43241-B4
Calculation and Analysis of Enzyme Bimodal Stability Curves from Novel Applications of Differential Scanning Calorimetry
Last year we achieved the aims of the proposed work. These aims were to construct stability curves (plots of the free energy of unfolding ΔGu versus temperature). The curves were constructed from a combination of isothermal titrations with guanidine hydrochloride and a novel application of differential scanning calorimetry which gives physiological thermodynamic data from measurements made under reversible but nonphysiological conditions. We found that every enzyme we have studied by this method – namely, bovine adenosine deaminase, bovine carbonic anhydrase, baker’s yeast phosphoglycerate kinase, hen egg white lysozyme, and papaya pain -- displayed an abrupt, temperature-dependent 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 suggesting the crystal structures are likely to be poor representations of the physiological structures.
During the last year our efforts have focused on a new but related direction relative to the proposed project. We are constructing partial phase diagrams – plots of the pressure dependence of the temperatures of nondenaturational conformational change and of unfolding -- of the enzymes studied in the original project. Application of the Clausius-Clapeyron equation will permit the determination of volume changes associated with each transition. Substantial volume changes in the nondenaturational conformational changes will support our conclusions from the original project that suggest there is a significant conformational change from a compact structure to a much more open structure. Furthermore, our use of slow-scan-rate differential scanning calorimetry will permit calculation of the activation volume ΔV‡ associated with attainment of the conformational change transition state. The temperature of nondenaturational conformational change at each pressure is determined by slow-scan-rate differential scanning calorimetry. 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 completed this study with bovine carbonic anhydrase. Here we found that the volume change associated with the low- to physiological-temperature conformational transition ΔVL→P = 15 ± 2 L/mole which contrasts with the partial unfolding of the physiological-temperature conformer to the molten globule state (ΔVP→MG = 26 ± 9 L/mole). Such a large ΔVL→P supports our earlier conclusions that the low-temperature conformation of bovine carbonic anhydrase is a much more compact structure than the physiological-temperature conformation. The activation volume for this process ΔV‡L→P = 51 ± 9 L/mole suggesting a signficant unfolding quality of the nondenaturational conformational change transition state.
Support for our research from the ACS PRF over the 3-year term of the grant has resulted in 5 peer-reviewed publications with 6 undergraduate students (I anticipate that there will be 2 more publications in the near future) and 16 poster presentations by these students at regional and national meetings. I sincerely thank the ACS for this opportunity.
Mark Britt
