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On the precision of experimentally determined protein folding rates and -values

¹ Department of Chemistry and Biochemistry, and Interdepartmental Program in Biomolecular Science and Engineering, University of California, Santa Barbara, Santa Barbara, California 93106, USA
² Biochemistry and Cell Biology Department and Chemistry Department, Rice University, Houston, Texas 77251, USA
³ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA
⁴ Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois 60637, USA
⁵ Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA

(RECEIVED October 3, 2005; FINAL REVISION November 14, 2005; ACCEPTED November 14, 2005)

-Values, a relatively direct probe of transition-state structure, are an important benchmark in both experimental and theoretical studies of protein folding. Recently, however, significant controversy has emerged regarding the reliability with which -values can be determined experimentally: Because is a ratio of differences between experimental observables it is extremely sensitive to errors in those observations when the differences are small. Here we address this issue directly by performing blind, replicate measurements in three laboratories. By monitoring within- and between-laboratory variability, we have determined the precision with which folding rates and -values are measured using generally accepted laboratory practices and under conditions typical of our laboratories. We find that, unless the change in free energy associated with the probing mutation is quite large, the precision of -values is relatively poor when determined using rates extrapolated to the absence of denaturant. In contrast, when we employ rates estimated at nonzero denaturant concentrations or assume that the slopes of the chevron arms (m_f and m_u) are invariant upon mutation, the precision of our estimates of is significantly improved. Nevertheless, the reproducibility we thus obtain still compares poorly with the confidence intervals typically reported in the literature. This discrepancy appears to arise due to differences in how precision is calculated, the dependence of precision on the number of data points employed in defining a chevron, and interlaboratory sources of variability that may have been largely ignored in the prior literature.

Keywords: -values; protein folding; stopped-flow mixing; FynSH3 domain

Abbreviations: GuHCl, guanidine hydrochloride

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051870506.

Reprint requests to: Ingo Ruczinski, Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA; e-mail: ingo{at}jhu.edu; fax: (410) 955-0958.

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