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 analyzing a complete set of results from kinetic experiments with the SH3 domain of alphaspectrin (Spc-SH3) and applying classical error methods and error-propagation formulas, we 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 multipoint Leffler plots via the analysis of pH dependencies of kinetic parameters. We calculated the definitive phi-values for a collection of single mutations previously designed to characterize the folding transition state of the alphaspectrin 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 multipoint Leffler plots, we conclude that the precision obtained for phi-values should be approximately 25%, a reasonable limit that takes into account the propagation of experimental errors.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Cobos ES,Candel AM,Martinez JCdoi
10.1529/biophysj.107.113241subject
Has Abstractpub_date
2008-06-01 00:00:00pages
4393-404issue
11eissn
0006-3495issn
1542-0086pii
S0006-3495(08)70094-8journal_volume
94pub_type
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