One- plus two-hybrid system, a novel yeast genetic selection for specific missense mutations disrupting protein/protein interactions

Ji Young Kim, Ok Gu Park, Jae Lee, Young Chul Lee

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

To facilitate analysis of protein/protein interaction interfaces, we devised a novel yeast genetic screening method, named the "one- plus two-hybrid system," for the efficient selection of missense mutations that specifically disrupt known protein/protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid system is first used to select the intact interacting partner (prey), resulting in the positive selection of informative missense mutants from a large library of randomly generated mutant alleles. Then in a second screening step, interaction-defective prey mutants for a given protein are selected using the two-hybrid reporter system among the isolated missense mutants. We used this method to characterize the interactions between unliganded nuclear receptors (NRs) and the conserved motif within the bipartite NR interaction domains (IDs) of the NR corepressor (N-CoR) and identified the specific residues of N-CoR-IDs required either generally for optimal NR binding or to interact with a particular NR. This efficient and rapid method should allow us to quickly analyze a large number of interaction interfaces.

Original languageEnglish (US)
Pages (from-to)1727-1740
Number of pages14
JournalMolecular and Cellular Proteomics
Volume6
Issue number10
DOIs
StatePublished - Oct 2007
Externally publishedYes

Fingerprint

Genetic Selection
Missense Mutation
Hybrid systems
Yeast
Cytoplasmic and Nuclear Receptors
Yeasts
Co-Repressor Proteins
Proteins
Screening
Two-Hybrid System Techniques
Genetic Testing
Libraries
Alleles

ASJC Scopus subject areas

  • Biochemistry

Cite this

One- plus two-hybrid system, a novel yeast genetic selection for specific missense mutations disrupting protein/protein interactions. / Kim, Ji Young; Park, Ok Gu; Lee, Jae; Lee, Young Chul.

In: Molecular and Cellular Proteomics, Vol. 6, No. 10, 10.2007, p. 1727-1740.

Research output: Contribution to journalArticle

@article{d503d5cff91b40c0a46b77869bdc36da,
title = "One- plus two-hybrid system, a novel yeast genetic selection for specific missense mutations disrupting protein/protein interactions",
abstract = "To facilitate analysis of protein/protein interaction interfaces, we devised a novel yeast genetic screening method, named the {"}one- plus two-hybrid system,{"} for the efficient selection of missense mutations that specifically disrupt known protein/protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid system is first used to select the intact interacting partner (prey), resulting in the positive selection of informative missense mutants from a large library of randomly generated mutant alleles. Then in a second screening step, interaction-defective prey mutants for a given protein are selected using the two-hybrid reporter system among the isolated missense mutants. We used this method to characterize the interactions between unliganded nuclear receptors (NRs) and the conserved motif within the bipartite NR interaction domains (IDs) of the NR corepressor (N-CoR) and identified the specific residues of N-CoR-IDs required either generally for optimal NR binding or to interact with a particular NR. This efficient and rapid method should allow us to quickly analyze a large number of interaction interfaces.",
author = "Kim, {Ji Young} and Park, {Ok Gu} and Jae Lee and Lee, {Young Chul}",
year = "2007",
month = "10",
doi = "10.1074/mcp.M700079-MCP200",
language = "English (US)",
volume = "6",
pages = "1727--1740",
journal = "Molecular and Cellular Proteomics",
issn = "1535-9476",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "10",

}

TY - JOUR

T1 - One- plus two-hybrid system, a novel yeast genetic selection for specific missense mutations disrupting protein/protein interactions

AU - Kim, Ji Young

AU - Park, Ok Gu

AU - Lee, Jae

AU - Lee, Young Chul

PY - 2007/10

Y1 - 2007/10

N2 - To facilitate analysis of protein/protein interaction interfaces, we devised a novel yeast genetic screening method, named the "one- plus two-hybrid system," for the efficient selection of missense mutations that specifically disrupt known protein/protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid system is first used to select the intact interacting partner (prey), resulting in the positive selection of informative missense mutants from a large library of randomly generated mutant alleles. Then in a second screening step, interaction-defective prey mutants for a given protein are selected using the two-hybrid reporter system among the isolated missense mutants. We used this method to characterize the interactions between unliganded nuclear receptors (NRs) and the conserved motif within the bipartite NR interaction domains (IDs) of the NR corepressor (N-CoR) and identified the specific residues of N-CoR-IDs required either generally for optimal NR binding or to interact with a particular NR. This efficient and rapid method should allow us to quickly analyze a large number of interaction interfaces.

AB - To facilitate analysis of protein/protein interaction interfaces, we devised a novel yeast genetic screening method, named the "one- plus two-hybrid system," for the efficient selection of missense mutations that specifically disrupt known protein/protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid system is first used to select the intact interacting partner (prey), resulting in the positive selection of informative missense mutants from a large library of randomly generated mutant alleles. Then in a second screening step, interaction-defective prey mutants for a given protein are selected using the two-hybrid reporter system among the isolated missense mutants. We used this method to characterize the interactions between unliganded nuclear receptors (NRs) and the conserved motif within the bipartite NR interaction domains (IDs) of the NR corepressor (N-CoR) and identified the specific residues of N-CoR-IDs required either generally for optimal NR binding or to interact with a particular NR. This efficient and rapid method should allow us to quickly analyze a large number of interaction interfaces.

UR - http://www.scopus.com/inward/record.url?scp=35648977580&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=35648977580&partnerID=8YFLogxK

U2 - 10.1074/mcp.M700079-MCP200

DO - 10.1074/mcp.M700079-MCP200

M3 - Article

C2 - 17609197

AN - SCOPUS:35648977580

VL - 6

SP - 1727

EP - 1740

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 10

ER -