help button home button Biophys. J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
Biophys. J. BioFAST: First Published January 25, 2008. doi:10.1529/biophysj.107.113241
© 2008 by the Biophysical Society.

A more recent version of this article appeared on June 1, 2008.
This Article
Right arrow Full Text (Rapid PDF)
Right arrow All Versions of this Article:
biophysj.107.113241v1
94/11/4393    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cobos, E. S
Right arrow Articles by Martinez, J. C
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cobos, E. S
Right arrow Articles by Martinez, J. C

PROTEINS

An error analysis for two-state protein folding kinetic parameters and {phi} values: progress towards precision by exploring pH dependencies on Leffler plots

Eva S Cobos 1, Adela M Candel 1 and Jose C Martinez 1*

1 University of Granada

* To whom correspondence should be addressed. E-mail: jcmh{at}ugr.es.

Submitted on May 19, 2007
Revised on August 14, 2007
Accepted on 20 November 2007


  Abstract
The interpretation of {phi} values has led to an understanding of the folding transition state ensemble of a variety of proteins. Although the main guidelines and equations for calculating {phi} are well established there remains some controversy about the quality of the numerical values obtained. By analysing a complete set of results from kinetic experiments with the SH3 domain of {alpha}-spectrin (Spc-SH3) and applying classical error methods and error-propagation formulas we have evaluated the uncertainties involved in two-state-folding kinetic experimental parameters and the corresponding calculated {phi} values. We show that kinetic constants in water and m values can be properly estimated from a judicious weighting of fitting errors and describe some procedures to calculate the errors in Gibbs energies and {phi} values from a traditional two-point Leffler analysis. Furthermore, on the basis of general assumptions made with the protein engineering method, we show how to generate multi-point Leffler plots via the analysis of pH dependencies of kinetic parameters. We have calculated the definitive {phi} values for a collection of single mutations previously designed to characterise the folding transition state of the {alpha}-spectrin SH3 domain. The effectiveness of the pH-scanning procedure is also discussed in the context of error analysis. Judging from the magnitudes of the error bars obtained from two-point and multi-point Leffler plots we conclude that the precision obtained for {phi} values should be around 25%, a reasonable limit that takes into account the propagation of experimental errors.

Key Words: Leffler plots, errors propagation, phi value analysis, protein folding kinetics, rate-equilibrium relationships, transition state






HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH
Copyright © 2008 by the Biophysical Society.