One avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. Among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry such peptide sequences interact with the target protein in the cell. With the protocols described in this chapter, a laboratory with skills in microbiology, molecular biology, and protein biochemistry can readily identify peptides in the library that bind selectively, and with micromolar affinity, to a given target protein on the time scale of 2 months. To illustrate this approach, we use a library of bacteriophage M13 particles, which display 12-mer combinatorial peptides, to affinity select different peptide ligands for two different targets, the SH3 domain of the human Lyn protein tyrosine kinase and a segment of the yeast serine/threonine protein kinase Cbk1. The binding properties of the selected peptide ligands are then dissected by sequence alignment, Kunkel mutagenesis, and alanine scanning. Finally, the peptide ligands can be used to predict cellular interacting proteins and serve as the starting point for drug discovery.

Mixed-lineage kinase 3 (MLK3; also known as MAP3K11) is a Ser/Thr protein kinase widely expressed in normal and cancerous tissues, including brain, lung, liver, heart, and skeletal muscle tissues. Its Src homology 3 (SH3) domain has been implicated in MLK3 autoinhibition and interactions with other proteins, including those from viruses. The MLK3 SH3 domain contains a six-amino-acid insert corresponding to the n-Src insert, suggesting that MLK3 may bind additional peptides. Here, affinity selection of a phage-displayed combinatorial peptide library for MLK3's SH3 domain yielded a 13-mer peptide, designated “MLK3 SH3–interacting peptide” (MIP). Unlike most SH3 domain peptide ligands, MIP contained a single proline. The 1.2-Å crystal structure of the MIP-bound SH3 domain revealed that the peptide adopts a β-hairpin shape, and comparison with a 1.5-Å apo SH3 domain structure disclosed that the n-Src loop in SH3 undergoes an MIP-induced conformational change. A 1.5-Å structure of the MLK3 SH3 domain bound to a canonical proline-rich peptide from hepatitis C virus nonstructural 5A (NS5A) protein revealed that it and MIP bind the SH3 domain at two distinct sites, but biophysical analyses suggested that the two peptides compete with each other for SH3 binding. Moreover, SH3 domains of MLK1 and MLK4, but not MLK2, also bound MIP, suggesting that the MLK1–4 family may be differentially regulated through their SH3 domains. In summary, we have identified two distinct peptide-binding sites in the SH3 domain of MLK3, providing critical insights into mechanisms of ligand binding by the MLK family of kinases.

Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated.

Phage-display is a convenient vehicle for the generation and screening of combinatorial peptide libraries for a variety of purposes. With standard molecular biology techniques, it is now possible to generate phage libraries displaying 10 10 different peptides, and screen them for peptide ligands to cell surfaces, metals, and proteins, or substrates of proteases, on the time frame of weeks.The first reported display of an exogenous protein fragment on the surface of bacteriophage occurred in 1985 [1]. In pioneering work by Professor George Smith, a fragment of the b-galactosidase protein of Escherichia coli was inserted into a gene encoding a minor capsid protein of M13 bacteriophage and the resulting phage were still infectious. Not only did the resulting chimeric phage particles display epitopes of the b-galactosidase protein, but also the fusion phage could be enriched more than 1000-fold over nonrecombinant phage with antibodies to the b-galactosidase protein.Protocols were subsequently developed that permitted a single fusion phage to be isolated, through affinity selection with antibodies, from an excess of 10 8 nonrecombinant phage particles [2].Combinatorial peptide libraries that were phage-displayed appeared in three simultaneous publications in 1990. In two of the publications, the linear epitopes of two different monoclonal antibodies were mapped by affinity selecting families of related peptides from libraries of 10 8 different peptides [3,4]. In the third publication, peptide ligands to streptavidin were selected from a library of 10 8 different peptides [5]. These three publications were unique in that libraries of such a large size were available for the first time and were sufficiently large enough to represent nearly all 6-mer peptide permutations. In addition, these combinatorial peptide libraries were proposed to be the source of peptide ligands to receptors, and thus serve as agonists and antagonists. Since then, phage-displayed combinatorial peptides have been the source of peptides that selectively bind cell and tissue surfaces [6-11], cytosolic proteins [12][13][14], receptors [15][16][17][18][19][20][21], inert materials [22,23], metals [24], and toxins [25][26][27].