When Monomers Are Preferred: a Strategy for the Identification and Disruption of Weakly Oligomerized Proteins

Overview

Journal Structure

Publisher Cell Press

Specialties Biochemistry
Biotechnology
Cell Biology
Molecular Biology

Date 2005 Jan 12

PMID 15642257

Citations 16

Authors

Yufeng Tong

David Hughes

Lisa Placanica

Matthias Buck

Affiliations

Soon will be listed here.

Abstract

Oligomerization is important for the structure and function of many proteins, but frequently complicates their characterization. It is often desirable to obtain the protein in monomeric form. Here, we report a strategy that allows the generation of monomers from weakly associated oligomers but does not require knowledge of the three-dimensional structure of the protein. The dynamics of protein association are used in solution NMR spectroscopy to identify regions of the polypeptide chain that are likely to be responsible for the interaction. Protein sequence analysis further refines the selection, as conserved sites with moderate hydrophobicity are targeted for modification. Gel filtration and activity assays straightforwardly reveal the consequences of the change and are used to screen for the desired mutants. The strategy is demonstrated for the Rac1 binding domain of plexin-B1. A monomeric variant is generated which preserves the Rac1 binding activity and the wild-type protein structure.

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Analysis of 15N-1H NMR relaxation in proteins by a combined experimental and molecular dynamics simulation approach: picosecond-nanosecond dynamics of the Rho GTPase binding domain of plexin-B1 in the dimeric state indicates allosteric pathways.

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