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43241-B4
Calculation and Analysis of Enzyme Bimodal Stability Curves from Novel Applications of Differential Scanning Calorimetry

Billy Britt, Texas Woman's University

This year we achieved the aims of the proposed work. These aims were to construct stability curves – plots of the free energy of unfolding ΔG(u) versus temperature -- for several enzymes using data obtained over a broad temperature range. The curves were constructed from a combination of isothermal titrations with guanidine hydrochloride and a novel application of differential scanning calorimetry (DSC) which gives physiological thermodynamic data from measurements made under reversible but nonphysiological conditions. We found that bovine carbonic anhydrase and baker's yeast phosphoglycerate kinase each displayed an abrupt conformational change at a temperature intermediate between the temperature where the enzyme crystals were formed for structure determination and the physiological temperatures. Thermodynamic characterization of each conformer pair reveals that the two structures differ markedly and suggest the crystal structures should never be confused with the physiological structures. We did not observe such a conformational change in our study of hen egg white lysozyme.

We are currently focusing our efforts on further understanding the nature of these nondenaturational conformational changes. First, we are characterizing the transition state thermodynamics of the low- to physiological-temperature conformational change by slow-scan-rate differential scanning calorimetry. Slow-scan-rate DSC was developed in our laboratory and employs scan rates as low as 0.010ºC/min. We find that the temperature of conformational transition is highly scan rate dependent indicating the transitions are under kinetic control. Rate constants are calculated from k = r(scan)/ΔT (where r(scan) is the instrument scan rate and ΔT is the temperature interval of the transition), an Arrhenius plot is constructed, and the transition state thermodynamics fully characterized by standard methods. To date we have examined bovine adenosine deaminase, yeast phosphoglycerate kinase, bovine carbonic anhydrase, and papaya papain. The analysis for all enzymes suggests the transition state possesses a substantial unfolding quality. We will study thermolysin next.

Second, we are constructing phase diagrams – plots of the pressure dependence of the temperatures of nondenaturational conformational change and unfolding -- of several enzymes. The temperature of nondenaturational conformational change at each pressure is determined from extrapolation to zero scan rate the apparent temperature of transition as obtained by slow-scan-rate DSC. Unfolding temperatures at each pressure are calculated from extrapolation of apparent unfolding temperatures in the presence of guanidine hydrochloride, which renders the transitions reversible, to zero molar denaturant. Pressures from 1.0 to 5.0 atm are employed. We have only begun this research and plan to study bovine carbonic anhydrase, bovine adenosine deaminase, thermolysin, and lysozyme.

Since September 1, 2006 our PRF grant has made possible the publication of one paper with three undergraduate co-authors, the submission of two manuscripts with three undergraduate co-authors, and the presentation of three posters by two undergraduate students.

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