On the precision of experimentally determined protein folding rates and phi-values.
Phi-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 phi-values can be determined experimentally: Because phi 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 phi-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 phi-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 (mf and mu) are invariant upon mutation, the precision of our estimates of phi 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.
de los Rios
Miguel A
MA
Department of Chemistry and Biochemistry, University of California, Santa Barbara 93106, USA.
Muralidhara
B K
BK
Wildes
David
D
Sosnick
Tobin R
TR
Marqusee
Susan
S
Wittung-Stafshede
Pernilla
P
Plaxco
Kevin W
KW
Ruczinski
Ingo
I
D005470:Fluorometry; D007700:Kinetics; D015588:Observer Variation; D017354:Point Mutation; D017510:Protein Folding; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry / Q000235:genetics; D013816:Thermodynamics; D018909:src Homology Domains
D051076:Proto-Oncogene Proteins c-fyn