A quantitative, high-throughput screen for protein stability
Open Access
- 11 July 2000
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 97 (15) , 8296-8301
- https://doi.org/10.1073/pnas.140111397
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.Keywords
This publication has 30 references indexed in Scilit:
- Screening Combinatorial Libraries of de Novo Proteins by Hydrogen−Deuterium Exchange and Electrospray Mass SpectrometryJournal of the American Chemical Society, 1999
- Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large ΔCp'sProtein Science, 1999
- Folding Kinetics of a Fluorescent Variant of Monomeric λ RepressorBiochemistry, 1998
- Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequencesJournal of Molecular Biology, 1997
- Microsecond Protein Folding Through a Compact Transition StateJournal of Molecular Biology, 1996
- Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfoldingProtein Science, 1995
- Guanidinium Chloride Induction of Partial Unfolding in Amide Proton Exchange in RNase AScience, 1993
- Genetic approach to the role of tryptophan residues in the activities and fluorescence of a bacterial periplasmic maltose-binding proteinJournal of Molecular Biology, 1990
- A novel genetic system to detect protein–protein interactionsNature, 1989
- THE THERMODYNAMIC STABILITY OF PROTEINSAnnual Review of Biophysics, 1987