Identifying Small‐Molecule Modulators of Protein‐Protein Interactions

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit outlines methods for identifying cyclic peptides that inhibit protein?protein interactions. Proteins of interest are cloned into a two?hybrid system engineered to operate in reverse, allowing the disruption of a protein complex to be coupled to cell growth. Cyclic peptide libraries are generated using an intein?based plasmid construct, and the cyclized sequence is randomized using a PCR procedure. By transforming plasmid libraries into host cells containing the two?hybrid fusions, cyclic peptide inhibitors can be identified by growing the cells under the appropriate selective conditions. A detailed procedure for performing the genetic selection and identifying false positives is provided. Methods for building the two?hybrid protein fusions and optimizing media conditions, as well as an additional protocol for constructing cyclic peptide libraries are also provided.

Keywords: small?molecule; protein?protein; cyclic peptide; two?hybrid; inhibitor

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Basic Protocol 1: Identification of Small‐Molecule Inhibitors of In Vivo Protein‐Protein Interactions by Genetic Selection
Support Protocol 1: Construction and Optimization of a Strain for Inhibitor Selection
Support Protocol 2: Construction of Cyclic Peptide Libraries
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Identification of Small‐Molecule Inhibitors of In Vivo Protein‐Protein Interactions by Genetic Selection

Materials

Selection medium (see recipe )
Luria‐Bertani (LB) agar plates ( appendix 4A ) supplemented with 25 µg/ml chloramphenicol
E. coli strain SNS‐126 containing the two‐hybrid reporter and protein domains of interest (or control protein) fused to the bacteriophage repressor DNA‐binding domains ( protocol 2 )
50 to 200 ng cyclic peptide library ( protocol 3 )
SOC broth (see recipe )
1× MMA: diluted from 5× minimal medium A ( appendix 4A )
LB broth ( appendix 4A ) with and without 25 µg/ml chloramphenicol
200 mM IPTG
Oligonucleotide that hybridizes to the intein fragment (in this case, pAR‐NdeI, 5′‐ GGAATTCCATATGGTTAAAGTTATCGGTCGTCGT‐3′)

245 × 245–mm culture plates (Nunc)
100 × 15–mm petri dishes
Electroporator and cuvettes
18 × 100–mm culture tubes
Toothpicks, sterile
Inoculating loops, sterile
Plasmid preparation kit, optional (e.g., QIAquick Spin Miniprep Kit; Qiagen)
96‐well microtiter plates
Multichannel pipettor
DNA sequencing facility

Additional reagents and equipment for growing bacteria ( appendix 4B ), preparing electrocompetent cells for electroporation (Seidman et al., ), and purifying plasmid DNA ( appendix 4C )

NOTE: All reagents used with E. coli cells must be sterile.

Support Protocol 1: Construction and Optimization of a Strain for Inhibitor Selection

Materials

Oligonucleotides (specifically designed)
Plasmid pTHCP16 (Fig. A, for making homodimeric fusions) or pTHCP14 (Fig. C, for making heterodimeric fusions)
DNA template (gene of interest)
2.5 U/ml proofreading PCR polymerase (e.g., Deep Vent, New England Biolabs; Pfu, Stratagene)
0.1 M EDTA, pH 8.0
10% SDS
2.5 mg/ml proteinase K
3 M sodium acetate, pH 5.2
70% (v/v) and 100% ethanol
PCR purification kit (e.g., QIAquick, Qiagen; optional)
DNA purification kit (e.g., QIAquick Spin Miniprep Kit, Qiagen; optional)
Restriction enzymes (specific to the cloning procedure)
Gel extraction kit (e.g., QIAquick gel extraction kit, Qiagen; optional)
T4 DNA ligase (New England Biolabs)
E. coli competent cells (e.g., DH5α): prepared ( appendix 4D ) or purchased from commercial suppliers
LB agar plates ( appendix 4A ) supplemented with 100 µg/ml ampicillin and with and without IPTG (0 to 1 mM)
E. coli strain SNS‐118 (homodimeric reporter) or SNS‐126 (heterodimeric reporter)
CRIM plasmids (E. coli Genetic Stock Center, Yale), optional
Z buffer (see recipe )
Chloroform
0.1% (w/v) SDS
4 mg/ml o ‐nitrophenyl‐β‐D‐galactopyranoside (ONPG): prepared fresh
1 M sodium bicarbonate (Na₂ CO₃ )

¾ in. sterile, optical glass culture tubes, optional
13 × 100–mm glass tubes (disposable)

Additional reagents and equipment for growing bacteria ( appendix 4B ), performing PCR ( appendix 4J ), separating DNA by agarose gel electrophoresis ( appendix 4F ), purifying plasmid DNA ( appendix 4C ), quantitating DNA ( appendix 4K )

NOTE: For more information about preparing, analyzing, and cloning DNA see appendix 44 .

Support Protocol 2: Construction of Cyclic Peptide Libraries

Materials

LB broth ( appendix 4A ) supplemented with 30 µg/ml chloramphenicol
E. coli culture containing plasmid pARCBD‐p (Scott et al., ), or another compatible plasmid
DNA purification kit (e.g., QIAquick Spin Miniprep Kit, Qiagen; optional)
2.5 U/µl proofreading PCR polymerase (e.g., Deep Vent; New England Biolabs) and 10× buffer
2 mM (each) dNTP mix
Oligonucleotide primers
- C+5 (5′‐GGAATTCGCCAATGGGGCGATCGCCCACAATTGTNNSNNSNNSNNSNNSTGCTTAAGTTTTGGC‐3′)
- CBD‐reverse (5′‐GGAATTCAAGCTTTCATTGAAGCTGCCACAAGG‐3′)
- zipper (5′‐GGAATTCGCCAATGGGGCGATCGCC‐3′)
PCR purification kit, optional (e.g., QIAquick,Qiagen)
100 mM EDTA, pH 8
10% (w/v) SDS
2.5 mg/ml proteinase K
Restriction enzymes: Bgl I and Hind III (e.g., New England Biolabs)
Phosphatase (e.g., shrimp alkaline phosphatase; United States Biochemical)
Gel extraction kit (e.g., QIAquick gel extraction kit, Qiagen)
T4 DNA ligase (e.g., Promega)
3 M sodium acetate, pH 5.2
100% ethanol

Thermal cycler
0.05 micron VM nitrocellulose membranes (Millipore), optional
250‐ml beaker, optional

Additional reagents and equipment for purifying plasmid DNA ( appendix 4C ), purifying DNA ( appendix 4E ), performing agarose gel electrophoresis ( appendix 4F ), and quantitating DNA by spectroscopy ( appendix 4K )

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Figures

Figure 19.15.1 Engineering the DnaE trans ‐intein of Synechocystis sp. PCC6803 to cyclize an intervening sequence. This strategy is utilized in the SICLOPPS method to make libraries of cyclic peptides (Scott et al., ).

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Figure 19.15.2 Scheme for genetic selection for cyclic peptide inhibitors of protein‐protein interactions (Horswill et al., ). A protein of interest is fused to a repressor DNA‐binding domain, which associates to block expression of three genes encoding HIS3 (IGPD, histidine biosynthesis), kan (aminoglycoside 3′‐phosphotransferase, kanamycin resistance), and lacZ (β‐galactosidase). Background expression can be chemically tuned with 3‐aminotriazole (3‐AT, competitive inhibitor of IGPD) and kanamycin. By placing E. coli host cells on minimal medium lacking histidine, a genetic selection is created that can be used to identify cyclic peptides that inhibit protein‐protein interactions. Reproduced from Horswill et al. () with permission, Copyright 2004 by National Academy of Sciences, U.S.A.

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Figure 19.15.3 Steps involved in the . The points at which Support Protocols 1 and 2 become relevant are indicated.

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Figure 19.15.4 Graphic map of the RTHS plasmids used for making repressor fusions and their respective promoter sequences: (A ) Plasmid pTHCP16 for constructing homodimeric fusions requires a strain containing the phage 434 wild‐type promoter ( E. coli strain SNS118 derivatives). (B ) The wild‐type 434 promoter. (C ) Plasmid pTHCP14 for constructing heterodimeric fusions requires a strain containing the chimeric phage 434·P22 promoter ( E. coli strain SNS126 derivatives). (D )The chimeric 434·P22 promoter.

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Videos

Literature Cited

Literature Cited
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Identifying Small‐Molecule Modulators of Protein‐Protein Interactions

Abstract

Table of Contents

Materials

Basic Protocol 1: Identification of Small‐Molecule Inhibitors of In Vivo Protein‐Protein Interactions by Genetic Selection

Support Protocol 1: Construction and Optimization of a Strain for Inhibitor Selection

Support Protocol 2: Construction of Cyclic Peptide Libraries

Figures

Videos

Literature Cited