TY - JOUR
T1 - On the precision of experimentally determined protein folding rates and φ-values
AU - De Los Rios, Miguel A.
AU - Muralidhara, B. K.
AU - Wildes, David
AU - Sosnick, Tobin R.
AU - Marqusee, Susan
AU - Wittung-Stafshede, Pernilla
AU - Plaxco, Kevin W.
AU - Ruczinski, Ingo
PY - 2006/3
Y1 - 2006/3
N2 - φ-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 φ-values can be determined experimentally: Because φ 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 φ-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 φ-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 mu) are invariant upon mutation, the precision of our estimates of φ 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. Published by Cold Spring Harbor Laboratory Press.
AB - φ-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 φ-values can be determined experimentally: Because φ 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 φ-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 φ-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 mu) are invariant upon mutation, the precision of our estimates of φ 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. Published by Cold Spring Harbor Laboratory Press.
KW - FynSH3 domain
KW - Protein folding
KW - Stopped-flow mixing
KW - φ-values
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U2 - 10.1110/ps.051870506
DO - 10.1110/ps.051870506
M3 - Article
C2 - 16501226
AN - SCOPUS:33644541811
SN - 0961-8368
VL - 15
SP - 553
EP - 563
JO - Protein Science
JF - Protein Science
IS - 3
ER -