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Protein Dynamics in a Family of Laboratory Evolved Thermophilic Enzymes

Wintrode, Patrick L. and Zhang, Deqiang and Vaidehi, Nagarajan and Arnold, Frances H. and Goddard, William A., III (2003) Protein Dynamics in a Family of Laboratory Evolved Thermophilic Enzymes. Journal of Molecular Biology, 327 (3). pp. 745-757. ISSN 0022-2836. doi:10.1016/S0022-2836(03)00147-5.

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Molecular dynamics simulations were employed to study how protein solution structure and dynamics are affected by adaptation to high temperature. Simulations were carried out on a para-nitrobenzyl esterase (484 residues) and two thermostable variants that were generated by laboratory evolution. Although these variants display much higher melting temperatures than wild-type (up to 18 °C higher) they are both >97% identical in sequence to the wild-type. In simulations at 300 K the thermostable variants remain closer to their crystal structures than wild-type. However, they also display increased fluctuations about their time-averaged structures. Additionally, both variants show a small but significant increase in radius of gyration relative to wild-type. The vibrational density of states was calculated for each of the esterases. While the density of states profiles are similar overall, both thermostable mutants show increased populations of the very lowest frequency modes (<10 cm^(−1)), with the more stable mutant showing the larger increase. This indicates that the thermally stable variants experience increased concerted motions relative to wild-type. Taken together, these data suggest that adaptation for high temperature stability has resulted in a restriction of large deviations from the native state and a corresponding increase in smaller scale fluctuations about the native state. These fluctuations contribute to entropy and hence to the stability of the native state. The largest changes in localized dynamics occur in surface loops, while other regions, particularly the active site residues, remain essentially unchanged. Several mutations, most notably L313F and H322Y in variant 8G8, are in the region showing the largest increase in fluctuations, suggesting that these mutations confer more flexibility to the loops. As a validation of our simulations, the fluctuations of Trp102 were examined in detail, and compared with Trp102 phosphorescence lifetimes that were previously measured. Consistent with expectations from the theory of phosphorescence, an inverse correlation between out-of-plane fluctuations on the picosecond time scale and phosphorescence lifetime was observed.

Item Type:Article
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Arnold, Frances H.0000-0002-4027-364X
Goddard, William A., III0000-0003-0097-5716
Additional Information:© 2003 Published by Elsevier Ltd. Received 14 June 2002, Revised 26 November 2002, Accepted 14 December 2002, Available online 6 March 2003. We thank Dr Anne Gershenson (Brandeis University) for her insight and helpful discussions. This work was supported in part by the Army Research Office and the Center for Science and Engineering of Materials at Caltech (NSF-MRSEC). The facilities of the Materials and Process Simulation Center used in this project are supported also by DOE (ASCI ASAP), NSF (CTS and MRI), NIH, ARO-MURI, Chevron Corp., MMM, Seiko-Epson, Dow Chemical, Avery-Dennison Corp., Kellogg's, General Motors, Asahi Kasei, the Beckman Institute, and ONR.
Funding AgencyGrant Number
Army Research Office (ARO)UNSPECIFIED
Department of Energy (DOE)UNSPECIFIED
Chevron CorporationUNSPECIFIED
Dow Chemical CompanyUNSPECIFIED
Avery-Dennison CorporationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Office of Naval Research (ONR)UNSPECIFIED
Subject Keywords:thermophile; protein stability; directed evolution; protein dynamics; molecular dynamics simulation
Issue or Number:3
Record Number:CaltechAUTHORS:20180823-075442841
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Official Citation:Patrick L Wintrode, Deqiang Zhang, Nagarajan Vaidehi, Frances H Arnold, William A Goddard, Protein Dynamics in a Family of Laboratory Evolved Thermophilic Enzymes, Journal of Molecular Biology, Volume 327, Issue 3, 2003, Pages 745-757, ISSN 0022-2836, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89075
Deposited By: Tony Diaz
Deposited On:23 Aug 2018 16:53
Last Modified:16 Nov 2021 00:32

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