ABSTRACT 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

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W610–W614 (2004). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS 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. AUTHOR INFORMATION Author notes * Eleanor

Campbell and Miriam Kaltenbach: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Research School of Chemistry, Australian National University, Canberra, Australia

Eleanor Campbell, Galen J Correy, Paul D Carr, Emma K Livingstone, Livnat Afriat-Jurnou & Colin J Jackson * Michael Smith Laboratories, University of British Columbia, Vancouver, British

Columbia, Canada Miriam Kaltenbach & Nobuhiko Tokuriki * Department of Biochemistry, University of Cambridge, Cambridge, UK Miriam Kaltenbach & Florian Hollfelder * Biomedicine

Discovery Institute, Monash University, Clayton, Victoria, Australia Benjamin T Porebski & Ashley M Buckle * Department of Biochemistry and Molecular Biology, Monash University, Clayton,

Victoria, Australia Benjamin T Porebski & Ashley M Buckle * Institut de Biologie Structurale, University Grenoble Alpes, Commissariat à l'Energie Atomique and Centre National de la

Recherche Scientifique, Grenoble, France Martin Weik Authors * Eleanor Campbell View author publications You can also search for this author inPubMed Google Scholar * Miriam Kaltenbach View

author publications You can also search for this author inPubMed Google Scholar * Galen J Correy View author publications You can also search for this author inPubMed Google Scholar * Paul

D Carr View author publications You can also search for this author inPubMed Google Scholar * Benjamin T Porebski View author publications You can also search for this author inPubMed Google

Scholar * Emma K Livingstone View author publications You can also search for this author inPubMed Google Scholar * Livnat Afriat-Jurnou View author publications You can also search for

this author inPubMed Google Scholar * Ashley M Buckle View author publications You can also search for this author inPubMed Google Scholar * Martin Weik View author publications You can also

search for this author inPubMed Google Scholar * Florian Hollfelder View author publications You can also search for this author inPubMed Google Scholar * Nobuhiko Tokuriki View author

publications You can also search for this author inPubMed Google Scholar * Colin J Jackson View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS

E.C., M.K., G.J.C., P.D.C., B.T.P., E.K.L. and L.A.-J. performed experiments and analyzed results; A.M.B. and M.W. analyzed results; F.H. conceived the project, designed experiments and

analyzed results; N.T. conceived the project, designed experiments, analyzed results and wrote the manuscript; and C.J.J. conceived the project, designed experiments, performed experiments,

analyzed results and wrote the manuscript. CORRESPONDING AUTHORS Correspondence to Nobuhiko Tokuriki or Colin J Jackson. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Results, Supplementary Figures 1–7 and Supplementary Tables 1–3. (PDF 1751 kb) RIGHTS

AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 21 January 2016 * Accepted: 17 June 2016 * Published: 12 September 2016 *

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