Enzymes must be ordered to allow the stabilization of transition states by their active sites, yet dynamic enough to adopt alternative conformations suited to other steps in their catalytic cycles. The biophysical principles that determine how specific protein dynamics evolve and how remote mutations affect catalytic activity are poorly understood. Here we examine a 'molecular fossil record' that was recently obtained during the laboratory evolution of a phosphotriesterase from Pseudomonas diminuta to an arylesterase. Analysis of the structures and dynamics of nine protein variants along this trajectory, and three rationally designed variants, reveals cycles of structural destabilization and repair, evolutionary pressure to 'freeze out' unproductive motions and sampling of distinct conformations with specific catalytic properties in bi-functional intermediates. This work establishes that changes to the conformational landscapes of proteins are an essential aspect of molecular evolution and that change in function can be achieved through enrichment of preexisting conformational sub-states.
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We thank D.S. Tawfik for stimulating discussions. C.J.J. thanks the Australian Research Council for a Future Fellowship (FT140101059) and Discovery Project (DP130102144). This research was undertaken on the MX1 and MX2 beamlines at the Australian Synchrotron, Victoria, Australia. F.H. thanks the Biotechnology and Biological Sciences Research Council and European Research Council (starting investigator grants). M.K. thanks the EU Innovative Training Network (ProSA) for a studentship. N.T. is funded as a Canadian Institutes of Health Research new investigator and a Michael Smith Foundation of Health Research (MSFHR) career investigator. N.T. thanks Natural Sciences and Engineering Research Council of Canada Discovery Grant RGPIN 418262-12. A.M.B. is funded as a National Health and Medical Research Senior Research Fellow (1022688). This work was supported by the Victorian Life Sciences Computation Initiative, an initiative of the Victorian Government, Australia.
The authors declare no competing financial interests.
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Campbell, E., Kaltenbach, M., Correy, G. et al. The role of protein dynamics in the evolution of new enzyme function. Nat Chem Biol 12, 944–950 (2016). https://doi.org/10.1038/nchembio.2175
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