- -C3-C2-Cl-Ccap-C'-C"-where Nl through CI are residues which contain a-helical phi, psi angles and the primed residues belong to the residues outside the helix on either end.

[0046] Preferred linkers of the invention are those possessing an ER/K motif. E, R, and K refer to the amino acids glutamic acid, arginine and lysine respectively. The ER/K motifs are polypeptides wherein at least 70% of the amino acid residues are combinations of E and/or R and/or K and are capable of forming an a helix. Often the ER/K motif is composed of stretches of 4 negatively charged amino acids, for example EEEE, EEED, EEDE, EDEE, and DEEE. In certain circumstances, the ER/K motif can be composed of one or more repeating sequences of four negatively charged amino acid residues followed by four positively charged amino acid residues. In certain circumstances, the ER/K motif can be composed of one or more repeating sequences represented by the formula (E₄(R/K)4)_n, wherein n can be 0 or any integer from 1 to 43, that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, or 43. However, a polypeptide need not include a sequence represented by the formula

(E₄(R/K)₄)_n in order to be considered as possessing an ER/K motif. For example, while polypeptide linker sequence SEQ. ID. NO. 1 :

EEEEKKKQQEEEAERLRRIQEEMEKERKRREEDEERRRKEEEERRMKLEMEAKRKQ EEEERKKREDDEKRKKK, derived from myosin VI of Sus scrofa, possesses an ER/K motif, with at least 70% of the amino acid residues being E, R, and K and capable of forming an a helix, the sequence does not possess a sequence represented by the formula (E₄(R/K)₄)_n Likewise, the polypeptide linker sequences, SEQ ID NO. 2:

EEEEKKKEEEEKKQKEEQERLAKEEAERKQKEEQERLAKEEAERKQKEEEERKQKE EEEERKQKEEEERKLKEEQERKAAEEKKAKEEAERKAKEEQERKAEEERKKKEEEE RLERERKE, and SEQ ID NO:3

EEEEKKKEEEEKKQKEEQERLAKEEAERKQKEEQERLAKEEAERKQKEEEERKQKE EEEERKQKEEEERKLKEEQERKAAEEKKAKEEAERKAKEEQERKAEEERKKKEEEE RLERERKEREEQEKKAKEEAERIAKLEAEKKAEEERKEKEEEERKAKEEEERKKKEE QERLAKEKEEAERKAAEEKKAKEEQERKEKEEAERKQR, both derived from a Kelch- motif family protein of Trichomonas vaginalis, possess an ER K motif, with at least 70% of the amino acid residues being E, R, and K and capable of forming an a helix, the sequence does not possess a sequence represented by the formula (E₄(R K)₄)_n The boundaries of the ER/K motif are defined by the presence of the amino acids C, F, G, H, N, P, S, T, W, and Y, that is, these amino acids identify and occupy N' and C. Thus, an ER/K motif can be defined in certain circumstances as a polypeptide composed of at least 70% of the amino acid residues being E, R, and K and capable of forming an a helix, with the ends of the ER/K motif defined by the first instance of anyone of the amino acids C, F, G, H, N, P, S, T, W, and Y.

[0047] Linkers of the invention are between 4 and 350 amino acids in length. Preferred linkers are at least about 16 amino acids in length, more usually at least about 30 amino acids in length and not more than about 346 amino acids in length.

[0048] In some embodiments, the linker of the present invention is a polypeptide of from about 30 to about 73 amino acids in length, having at least about 90% or at least about 95% sequence identity to SEQ ID NO. 1. In other embodiments, the linker of the present invention is a polypeptide of from about 30 to about 120 amino acids in length, having at least 90% or at least 95% sequence identity to SEQ ID NO. 2. In other embodiments, the linker of the present invention is a polypeptide of from about 30 to about 346 amino acids in length, having at least 90% or at least 95% sequence identity to SEQ ID NO. 3.

[0049] The terms "identical" or percent "identity," in the context of two or more polypeptide or nucleic acid sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0050] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0051] Optimal alignment of sequences for comparison can be conducted, e.g. , by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444

(1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0052] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 1 1 , an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0053] Linkers of the invention do not include polypeptide sequences which can be represented by the formula A(EAAAK)_nA, wherein n can be 2, 3, 4, or 5.

[0054] Effective concentration refers to a measure of strength of the interaction between a protein pair within the biosensor and can be estimated using a competitive binding assay (see Methods).

[0055] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g. , cDNA or genomic DNA), RNA molecules (e.g. , mRNA), analogs of the

DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule can be single-stranded or double-stranded, but

advantageously is double-stranded DNA. An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. A "nucleoside" refers to a base linked to a sugar. The base may be adenine (A), guanine (G) (or its substitute, inosine (I)), cytosine (C), or thymine (T) (or its substitute, uracil (U)). The sugar may be ribose (the sugar of a natural nucleotide in RNA) or 2- deoxyribose (the sugar of a natural nucleotide in DNA). A "nucleotide" refers to a nucleoside linked to a single phosphate group..

[0056] As used herein, the term "polynucleotide" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. Polynucleotide encompasses the terms "nucleic acid," "nucleic acid sequence," or "oligonucleotide."

[0057] As used herein, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein.

[0058] "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. [0059] The term "expression" is used herein to mean the process by which a polypeptide is produced from a nucleic acid. When the nucleic acid is DNA, this process involves the transcription of the gene into mRNA and the translation of this mRNA into a polypeptide. Depending on the context in which it is used, "expression" may refer to the production of RNA, protein, or both. [0060] The term "gene product" as used herein means an RNA (for example, a messenger RNA (mRNA) or a micro RNA (miRNA)) or protein that is encoded by the gene. [0061] As used herein, the term "isolated" refers to a molecule that is substantially pure. An isolated protein may be substantially pure if it is, for example, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 98%, 99% or more free of a polypeptide or polypeptides with less than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 98%, 99% identity to the protein to be isolated. [0062] As used herein, the term "reporter" refers to a signal generating compound. Suitable signal-generating compounds include, for example, chromagens, catalysts such its enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums and luminol, radioactive elements, and direct visual labels. Suitable enzymes include, for example, alkaline phosphatase, horseradish peroxidase, β-lactamase β-galactosidase can be used as the reporter molecule. The selection of particular label is not critical, but it must be capable of producing a signal either by itself or in conjunction with one or more additional substances.

[0063] The term "RET" stands for resonance energy transfer, and refers to the transmission (e.g., radiationless) of an energy quantum from its site of absorption (the donor) to the site of its utilization (the acceptor) in a molecule, or system of molecules, by resonance interaction between donor and acceptor species, over distances considerably greater than interatomic. A donor is a moiety that initially absorbs energy (e.g. , optical energy or electronic energy). RET is sometimes referred to as fluorescent resonance energy transfer or Forster resonance energy transfer (both abbreviated FRET). RET includes TR-FRET, FRET and LRET. LRET uses the term "luminescence" which is more general than the term "fluorescence" (as in FRET).

[0064] The term "energy transfer pair" or RET pair as used herein refers to any two moieties that participate in energy transfer. In some embodiments, one of the moieties acts as a fluorescent reporter, e.g. , a donor, and the other acts as an acceptor, which may be a quenching compound or a compound that absorbs and re-emits energy in the form of a detectable signal, e.g. , a fluorescent signal ("Fluorescence resonance energy transfer." Selvin P. (1995) Methods Enzymol 246:300-334; dos Remedios C. G. (1995) J. Struct. Biol.

1 15 : 175-185; "Resonance energy transfer: methods and applications." Wu P. and Brand L. (1994) Anal Biochem 218: 1-13). RET is a distance-dependent and/or orientation-dependent interaction between two moieties in which excitation energy, (e.g. , light) is transferred from a donor to an acceptor without emission of a photon. Deuschle et al. (Protein Sci. 2005 14: 2304-2314) and Smith et al. (Protein Science, 2005, 14:64-73) describes how the distance between the RET pair and their orientation affect RET. [0065] "Fluorescence resonance energy transfer" is used interchangeably with and "LRET" and refers to an energy transfer phenomenon in which the excited state energy (e.g. , light) emitted by an excited luminescent group is absorbed at least partially by a luminescence- modifying group of the invention and re-emitted at a different (e.g. , longer) wavelength by the luminescence-modifying group. FRET depends on energy transfer between the luminescent group and the luminescence-modifying group. The efficiency of FRET depends at least in part on the distance between the luminescence modifying group and the

luminescent group. In contrast to excimers and exciplex fluorescence, FRET pairs do not require the dye molecules forming the complexes to be in very close proximity. FRET is commonly used in several detection modes to detect, characterize or identify a variety of biologically active molecules including nucleic acids, e.g. , oligonucleotides, peptides (e.g. , peptides including one or more protease cleaveage site) and proteins. One of the advantages of FRET is that fluorescence arises under physiologically relevant conditions (e.g., pH between about 7 and about 8, e.g., 7.3-7.5). [0066] The acceptor may be fluorescent and emit the transferred energy at a longer wavelength, or it may be non- fluorescent, e.g., serves to diminish the detectable fluorescence of the reporter molecule (quenching). RET may be either an intermolecular or intramolecular event, and is typically dependent on the inverse sixth power of the separation of the donor and acceptor, making it useful over distances comparable with the dimensions of biological macromolecules. Thus, the spectral properties of the energy transfer pair as a whole, change in some measurable way if the distance and/or orientation between the moieties is altered. Self-quenching probes incorporating fluorescent donor-non-fluorescent acceptor

combinations have been developed for detection of proteolysis (Matayoshi, (1990) Science 247:954-958) and nucleic acid hybridization ("Detection of Energy Transfer and

Fluorescence Quenching" Morrison, L., in Nonisotopic DNA Probe Techniques, L. Kricka, Ed., Academic Press, San Diego, (1992) pp. 31 1 -352; Tyagi S. (1998) Nat. Biotechnol. 16:49-53; Tyagi S. (1996) Nat. 14(8):947-8).

[0067] The term "acceptor" or "acceptor moiety" refers to a chemical or biological moiety that accepts energy via RET. In RET applications, acceptors may re-emit energy transferred from a donor moiety (e.g. , fluorescent or luminescent moiety), for example as fluorescence. In some embodiments, the transfer is via RET or TR-RET. As used herein, a donor moiety (e.g., fluorescent or luminescent moiety) and an acceptor moiety (e.g. , fluorescent moiety) are referred to as a "RET pair." Examples of acceptors include coumarins and related fluorophores; xanthenes such as fluoresceins and fluorescein derivatives; fluorescent proteins such as GFP and GFP derivatives; rhodols, rhodamines, and derivatives thereof; resorufins; cyanines; difluoroboradiazaindacenes; and phthalocyanines. Acceptors, including fluorescent acceptor moieties, can also be useful as fluorescent probes in FP assays. In most applications, the donor and acceptor dyes are different, in which case RET can be detected by the appearance of sensitized fluorescence of the acceptor or by quenching of donor fluorescence.

[0068] In some embodiments of the invention, RET increases upon binding or release of the ligand by a modified ligand binding molecule. In one embodiment, RET increases upon ligand binding. In some embodiments, this increase will be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 100%, greater than 200%, greater than 300%, greater than 400%, greater than 750%, greater than 1000%, greater than 2000%, greater than 3000%, or greater than 5000%). In some embodiments, this change in RET is greater than 10% and less than 1000%), greater than 100% and less than 400%, greater than 20% and less than 200%, greater than 200% and less than 750%, greater than 400% and less than 1000%, greater than 750% and less than 3000%, or greater than 1000% and less than 5000%.

[0069] The term "complementing fragments" or "complementary fragments" when used in reference to a reporter polypeptide refer to fragments of a polypeptide that are individually inactive (e.g. , do not express the reporter phenotype), wherein binding of the complementing fragments restores reporter activity. The terms "self-complementing", "self-assembling", and "spontaneously-associating", when used to describe two fragments of the same protein, mean that the fragments are capable of reconstituting into an active protein when the individual fragments are soluble and are sufficiently close to or contact one another. Fusion Protein Constructs

[0070] The instant disclosure provides biosensors, these biosensors are fusion protein constructs encompassing a first polypeptide and a second polypeptide that are joined by a linker as described above to form a fusion polypeptide. By "fused" or "operably linked" herein is meant that the polypeptides are linked together to form a continuous polypeptide chain.

[0071] As outlined below, the fusion polypeptide (or fusion polynucleotide encoding the fusion polypeptide) can comprise further components as well, including multiple peptides at multiple loops, fusion partners, etc. The precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion or binding characteristics of the binding partner. The optimal site will be determined by routine experimentation. [0072] The first and second polypeptide components, which are separated by the linker, each provide for a distinct functional entity. Such functional entities will typically correspond to one or more polypeptide domains. As is known in the art, a protein domain is a substructure produced by any part of a polypeptide chain that can fold independently into a compact, stable structure. A domain usually contains between about 35 to about 350 amino acids, and it is the modular unit from which many larger proteins are constructed. The different domains of a protein are often associated with different functions. The smallest protein molecules contain only a single domain, whereas larger proteins can contain as many as several dozen domains. The central core of a domain can be constructed from a helices, from β sheets, or from various combinations of these two fundamental folding elements. [0073] The invention further provides nucleic acids encoding the fusion polypeptides of the invention. As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the fusion proteins of the present invention. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the fusion protein.

[0074] A variety of nucleic acid constructs can be made to express the fusion proteins of the instant invention. The expression constructs may be self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Alternatively, for purposes of cell-free expression the construct may include those elements required for transcription and translation of the desired polypeptide, but may not include such elements as an origin of replication, selectable marker, etc. Cell-free constructs may be replicated in vitro, e.g. by PCR, and may comprise terminal sequences optimized for amplification reactions.

[0075] Generally, expression constructs include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the fusion protein. The term

"control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular expression system, e.g. mammalian cell, bacterial cell, cell-free synthesis, etc. The control sequences that are suitable for prokaryote systems, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cell systems may utilize promoters, polyadenylation signals, and enhancers.

[0076] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Linking is accomplished by ligation or through amplification reactions. Synthetic oligonucleotide adaptors or linkers may be used for linking sequences in accordance with conventional practice.

[0077] In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.

[0078] Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention. In a preferred embodiment, the promoters are strong promoters, allowing high expression in in vitro expression systems, such as the T7 promoter.

[0079] In addition, the expression construct may comprise additional elements. For example,^' the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification. In addition the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used. [0080] "Host cell" refers to a prokaryotic or eukaryotic cell that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector. When host cells are "transfected" or "transformed" with nucleic acid molecules, they are referred to as "engineered" or "recombinant" cells or host cells, e.g. , a cell into which an exogenous nucleic acid sequence, such as, for example, a vector, has been introduced. Therefore, recombinant cells are distinguishable from naturally-occurring cells which do not contain a recombinantly introduced nucleic acid. [0081] Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Cell types available for vector replication and/or expression include, but are not limited to, bacteria, such as E. coli (e.g. , E. coli strain RR1 , E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coli W31 10 (F-, lambda-, prototrophic, ATCC No. 273325), DH5a, JM109, and KC8, bacilli such as Bacillus subtilis; and other enterobacteriaceae such as Salmonella typhimurium, Serratia marcescens, various Pseudomonas specie, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells (STRATAGEN®, La Jolla). In certain embodiments, bacterial cells such as E. coli LE392 are particularly contemplated as host cells for phage viruses.

[0082] Examples of eukaryotic host cells for replication and/or expression of a vector include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, PerC6, CHO and PC 12 cells and derivatives thereof. Many host cells from various cell types and organisms are available and would be known to one of skill in the art. Similarly, a viral vector may be used in conjunction with either a eukaryotic or prokaryotic host cell, particularly one that is permissive for replication or expression of the vector. [0083] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. The host cells generally will have been engineered to express a screenable or selectable marker. [0084] Suitable methods for nucleic acid delivery to the host cell with the current invention include virtually any method by which a nucleic acid molecule (e.g. , DNA) can be introduced into a cell as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo

transfection (Wilson, et al, Science, 244: 1344-1346 (1989), Nabel et al, Science, 244: 1342- 1344 (1989), by injection (U.S. Patent Nos. 5,994,624, 5,981 ,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, including microinjection (Harlan and Weintraub, J. Cell Biol., 101(3): 1094-1099 (1985); U.S. Patent No. 5,789,215; by electroporation (U.S. Patent No. 5,384,253; Tur-Kaspa, et al., Mol: Cell Biol., 6:716-718

(1986) ; Potter, et al., Proc. Natl. Acad Sci. USA, 81 :7161 -7165 (1984); by calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467 (1973); Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752 (1987); Rippe, et al., Mol. Cell Biol., 10:689-695 (1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell Biol., 5: 1 188-190 (1985); by direct sonic loading (Fechheimer, et al., Proc. Natl. Acad Sci. USA, 89(17):8463- 8467 (1987); by liposome mediated transfection (Nicolau and Sene, Biochem. & Biophys. Acta., 721 : 185-190 (1982); Fraley, et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352 (1979); Nicolau, et al, Meth. Enzym., 149: 157-176 (1987); Wong, et al., Gene, 10:879-894 (1980); Kaneda, et al., Science, 243:375-378 (1989); Kato, et al, .J. Biol. Chem., 266:3361 -3364 (1991) and receptor-mediated transfection (Wu and Wu, J. Biol. Chem., 262:4429-4432

(1987) ; Wu and Wu, J Biol Chem. 263(29): 14621 -4. (1988); by PEG-mediated

transformation of protoplasts (Omirulleh, et al, Plant Mol. Biol, 21(3):415-428 (1987); U.S. Patent Nos. 4,684,61 and 4,952,500 ; by desiccation/inhibition-mediated DNA uptake (Potrykus, et al. Mol. Gen. Genet., 199(2): 169- 177 (1985), and any combination of such methods.

[0085] In some embodiments, the fusion construct comprises a protease recognition site. Proteases are well characterized enzymes that cleave other proteins at a particular site. One family, the Ser/Thr proteases, cleaves at serine and threonine residues. Other proteases include cysteine or thiol proteases, aspartic proteases, metalloproteinases, aminopeptidases, di & tripeptidases, carboxypeptidases, and peptidyl peptidases. The choice of these is left to the skilled artisan and certainly need not be limited to the molecules described herein. It is well known that enzymes have catalytic domains and these can be used in place of full length proteases. Such are encompassed by the invention as well. [0086] In one embodiment, the biosenor fusion protein possesses an engineered protease recognition site for the Tev protease. Other specific cleavage sites for proteases may also be used, as will be clear to the skilled artisan. Assays for the Detection of Protein-Protein Interactions

[0087] A number of methods for studying and assaying protein-protein interactions are known. Approaches include the use of assay based on the classical complementation of and ω fragments of β-galactosidase, a protein complementation assay, described by Rossi F , C A Charlton and H M Blau (1997) Proc Nat Acad Sci (USA) 94 8405-8410 and resonance energy transfer between dye-coupled proteins, e.g. FRET.

Protein Complementation Assays (PCA)

[0088] Protein complementation methods are a common method for studying the dynamics of protein-protein interactions in cells. (Remy and Michnick, Nature Methods 3(12):977-979 (2006)). In this strategy, two proteins of interest are fused to complementary fragments of a reporter protein. If the proteins interact, the reporter fragments are brought together, fold into the native structure and the PCA reporter activity is reconstituted {e.g., see Michnick, Curr. Opin. Struct Biol. 11 :472-477 (2001). Some related methods are based on fluorescent proteins because a signal is provided by the intrinsic fiuorophore {e.g. see Ghosh et al., Am. Chem. Soc. 122:5658-5659 (2000); Hu et al., Mo/. Cell 9:789-798 (2002); Remy &

Michnick, Methods 32:381-388 (2004); Remy et al., Nat Cell Biol. 6:358-365 (2004);

Magliery et al, J. Am. Chem. Soc. 127: 146-157 (2005); and Macdonald et al, Nat Chem. Biol. 2:329-337 (2006)).

[0089] In some embodiments, the interaction of the test proteins is reported by applying a protein complementation assay.

[0090] WO 2005/007822 discloses a method for determining if a test compound, or a mix of compounds, modulates the interaction between two proteins of interest. The determination is made possible via the use of two recombinant molecules, one of which contains the first protein a cleavage site for a proteolytic molecules, and an activator of a gene. The second recombinant molecule includes the second protein and the proteolytic molecule. If the test compound binds to the first protein, a reaction is initiated whereby the activator is cleaved, and activates a reporter gene.

[0091] WO 03/076932 describes a method for detecting and analyzing protein interactions in a cell, comprising the following steps: a) providing the activity of at least one enzyme selected from the group consisting of recombinases and proteases in the cell following a protein interaction; b) continued generation of an active reporter protein in the respective cell following the enzyme activity from step a) for a period of time exceeding that of the protein interaction from step a), and; c) producing a detection signal using the reporter proteins generated in b). It also describes reverse configurations of the above method for detecting and analyzing protein interactions in a cell, whereby following the induced dissociation of a defined interaction between proteins, the activity of at least one enzyme selected from the group consisting of recombinases and proteases is provided in the cell and is converted into a permanent detection signal of the cell.

FRET

[0092] In some embodiments, a biosensor for detecting protein-protein interactions is disclosed wherein the interaction of the proteins is detected by FRET. [0093] FRET (Forster Resonance Energy Transfer) represents a distance dependent, non- radiative transfer of energy from an excited donor (D) to an acceptor molecule (A). FRET occurs when the emission spectrum of a fluorescent molecule, an energy donor, overlaps with the absorption spectrum of another molecule, an energy acceptor. When the donor is excited, FRET results in reduction of the intensity of donor emission, as energy from the donor in its excited state is transferred to the acceptor. The acceptor can be either fluorescent or non- fluorescent. If the acceptor is fluorescent, the intensity of acceptor emission is increased as a result of FRET. A second critical criterion of energy transfer in FRET is close proximity between energy donor and acceptor. The efficiency of energy transfer in FRET decreases with or nearly to the 6th power of the distance between the donor and acceptor. A principal factor in the strong dependence of FRET on distance is believed to be that energy transfer between donor and acceptor requires a coupled dipole-dipole interaction, wherein transfer occurs when the dipole orientations are aligned, with no intermediate photon involved.

Regardless of mechanism, the distance between donor and acceptor molecules is a dominant factor in determining the extent of energy transfer, with energy transfer dropping off precipitously as the distance is increased beyond the optimal separation.

[0094] FRET has been used in biochemical applications within the 1 to 10 nm scale (K. E. Sapsford et al., Angew. Chem. Int. Ed., 45, 4562, 2006) (e.g. protein-protein binding, protein folding, molecular interactions at and in cell membranes, DNA hybridization and sequencing, immu-noreactions of antigens and antibodies). A FRET measurement is a distance between about 1 and 20 nm or a concentration of a specific substance.

[0095] Since the first genetically encoded protein FRET biosensor to detect calcium signaling over a decade ago (31 ), a large number of similar designs have been developed to directly detect the activation of proteins involved in cell signaling pathways (21 , 32, 33). These FRET biosensor designs are based on a single polypeptide chain with a CFP and YFP variant separated by two protein domains that interact in response to cell signaling events such as calcium influx or phosphorylation of one of the protein domains. The two protein domains in these biosensors are separated by a short ~ 3 amino acid linker composed of Gly and Ser. As a consequence, the CFP/YFP pair is in close proximity with substantial FRET under basal conditions. Activation of the sensor causes the protein domains to interact, which results in a decrease in distance and rotational freedom between the FRET pair and therefore increased FRET. The change in FRET signal, assessed from the emission spectrum following donor excitation, is limited by the close proximity of the CFP/YFP pair enforced by the short linker between the protein domains. Enhancing the change in FRET signal involves either changing the specific linker sequence by trial and error (34) or altering the arrangement of protein elements in the biosensor using circular permutation (35). In this study, we find that the ER/K a-helix minimizes baseline FRET by virtue of its extended structure (Fig. 3 a), resulting in a substantial increase in the dynamic range of the biosensor following activation (Fig. 3b). The availability of different length ER/K ot-helices allows systematic alteration of the effective concentration of the interacting proteins (Fig. 5b), such that the ER/K a-helix provides an affinity barrier that separates the protein domains in the inactive conformation. The biosensor constructs with the ER K a-helix are designed with (Gly-Ser-Gly)₄ linkers between the proteins to ensure rotational freedom between the protein domains. This feature combined with the wide range of conformations explored by the ER/K a-helix (Fig. 1) should allow protein domains with diverse binding interfaces to interact without steric hindrance. Further, the plasmid constructs for the biosensors containing the ER K a-helix have been designed with unique restriction enzyme sites flanking different protein domains to allow easy cut-and-paste cloning using polymerase chain reaction (PCR).

[0096] A component of a binding assay is a detectable label (also termed a tracer) that can be measured to provide an indication of the amount of binding. Labels employed in binding assays include radioactive elements, chemically reactive molecules, chromogenic molecules and nanoscale molecular assemblies, inorganic catalysts, and enzymes, among others. Labels based on luminescence, including chemiluminescence and fluorescence, are finding wide usage due to several advantages associated with these labels, including sensitivity, simplicity, linear range, and low toxicity. Luminescence is the emission of light from molecules in electronically excited states. Fluorescence is luminescence resulting from absorption of excitation light. The light emitted in fluorescence typically is at a lower energy or longer wavelength than the excitation light, with the difference between excitation and emission wavelengths termed the Stokes shift. A single fluorescent molecule can go through many cycles of excitation and emission to produce thousands of photons. This phenomenon, combined with the advance of technologies such as photomultiplier tubes (PMT) for the sensitive detection of photons, contributes to the high sensitivity associated with fluorescence technologies.

[0097] Further extension of these methods to noninvasively detect, localize, and quantify protein-protein and/or polypeptide domain interactions in the setting of an intact living experimental or clinical subject could have important implications for a wide variety of biological research endeavors, drug discovery, and molecular medicine. In particular, the visual representation, characterization, quantification, and timing of these biological processes in living subjects could create unprecedented opportunities to complement available in vitro or cell culture methodologies, in order: (i) to accelerate the evaluation in living subjects of novel drugs that promote or inhibit active homodimeric or heterodimeric protein assembly or polypeptide domain interaction, and (ii) to characterize more fully known protein-protein or polypeptide domain interactions (e.g., the reasons for, and the factors that drive their association) in the context of whole-body physiologically-authentic environments.

[0098] Accordingly, in some embodiments a nucleic acid and/or nucleic acids encoding and capable of expressing proteins or protein domains separated by an ER/K helix are introduced into an organism.

[0099] In some embodiments, the ER/K biosensor and methods described herein can be used to monitor and assess biological interactions in a transgenic organism.

[00100] As used herein, the term "subject" and "organism" includes humans, mammals (e.g. , cats, dogs, horses, etc.), living cells, and other living organisms. A living organism can be as simple as, for example, a single eukaryotic cell or as complex as a mammal. Typical organisms to which embodiments of the present disclosure may be administered will be mammals, particularly primates, especially humans. A wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g. , mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs.

[00101] In this regard, the present disclosure also includes transgenic animals comprising exogenous DNA incorporated into the animal's cells to effect a permanent or transient genetic change, preferably a permanent genetic change. Permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACS, and the like. Generally, transgenic animals are mammals, most typically mice.

[00102] The exogenous nucleic acid sequence may be present as an extrachromosomal element or stably integrated in all or a portion of the animal's cells, especially in germ cells.

[00103] Unless otherwise indicated, a transgenic animal includes stable changes to the germline sequence. During the initial construction of the animal, chimeric animals (chimeras) are generated, in which a subset of cells has the altered genome. Chimeras may then be bred to generate offspring heterozygous for the transgene. Male and female heterozygotes may then be bred to generate homozygous transgenic animals.

[00104] For example, an introduced transgene may include genes corresponding to the disclosed biosensor, which may become functional via complementation or reconstitution when exposed to appropriate test proteins or, alternatively, which may become nonfunctional when exposed to a particular test compound or protein. Such a transgene, when introduced into a transgenic animal or cells in culture, is useful for testing potential therapeutic agents known or believed to interact with a particular target protein implicated in a disease or disorder. Where the introduced gene is a coding sequence, it is usually operably linked to a promoter, which may be constitutive or inducible, and other regulatory sequences required for expression in the host animal. [00105] Transgenic animals can be produced by any suitable method known in the art, such as manipulation of embryos, embryonic stem cells, etc. Transgenic animals may be made through homologous recombination, where the endogenous locus is altered.

Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACS, and the like.

[00106] Numerous methods for preparing transgenic animals are now known and others will likely be developed. See, e.g. , U.S. Pats. Nos. 6,252,131 , 6,455,757, 6,028,245, and 5,766,879, all incorporated herein by reference. The microinjection technique is particularly useful for incorporating transgenes into the genome without the accompanying removal of other genes.

[00107] In some embodiments, the transgenic organism is a plant. [00108] Additionally, for diagnostic and research applications, body fluids and cell samples of the above subjects will be suitable for use, such as, without limitation, blood, urine, or tissue samples.

[00109] The term "sample" can refer to a tissue sample, cell sample, a fluid sample, and the like. The tissue sample can include hair (including roots), buccal swabs, blood, saliva, spinal fluid, lymph, semen, muscle, or from any internal organs. The body tissue can include, but is not limited to, skin, or any organ, including, without limitation, heart, lung, liver, intestine, bone marrow, and/or brain. In the present disclosure, the source of the sample is not critical.

[00110] In some embodiments, nucleic acids encoding fusion proteins encompassing two or more proteins or polypeptide domains separated by an ER/K linker is administered to a subject. In other embodiments, fusion proteins encompassing two or more proteins or polypeptide domains separated by an ER/K linker is administered to a subject.

[00111] For in vivo delivery, the fusion proteins of the invention containing an ER/K motif will be formulated into pharmaceutical compositions and will generally be

administered parenterally. The pharmaceutical compositions will also contain a

pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S

PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J., 1991). As is apparent to those skilled in the art in view of the teachings of this specification, an effective amount of viral vector which must be added can be empirically determined.

[00112] Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. [00113] In some embodiments, a nucleic acid encoding a fusion protein, wherein at least two proteins or polypeptide domains is separated by at least one ER/K motif is administered to a subject in need thereof. In some embodiments, the nucleic acid is deleivered to the subject as a viral vector. Methods of determining the most effective means and dosages of administration are well known to those of skill in the art and will vary with the viral vector, the composition of the therapy, the target cells, and the subject being treated. Single and multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

[00114] Pharmaceutical compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the protein of interest, e.g. , an amount sufficient to reduce or ameliorate symptoms of the disease state in question or an amount sufficient to confer the desired benefit. In animal models for gene transfer or of disease, or in cell culture-based applications, effective amount of the exogenous nucleic acid composition is such as to produce the desired effect in a host that can be monitored using several end- points known to those skilled in the art. Effective gene transfer of an exogenous nucleic acid to a host cell is confirmed by evidence of the transferred nucleic acid or expression of the exogenous nucleic acid within the host (e.g. , using the polymerase chain reaction in conjunction with sequencing, Northern or Southern hybridizations, or transcription assays to detect the nucleic acid in host cells, or using immunoblot analysis, antibody-mediated detection, mRNA or protein half-life studies, or particularized assays to detect protein or polypeptide encoded by the transferred nucleic acid, or impacted in level or function due to such transfer). One such particularized assay includes the Western immunoassay for the detection of the protein encoded by the exogenous nucleic acid. These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. [00115] By "administration" is meant introducing a compound of the present disclosure into a subject. The preferred route of administration of the compounds is intravenous. However, any route of administration, such as oral, topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments can be used.

[00116] "Treating" or "treatment" of a disease (or a condition or a disorder) includes preventing the disease from occurring in an animal that may be predisposed to the disease but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), inhibiting the disease (slowing or arresting its development), providing relief from the symptoms or side-effects of the disease (including palliative treatment), and relieving the disease (causing regression of the disease). With regard to cancer, these terms also mean that the life expectancy of an individual affected with a cancer will be increased or that one or more of the symptoms of the disease will be reduced.

PROBES

[00117] Examples of probes that can be used in the disclosed methods and

compositions, include, without limitation fluorescein, rhodamine, FITCs (e.g. , fluorescein- 5- isothiocyanate), 5 -carboxy fluorescein, 6-carboxyfluorescein, 5,6-carboxyfluorescein, 7- hydroxycoumarin-3-carboxamide, 6-chloro-7-hydroxycoumarin-3-carboxamide,

dichlorotriazinylaminofluorescein, tetramethylrhodamine-5-isothiocyanate,

tetramethylrhodamine-6-isothiocyanate, succinimidyl ester of 5-carboxyfluorescein, succinimidyl ester of 6-carboxyfluorescein, 5-carboxytetramethylrhodamine, 6- carboxymethylrhodamine, and 7-amino-4-methylcoumarin-3-acetic acid. Other suitable fluorophores include the Cy family of fluorophores (Cy 3, Cy3B, Cy3.5, Cy5; available from GE Healthcare, Chalfont St. Giles, United Kingdom); the Alexa Fluor family (available from Invitrogen, Carlsbad, Calif.); the BODIPY family (available from Invitrogen, Carlsbad, Calif.); carbopyronins; squarines; cyanine/indocyanines; benzopyrylium heterocyles; and amide-bridged benzopyryliums. [00118] Fluorescent proteins and mutants can also be used as fluorescent acceptor moieties. Examples include firefly, bacterial, or click beetle luciferases, aequorins, and other photoproteins (for example as described in U.S. Pat. Nos. 5,221 ,623; 5,683,888; 5,674,713; 5,650,289; and 5,843,746. GFP and GFP mutants are particularly useful in applications using Tb(III)-containing metal complexes. A variety of mutants of GFP from Aequorea victoria have been created that have distinct spectral properties, including improved brightness, and enhanced expression and folding in mammalian cells compared to the native GFP (e.g. , see Table 7 of U.S. Pat. No. 6,410,255; Green Fluorescent Proteins, Chapter 2, pages 19 to 47, edited by Sullivan and Kay, Academic Press; and U.S. Pat. Nos. 5,625,048; 5,777,079; and 5,804,387. Fluorescent proteins can be attached chemically or a ligand binding protein coding region can be engineered to express a fusion protein comprised of a fluorescent protein. Fluorescent proteins can be used as the acceptor moiety, donor moiety or both. [00119] In some embodiments, an acceptor moiety (e.g., fluorescent) for use in multiplex assays exhibits characteristics useful for RET applications. Examples of suitable acceptor fluorophores in RET assays using Tb(III)-containing luminescent metal complexes include: fluorescein (and its derivatives); rhodamine (and its derivatives); Alexa Fluors 488, 500, 514, 532, 546, 555, 568 (available from Molecular Probes); BODIPYs FL, R6G, and TMR (available from Molecular Probes); Cy3 and Cy3B (available from Amersham

Biosciences), and IC3 (available from Dojindo Molecular Technologies, Gaithersburg, Md.). Examples of suitable acceptor fluorophores in RET assays using Eu(III)-containing luminescent metal complexes include: Alexa Fluors 594, 610, 633, 647, and 660 (available from Molecular Probes); BODIPYs TR, 630/650, and 650/665 (available from Molecular Probes); Cy5 (available from Amersham Biosciences) and IC5 (available from Dojindo Molecular Technologies). Any fluorescent protein from any species could also serve as a suitable acceptor e.g. , wild type (native or recombinant) or a mutant of Green Fluorescein Protein e.g. , from Aequorea Victoria, a blue fluorescent protein (BFP), a red fluorescent protein (RFP), a cyan fluorescent protein (CFP), or a yellow fluorescent protein (YFP).

Suitable fluorophores for use in the present invention are commercially available, e.g. , from Invitrogen, Carlsbad, Calif., Attotec (Germany), Amersham, and Biosearch Technologies (Novato, Calif.). Methods for incorporating fluorophores into proteins are known to those of skill in the art; e.g. , U.S. Pat. Nos. 5,898,069 and 6,410,255.

[00120] In some embodiments, the acceptor moiety is not a GFP. In some

embodiments, the acceptor moiety is not an aequorin protein.

[00121] In some embodiments, the donor moiety is not a GFP. In some embodiments, the donor moiety is not an aequorin protein.

[00122] The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein. [00123] Whereas, particular embodiments of the invention have been described herein for purposes of description, it will be appreciated by those skilled in the art that numerous variations of the details may be made without departing from the invention as described in the appended claims.

EXAMPLES

EXAMPLE 1: Estimation of interaction rate of two polypeptides separated by a ER/K linker

[00124] In order for a linker to regulate the frequency of interaction of two proteins or protein domains at its ends, the linker must either limit the on-rate (k_ci_ose) or off-rate (k_open) of the interaction. Linkers possessing the ER/K motif were tested to determine whether either of these parameters was limited.

[00125] A Monte Carlo simulation of the ER/K a-helix, modeled as an ideal wormlike-chain with a length (L) of 15 nm, provided a distribution of conformations adopted by different length ER/K a-helices. Extensive conformation sampling (> 20 million) was used to estimate the probability of the two ends of the ER/K a-helix being close enough to facilitate interaction between two proteins. This probability is defined as the fraction of total number of conformations sampled with end-to-end distance less than 2 nm. 2 nm was used as the distance between the ends of the ER/K a-helix when two small proteins (~ 20 kDa with hydrodynamic radius (R_h) ~ 2 nm) bind to each other. However, the conclusions of different models do not vary for lengths ranging from 1 -3 nm. This probability was computed over a range of ER/K a-helix lengths (L) and was found to be vanishingly small (< 10^"5) for L comparable to the L_p. Mehraeen et al. (15) have previously reported theoretical distributions for end-to-end distance distributions for WLCs. Their theoretical distribution show that the cumulative distribution of end-to-end distances less than 2 nm are also on the order of 10^"5 for L ~ L_p. These results suggest that for a previously estimated L_p of 15 nm (1 1) and L ranging from 10-30 nm, the ER/K a-helix could limit the k_ci_ose of interaction of proteins at its ends.

[00126] Given the rarity of Monte Carlo-derived conformations with end-to-end distance less than 2 nm, this approach cannot accurately estimate the effect of ER/K a-helix length on the frequency of interaction. The theoretical distributions of Mehraeen et al. ( 15) suggest that for an ideal WLC with L_p = 15 nm the number of conformations with end-to-end distance less than 2 nni increases with ER K a-helix length ranging from 10-30 nm.

However, given the low frequency of occurrence of these conformations it is possible that any deviations of the ER/K a-helix from an ideal WLC will likely influence this trend.

Nonetheless, the ER/K a-helix is a good candidate linker to limit k_ci_ose. [00127] The ER/K a-helix could also alter k_ope„. For the ER/K a-helix as an ideal WLC, the additional energy stored in the closed state relative to the open state decreased from 9 kcal/mol for L = 10 nm to 5 kcal/mol for L = 20 nm and 3 kcal/mol for L = 30 nm (see Supplementary data). For comparison, the strength of a single protein salt-bridge between proteins has been estimated to range from 0.5-4 kcal/mol (16). The probability of dissociation of the protein-protein interaction will therefore increase, as the energy needed to hold the ER/K a-helix in the closed state increases. Therefore, the ER/K a-helix modeled as an ideal WLC is a good candidate to increase k_ope„ relative to that of the free bi-molecular interaction.

EXAMPLE 2: Design of single polypeptide chain used to detect ER K a-helix modulation

[00128] Fig. 1 a shows a schematic of a single polypeptide chain used to detect ER/K a-helix modulation of the interaction between calmodulin (CAM) and its binding peptide (peptide). This single polypeptide chain is termed the Systematic Protein Affinity Strength Modulation (SPASM) FRET sensor to emphasize the presence of the ER/K a-helix and the FRET pair for sensing the frequency of interaction of the protein and protein/peptide at the ends of the ER/K a-helix.

[00129] A modular cloning approach was used to integrate elements of the SPASM sensor. This approach allows easy restriction enzyme cloning of any two interacting proteins or protein domains to form a SPASM sensor. All elements were cloned into the multiple cloning sites (MCS) of the pET47b vector (Novagen). A (Gly-Ser-Gly)4 linker is inserted between all protein domains as part of the primer sequence. The vector has an N-terminal

His6 tag followed by a PreScission protease site. A fusion between monomeric eCFP (A206K mutation) and CAM (pan troglodytes), containing a (GSG)4 linker in between, was amplified by crossover polymerase chain reaction (PCR) and cloned between BamHI and EcoRI sites. The placement of protein domains on either side of the ER/K a-helix was based on the modeled structure (Fig. lb). ER/K a-helices (see Fig. l c for details) with N-terminal ENFLYQG Tev-protease recognition site were cloned between EcoRI and Ascl sites.

mCitrine was cloned between Ascl and Sail sites. Oligos that translate into the different calmodulin binding peptide sequences were purchased from IDT, corresponding to the forward and reverse strands and containing 5' phosphate groups. CAM peptide sequences were cloned between Sail and Pad sites. Sequences of peptides used are (1) C15W - LPvRGQILWFRGLNRI (2) Trp3 - LKWKKLLKLLKKLLKLG (3) Melittin - GIGAVLKVLTTGLPALISWIKRKRQQ (4) Control - (GSG)4 extension.

[00130] From the N-terminus to the C-terminus, the sensor contained enhanced Cyan Fluorescent Protein (eCFP, the FRET donor), CAM, ER/K a-helix, mCitrine (mCit, the

9+

FRET acceptor) and a peptide with known affinity for binding Ca -activated CAM. All components were separated by a (Gly-Ser-Gly)₄ linker to ensure rotational freedom. [00131] CAM is a signaling molecule that binds to Ca²⁺ and switches from an inactive state (apocalmodulin (Apo CAM)) to an active state (CAM) (17). Ca²⁺ binding causes a dramatic increase in its binding affinity for a variety of effectors including protein kinases (CAMKII), phosphatases (calcineurin), ion channels and active transporters (Ca²⁺ pump).

9+

CAM binding to these proteins triggers their activation in response to Ca influx. A wide variety of small peptides that bind specifically to Ca²⁺ bound CAM and Apo CAM have been identified, and their affinities for binding CAM have been systematically quantified (18). CAM-peptide interactions are a model system to study ER/K a-helix modulation of protein- protein interactions. CAM-peptide binding affinity can be easily controlled in vitro by the presence or absence (chelation with EGTA) of Ca²⁺. The wide range of interaction affinities derived from CAM effector binding sites, both in the Ca bound and Apo states, can be used to quantify the contribution of the ER/K a-helix to the on and off rates of protein-protein interactions.

94-

[00132] FRET was used as a measure of the interaction between CAM and Ca . FRET occurs between eCFP and mCitrine, variants of Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP), which are used because of their increased brightness and fluorophore stability (19). When CFP and YFP are in close proximity (<8 nm) excitation of CFP results in significant fluorescence resonance energy transfer (FRET) from CFP to YFP. FRET between CFP and YFP has been used extensively to assess interaction between two proteins that are either free in solution or tethered together with a linker (20, 21). In the absence of Ca²⁺, the ER/K a-helix was designed to separate eCFP and mCit with the sensor adopting an Open' state (Fig. lb, left panel). In the presence of Ca²⁺ the high affinity of the peptide for CAM was designed to hold the ER/K a-helix in a 'closed' state (Fig. 2b, right panel) and bring eCFP and mCit within FRET range. The emission spectrum of the sensor sample following excitation of the donor, referred to as the FRET spectrum, is a function of the relative number of sensor molecules in the open and closed states. There are several ER/K a-helical protein motifs with L = 10, 20 and 30 nm in naturally occurring proteins (10). In this study, a 10 nm helix from myosin VI, and a 20 and 30 nm helix derived from the Kelch- motif family protein were used (Fig. lc) for our SPASM sensor constructs. Sensor was purified using a combination of affinity (Ni-NTA) and gel filtration and the purity was assessed using laser fluorescence gel scanning and coomassie staining of the PAGE run samples (Fig. 2d) as described below. [00133] pET47b vectors with different SPASM sensors were transformed into

JM109(DE3). Single colonies were inoculated in TB media (Invitrogen) and grown at 30 oC to an O.D. (600 nm) of 1.0 and transferred to room temperature (22 °C) where protein expression was induced with 500 DM IPTG. Cells were incubated at 22 °C for 4 hours and harvested by spinning at 4000xg for 20 minutes. Cells were resuspended in lysis buffer (20 mM phosphate pH 7.4, 0.5 M NaCl, 10 mM imidazole pH 7.5, 10% glycerol plus Complete Protease Inhibitor Tabs (Roche) and lysed by adding lysozyme (Sigma) to 1 mg/ml, incubating for 15 minutes, and sonicating. Lysates were clarified by centrifugation at 100,000xg for 30 minutes and supernatants were bound to Ni-NTA resin (Qiagen) for 30 minutes. The resin was washed with 8 column volumes of 20 mM phosphate pH 7.4, 0.5 M NaCl, 20 mM imidazole pH 7.5 and eluted with 20 mM phosphate pH 7.4, 0.5 M NaCl, 0.5 M imidazole pH 7.5. Ni-NTA eluate was desalted on PD10 column (GE Healthcare) into buffer A (20 mM Tris pH 8, 50 mM NaCl, 0.5 mM EDTA). Sample was concentrated 4-8 fold using 30 kDa Vivaspin concentrators (GE Healthcare). For control and C15W peptide sensors sample was purified by size-exclusion on superdex 200 column in buffer A. For high affinity constructs (Trp3 and Melittin) the sample was mixed with equal volume of 20 mM Tris pH 8, 50 mM NaCl, 0.5 mM EDTA, 8 M Urea. Urea is used to reversibly denature the proteins and ensure separation between CAM and peptide that interact outside of the intramolecular interaction. The samples were then purified by size-exclusion on superdex 200 column in buffer B (20 mM Tris pH 8, 50 mM NaCl, 0.5 mM EDTA, 4 M Urea). Gel filtration fractions were run on SDS PAGE gels and the fractions with a single band on coomassie gel, that contain both eCFP and mCit (assessed by gel scan on Typhoon gel image (GE Healthcare)) were pooled. Samples in buffer B were dialyzed into buffer A (three sequential steps with 500-fold dilution each step). Samples were spin concentrated to obtain the final fraction that was used for fluorometer measurements. The FRET spectrum is sensitive to the stoichiometry of eCFP and mCit (22). Degradation of the sensor during protein purification or an excess of eCFP resulting from partial translation of the sensor will affect the relative signal from eCFP and mCit. Furthermore, proteolysis of the sensor during protein purification such that the eCFP and mCit are no longer tethered can lead to misinterpretation of the contribution of the ER/K a-helix to the FRET spectrum. Hence, the sensor purity was assessed using laser fluorescence gel scanning and coomassie staining of the PAGE run samples (Fig. Id), in addition to the sample absorption spectrum showing peaks at 433 nm (eCFP) and 514 nm (mCit) (Fig. le). Sample concentrations were obtained from absorption spectra (Fig. l e) based on extinction coefficients of eCFP (32,500 M-1 cm-1 at 475 nm) and mCitrine (77,000 M-1 cm-1 at 526 nm). Concentrations obtained using eCFP and mCitrine were within 10% of each other for all samples. In fact, the ratio of eCFP to mCitrine absorption correlated well to the purity of the samples observed on SDS PAGE. CAM was cloned into pET28a vector (Novagen). CAM construct was transformed into Rosetta(DE3) (EMDChemicals) cells. Single colonies were inoculated in TB media

(Invitrogen) and grown at 37 °C to an O.D. (600 nm) of 1.0 and transferred to room temperature (22 °C) where protein expression was induced with 500 μΜ IPTG. Cells were incubated at 22 °C for 4 hours and harvested by spinning at 4000xg for 20 minutes. Cells were resuspended in 50 mM Tris pH 7.5, 2 mM EDTA, 1 mM DTT and lysed by high pressure homogenization (EmulsiFlex, Avestin) and centrifuged 35,000g for 20 min. CaC12 was added to the supernatant to a final concentration of 5 mM and bound to a

phenylsepharose column equilibrated in C-buffer (50 mM Tris pH 7.5, 0.5 mM DTT, 0.1 mM CaC12). Column was washed sequentially with C-buffer followed by C-buffer with 0.5 M NaCl. CAM was eluted with 50 mM Tris pH 7.5, 0.5 mM DTT, 1 mM EDTA, 150 mM NaCl. Fractions containing purified CAM were pooled and spin concentrated (Vivaspin, 10 kD cut-off). CAM concentration was measured by O.D. 280 nm. His6-tagged Tev protease was expressed and purified also.

[00134] In addition to the sample absorption spectrum showing peaks at 433 nm

(eCFP) and 514 nm (mCit) (Fig. 2e). [00135] For an ideal WLC with L_p = 15 nm, the mean end-to-end distances for ER/K a-helices with L = 10, 20 and 30 nm are 9, 16 and 23 nm, respectively (23). Therefore, in the absence of CAM-peptide interactions no significant FRET between eCFP and mCit is expected (R₀ ~ 5 nm (24)). Accordingly, sensor constructs with 10, 20 and 30 nm helices that have the CAM binding peptide substituted by an extended linker (GSG)x4 with no known affinity for CAM showed no measurable FRET (Fig. la).

[00136] Fluorometer data acquisition was accomplished as outlined below. The bandpass width was selected to maximize signal without saturation of the detector. PMT voltage was varied from 600 - 950 V depending on signal strength. For measurement of Kd of bi- molecular interactions, serial dilutions were used. Serial dilution minimizes pipetting error from samples prepared at different concentrations. Quartz sub-micro cuvettes allow measurements with small volumes (~ 50 μΐ) on a standard fluorometer were used. These cuvettes limit the volume of sample excited during each scan. Serial dilution was performed by addition of sample buffer directly to the cuvette. Therefore, the fraction of sample that is subject to repeated scans, decreases with each subsequent scan and minimizes photobleaching to the sample. Further, an equal number of repetitive scans on a 50 μΐ sample without serial dilution showed that while there is photobleaching of the sample over the course of the experiment, the R values do not change unless the concentration is altered. For measurement of Kd of high affinity interactions, the purified CAM-Trp3 FRET protein is digested with Tev protease to unlink the eCFP-tagged CAM and mCit-tagged Trp3, and then diluted to 5 nM concentration. The sample is mixed with increasing concentration (1 nM to 20 μΜ) of unlabeled vasoactive intestinal peptide (VIP), which binds Ca2+ activated CAM with 50 nM affinity. Samples with increasing concentration of VIP are scanned on the fluorometer. The inverse FRET ratio (IR), defined as the ratio of eCFP (475 nm) to mCit (525 nm) emission maxima. Similar to the FRET ratio (R₀bs)_> IRobs can be expressed as a linear combination of IR for the closed and open states. For known concentrations of CAM, Trp3, VIP and previously measured Kd of one of the interactions, the Kd of the other interaction can be computed by an iterative fit of equilibrium competitive binding equations to the experimental data.

[00137] Selective excitation of the donor (eCFP at 433 nm) resulted in donor emission (eCFP emission maximum at 475 nm) without significant acceptor emission (mCit emission maximum at 525 nm) (Fig. 2a). A CAM binding peptide that selectively binds to the activated Ca bound CAM underwent a dramatic increase in FRET when CAM was transitioned from the Apo (Fig. 2b, EGTA) to Ca²⁺ bound state (Fig. 2b, Calcium). The change in signal was found to be similar for 10, 20 and 30 nm ER/K ot-helices. It must be noted that the concentrations of sensors used in these experiments ranged from 5-50 nM, well below the previously reported free solution Kd (equilibrium dissociation constant) of this CAM-peptide interaction (~ 1 μΜ) (25). Therefore, the change in FRET is derived primarily from intramolecular interactions with minimal contribution from intermolecular interactions. This observation was also confirmed by showing that serial dilution of the sample does not significantly alter the ratio of mCit (acceptor) to eCFP (donor) emission (see below). [00138] The relative number of sensors in the closed state, also termed f_c for fraction closed (fraction open (f₀) is l-f_c), is related to the ratio of the intramolecular on and off rates by the following equation

k close fc (1)

[00139] Here k_open and k_ci_0Se are the intramolecular off and on rates respectively. In order to derive f_c from the FRET spectrum, the equivalent bi-molecular system was used where f_c can be varied systematically by changing the sensor concentration (see

Supplementary discussion). A Tev protease site was built into the sensor between the CAM and the ER/K a-helix. Following purification of the sensor as a single polypeptide chain (see Methods), Tev protease was used to obtain stoichiometrically equivalent amounts of eCFP- tagged CAM and mCit-tagged binding peptide that participate in a bimolecular interaction. Complete digestion with Tev was verified by laser fluorescence gel scanning (Fig. Id). Tev- digested sample was concentrated to over ten- fold (16 μΜ) above the K (~ 1 μΜ) to

9

facilitate the bi-molecular interaction. Bi-molecular interactions between Ca activated CAM and peptide resulted in high FRET (Fig. 3a). Serial dilutions of the sample were used to reduce the equilibrium fraction bound in accordance with the Kj, and therefore the fraction of molecules in the high FRET state. This in turn resulted in a reduction in mCit emission with decreasing concentration of the sample (Fig. 3b). Given the Kj of the interaction, the f_c for a given concentration of the sample is directly calculated from equation (2) (Note Fig. 3b top vs bottom abscissa).

[00140] The FRET ratio (mCitmax/eCFPmax) varied -linearly with f_c (Fig. 3b,

R²=Q.99?> for linear regression fit). The -linear relationship between FRET ratio (R) df_c consistent with a two-state model of the FRET sensor (see Methods). R for a given f_c was therefore expressed as a linear combination of the R₀bs of the unbound (open) state (R_open) and the bound (closed) state (R_ciose_<d-

^Robs = ^closed ^X fc + ^Ropen ^X 0 ^~ fc ) (3)

[00141] R_obs unlike the FRET spectrum, is normalized for the number of sensor molecules present in the sample. While R_ci_0Sed and R_ope„ cannot be directly measured, they can be estimated from the equivalent bi-molecular interaction as discussed in the next section.

EXAMPLE 3: FRET ratio (R) can be used to measure K_d of medium affinity

interactions (~ μΜ) by SPASM FRET

[00142] The use of R to measure f_c was independently validated using another approach. Given, the linear relationship between f_c and FRET ratio (Robs) the data for the bi- molecular interaction (Fig. 4a) can be fit to equations (2) and (3) with Kd, Ropen and R_ci_0Sed as fit parameters. The fit yielded a Kd of 1.3 μΜ, which agrees well with a previous

measurement of 1.7 μΜ for this CAM-peptide interaction pair (25). While Ro_pen and R_ci₀sed were allowed to float for the fit, they are constrained by Robs at high and low Ct. However, the Kd measurement and the quality of the fit (R-square) were not affected significantly (< 10% difference) when R_open and Rciosed were constrained to Robs at low and high

concentrations, respectively. Therefore, a single parameter fit of the experimental data to equations (2) and (3) was sufficient to measure Kd. This approach to measure Kd using R vs C can be universally applied to any protein-peptide or protein-protein interaction. A single concentrated protein sample (~ 20-fold higher than the expected Kd with 50 μΐ volume) is sufficient to obtain the entire curve using serial dilution (Fig. 4 represents the mean+STD for four samples, see Methods). Co-expression of the protein and protein/peptide with both a Tev-protease site and an ER/K a-helix between them provides an easy method to obtain stoichiometrically equivalent amounts of the two binding partners from a single purified sample. The ER/K a-helix acts as a spacer that allows proper folding of the two proteins. In order to accurately estimate the Kd of two interacting proteins using this approach, it is essential to measure R₀b_S over a range of bi-molecular concentrations that bracket the Kd. For very high affinity interactions (< 10 nM) the FRET spectrum from the fluorimeter has lower signal to noise and the accuracy of R^bs is reduced. For such high affinity interactions, competitive binding of either of the fluorescently tagged proteins to a non-fluorescent partner with known affinity can be used, as described in the next section. EXAMPLE 4: FRET ratio (R) can be used to measure the K_d of high affinity

interactions (~ nM) by SPASM FRET

[00143] Competitive binding assays have been used to determine the Kd of high affinity interactions. However, the techniques for detecting competitive binding require multiple steps, for instance centrifugation followed by either quantitative analysis of PAGE gels by Coomassie stain, immunoblotting or a radioimmunoassay (27). In this study FRET was used as a quantitative readout of competitive binding. Inverse FRET ratio (IR), defined as the ratio of eCFP (475 nm) to mCit (525 nm) emission maxima, decreases as the human vasoactive intestinal peptide (VIP) peptide competes for binding in the bi-molecular FRET interaction between eCFP-tagged CAM and mCit-tagged Trp3 (Fig. 5b). A measured Kd of 53 nM for the VIP peptide based on a 0.3 nM affinity of the Trp3 peptide (see Methods), which is nearly identical to the previously reported 54 nM affinity for this interaction (28).

EXAMPLE 5: Effective concentration of the intramolecular interaction decreases with increasing ER/K a-helix length [00144] Studies on the equivalent bi-molecular interaction can be used to measure the

FRET ratio corresponding to the open {R_open) and closed (R_cbsed) states. This in turn can be used to calculate f_c (equation (3)) and hence k_openlk_ci_ose for different SPASM sensors. One drawback of this approach is that as f_c approaches 1 , the k_openlk_c\_ose is very sensitive to the accuracy of f_c (equation (l));f_c approaches 1 for protein/peptide pairs that interact with high affinity (~ nM). In addition, for such high affinity interactions we cannot use the serial dilution approach, outlined earlier, to measure R_open and R_chsed- Hence, an alternative approach to measure f_c based competitive binding of the sensor to free unlabeled CAM was used (Fig. 4c, Methods). This technique of measuring^ can be applied to both weak and strong affinity interactions between the protein and protein/peptide within the sensor. [00145] An alternate measure of the strength of interaction between the two proteins is the effective concentration (C_ejj). Effective concentration is the concentration of the protein/peptide pair in the equivalent bi-molecular interaction that has an f_c equal to that of the intramolecular interaction within the SPASM sensor. The effective concentration can be estimated from the competitive binding assay. The higher the C_e//or f_c, the stronger the interaction between the protein-peptide pair within the sensor and vice versa. The advantage of using C_eff over f_c is that as f_c approaches 1 , small changes in f_c can cause large changes in C_eff. Therefore, comparing interaction strengths based on C_e//is easier than with f_c. Fig. 5d is a summary of ^for the intramolecular interaction between CAM and corresponding peptide, measured using five different interaction affinities (four different peptides with one in the Apo CAM state), for three ER/K a-helices with length 10, 20 and 30 nm. Regardless of the affinity of interaction between CAM and peptide, or the state of activation of CAM, the C_ejf for a given interaction was found to decrease with increasing ER/K a-helix length (Fig. 5d). Specifically, C_ef decreased about ten-fold when helix length increased from 10 to 20 nm and about three-fold when the helix length increased from 20 to 30 nm. Hence the ER/K a-helix can be used to systematically effect large changes in C_e// between interacting proteins.

Further, we estimated the off-rate (k_ope„) of the CAM-Trp3 peptide sensor by monitoring the quench of FRET signal following incubation of vast excess of unlabeled CAM. These measurements show the ER/K a-helix does not affect the off-rate (k_open). Therefore the effect of the ER/K a-helix on the effective concentration is mediated through the on-rate (k_c/ose).

[00146] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

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Claims

WE CLAIM;

1. A biosensor comprising at least two test proteins, a polypeptide linker, and a reporter, wherein the at least two test proteins are covalently linked to, and separated by, the polypeptide linker, and wherein the polypeptide linker comprises an ER K motif, and wherein the interaction of the at least two test proteins results in a detectable signal.

2. The biosensor of claim 1 , wherein the detectable signal is a fluorescent signal.

3. The biosensor of claim 1 , wherein the reporter is a Forster's resonance energy transfer (FRET) pair comprising a donor moiety and an acceptor moiety.

4. The biosensor of claims 3, wherein the donor moiety is covalently linked to one of the at least two test proteins and the acceptor moiety is covalently linked to one of the at least two test proteins.

5. The biosensor of claim 1 , wherein the polypeptide linker comprises the sequence of SEQ ID NO: l , SEQ ID NO:2, or SEQ ID NO:3.

6. A biosensor comprising at least two test proteins, a polypeptide linker, and a reporter, wherein the at least two test proteins are covalently linked to, and separated by, the polypeptide linker, and wherein the amino acid sequence of the polypeptide linker is such that an at least three-fold increase in the length of the linker results in at least a 30 fold decrease in the effective concentration of at least two test proteins.

7. A nucleic acid encoding a biosensor of any one of the preceding claims.

8. The nucleic acid of claim 7, wherein the biosensor is operatively linked to a promoter.

9. A host cell comprising the nucleic acid of claim 8.

10. The host cell of claim 9, wherein the nucleic acid is expressed.

1 1. A method for measuring the equilibrium dissociation constant of a protein - protein or protein-ligand interaction, the method comprising detecting a signal from a biosensor at two or more different concentrations of the biosensor wherein the biosensor comprises at least two test proteins, a polypeptide linker, and a reporter, wherein the at least two test proteins are covalently linked to, and separated by, the polypeptide linker, and wherein the

polypeptide linker comprises an ER/K motif, and wherein the interaction of the at least two test proteins results in said detectable signal.

12. The method of claim 1 1, wherein the equilibrium dissociation constant is in the micromolar range.

13. A method for measuring the equilibrium dissociation constant of a protein - protein or protein-ligand interaction, the method comprising detecting the signal from a biosensor in the presence of two or more concentrations of a competitive binding ligand, wherein the biosensor comprises at least two test proteins, a polypeptide linker, and a reporter, wherein the at least two test proteins are covalently linked to, and separated by, the polypeptide linker, and wherein the polypeptide linker comprises an ER/K motif, and wherein the interaction of the at least two test proteins results in a detectable signal.

14. The method of claim 10, wherein the equilibrium dissociation constant is in the nanomolar range.

15. An assay for identifying compounds that alter a protein-protein interaction, the assay comprising detecting a signal from a biosensor in the absence and in the presence of a test compound, wherein the biosensor comprises a test protein A, a test protein B, a polypeptide linker, and a report, wherein the test protein A and the test protein B are covalently attached, and separated by, the polypeptide linker, wherein the polypeptide linker comprises an ER/K domain, and wherein a difference in the signal detected in the presence of the test compound as compared to the absence of the test compound is indicative of a compound that alters a protein-protein interaction.