Construction and evaluation of an automated light directed protein-detecting microarray synthesizer.

We have designed, constructed, and evaluated an automated instrument that has produced high-density arrays with more than 30 000 peptide features within a 1.5 cm(2) area of a glass slide surface. These arrays can be used for high throughput library screening for protein binding ligands, for potential drug candidate molecules, or for discovering biomarkers. The device consists of a novel fluidics system, a relay control electrical system, an optics system that implements Texas Instruments' digital micromirror device (DMD), and a microwave source for accelerated synthesis of peptide arrays. The instrument implements two novel solid phase chemical synthesis strategies for producing peptide and peptoid arrays. Biotin-streptavidin and DNP anti-DNP (dinitrophenol) models of antibody small molecule interactions were used to demonstrate and evaluate the instrument's capability to produce high-density protein detecting arrays. Several screening assay and detection schemes were explored with various levels of efficiency and assays with sensitivity of 10 nM were also possible.

Photo-induced oxidative cross-linking as a method to evaluate the specificity of protein-ligand interactions.

The isolation of protein-binding synthetic molecules from combinatorial libraries or compound collections is now a common practice in chemical biology. An important, but underdeveloped, aspect of characterizing the binding properties of such molecules is their level of binding specificity. This is often evaluated by simply measuring the equilibrium binding affinity of the compound of interest with its target protein and comparing this value with its affinity to one or a few other purified proteins selected at random. These measurements may not reflect accurately the ability of the compound to seek out its target in a complex mixture of proteins such as a cell extract or serum. A more desirable alternative would be to develop solution assays that measure directly the binding of the molecule of interest to both target and competitor proteins in complex solutions. In this report, we evaluate a rapid and efficient photo-triggered cross-linking reaction for assessing binding specificity of synthetic molecules in protein mixtures. Using peptide-protein complexes, we demonstrate that this reaction provides an unbiased view of the peptide-protein contacts present in solution under a given set of conditions and thus is useful for assessing binding specificity. We also discuss the potential application of this chemistry to the related, but more difficult, problem of the identification of protein targets of bioactive molecules.

Evidence that Gal11 protein is a target of the Gal4 activation domain in the mediator.

The mediator is an approximately 20 protein complex that is essential for the transcription of most genes in yeast. It is contacted by a number of gene-specific activators, but the details of these interactions are not well understood in most cases. Here, evidence is presented that the mediator component Gal11 represents at least one target of the Gal4 activation domain (AD). Deletion of Gal11 is shown to decrease the affinity of the Gal4 AD for the mediator, and direct binding of an N-terminal domain of Gal11 with the Gal4 AD is demonstrated. Quantitative studies, however, indicate that the K(D) of the 1:1 Gal4 AD--Gal11 complex is modest. Combined with in vivo data showing that Delta gal11 cells exhibit reduced, but still significant, Gal4-mediated gene expression, these results suggest that the dimeric activator might also contact another protein in the mediator in addition to Gal11.

The 19S regulatory particle of the proteasome is required for efficient transcription elongation by RNA polymerase II.

It is generally thought that the primary or even sole activity of the 19S regulatory particle of the 26S proteasome is to facilitate the degradation of polyubiquitinated proteins by the 20S-core subunit. However, we present evidence that the 19S complex is required for efficient elongation of RNA polymerase II (RNAP II) in vitro and in vivo. First, yeast strains carrying alleles of SUG1 and SUG2, encoding 19S components, exhibit phenotypes indicative of elongation defects. Second, in vitro transcription is inhibited by antibodies raised against Sug1, or by heat-inactivating temperature-sensitive Sug1 mutants with restoration of elongation by addition of immunopurified 19S complex. Finally, Cdc68, a known elongation factor, coimmunoprecipitates with the 19S complex, indicating a physical interaction. Inhibition of the 20S proteolytic core of the proteasome has no effect on elongation. This work defines a nonproteolytic role for the 19S complex in RNAP II transcription.

The Gal4 activation domain binds Sug2 protein, a proteasome component, in vivo and in vitro.

An in vivo protein interaction assay was used to search a yeast cDNA library for proteins that bind to the acidic activation domain (AD) of the yeast Gal4 protein. Sug2 protein, a component of the 19 S regulatory particle of the 26 S proteasome, was one of seven proteins identified in this screen. In vitro binding assays confirm a direct interaction between these proteins. SUG2 and SUG1, another 19 S component, were originally discovered as a mutation able to suppress the phenotype of a Gal4 truncation mutant (Gal4(D)p) lacking much of its AD. Sug1p has previously been shown to bind the Gal4 AD in vitro. Taken together, these genetic and biochemical data suggest a biologically significant interaction between the Gal4 protein and the 19 S regulatory particle of the proteasome. Indeed, it is demonstrated here that the Gal4 AD interacts specifically with immunopurified 19 S complex. The proteasome regulatory particle has been shown recently to play a direct role in RNA polymerase II transcription and the activator-19 S interaction could be important in recruiting this large complex to transcriptionally active GAL genes.

An inhibitor of sequence-specific proteolysis that targets the substrate rather than the enzyme.

BACKGROUND: Traditional protease inhibitors target the active site of the enzyme. However, since most proteases act on multiple substrates, even the most specific protease inhibitors will affect the levels of a number of different proteins. However, if substrate-targeted inhibitors could be developed, much higher levels of specificity could be achieved. In theory, compounds that bind the cleavage site of a particular substrate could block its interaction with a protease without having any effect on the processing of other substrates of that protease. RESULTS: A model system is presented that demonstrates the feasibility of substrate-targeted inhibition of proteolysis. A peptide selected genetically to bind a 14-residue epitope that encompasses the cleavage site of human pro-IL-1beta was shown to inhibit interleukin-converting enzyme (ICE)-mediated proteolysis of model substrates containing the 14-mer target sequence. However, the peptide had no effect on the cleavage of other ICE substrates with different amino acids flanking the minimal cleavage site. CONCLUSIONS: This study demonstrates the feasibility of substrate-targeted inhibition of proteolysis. More potent compounds must be developed before substrate-targeted inhibitors can be used routinely. Nonetheless, this novel strategy for protease inhibition seems promising for the development of extremely selective molecules with which to manipulate the maturation of many important pro-hormones, -cytokines and -proteins.

Protein microarrays: prospects and problems.

Protein microarrays are potentially powerful tools in biochemistry and molecular biology. Two types of protein microarrays are defined. One, termed a protein function array, will consist of thousands of native proteins immobilized in a defined pattern. Such arrays can be utilized for massively parallel testing of protein function, hence the name. The other type is termed a protein-detecting array. This will consist of large numbers of arrayed protein-binding agents. These arrays will allow for expression profiling to be done at the protein level. In this article, some of the major technological challenges to the development of protein arrays are discussed, along with potential solutions.

TATA-binding protein and the Gal4 transactivator do not bind to promoters cooperatively.

The yeast Gal4 protein, like many activators, binds TATA-binding protein (TBP) directly in vitro. It has been speculated that this protein-protein interaction is important for Gal4p-mediated activation of transcription, but little work has been done to test specific models involving this interaction. In this study, the effect of Gal4p on TBP-TATA binding is addressed. Specifically, it is asked if the Gal4p-TBP interaction can support cooperative binding of the two factors to promoters. It is easy to see how such an event could stimulate transcription, particularly from promoters with a non-consensus TATA box. In vitro, however, a derivative of Gal4p (Gal4-(1-93+768-881)) containing the DNA-binding, dimerization, and activation domains does not bind to promoter DNA cooperatively with either recombinant, purified TBP, or with protein from a yeast crude extract. In vivo, reporter gene experiments using promoters with differing TBP affinities reveal no major Gal4p-mediated stimulation of TBP function from weak TATA boxes, as would be predicted if the proteins bind cooperatively. Furthermore, native Gal4p and a potent Gal4p-based artificial activator lacking a TBP-binding activation domain support similar ratios of transcription from a series of promoters identical except for mutations in the TATA box. It is concluded that Gal4p and TBP do not bind cooperatively to promoters and that this mechanism does not contribute substantially to Gal4p-mediated transcriptional activation.

Scope, limitations and mechanistic aspects of the photo-induced cross-linking of proteins by water-soluble metal complexes.

BACKGROUND: Chemical cross-linking is a valuable tool with which to study protein-protein interactions. Recently, a new kind of cross-linking reaction was developed in which the photolysis of associated proteins with visible light in the presence of ammonium persulfate and tris(2,2'-bipyridyl)ruthenium(II) dication or palladium(II) porphyrins results in rapid and efficient covalent coupling (Fancy, D.A. & Kodadek, T. (1999). Proc. Natl. Acad. Sci. USA 96, 6020-6024 and Kim, K., Fancy, D.A. & Kodadek, T. (1999). J. Am. Chem. Soc. 121, 11896-11897). Here, mechanistic and practical aspects of the reaction of importance for its application to biochemical problems are examined. RESULTS: It is shown that the photo-initiated cross-linking chemistry can be optimized for the analysis of protein-protein interactions in crude cell extracts. A number of commonly used epitope or affinity tags survive the reaction in functional form, allowing the simple visualization of the cross-linked products, or their isolation. It is shown that very little light-independent oxidation of protein residues occurs and that significant perturbation of complexes of interest prior to the brief photolysis period does not occur. Finally, evidence is presented that is consistent with a mechanistic model in which ammonium persulfate functions simply as an electron acceptor, facilitating the generation of the key high valent metal complex from the photoexcited species by electron transfer. In the absence of an electron acceptor, a much lower efficiency reaction is observed that appears to involve products resulting from reaction of the excited state metal complex with molecular oxygen. CONCLUSIONS: These results provide useful practical information for chemists and biochemists who may wish to employ this new cross-linking chemistry for the analysis of protein complexes. They also shed new light on the mechanism of this interesting reaction.

Biochemical characterization of the TATA-binding protein-Gal4 activation domain complex.

It has been suggested that complexes between gene-specific activators and the TATA-binding protein (TBP) play an important role in the expression of many genes. However, few detailed studies of well defined activator-TBP complexes have been reported. An analysis of the biochemical properties of the complex formed by the acidic activation domain (AAD) of the yeast activator Gal4 and TBP is presented here. This is shown to be composed of two AAD and one TBP molecule. DNA binding experiments reveal that TATA-containing DNAs and the Gal4 AAD bind TBP competitively, suggesting that the AAD and TATA boxes recognize overlapping surfaces of TBP. The kinetics of the formation and dissociation of the AAD(2)-TBP complex is also probed. The impact of these findings on models for Gal4-mediated transcriptional activation is considered.

A CDC6 protein-binding peptide selected using a bacterial two-hybrid-like system is a cell cycle inhibitor.

Peptides or small molecules able to modulate protein-protein interactions hold promise as tools with which to probe and manipulate biological pathways. An important issue in this nascent field is to evaluate different methods with which to search libraries for molecules that modulate the function of specific target proteins. One strategy is to screen libraries for molecules that bind specifically to a protein known to be critical in the pathway of interest, with the expectation that the molecules isolated will recognize regions of the target protein important for its function and thereby exhibit biological activity. Here, a peptide library was screened using a two-hybrid-like system for molecules able to bind human CDC6 protein (CDC6p), required for the initiation of DNA replication in eukaryotic cells. From a collection of over a million peptides, a single species that exhibited good affinity and specificity for binding CDC6p was obtained. When expressed in human cells, the peptide inhibited cell cycle progression and exhibited other properties expected of a CDC6p inhibitor. This approach, which does not require detailed knowledge of the mechanism of action of a protein target, may be generally useful for isolating peptides capable of manipulating biological pathways.

Peptides selected to bind the Gal80 repressor are potent transcriptional activation domains in yeast.

The activation domain of the yeast Gal4 protein binds specifically to the Gal80 repressor and is also thought to associate with one or more coactivators in the RNA polymerase II holoenzyme and chromatin remodeling machines. This is a specific example of a common situation in biochemistry where a single protein domain can interact with multiple partners. Are these different interactions related chemically? To probe this point, phage display was employed to isolate peptides from a library based solely on their ability to bind Gal80 protein in vitro. Peptide-Gal80 protein association is shown to be highly specific and of moderate affinity. The Gal80 protein-binding peptides compete with the native activation domain for the repressor, suggesting that they bind to the same site. It was then asked if these peptides could function as activation domains in yeast when tethered to a DNA binding domain. Indeed, this is the case. Furthermore, one of the Gal80-binding peptides binds directly to a domain of the Gal11 protein, a known coactivator. The fact that Gal80-binding peptides are functional activation domains argues that repressor binding and activation/coactivator binding are intimately related properties. This peptide library-based approach should be generally useful for probing the chemical relationship of different binding interactions or functions of a given native domain.

Selection and application of peptide-binding peptides.

Peptide-binding ligands would be useful for directing reagents to particular epitopes in a protein, the detection of peptide hormones, and many other applications. Here we show that peptides of modest size isolated from a library using a simple genetic assay can act as specific receptors for other peptides. The equilibrium dissociation constants of these peptide-peptide complexes are higher than those of typical monoclonal antibody-epitope complexes. Nonetheless, as shown here, these peptide-binding peptides can be used to detect or purify proteins containing the partner peptide.

Chemistry for the analysis of protein-protein interactions: rapid and efficient cross-linking triggered by long wavelength light.

Chemical cross-linking is a potentially useful technique for probing the architecture of multiprotein complexes. However, analyses using typical bifunctional cross-linkers often suffer from poor yields, and large-scale modification of nucleophilic side chains can result in artifactual results attributable to structural destabilization. We report here the de novo design and development of a type of protein cross-linking reaction that uses a photogenerated oxidant to mediate rapid and efficient cross-linking of associated proteins. The process involves brief photolysis of tris-bipyridylruthenium(II) dication with visible light in the presence of the electron acceptor ammonium persulfate and the proteins of interest. Very high yields of cross-linked products can be obtained with irradiation times of <1 second. This chemistry obviates many of the problems associated with standard cross-linking reagents.

Genetic selection of short peptides that support protein oligomerization in vivo.

An important goal in protein engineering is to control associations between designed proteins. This is most often done by fusing known, naturally occurring oligomerization modules, such as leucine zippers [1] [2] [3], to the proteins of interest [4] [5] [6]. It is of considerable interest to design or discover new oligomerization domains that have novel binding specificities [7] [8] [9] [10] [11] in order to expand the 'toolbox' of the protein engineer and also to eliminate associations of the designed proteins with endogenous factors. We report here a simple genetic selection scheme through which to search libraries for peptides that are able to mediate homodimerization or higher-order self-oligomerization of a protein in vivo. We found several peptides that support oligomerization of the lambda repressor DNA-binding domain in Escherichia coli cells, some of them as efficiently as the endogenous dimerization domain or the GCN4 leucine zipper. Many are very small, comprising as few as six residues. This study strongly supports the notion that peptide sequence space is rich in small peptides, which might be useful in protein engineering and other applications.

TATA element recognition by the TATA box-binding protein has been conserved throughout evolution.

Cocrystal structures of wild-type TATA box-binding protein (TBP) recognizing 10 naturally occurring TATA elements have been determined at 2.3-1.8 A resolution, and compared with our 1.9 A resolution structure of TBP bound to the Adenovirus major late promoter (AdMLP) TATA box (5'-TATAAAAG-3'). Minor-groove recognition by the saddle-shaped protein induces the same conformational change in each of these oligonucleotides, despite variations in promoter sequence that reduce the efficiency of transcription initiation. Three molecular mechanisms explain assembly of diverse TBP-TATA element complexes. (1) T --> A and A --> T transversions leave the minor-groove face unchanged, permitting formation of TBP-DNA complexes on many A/T-rich core promoter sequences. (2) Cavities in the interface between TBP and the minor-groove face of the AdMLP TATA box accommodate the exocyclic NH(2) groups of G in a TACA box and in a TATAAG box. (3) Formation of a C:G Hoogsteen basepair in a TATAAAC box eliminates steric clashes that would be produced by the Watson-Crick base pair. We conclude that the structure of the TBP-TATA box complex found at the heart of the polymerase II (pol II) transcription machinery has remained constant over the course of evolution, despite variations in TBP and its DNA targets.

Protein cross-linking mediated by metalloporphyrins.

A biomimetic protein cross-linking reaction is described which employs oxidatively-activated manganese and iron porphyrins as the reactive species. A wide range of proteins cross-link under these conditions, but only if they are intimately associated in solution. The reaction is rapid, efficient, and will be useful for the suprastructural analysis of multiprotein complexes.

Small-molecule-based strategies for controlling gene expression.

A central goal in chemical biology is to gain control over biological pathways using small molecules, and the mRNA-synthesizing machinery is a particular important target. New advances in our understanding of transcriptional regulation suggests strategies to manipulate these pathways using small molecules.

A critical role for tyrosine residues in His6Ni-mediated protein cross-linking.

A new type of affinity cross-linking strategy has been developed in which His6-tagged proteins can be cross-linked to their binding partners in the presence of unmodified proteins (D. Fancy, K. Melcher, S. A. Johnston, and T. Kodadek, 1996, Chem. Biol. 3, 551-559). The chemistry involves the addition of Ni(II) to the His6 tag, followed by oxidation of the metal with a peracid. It is shown here that, in addition to the His6 tag, a tyrosine residue placed in close proximity to the metal-binding site can strongly stimulate the yield of cross-linked product. This finding has important practical implications in the use of the His6-Ni-based cross-linking reaction for the analysis of multiprotein complexes.