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Repacking the Core of T4 lysozyme by automated design

Mooers, Blaine H. M. and Datta, Deepshikha and Baase, Walter A. and Zollars, Eric S. and Mayo, Stephen L. and Matthews, Brian W. (2003) Repacking the Core of T4 lysozyme by automated design. Journal of Molecular Biology, 332 (3). pp. 741-756. ISSN 0022-2836. doi:10.1016/S0022-2836(03)00856-8.

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Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective. Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8 Å relative to both the designed structure and wild-type. Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone.

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Mayo, Stephen L.0000-0002-9785-5018
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Additional Information:© 2003 Elsevier Ltd. Received 25 March 2003; revised 1 July 2003; Accepted 1 July 2003. Edited by F. E. Cohen. Available online 2 September 2003. We thank Hong Xiao, Leslie Gay, and Andy Fields for making the mutant proteins and for crystallizing them, Cathy Sarisky for help with the calculations, Doug Juers for collecting preliminary X-ray data for the mutant proteins V103I and Core-7, and the user support staff at SSRL and ALS for their assistance. This work was supported in part by grants from the NIH (GM21967 to B.W.M.), the Howard Hughes Medical Institute (to S.L.M. & B.W.M.), the Ralph M. Parsons Foundation (to S.L.M.) and an IBM Shared University Research grant (to S.L.M.).
Funding AgencyGrant Number
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Ralph M. Parsons FoundationUNSPECIFIED
Subject Keywords:Crystallography: X-Ray; Solvents; Models: Molecular; Protein Conformation; Enzyme Stability; Escherichia coli; Bacteriophage T4; Thermodynamics; Mutation; Methionine; Automation; Software; Water; Muramidase; Temperature; ORBIT; single-site revertants; T4 lysozyme; repacking
Issue or Number:3
Record Number:CaltechAUTHORS:20110913-173426073
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Official Citation:Blaine H.M. Mooers, Deepshikha Datta, Walter A. Baase, Eric S. Zollars, Stephen L. Mayo, Brian W. Matthews, Repacking the Core of T4 Lysozyme by Automated Design, Journal of Molecular Biology, Volume 332, Issue 3, 19 September 2003, Pages 741-756, ISSN 0022-2836, 10.1016/S0022-2836(03)00856-8.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:25333
Deposited By: Marie Ary
Deposited On:23 Sep 2011 22:51
Last Modified:09 Nov 2021 16:32

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