A quantitative, high-throughput screen for protein stability

A quantitative, high-throughput screen for protein stability

^*Department of Biochemistry, Duke University Medical Center, Durham, NC 27710; and ^†Department of Chemistry, Duke University, Durham, NC 27710

Edited by Alexander M. Klibanov, Massachusetts Institute of Technology, Cambridge, MA, and approved June 6, 2000 (received for review March 14, 2000)

Abstract

In proteomic research, it is often necessary to screen a large number of polypeptides for the presence of stable structure. Described here is a technique (referred to as SUPREX, stability of unpurified proteins from rates of H/D exchange) for measuring the stability of proteins in a rapid, high-throughput fashion. The method uses hydrogen exchange to estimate the stability of microgram quantities of unpurified protein extracts by using matrix-assisted laser desorption/ionization MS. The stabilities of maltose binding protein and monomeric λ repressor variants determined by SUPREX agree well with stability data obtained from conventional CD denaturation of purified protein. The method also can detect the change in stability caused by the binding of maltose to maltose binding protein. The results demonstrate the precision of the method over a wide range of stabilities.

Footnotes

↵ ‡ To whom reprint requests should be addressed. E-mail: T.Oas{at}duke.edu.
This paper was submitted directly (Track II) to the PNAS office.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.140111397.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.140111397
Abbreviations:

MALDI,

matrix-assisted laser desorption/ionization;

SUPREX,

stability of unpurified proteins from rates of H/D exchange;

MBP,

maltose binding protein;

λ6–85,

monomeric λ repressor;

GdmCl,

guanidinium monochloride