PubMed · 4680722
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M C Tobin. 1972. Raman spectroscopy.. https://doi.org/10.1016/s0076-6879(72)26025-6
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We have examined the role of a salt bridge between Lys242 and Glu246 in loop L4 of procaspase 3 and of mature caspase 3, and we show that the interactions are required for stabilizing the active site. Replacing either of the residues with an alanine residue results in a complete loss of procaspase 3 activity. Although both mutants are active in the context of the mature caspase 3, the mutations result in an increase in K(m) and a decrease in kcat when compared with the wild-type caspase 3. In addition, the mutations result in an increase in the pK(a) value associated with a change in kcat with pH, but does not affect the transition observed for Km versus pH. The mutations also affect the accessibility of the active-site solvent as measured by tryptophan fluorescence emission in the presence of quenching agents and as a function of pH. We show that, as the pH is lowered, the (pro)caspase dissociates, and the mutations increase the pH-dependent instability of the dimer. Overall, the results suggest that the contacts lost in the procaspase as a result of replacing Lys242 and Glu246 are compensated partially in the mature caspase as a result of new contacts that are known to form on zymogen processing
The effects of solvation on the conformations and energies of alanine dipeptide (AD) have been studied by ab initio calculations up to MP2/cc-pVTZ//MP2/6-31G**, utilizing the polarizable continuum model (PCM) to mimic solvation effects. The energy surfaces in the gas phase, ether, and water bear similar topological features carved by the steric hindrance, but the details differ significantly due to the solvent effects. The gas-phase energy map is qualitatively consistent with the Ramachandran plot showing seven energy minima. With respect to the gas-phase map, the significant changes of the aqueous map include (1) the expanded low-energy regions, (2) the emergence of an energy barrier between C5-beta and alpha(R)-beta(2) regions, (3) a clearly pronounced alpha(R) minimum, a new beta-conformer, and the disappearance of the gas-phase global minimum, and (4) the shift of the dominant region in LEII from the gas-phase C7(ax) region to the alpha(L) region. These changes bring the map in water to be much closer to the Ramachandran plot than the gas-phase map. The solvent effects on the geometries include the elongation of the exposed N-H and C=O bonds, the shortening of the buried HN--CO peptide bonds, and the enhanced planarity of the peptide bonds. The energy surface in ether has features similar to those both in the gas phase and in water. The free energy order computed in the gas phase and in ether is in good agreement with experimental studies that concluded that C5 and C7(eq) are the dominant species in both the gas phase and nonpolar solvents. The free energy order in water is consistent with the experimental observation that the dominant C7(eq) in the nonpolar solvent was largely replaced by P(II)-like (i.e., beta) and alpha(R) in the strong polar solvents. Based on calculations on AD + 4H(2)O and other AD-water clusters, we suggest that explicit water-AD interactions may distort C5 and beta (or alpha(R) and beta) to an intermediate conformation. Our analysis also shows that the PCM calculations at the MP2/cc-pVTZ//MP2/6-31G** level give good descriptions to the bulk solvent polarization effect. The results presented in this article should be of sufficient quality to characterize the peptide bonds in the gas phase and solvents. The energy surfaces may serve as the basis for developing of strategies enabling the inclusion of solvent polarization in the force field.
Beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus is a homotetramer with a higher number of ion pairs compared with mesophilic glycoside hydrolases. The ion pairs are arranged in large networks located mainly at the tetrameric interface of the molecule. In the present study, the structure and thermal stability of the wild-type beta-glycosidase and of three mutants in residues R488 and H489 involved in the C-terminal ionic network were examined by FTIR (Fourier-transform IR) spectroscopy. The FTIR data revealed small differences in the secondary structure of the proteins and showed a lower thermostability of the mutant proteins with respect to the wild-type. Generalized 2D-IR (two-dimensional IR correlation spectroscopy) at different temperatures showed different sequences of thermal unfolding events in the mutants with respect to the wild-type, indicating that punctual mutations affect the unfolding and aggregation process of the protein. A detailed 2D-IR analysis of synchronous maps of the proteins allowed us to identify the temperatures at which the ionic network that stabilizes the quaternary structure of the native and mutant enzymes at the C-terminal breaks down. This evidence gives support to the current theories on the mechanism of ion-pair stabilization in proteins from hyperthermophilic organisms.