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Tetragonal Lysozyme Interactions Studied by Site Directed MutagenesisA number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk'solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 4(sub 3) axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to greater than 500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 yields PHE or ALA and ASN 113 yields ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 4(sub 3) helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.
Document ID
19980236895
Acquisition Source
Marshall Space Flight Center
Document Type
Reprint (Version printed in journal)
Authors
Crawford, Lisa
(Toledo Univ. OH United States)
Karr, Laurel
(NASA Marshall Space Flight Center Huntsville, AL United States)
Pusey, Marc
(NASA Marshall Space Flight Center Huntsville, AL United States)
Date Acquired
August 18, 2013
Publication Date
January 1, 1998
Subject Category
Solid-State Physics
Meeting Information
Meeting: Crystallization of Biological Macromolecules
Location: Granada
Country: Spain
Start Date: May 3, 1998
Distribution Limits
Public
Copyright
Work of the US Gov. Public Use Permitted.

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