Engineering Enzymes for Enhanced Thermostability
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ENGINEERING ENZYMES FOR ENHANCED THERMOSTABILITY
Phoebe Shih, Bruce A. Malcolm, and Jack F. Kirsch Department of Molecular and Cellular Biology, Barker Hall, University of California, Berkeley, CA 94720 and Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, CA 94720 ABSTRACT
Chicken egg-white lysozyme (CEWL) is used as a model to attempt to engineer proteins for enhanced thermostability. Site-directed mutagenesis is employed for selective amino acid substitution to probe the contribution of an individual amino acid in a given sequence to thermostability. A linear correlation is found between the side-chain volume of a triplet of amino acid residues located at the interior core of the protein and its thermostability. Additional mutant constructs at the core position reveal that hyperpacking can disrupt other intramolecular contacts and offset the hydrophobic stabilization due to denser packing. Multiple substitutions at different loci of the protein are combined to analyze the additivity of thermostability mutations. INTRODUCTION A major unsolved problem in protein chemistry is to understand the physical forces that direct a given amino acid sequence to its native structure in solution. A complex balance of intramolecular noncovalent interactions: e.g., hydrogen bonds, hydrophobic, ionic, and Van der Waals interactions is responsible for the stabilization of the folded protein structure. Examples that demonstrate increases in protein stability by enhancing one or more of the above forces are known (1-4). However, due to the large size of a protein molecule and its complex interaction with solvent, general methods for increasing protein stability are lacking. A better understanding of the rules underlying the stability of protein architecture is desirable because it will improve our knowledge of the protein folding problem which is sometimes called the second half of genetic code. A general strategy for designing thermostable enzymes is also an highly attractive goal for industrial application. The present investigation is concerned with the analysis of protein thermostability with particular emphasis on hydrophobic or nonpolar packing. Site-directed mutagenesis technology was employed to modify chicken egg-white lysozyme (CEWL) with single or multiple amino acid substitution(s). The effect of mutation(s) on the thermostability of the modified lysozymes was analyzed by monitoring the difference in tryptophan environment by changes in absorbance at 292 nm (Fig 1). CEWL is a small globular monomeric protein composed of 129 amino acids with four disulphide cross links. It catalyzes the hydrolysis of the peptidyl-glycan component of the bacterial cell wall, and has an inferred bacteriocidal role in the avian egg-white. CEWL has been demonstrated to undergo a reversible unfolding reaction which obeys a two-state model to a good approximation (6). Correlation between side-chain volume and thermostability of mutant constructs in the interior of the protein Mat. Res. Soc. Symp. Proc. Vol. 218. @1991 Mat
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