Mutagenesis site-specific, lysozyme

Using both the X-ray structure of the Fab lysozyme complex (Sheriff et al., 1987, 1988 Amit et al., 1986) and the site-specific mutagenesis of... 

Lavoie, T. B., Drohan, W. N. and Smith-Gill, S. J. (1992), Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme , f. Immunol., 148, 503-513. 

In summary, we have shown that site-directed mutagenesis is an important tool which allows precise investigation into the detailed chemistry of protein-protein interaction. HEL has proved to be ideal for these studies. Monoclonal antibodies, X-ray crystal structures of protein complexes, and evolutionary lysozyme variants found in nature provide an excellent system for understanding the molecular basis of protein recognition. Site-directed mutagenesis offers the ability to alter a protein at a specific site. Interpretation of the results within the context of an X-ray crystal structure allows the quantitative assignment of the free energy contributions from individual amino adds to the stability of the complex. 

Before the introduction of site-directed mutagenesis, chemical modification was the predominant means of altering a specific amino acid the earliest report with lysozyme was from Parsons and Raftery (241), who prepared the ethyl ester derivative of Asp-52 by reaction with triethyloxonium fluoroborate the modified enzyme lost catalytic function but not substrate affinity. An ethyleneimine reaction product of Asp-52 has also been prepared with similar effects on catalysis (242). Sharon and co-workers modified and then regenerated Asp-52 to eliminate concern that inactivation results from experimental manipulation rather than specific amino acid modification (243, 244). Thus, Asp-52 was first esteri-fied with an epoxypropyl-jS-glycoside derivative of di-(/V-acetyl-D-glucosamine), then reduced to homoserine or hydrolyzed to return the free aspartate. Both the... 

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