Dr. Royer will use a combination of steady-state and time-resolved fluorescence spectroscopy in order to study the effect of amino acid substitution on the high pressure denaturation and native and denatured state dynamics of staphylococcal nuclease (Snase). Dr. Royer will characterize the contributions of the exposure of hydrophobic surface area in the denatured state and internal cavities and proline cis/trans isomerization in the native state to the pressure stability, internal dynamics and function of the protein. Three categories of mutant Snases will be investigated; 1) mutants which exhibit large changes in the cooperativity of unfolding by guanidine hydrochloride; 2) cavity mutants, in which large hydrophobic residues have been replaced by alanine or glycine; and 3) mutants in which the native stat cis/trans isomerization about the peptide bond preceding proline 117 has been altered or mutants in which the number of proline residues has been altered. For the third class of mutants Dr. Royer will perform kinetic studies of the pressure denaturation/renaturation reactions. Finally, she will use time-resolved and steady-state fluorescence intensity, energy and anisotropy measurements to characterize the native and denatured states of all three classes of mutants. These results will be correlated to the enzymatic activity of these proteins. %%% Proteins are increasingly being used in many areas of biotechnology. It has become apparent for a number of reasons that the industrial uses of proteins would benefit from a better understanding of the structural and dynamic factors affecting the stability and function of the protein. For example, it would be of interest to be able to design proteins which were more heat and pressure stable, yet with similar or even enhanced functional properties. It is the object of the research in the present proposal to study three categories of single amino acid mutants of SNase, a small well-characterized enzyme which catalyzes the hydrolysis of ribo and deoxyribonucleotides. Its three dimensional structure is known from X-ray crystallographic studies. By examining the high pressure equilibrium and kinetic denaturation profiles of three classes of mutant SNase proteins Dr. Royer will be able to identify any correlations between the three dimensional packing of the native proteins, the characteristics of their unfolding states, their dynamic properties, their stability and their enzymatic activity. It is hoped that determining the effect of amino-acid substitution on these properties of the protein will help to develop guidelines for engineering more efficient and effective proteins for a large number of biotechnological applications.
