Abstract:
Weak binding motifs were transformed into a high affinity ligand surface by using a heterologous self-assembled monolayer (SAM) as a rigid scaffold to present discrete binding moieties, in a controlled geometry, to a target molecule. At a critical ligand density, the discrete binding moieties simulated a multivalent ligand and promoted high-affinity, cooperative binding of the target molecule. Statistical calculations were applied to SAM components in solution and gold-sulfur packing dimensions to extract the inter-ligand-distance within the SAM. This distance information is valuable to the rational design of multivalent drugs.

Description:
RELATED APPLICATIONS  
       [0001]     This application is a continuation of U.S. patent application Ser. No. 09/324,258, filed Jun. 2, 1999, which claims priority to U.S. Provisional Patent Application No. 60/087,766, filed on Jun. 2, 1998. 
     
    
     STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT  
       [0002]     This work was supported, in part, by NIH Grants 5T32EM-07598-18 and GM-32308. The government of the United States of America may have some rights in this invention. 
     
    
     FIELD OF INVENTION  
       [0003]     The present invention relates to the use of self-assembled monolayers attached to surfaces for the detection and probing of target molecule structure and function.  
       BACKGROUND OF THE INVENTION  
       [0004]     Combinatorial chemistry techniques are used to synthesize diverse “libraries” of unique chemical compounds. These small molecule libraries often yield drug candidates that are capable of binding a specific biological target but because of their small size and relative simple chemical makeup, they characteristically interact with the target in a low affinity interaction. These low affinity interactions cannot adequately compete with larger more diverse natural ligands, like proteins and protein complexes, and thus provide little therapeutic value. Natural products, which are naturally occurring organisms isolated from soils, yeast, marine organisms, and the like are larger and chemically more interesting than small molecules from combinatorial libraries. Natural products are routinely screened for therapeutic activity against disease-related organisms. Many cancer drugs have been identified in this way. The problem with developing a natural product for the drug marked is that they are large and chemically complicated, which means that elaborate and expensive schemes for their synthesis must be developed. Identifying a synthetic scheme that is commercially feasible is a technical challenge that at best takes years and millions of dollars to accomplish and at worst cannot be done. For this reason, there is interest in enhancing the affinity between small molecule drugs and their biologically relevant targets.  
         [0005]     Knowles and colleagues, at Harvard, reported that they could enhance the binding affinity of a small molecule for a particular target by attaching a “greasy tail” to the small molecule. This hydrophobic tail was later shown to interact with a hydrophobic patch on the target molecule adjacent to the binding site.  
         [0006]     Many biologically relevant target molecules present more than one binding site for a particular ligand. Some present pseudo identical binding sites with which they bind natural ligands that contain “repeats” of a binding motif. It is known that bivalent interactions (like antibody interactions) are higher affinity interactions than monovalent interactions, due to the cooperative binding effect. Therefore, one would like to link several small molecule drugs together to form a pseudo multivalent drug that would interact more strongly with a multi-binding-site target molecule. The problem with this logic is that the enthalpic advantage of the additional binding energy is offset by the large entropic energy cost of ordering the connected binding moieties. However, making the linker between the binding moieties a rigid linker would introduce order and thus minimize the entropic cost to yield a higher affinity interaction. In order to connect two binding moieties (the small molecule drugs) with a rigid linker, in a geometry that would encourage its binding to the target molecule, one would need to know apriori the distance between the binding sites on the target molecule. This inter-binding-site distance information is currently derived from X-ray or NMR structure determination of the target molecule. This process is time-consuming (years) and expensive.  
         [0007]     The subject of this invention is how self-assembled monolayers (SAMs) can be used to present discrete binding moieties, at varying densities, in a rigid 2-dimensional array, to multivalent target molecules in order to promote a higher affinity, cooperative interaction. Ligand densities within the SAM are varied to determine the critical distance between binding moieties that will promote simultaneous, cooperative binding of the target molecule. By monitoring the kinetics of binding events between the target molecule and the variable density ligand surfaces, one can empirically determine the lowest surface density that prompts a large shift in affinity for the multivalent target molecule. One can then use Poisson statistics to infer the distance between surface-immobilized ligands and thus also the distance between the binding sites on the target molecule. Once this distance information has been deduced, it can be used to rationally design bi- or multi-valent drugs or rigid linkers to connect two binding moieties. Alternatively, the SAM itself can become a part of the “drug”; in this case, the SAM is used as the “rigid linker” between binding moieties to present multiple binding motifs, at the empirically determined critical density, to promote the higher affinity cooperative interaction. The SAM, presented ligands and underlying gold (may be gold colloids) are both the drug and the drug delivery system. Inert thiols of the SAMs can be terminated with lipid-like groups to facilitate drug delivery. Similarly, a biospecific ligand could be incorporated (at varying densities) into a liposome, at the critical presentation density determined, and used directly as a multivalent drug in its own delivery system.  
       SUMMARY OF THE INVENTION  
       [0008]     Self-assembled monolayers are used as a rigid 2-dimensional matrix for presenting binding moieties, at varying distances from each other, to a target molecule. Two-component SAMs incorporate an inert spacer molecule and a biospecific molecule that can directly or indirectly present a binding moiety to a target molecule. The distance between the biospecific molecules in the array, the ligand density, is controlled by manipulating the concentrations of the two component thiols in solution before deposition onto gold. The affinity of the interaction between the surface immobilized ligands and the multivalent target molecule is monitored as a function of ligand density. The lowest ligand surface density that elicits a jump in affinity for the target molecule contains the critical information needed to extract the distance between binding sites on the target molecule. The dimensions of the hexagonal tiling pattern formed when the sulfurs from the thiols bind to gold solid are known. Therefore, Poisson statistics can be used to infer the distance between surface immobilized ligands, and thus the inter-binding-site distance on the target molecule, from the concentrations of the thiols in solution. Further, the gold surface itself and the attached SAM can be used as a scaffold to present binding moieties, in a controlled, higher affinity geometry, to a target molecule.  
         [0009]     In a preferred embodiment, SAMs are generated that incorporate two thiol types: 1) an inert tri-ethylene glycol-terminated thiol; and 2) a nitrilo tri-acetic acid (NTA) terminated thiol that when complexed with Ni, captures histidine-tagged proteins or peptides. The density of NTA-thiol within the SAM is varied to present varying densities of a histidine-tagged binding moiety to a multi-valent target molecule. The affinity of the interaction is plotted as a function of ligand density within the SAM. A dramatic increase in the binding affinity occurs at a critical surface density when the presented ligands are close enough to each other to simultaneously bind to a common target molecule. The solution concentrations of the two thiol types and the dimensions of the tiling pattern that the thiols form on the gold substrate are input into Poisson distribution equations to extract the probable distance between binding sites on a target molecule. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0010]      FIG. 1  shows the predicted structure of mixed self-assembled monolayers made by doping a thiol solution with a nitriloacetic acid terminated thiol.  
         [0011]      FIG. 2  shows the results of the binding of hTBPc to GST-2× peptide surfaces of differing densities.  
         [0012]      FIG. 3  shows the binding of TBP target protein as a function of peptide surface density.  
         [0013]      FIG. 4  shows that the binding of hTBPc to surface immobilized GST-2× is a non-linear function of the surface density of the peptide.  
         [0014]      FIG. 5  shows the three possible mechanistic models for describing the interaction of TBP with reiterated peptide activation motifs.  
         [0015]      FIG. 6  shows titration curves summarizing competitive inhibition experiments designed to measure the kinetics of hTBPc-peptide activation motif binding.  
         [0016]      FIG. 7  shows that TATA sequence DNA bound to hTBP does not inhibit the interaction of hTBP with GST-4×.  
         [0017]      FIG. 8  shows that there is a synergistic increase in affinity between hTBPc in solution and surface-bound GST-2× when the density of immobilization is increased from 3.8% to 5.7%.  
         [0018]      FIG. 9  shows competitive inhibition experiments demonstrating that 2× ligands behave very differently in solution versus when immobilized.  
     
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS  
       [0019]     Variable density nitrilotriacetic acid (NTA)-SAMs were used to probe the binding site(s) of a biologically important molecule, the human general transcription factor TATA box binding protein (hTBP) [Burley, S. K. and Roeder, R. G. (1996) Biochemistry and structural biology of transcription factor IID (TFIID).  Annu. Rev. Biochem.  65:769-799]. This transcription factor has been implicated as a direct target of transcriptional activators such as VP16 [Ingles, J. C., M. Shales, W. D. Cress, S. J. Triezenberg and J. Greenblatt. (1991) Reduced binding of TFIID to transcriptionally compromised mutants of VP16.  Nature.  351:588-590]. In fact, the need for an activator is eliminated when TBP is artificially tethered to a DNA promoter [Xiao, H., J. D. Friesen and J. T. Lis. 1995. Recruiting TATA-binding protein to a promoter: transcriptional activation without an upstream activator.  Mol. and Cell. Biol.  15(10):5757-5761].  
         [0020]     Transcriptional activator proteins are modular in that they have functionally separable domains [Brent, R. and M. Ptashne. (1985) A Eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor.  Cell.  43:729-736], a DNA binding domain, and an activating region. The structures of TBP [Nikolov, D. B., H. Chen, E. D. Halay, A. A. Usheva, K. Hisatake, D. K. Lee, R. G. Roeder and S. K. Burley. (1995) Crystal structure of a TFIIB-TBP-TATA element ternary complex.  Nature.  377:119-128] and several activator DNA binding domains [Marmorstein, R., M. Carey, M. Ptashne, and S. C. Harrison. 1992. DNA recognition by Gal4: structure of a protein/DNA complex.  Nature.  356:408-414; Ellenberger et al., 1992; and Baleja, J. D., R. Marmorstein, S. C. Harrison and G. Wagner. 1992]. The structure of the DNA-binding domain of Cd2-Gal4 from  Saccaromyces cervisiae  in solution has been solved, yet the structure of an activating region, alone or complexed with a target molecule has remained elusive. Fundamental questions as to how an activating region effects gene transcription remain unanswered. One mechanistic model of gene activation proposes that DNA-bound activators trigger transcription by merely “recruiting” some necessary factor, perhaps TBP, to the promoter through direct contact with the activating region [Triezenberg, S. J. 1995. Structure and function of activation domains.  Curr. Opin. Genet. Dev.  5(2):190-196]. Another model proposes that activating regions induce a conformational change in a target protein(s) [Sheldon and Reinberg, 1995] or sequentially perform some function until a threshold is reached which catalyzes gene transcription.  
         [0021]     In eukaryotes, more than one DNA-tethered activator is typically required to achieve activated transcription and that multiply bound activators transcribe synergistically [Lin, Y. S., M. Carey, M. Ptashne and M. R. Green. (1990) How different eukaryotic transcriptional activators can cooperate promiscuously.  Nature  345:359-361]. Cryptic repeats of minimal activation motifs have been identified in eukaryotic activators that, when tandemly reiterated and tethered to DNA, efficiently activate transcription in vitro [Blair et al., 1994; Tanaka, M. and W. Herr, (1994) Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain.  Mol. Cell. Biol.  14(9):6056-6067]. An eight amino acid minimal activation motif (DFDLDMLG) derived from the prototypic mammalian activator VP16 was recently identified [Tanaka, M. (1996) Modulation of promoter occupancy by cooperative DNA binding and activation-function is a major determinant of transcriptional regulation by activators in vivo.  Proc. Natl. Acad Sci. USA.  93(9):4311-4315]. As an exemplary embodiment, this invention describes novel biophysical methods to quantitate the kinetics, as well as investigate the mechanism, of the interaction between hTBP and tandem repeats of the VP 16 minimal motif.  
         [0022]     The interactions were characterized by SPR in a BIAcore instrument. SPR is a fairly new optical technique for the real time detection and kinetic analysis of intermolecular interactions [Liedberg, B., C. Nylander and L. Lundstrom. (1983) Surface plasmon resonance for gas detection and biosensing.  Sens. Actuators.  4(2):299-304.; Daniels et al., 1988; Lofas, S. and Johnsson, B. (1990) A novel hydrogel matrix on gold surfaces in surface plasmon resonance sensors for fast and efficient covalent immobilization of ligands. J Chem. Soc., Chem. Commun.: 1526-1528]. The basis of the technology is as follows: ligands are immobilized on a surface; putative target molecules are flowed over this surface; the protein concentration at the solution-surface interface changes as target binds ligand. The increased protein mass at the interface causes a change in the optical properties of the system. The amount of new protein recruited to the interfacial region can be quantitated by measuring the change in the angle at which light reflected off the interface is a minimum [for a review see Bamdad, C. 1997. Surface plasmon resonance for measurements of biological interest.  Current Protocols in Molecular Biology  20.4.1-20.4.12.]. Changes in this angle are measured in resonance units (RUs) where 1 RU is defined as a change of {fraction (1/10,000)}th of a degree. A rule of thumb is that for a distance of about 150 mn from the interface, 1 ng protein/mm 2  registers 10 3  RUs.  
         [0023]     SAMs were generated that incorporated an NTA group for the specific binding of histidine-tagged peptides. The density of NTA in the SAM was varied so that different amounts of a His-tagged activation motif could be presented to TBP, in solution. SPR was used to quantitate avidity effects between TBP and surface-bound peptides as a function of peptide density.  
         [0024]      FIG. 1  shows mixed self-assembled monolayers (SAMs) that were generated by doping a thiol solution with an NTA-terminated thiol and designed to capture histidine-tagged proteins. Sulfur atoms deposit on gold substrates in a hexagonal tiling pattern 4.99 Å on edge with three possible positions for thiol deposition per hexagon. If it is assumed that in a well-ordered SAM all sites are occupied, Poisson statistics can be used to calculate an average distance between NTA-thiols for a given NTA concentration. Equation (1) calculates how many hexagons must be filled before two NTA-thiols are deposited. For a 3.8% NTA-thiol concentration in solution, relative to EG 3 -thiol, an average of 17.5 hexagons must be filled before 2 NTA ligands appear. For a 5.7% NTA solution, 11.7 hexagons must be filled before an average of two NTA ligands are deposited. The area of a hexagon 4.99 Å on edge is 64.69 Å 2  which is equal to the area of a square, 8.04 Å on edge. NTA ligands on SAMs formed from a 3.8% NTA-thiol solution would be an average of 29 Å apart, while NTA ligands in a SAM formed from a 5.7% NTA-thiol solution would be 23 Å apart. It was assumed that the concentration of NTA-thiol in solution was equal to its concentration in the SAM; see  FIG. 2  of Sigal et al., 1996.  
         [0025]      FIG. 2  shows that hTBPc in solution will not bind to GST-2× peptide surfaces unless peptides are immobilized close to one another. The BIAcore SPR instrument records changes in the angle of minimum reflectance (RUs) as a function of time. Reagents are flowed over individual flow cells of the SAM. The “square waves” represent injections of protein “plugs” that interrupt the constant buffer flow. An association constant can be derived from an analysis of the initial phase of the injection and a dissociation rate can be extracted from analysis of the system as it returns to buffer flow. GST-2× or 4× fusion proteins (X=DFDLDMLG) were separately immobilized on NTA-SAMs via histidine-tags then hTBPc (124 nM) was injected over the surfaces. An overlay of two SPR sensorgrams shows that hTBPc does not bind to GST-2× immobilized on a 3.8% NTA-SAM (dashed line) but binds very tightly when immobilized on a 5.7% NTA-SAM (solid line).  
         [0026]      FIG. 3  shows the binding of target protein TBP measured by SPR as a function of peptide surface density. A series of NTA-SAMs were generated to display peptides at low to high density. When two tandem repeats of the minimal activation peptide (GST2×) were displayed at low density (1.3%-3.8%), human TBPc did not bind to the surface. In contrast, a more dense GST-2× surface (5.7%-11.4%), bound significant amounts of human TBPc. Fusion proteins bearing four tandem repeats of the minimal activation peptide (GST-4×) bound hTBPc whether the peptides were displayed at low or high density. The stoichiometry of the interaction was a constant, independent of the immobilization density. Notably, at corresponding surface concentrations, GST-2× bound half as much hTBPc as GST-4×, suggesting that two -2× modules immobilized at close proximity to each other (high density) simultaneously contact one hTBPc molecule.  
         [0027]      FIG. 4  shows that the binding of hTBPc to surface immobilized GST-2× is a non-linear function of the surface density of the peptide. Histidine-tagged peptides were separately immobilized on SAMs presenting NTA over a wide range of surface densities. SPR was used to quantitate the amount of target protein, hTBPc that bound to each surface. The mass ratios of captured hTBPc to surface immobilized peptide (GST-2× or -4×) was plotted as a function of peptide concentration. The binding of hTBPc to GST-4× (dashed line) is roughly constant over the range of surface peptide densities. However, the binding of hTBPc to GST-2× (solid line) approximates a step function of GST-2× surface concentration.  
         [0028]      FIG. 5  shows experiments that were designed to discriminate between three possible mechanistic models to explain how reiterated peptide activation motifs synergistically effect transcription of a nearby gene. Model 1: two connected peptide motifs must be positioned such that they can simultaneously bind to quasi-identical sites on TBP. The bivalent, high affinity interaction would keep the general transcription factor tethered near the start site of transcription awaiting other steps in the transcriptional activation process. Model 2: the binding of one or two peptide activation motifs causes a conformational change in TBP. The allosteric effect enhances the subsequent binding of additional peptide motifs and a high affinity interaction results. Model 3: a high affinity interaction occurs between the peptide repeats and TBP but rather than resulting from a “bivalent” interaction or an allosteric effect, it results from the simple summation of multiple interactions between TBP and the entire length of the activation peptide.  
         [0029]      FIG. 6  shows titration curves, summarizing competitive inhibition experiments, that yield IC 50 s that show the 4× peptide binds hTBPc 250 times tighter than the 2× peptide. In order to quantitate the solution kinetics of hTBPc binding to synthetic 4× peptides (4 tandem repeats of DFDLDMLG) or 2× peptides (2 repeats), aliquots of hTBPc (124 nM) were incubated with increasing concentrations of either peptide at 4° C. for 1 hour. The mixtures were then separately injected over identical SAMs that were pre-bound with GST-4×. Percent inhibition is plotted against the concentration of the blocking peptide in solution. 0% inhibition was taken to be the amount of hTBPc that bound to GST-4× surfaces when it was incubated with buffer alone. Background levels of binding were determined by injection of protein mixtures over naked GST surfaces. An IC 50  of 370 nM and 90 μM describe the equilibrium kinetics of hTBPc binding to 4× and 2× peptides, respectively.  
         [0030]      FIG. 7  shows that TATA sequence DNA bound to hTBP does not inhibit the hTBP/GST-4× interaction. N-terminally histidine-tagged hTBP was bound to NTA-SAMs and the mass of bound protein was quantitated and recorded by a BIAcore SPR instrument. The SAMs, bound with hTBP, were then removed from the instrument and separately incubated at RT for 15 minutes with solutions containing equal mass amounts of either DNA bearing the hTBP TATA recognition sequence or random sequence DNA (150 MM NaCl; 50 nanomoles DNA). The SAMs were then washed in running buffer and re-docked in the SPR instrument. The increase in absolute RUs of the baseline indicated that the TATA sequence DNA bound to surface immobilized hTBP with roughly 1:1 stoichiometry while the random DNA bound only nonspecifically. Protein plugs of GST4× were separately injected over these surfaces; the presence of DNA, bound nonspecifically or specifically, was not inhibitory to the subsequent binding of GST-4× to hTBP. Additionally, the measured association and dissociation rates, which were not affected by DNA-binding, were identical to those measured with GST-4× bound to the SAM and TBP in solution.  
         [0031]      FIG. 8  shows that there is a synergistic increase in affinity between hTBPc in solution and surface-bound GST-2× when the density of immobilization is increased from 3.8% to 5.7%. Low (3.8% NTA) then high (5.7% NTA) density SAMs were docked in an SPR device. Histidine-tagged GST-2× and GST-4× fusion proteins (0.3 mg/ml) were separately immobilized on individual flow cells of the SAMs. The mass of the immobilized species is recorded in resonance units (RUs), where 1000 RUs=1 ng protein/mm 2 . One RU results from a net change of {fraction (1/10,000)} of a degree in the angle of minimum reflectance off of the differential dielectric interface of the sensing wave. hTBPc (124 nM) was then injected over the derivatized surfaces. The mass of the captured analyte was obtained by taking the difference between RUs recorded 10 seconds prior to and 25 seconds after the injection. When GST-2× was immobilized at low density it was not able to bind hTBP. However, when immobilized at slightly higher density, a high affinity interaction resulted. The stoichiometry of surface immobilized GST-4× binding to hTBPc was relatively constant but, notably, twice that of GST-2× binding to hTBPc which reinforces the idea that two −2× ligands bind one hTBPc molecule.  
         [0032]      FIG. 9  shows competitive inhibition experiments in which 2× ligands behave very differently in solution than when surface immobilized and that reiterated minimal activation motifs effectively compete for the same binding site(s) on hTBP as the parent protein. Histidine-tagged GST-4× or GST-2× were separately immobilized on NTA-SAMs docked in a BIAcore SPR instrument. hTBPc(residues 155-335) or hTBP (full length) was pre-incubated at high concentration (35 μM) with either buffer, a synthetic 2× peptide (X=DFDLDMLG) at 1:4 stoichiometry, a 4× peptide at 1:2 stoichiometry, or a 1X-linker-1X peptide DFDLDMLG-((Ser) 4 Gly 1 ) 3 -DFDLDMLG) at 1:2 stoichiometry for 1 h at 40° C. Just prior to injection over the derivatized surfaces, the pre-incubation mixtures were diluted such that the final hTBP concentration was (124 nM). The synthetic 4× and 1X-linker-1X peptides blocked the interaction of hTBP with surface immobilized ligands but 2× peptides were not inhibitory. Histidine-tagged Gal4(1147)+VP16(413-490) were similarly immobilized on NTA-SAMs. hTBP was preincubated, as described above, with either buffer or 4× peptide then diluted and injected over the VP16 presenting surfaces. The 32 amino acid 4× peptide effectively blocked the interaction of hTBP with the 78 amino acid VP16 activation domain.  
         [0033]     A panel of variable density NTA-SAMs were prepared by diluting the concentration of the active component, NTA-thiol, relative to that of the inert component, EG 3 -thiol, in ethanol solutions. Gold-coated glass slides were incubated in solutions containing 1.3%, 3.8%, 5.7%, or 11.4% NTA-thiol, with the total thiol concentration constant at 1 mM. The SAMs were glued onto blank CM-5 SPR chip cassettes and docked into a BIAcore instrument. A 16-mer peptide comprised of two repeats of the eight amino acid minimal activation motif (X=DFDLDMLG), derived from the human activator VP16, was fused to histidine-tagged GST (GST-2×). The fusion proteins were then immobilized on variable density SAMs through complexation of the NTA group by the protein&#39;s histidine tag. This generated a series of surfaces that displayed peptides at incrementally decreasing distances from each other. The core region of human TBP (hTBPc: residues 155-335) (Nikolov et al., 1995) was injected over the peptide surfaces. GST-2× immobilized at low density (1.3%-3.8%), was unable to bind hTBPc. In contrast, when the same concentration hTBPc was injected over a more dense (5.7%-11.4%) GST-2× surface, where the average distance between peptide motifs would be smaller, a high affinity interaction resulted (see  FIGS. 2 and 8 ). As a control, fusion proteins bearing four iterations of the minimal motif (GST-4×) were immobilized on the different density SAMs and assayed for the ability to bind the target molecule. Human TBPc, in solution, bound identically to GST-4× surfaces irrespective of the peptide density (see  FIG. 3  and  FIG. 8 ).  
         [0034]     As the graph of  FIG. 4  shows, the stoichiometry of hTBPc binding to GST-4× derivatized surfaces is a constant, independent of the immobilization density. In contrast, the binding of hTBPc to GST-2× surfaces is a non-linear function of the surface density. Notably, at corresponding surface concentrations, GST-2× bound half as much hTBPc as GST-4×, suggesting that two 2× modules immobilized at close proximity to each other (high density) simultaneously contact one hTBPc molecule. Kinetic rate constants were extracted by analyzing association and dissociation phases of sensorgram curves using a non-linear regression curve fitting program: BIAevaluation, version 2.1. The analysis assumed pseudo-first order reactions. The interaction between GST4× and hTBPc was characterized by an average association rate of 2.5×10 4  s −1  M −1  and an average dissociation rate of 4×10 −4  s −1 , yielding a calculated average k d  of 16×10 −9  M. Standard errors obtained for each SPR experiment were considerably smaller than the variation in kinetic rates measured among several experiments using a wide range of NTA concentrations. There could be as much as a two-fold variation in the calculated k d . Sensorgram association curves from the binding of hTBPc to GST-2× could not be fit by pseudo first order kinetics, again consistent with the idea that two −2× modules bind one hTBPc molecule. However, the dissociation phase of the sensorgram was well fit and yielded an average k d  of 1.5×10 −3 +/−0.13 s −1  for the interaction. The almost ten-fold difference between the 4× k d  and 2× k d  may indicate that the 2× dissociation curve is the superposition of two decay rates, corresponding to two dissociating species.  
         [0035]     Note that at high NTA density, the chip surface acted as a rigid linker between two −2× modules to mimic a 4× module, thus creating a higher affinity ligand. Three possible models might explain why the 4× peptide is a higher affinity ligand for hTBPc than a 2× peptide (See  FIG. 5 ). Model 1 proposes that the 4× peptide is a “bivalent” ligand that simultaneously and cooperatively binds more than one site on the target protein, producing a high affinity interaction characterized by a slower off-rate (Jencks, W. P. 1981. On the attribution and additivity of binding energies.  Proc. Natl. Acad Sci. USA.  78(7):4046-4050.). Model 2 says the binding of one recognition motif causes an allosteric effect that enhances the binding of subsequent motifs. Four connected minimal motifs provide for an increased local concentration of ligand available for the second higher affinity interaction. Model 3 proposes that the higher affinity interaction is the result of the summation of multiple interactions of equal strength between the target protein and the entire length of the peptide. A prediction of Model 1 is that 2× peptides, free in solution, will interact with hTBPc independently and exhibit a faster off-rate which is characteristic of monovalent binding. Therefore, if hTBPc is pre-bound by peptide in solution, the 4× peptide should be a much better inhibitor of hTBPc binding to surface immobilized ligand than the 2× peptide. Model 2 predicts that hTBPc pre-bound by 4× or 2× peptides (at twice the concentration) would be similarly inhibited, so long as incubation concentrations were high enough to compensate for the 4× local concentration advantage. Model 3 implies that mutation of amino acids within the peptide would decrease its affinity for TBP as an approximately linear function of the number of mutations.  
         [0036]     In order to compare dissociation rates, aliquots of hTBPc were pre-incubated at very high concentration (35 μM) with either buffer, 2× peptide (1:4 stoichiometry), or 4× peptide (1:2 stoichiometry), then diluted to the usual hTBPc concentration (124 nM) before injection over GST-4× surfaces. Synthetic 2× (16-mer) and 4× (32-mer) peptides were used to eliminate possible interference from GST.  FIG. 9  shows that the preincubation of hTBPc with 2× peptide was in no way inhibitory to its interaction with surface immobilized GST-4×. In contrast, preincubation of hTBPc with 4× peptide completely abolished the interaction. Additional experiments showed that the 32-mer, but not the 16-mer peptide, also blocked the binding of hTBPc to high density GST-2× surfaces, again demonstrating that GST-2×, immobilized at high density, behaves like GST-4×.  
         [0037]     The experiments tabulated in  FIG. 9  argue against the allosteric effect model but are consistent with Models 1 and 3. The question is, does the increased binding energy of the hTBP-4× interaction result from the cumulative effect of multiple bonds along the length of the peptide or from the synergistic effect of two minimal motifs simultaneously binding to the target molecule, with the intervening amino acids merely serving as a tether between the two? A synthetic 31 amino acid peptide consisting of two minimal motifs (DFDLDMLG) separated by a flexible linker ((Ser 4  Glyl) 3 ) was generated. This peptide, 1X-linker-1X, when preincubated with hTBP (under the same conditions described above) inhibited by 83% the complex&#39;s ability to bind to surface immobilized GST-4× (see  FIG. 9 ). These results reinforce the premise of Model 1 and imply that the enhanced strength of binding between hTBP and the 4× peptide is due to a synergistic effect caused by two connected minimal activation motifs simultaneously binding to two separate and discrete sites on hTBP. One may also infer, from the last experiment, that the interaction between minimal activation motifs and hTBP is specific.  
         [0038]     Next the kinetics of the surface interaction to analogous interactions in solution were compared. A series of equilibrium inhibition experiments were performed to characterize the solution interactions between hTBPc and 2× or 4× peptides. Aliquots of hTBPc, (124 nM), were mixed with increasing amounts of synthetic 2× or 4× peptide then incubated at 4° for 1 hour prior to injection over GST-4× surfaces. Titration curves (see  FIG. 6 ) yield an IC 50  of 370 nM for the 4× peptide and 90 μM for the 2× peptide binding to hTBPc. In summary, the 4× peptide binds hTBPc about 250-times better than the 2× peptide. This is the relative difference between monovalent and bivalent binding of hTBPc. The interaction between the 4× peptide and hTBPc in solution is about 20-times weaker than the comparable surface interaction where diffusion is limited.  
         [0039]     The physiological relevance of the interaction between hTBP and the reiterated minimal motifs was investigated. It has been argued that the widely observed in vitro interactions between TBP and activation domains are artifacts resulting from a nonspecific interaction between TBP&#39;s basic DNA-binding region and the acidic peptides. To rule out this possibility, N-terminally histidine-tagged hTBP was immobilized on NTA-SAMs then separately incubated with either: a) TATA sequence DNA; or b) DNA that did not contain a hTBP recognition sequence. GST-4× was then injected over the derivatized surfaces. DNA that did not contain a TATA sequence did not bind to the immobilized hTBP significantly. DNA containing a TATA sequence bound to immobilized hTBP with approximate 1:1 stoichiometry but was in no way inhibitory to the subsequent binding of GST-4× (see  FIG. 7 ). In fact, hTBPc complexed by its cognate DNA bound roughly twice as much GST-4× as the uncomplexed hTBPc. This result is consistent with the observation that hTBPc exists as a dimer that is disrupted upon DNA binding (Taggart, A. K. P. and B. F. Pugh. 1996. Dimerization of TFIID when not bound to DNA.  Science.  272:1331-1333.). The binding of an activating region does not seem to disrupt hTBPc dimerization.  
         [0040]     A competitive inhibition experiment was performed to determine whether the 4× peptide could block the interaction between hTBP and the native activation domain of VP16. A histidine-tagged Gal4(1-147)+VP16(413-490) fusion protein was immobilized on NTA-SAMs. hTBP was incubated with buffer or 4× peptide then injected over VP16 derviatized surfaces. The last two lines of  FIG. 9  show that preincubation of hTBP with the 4× peptide (32 amino acids) completely abolished the hTBP-VP 16 (78 amino acids) interaction. This result is consistent with the idea that minimal activation motifs recognize the same binding site(s) on hTBP as the parent activator.  
         [0041]     In conclusion, SAMs were used to form biospecific rigid, nano-scale probe arrays of known surface density and then utilized to determine the number of binding sites on a target molecule and an approximate distance between sites. This approach is not hampered by the vagaries of secondary or tertiary structures that would be encountered by using DNA or peptide spacers to determine distances between active sites. SPR was used to show that the avidity between TBP, in solution, and surface immobilized peptides was a non-linear function of peptide surface density.  
         [0042]     Peptides immobilized on a 3.8% NTA-SAM were not able to bind hTBP, while peptides presented on a 5.7% NTA-SAM bound TBP with nano-molar affinity. The findings are consistent with the idea that this large increase in binding strength marks the transition between mono- and bivalent binding of the target protein. Individual 8 amino acid minimal activation motifs separated by a 15 amino acid flexible linker bound hTBP nearly as well as four tandem repeats of the motif, leading to the conclusion that hTBP has at least two discrete sites capable of simultaneously interacting with the 8 amino acid motif. Calculations based on an assumed Poisson distribution of NTA in the SAM indicate that the surfaces that did not bind hTBP (3.7% NTA) presented peptides an average distance of 29 Å apart while peptides in denser arrays (5.7% NTA) that bound hTBP with high avidity were on average 2 Å apart.  
         [0043]     The crystal structure of hTBPc has been solved (Nikolov et al., 1995). The peptide consists of two imperfect repeats that form a two-domain saddle shaped DNA-binding protein with two-fold intramolecular symmetry. TBP binds DNA with the concave underside of its “saddle” shape. The general transcription factor TFIIB binds near the TBP/DNA complex at the downstream end leaving the convex “seat” of the saddle available for other intermolecular interactions. Quasi-identical structures composed of basic helices and P sheets flank the seat of the saddle. Mirror image helices H2 and H2′ are separated by distances on the order of 20 Å. It is conceivable that the minimal activation motifs, described herein, simultaneously bind to two-fold related pseudo-identical recognition sites that may be separated by approximately 23 Å.  
         [0044]     Similar schemes can be devised to determine distances between active sites on other bivalent molecules or complexes. Of particular interest are dimeric hormone receptors whose signaling activity depends on its association state. Detailed knowledge of distances between active sites would allow for the rational design of agonist or antagonist drugs.  
       Experimental Methods  
       [0045]     Protein preparation: hTBPc was prepared according to Nikolov et al., 1996 and full length histidine-tagged hTBP according to Lee et al. [Lee, W. S., C. C. Kao, G. O. Bryant, X. Liu and A. J. Berk. (1991) Adenovirus EIA activation domain binds the basic repeat in the TATA box transcription factor.  Cell  67:365-376]. Glutathione S-transferase (GST) fusion proteins were prepared according to Tanaka, 1996. The preparation of Gal4-VP 16 is described by Hori, R., S. Pyo and M. Carey, 1995. Protease footprinting reveals a surface on transcription factor TFHB that serves as an interface for activators and co-activators.  Proc. Natl. Acad Sci. USA.  92(13):6047-6051.  
         [0046]     DNA: TATA sequence DNA was prepared according to Parvin et al. [Parvin, J. D., R. J. McCormick, P. A. Sharp, and D. E. Fisher. 1995. Pre-bending of a promoter sequence enhances affinity for the TATA-binding factor.  Nature.  373:724-727] with the exception that it was not circularized. A 50 base-pair double stranded oligo containing 2 Gal4 binding sites, synthesized and quantitated by GibcoBRL, Life Technologies Inc., Grand Island, N.Y., was used as non-specific control DNA. Equal mass amounts of specific vs. non-specific DNA were added.  
         [0047]     Synthetic peptides: Peptides were generated by F-MOC synthesis and quantitated by amino acid analysis, analytical HPLC and mass spectroscopy.  
         [0048]     The preparation self-assembled monolayers: NTA-SAMs were prepared according to Sigal et al., 1996. A panel of incrementally different density NTA surfaces was generated by serial dilution of a stock solution containing 11.4% NTA-thiol, relative to tri-ethylene glycol terminated thiol, into solutions containing the tri-ethylene glycol terminated thiol alone. Total thiol concentration was kept constant at 1 mM. NTA-SAMs were stored under argon for up to 1 week prior to use. Background levels of binding were assessed by passing reactants over underivatized GST surfaces and subtracted.  
         [0049]     Surface plasmon resonance: Experiments were carried out in a BIAcore instrument at room temperature in phosphate buffered saline (PBS) (137 mM NaCl) running at a constant flow rate of 5 μl/min. Sample injection volumes (plugs) were 35 ∥l. Association and dissociation rate constants were extracted from the data with BIAevaluation software, version 2.1, assuming a pseudo first order kinetics model: A+B         AB. Error rates were taken from the deviation of measurements among multiple experiments performed on surfaces of different NTA densities with a range of protein concentrations and using several different protein preparations, of the same species, to account for variation of the active concentration of a component.  
         [0050]     Statistical calculations: Sulfur atoms bind to gold to form a face-centered hexagonal tiling pattern 4.99 Å on edge. In an ordered monolayer, all the positions of the hexagon are occupied by a thiol. Each vertex is shared by three hexagons, so there are three possible positions for thiol deposition per hexagon. If the thiol solution is doped with a derivatized species of thiol, such as ours is, the average number of NTA-thiols deposited per some number of hexagons (λ), can be calculated, assuming Poisson statistics, for a given NTA-thiol concentration. (It was assumed that the concentration of NTA-thiol in solution was equal to its concentration in the SAM; see  FIG. 2  of Sigal et al., 1996). Equation (1) of  FIG. 1  calculates how many hexagons, on average, must be filled before two NTA-thiols are deposited. For a 3.8% NTA-thiol concentration in solution, relative to EG 3 -thiol, an average of 17.5 hexagons must be filled before 2 NTA ligands appear. For a 5.7% NTA solution, 11.7 hexagons must be filled before an average of two NTA ligands are deposited. The area of a hexagon 4.99 Å on edge is 64.69 Å 2  which is equal to the area of a square, 8.04 Å on edge. 17.5 hexagons would occupy the same area as a square (17.5×8.04 2 ) 1/2  Å on edge, which equals 33.6 Å. Two NTA ligands were arbitrarily placed in a square representing 17.5 hexagons either 33.6 Å or 23.8 Å apart (See  FIG. 1 ).  
         [0051]     Since there are equal numbers of nearest and next-nearest neighbors, the average of these two distances is a first order approximation of the average distance between ligands resulting from a random distribution. According to this model, NTA ligands on SAMs formed from a 3.8% NTA-thiol solution would be an average of 29 Å apart, while NTA ligands in a SAM formed from a 5.7% NTA-thiol solution would be 23 Å apart. Calculations were done to evaluate the contribution of clustering using Poisson statistics.  
         [0052]     Equation 2 calculates the probability, P, of having n NTA ligands per unit area, where λ, equals the average number of NTAs per unit area. Equation 3 calculates the ratio of the probabilities of having one NTA ligand to two NTA ligands deposited per unit area. It is 17-times more likely to get one NTA than two, per unit area, for 3.8% NTA-thiol SAMs and 11 times more likely at 5.7% NTA concentration. 
 
 P ( n )= e   −λ λ n   /n!   (2) 
 
                 P   ⁡     (   1   )         P   ⁡     (   2   )         =             ⅇ       -     (     3   ⁢           ⁢   sites     )       ⁢     (       [   0.038   ]     ⁢           ⁢   NTA     )         ⁡     [       (   3   )     ⁢     (   0.038   )       ]       1     /     1   !               ⅇ       -     (     3   ⁢           ⁢   sites     )       ⁢     (       [   0.038   ]     ⁢           ⁢   NTA     )         ⁡     [       (   3   )     ⁢     (   0.038   )       ]       2     /     2   !                 (   3   )             
 
         [0053]     All publications cited in this application are hereby incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.  
         [0054]     It is to be understood that the above invention is not limited to the particular embodiments described which are meant to be for illustrative purposes only. Variations and modifications of these embodiments may be made that are still included in the description of this invention and fall within the scope of the appended claims.