Utilizing Peptide SPOT Arrays to Identify Protein Interactions

互联网2013-12-31

2433

Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

SPOT arrays consist of synthesized peptides 12? to 18?amino acids long, with overlapping sequences that cover the entire sequence of a protein, covalently linked to a solid support. This unit describes how to construct peptide SPOT arrays, biotinylate recombinant proteins, and conduct overlay assays to identify binding interactions. In addition, directions describing how to analyze results to determine single amino acid binding contributions are included. The two techniques in this unit describe how to scan protein sequences to find binding motifs and how to conduct site?directed mutagenesis studies. Curr. Protoc. Protein Sci. 51:18.10.1?18.10.9. © 2008 by John Wiley & Sons, Inc.

Keywords: peptides; arrays; protein?protein interactions

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Introduction
Basic Protocol 1: Utilizing Peptide Spot Arrays to Define Binding Sites
Commentary
Literature Cited
Figures

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Utilizing Peptide Spot Arrays to Define Binding Sites

Materials

Purified recombinant protein
PBS ( appendix 2E )
2‐Mercaptoethanol (2‐ME)
Sulfo‐NHS‐LC‐biotin (Pierce)
1 M Tris·Cl, pH 8.0 ( appendix 2E )
Dialysis buffer: 20 mM HEPES, pH 7.3, 100 mM potassium acetate, and 1 mM DTT
Fmoc‐amino acids (set of 20 amino acid cartridges; Intavis AG)
Amino acid stock solution: 2.3 g 1‐hydroxybenzotriazole hydrate (HoBt) dissolved in 20 ml 1‐methyl‐2‐pyrrolidinone (NMP) store up to 1 week at 4°C
1‐Methyl‐2‐pyrrolidinone (NMP)
Activator solution: 4.2 ml NMP and 0.8 ml N ,N ′‐diisopropylcarbodiimide (DIC), prepare fresh each day
N ,N ‐dimethyl formamide (DMF)
Ethanol
Capping solution: 15 ml DMF and 300 µl acetic anhydride
Fmoc deprotection solution: 20% piperidine in DMF
Side chain deprotection solution: 7.5 ml trifluoroacetic acid (TFA), 7.5 ml dichloromethane (DCM), 450 µl triisopropylsilane, and 4.6 ml H₂ O
Dichloromethane (DCM)
Blocking solution: TBST containing 5% nonfat dry milk (prepare on day of use)
Avidin‐HRP (Pierce)
Tris‐buffered saline/0.3% Tween (TBST): 20 mM Tris·Cl, pH 7.5, 137 mM NaCl, 0.3% Tween‐20, store at 4°C
ECL substrate (Amersham Biosciences)

Centricon‐10 concentrators (Millipore)
Rotator
NAP 5 column (G‐25 desalting column; Amersham Biosciences)
Auto Spot Robot ASP 222 peptide synthesizer (Intavis AG)
Amino‐PEG₅₀₀ ‐UC450 sheets (acid‐hardened; Intavis AG)
Platform shaker
X‐ray film
Versadoc imaging system (Bio‐Rad) or an equivalent densitometry system
Quantity One densitometry program (Bio‐Rad) or equivalent densitometry program

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 18.10.1 Schematic representation of peptide overlay procedure.

View Image

Figure 18.10.2 Determine binding contribution of a single amino acid. To determine the contribution of Q in this sequence, the binding intensities of the 12 peptides (SPOTs 25 to 36) containing Q are averaged.

View Image

Figure 18.10.3 Binding site determination. (A ) Example of autoradiogram. (B ) Example of graphical representation of binding data.

View Image

Figure 18.10.4 Site‐directed mutagenesis. At every position, an alanine, glutamine, or tyrosine was substituted for the naturally occurring amino acid. Arrows mark critical residues where binding was significantly reduced upon mutagenesis.

View Image

Videos

Literature Cited

Literature Cited
	Alto, N.M., Soderling, S.H., Hoshi, N., Langeberg, L.K., Fayos, R., Jennings, P.A., and Scott, J.D. 2003. Bioinformatic design of A‐kinase anchoring protein‐in silico: A potent and selective peptide antagonist of type II protein kinase A anchoring. Proc. Natl. Acad. Sci. U.S.A. 100:4445‐4450.
	Cushman, I., Palzkill, T., and Moore, M.S. 2006. Using peptide arrays to define nuclear carrier binding sites on nucleoporins. Methods 39:329‐341.
	Frank, R. 2002. The SPOT‐synthesis technique. Synthetic peptide arrays on membrane supports—Principles and applications. J. Immunol. Methods 267:13‐26.
	Knoblauch, N.T., Rudiger, S., Schonfeld, H.J., Driessen, A.J., Schneider‐Mergener, J., and Bukau, B. 1999. Substrate specificity of the SecB chaperone. J. Biol. Chem. 274:34219‐34225.
	Reineke, U., Volkmer‐Engert, R., and Schneider‐Mergener, J. 2001. Applications of peptide arrays prepared by the SPOT‐technology. Curr. Opin. Biotechnol. 12:59‐64.
	Rudiger, S., Germeroth, L., Schneider‐Mergener, J., and Bukau, B. 1997. Substrate specificity of the DnaK chaperone determined by screening cellulose‐bound peptide libraries. EMBO J. 16:1501‐1507.
	Rudiger, S., Mayer, M.P., Schneider‐Mergener, J., and Bukau, B. 2000. Modulation of substrate specificity of the DnaK chaperone by alteration of a hydrophobic arch. J. Mol. Biol. 304:245‐251.
	Wenschuh, H., Volkmer‐Engert, R., Schmidt, M., Schulz, M., Schneider‐Mergener, J., and Reineke, U. 2000. Coherent membrane supports for parallel microsynthesis and screening of bioactive peptides. Biopolymers 55:188‐206.
	Yaffe, M.B. and Cantley, L.C. 2000. Mapping specificity determinants for protein‐protein association using protein fusions and random peptide libraries. Methods Enzymol. 328:157‐170.