期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：26
页码：7966-7971
DOI：10.1073/pnas.1422683112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceFast-protein-folding experiments and molecular-dynamics simulations can nowadays be compared on the same timescale. The comparison is limited by the lack of time-resolved structural information from experiment, which usually provides only information on global kinetics or stability. Structural data from microsecond folding experiments enables more rigorous testing of the detailed predictions available from simulations, which will in turn make future simulations more reliable. Here, we tackled this long-standing problem by measuring tertiary contact formation between three helices in a fast-folding protein. The folding of this protein is not as simple as it seems but matches up nicely with a long all-atom simulation trajectory that monitors multiple folding and unfolding events. Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein {lambda}6-85 by engineering into it three fluorescent tryptophan-tyrosine contact probes. The probes report on distances between three different helix pairs: 1-2, 1-3, and 3-2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1-3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same "slow" and "fast" distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1-2 and 3-2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test.
