1. Grutter, M.G., Weaver, L.H., Gray, T.M. and Matthews, B.W., "Structure, Function, and Evolution of the Lysozyme from Bacteriophage T4", In Bacteriophage T4 (Mathews, C.K., Kutter, E.M., Mosig, G. and Berget, P.M., eds.), American Society for Microbiology, Washington, D.C., 356-360 (1983).
2. Gray, T.M. and Matthews, B.W., "Intrahelical Hydrogen Bonding of Serine, Threonine and Cysteine Residues Within a-Helices and its Relevance to Membrane-bound Proteins", J. Mol. Biol. 175, 75-81 (1984).
3. Weaver, L.H., Grütter, M.G., Remington, S.J., Gray, T.M., Isaacs, N.W. and Matthews, B.W., "Comparison of Goose-Type, Chicken-Type, and Phage-Type Lysozymes Illustrates the Changes that Occur in Both Amino Acid Sequence and Three-Dimensional Structure during Evolution", J. Mol. Evol. 21, 97-111 (1985).
4. Alber, T., Gray, T.M., Weaver, L.H., Bell, J.A., Wozniak, J.A., Doapin, S., Wilson, K., Cook, S.P., Baker, E.N. and Matthews, B.W., "Selected and Directed Mutants of T4 Phage Lysozyme", In Crystallography in Molecular Biology: Bischenberg, September 1985, (Moras, D., ed.) Plenum Publishing Corp., New York (1985).
5. Alber, T., Grutter, M.G., Gray, T.M., Wozniak, J.A., Weaver, L.H., Chen, B.-L., Baker, E.N. and Matthews, B.W., "Structure and Stability of Mutant Lysozymes from Bacteriophage T4", In UCLA Symposia on Molecular and Cellular Biology, New Series, Vol. 39. "Protein Structure, Folding and Design" (Oxender, D.L., ed.), Alan R. Liss, Inc., New York (1986).
6. Grütter, M.G., Gray, T.M., Weaver, L.H., Alber, T., Wilson, K.
and Matthews, B.W., "Structural Studies of Mutants of the Lysozyme
of Bacteriophage T4: The Temperature-Sensitive Mutant Protein Thr 157-->Ile",
J. Mol. Biol. 197, 315-329 (1987).
7. Gray, T.M. and Matthews, B.W., "Structural Analysis of the Temperature-Sensitive
Mutant of Bacteriophage T4, Glycine 156-->Aspartic Acid", J.
Biol. Chem. 262, 16858-16864 (1987).
8. Weaver, L.H, Gray, T.M., Grütter, M.G., Anderson, D.E., Wozniak,
J.A., Dahlquist, F.W., and Matthews, B.W., "High Resolution Structure
of the Temperature-Sensitive Mutant of Phage Lysozyme, Arg 96-->His",
Biochem. 28, 3793-3797 (1989).
9. Gray, T.M., Arnoys, E., Blankespoor, S. & Plowman, D., "Leu 91-->Pro:
A Tight Temperature Sensitive Mutant of T4 Lysozyme", J. Cell.Bioch.
Supplement 15G, 194 (1991). {Poster abstract}
10. Gray, T.M., "The Mistrial of Evolution", The Banner,
April 13, 1992, 12-13. {Book review}
12. Muyskens, M.A. and Gray, T.M., "Data Acquisition in the Chemistry Laboratory Using LabVIEW® Software", J. Chem. Ed. 73, 1112-1114 (1996).
Engineering Salt Bridges into T4 Lysozyme
We propose to engineer six salt bridges into buried or partially buried regions of T4 lysozyme in order to assess their role in the stability and specificity of folding of proteins. In the present wild-type structure the residues that will be mutated are involved in close-packing contacts in hydrophobic regions of the structure. The six pairs are:
Tyr 18--Thr 26 --> Lys 18--Asp 26
Leu 33--Leu 46 --> His 33--Asp 46
Leu 66--Ile 50 --> His 66--Asp 50
Ala 98--Phe 153 --> Asp 98--His 153
Met 102--Phe 114 --> Lys 102--Glu 114
Met 106--Trp 138 --> Lys 106--Glu 138
These salt bridges are scattered throughout the T4 lysozyme with three found in the hydrophobic core of the amino terminal domain and three in the hydrophobic core of the carboxyl terminal domain. They represent six different contexts from which to explore the role of salt bridges in hydrophobic environments.
Each of the twelve mutations will be made singly using site-directed mutagenesis, then in pairs as detailed above. We will use solvent-induced or thermal denaturation monitored by fluorescence spectroscopy to determine the thermodynamic parameters of the folding reaction. Since the charge on ionizable amino acids is pH dependent, the folding reactions will be performed at various pH's. We will attempt to crystallize the synthesized protein, and, if successful, we will determine the three-dimensional structure by means of x-ray crystallography. We will model the structures of mutants that we are unable to crystallize using computational methods.
A secondary objective for this project is to ask the question "How hydrophilic can the interior of a protein become before it adopts a completely different fold?" If protein folding is dominated in general by a non-polar inside/polar outside rule, then if enough non-polar residues are replaced by polar residues, the protein, if it adopts any fold at all, will adopt a different fold so that the general partitioning of non-polar inside/polar outside it obtained. We will explore this question by combining the pairwise mutants described above so that multiple salt bridges are introduced into the hydrophobic core of T4 lysozyme. These multiple mutants will be studied using the same thermodynamic and structure determination techniques as described above.
Generalizing from these results will contribute to the solution of the protein folding problem, i.e.being able to predict correctly the structure of a protein based on its amino acid sequence. In addition, the results of this study will expand the database for protein designers. Many naturally occurring proteins do have buried or partially buried salt bridges. What role do they play? How important are they in stabilizing the protein structure? What specific aspects of the structural context result in stabilizing effects? The secondary objective provides a means to address the question of the specificity of protein folding, i.e. why a protein of one sequence folds up one way but a protein of a different sequence folds up differently. What the structural consequences might be of such a radical alteration of the hydrophobic character of the core of a protein as being proposed here are completely unknown, although it is likely that the protein would adopt a different fold and thus have a different specificity of folding.