Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Free energies of protein–protein association determined by electrospray ionization mass spectrometry correlate accurately with values obtained by solution methods

Free energies of protein–protein association determined by electrospray ionization mass... The advantages of electrospray ionization mass spectrometry (ESIMS) to measure relative solution‐phase affinities of tightly bound protein–protein complexes are demonstrated with selected variants of the Bacillus amyloliquefaciens protein barstar (b*) and the RNAase barnase (bn), which form protein–protein complexes with a range of picomolar to nanomolar dissociation constants. A novel chemical annealing procedure rapidly establishes equilibrium in solutions containing competing b* variants with limiting bn. The relative ion abundances of the complexes and those of the competing unbound monomers are shown to reflect the relative solution‐phase concentrations of those respective species. No measurable dissociation of the complexes occurs either during ESI or mass detection, nor is there any evidence for nonspecific binding at protein concentrations <25 μM. Differences in ΔΔG of dissociation between variants were determined with precisions <0.1 kcal/mol. The ΔΔG values obtained deviate on average by 0.26 kcal/mol from those measured with a solution‐phase enzyme assay. It is demonstrated that information about the protein conformation and covalent modifications can be obtained from differences in mass and charge state distributions. This method serves as a rapid and precise means to interrogate protein–protein‐binding surfaces for complexes that have affinities in the picomolar to nanomolar range. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Protein Science Wiley

Free energies of protein–protein association determined by electrospray ionization mass spectrometry correlate accurately with values obtained by solution methods

Download PDF
11 pages

Loading next page...
 
/lp/wiley/free-energies-of-protein-protein-association-determined-by-Ekw4Z9E0hu

References (49)

Publisher
Wiley
Copyright
Copyright © 2006 The Protein Society
ISSN
0961-8368
eISSN
1469-896X
DOI
10.1110/ps.062083406
pmid
16731980
Publisher site
See Article on Publisher Site

Abstract

The advantages of electrospray ionization mass spectrometry (ESIMS) to measure relative solution‐phase affinities of tightly bound protein–protein complexes are demonstrated with selected variants of the Bacillus amyloliquefaciens protein barstar (b*) and the RNAase barnase (bn), which form protein–protein complexes with a range of picomolar to nanomolar dissociation constants. A novel chemical annealing procedure rapidly establishes equilibrium in solutions containing competing b* variants with limiting bn. The relative ion abundances of the complexes and those of the competing unbound monomers are shown to reflect the relative solution‐phase concentrations of those respective species. No measurable dissociation of the complexes occurs either during ESI or mass detection, nor is there any evidence for nonspecific binding at protein concentrations <25 μM. Differences in ΔΔG of dissociation between variants were determined with precisions <0.1 kcal/mol. The ΔΔG values obtained deviate on average by 0.26 kcal/mol from those measured with a solution‐phase enzyme assay. It is demonstrated that information about the protein conformation and covalent modifications can be obtained from differences in mass and charge state distributions. This method serves as a rapid and precise means to interrogate protein–protein‐binding surfaces for complexes that have affinities in the picomolar to nanomolar range.

Journal

Protein Science – Wiley

Published: Jun 1, 2006

There are no references for this article.