Thermal Stability of Proteins

The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. In turn, such models rely on a handful of physical laws: the laws of mass action and conservation, the law that relates the protein signal and concentration, and the one that relates stability and denaturant value. So far, equations have been derived mainly for the denaturation profiles of homomeric proteins. Here, we review the underlying basic physical laws and show in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates, and give full demonstrations. We show that such equations cannot be derived for pentamers or higher oligomers except in special degenerate cases. We expand the method to signals that do not correspond to extensive protein properties. We review and expand methods for uncovering hidden intermediates of unfolding. Finally, we review methods for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins. This work should provide a robust theoretical basis for measuring the stability of complex proteins.

New Delhi Metallo beta-lactamase (NDM) is of significant public health concern due to its enormous potential to hydrolyse all major beta-lactams including carbapenems. Amino acid substitutions outside the active site reportedly affect NDM beta-lactamase activities. Here, the effect of amino acid substitutions in the possible omega-like loop region of NDM-5 has been elucidated. Overall, three substitution mutations near active site of NDM-5 were done, namely, E152A, S191A and D223A and subsequently, the change in antimicrobial resistance was monitored upon expressing each mutant in a suitable host. Among the three mutants, E152A substitution on a loop near the active site resulted in significant reduction in beta-lactam antibiotic resistance as compared to NDM-5 that compelled us to conduct further studies on the E152A-substituted NDM-5. The purified NDM-5 was able to hydrolyse all the beta-lactams tested whereas the E152A mutation suppressed its activities. NDM-5 showed maximum k cat /K m ratio against penicillins and carbapenems and had lower K m as compared to NDM-5 E152A. Though, the amino acid substitution did not affect the overall folding pattern of NDM-5, significant differences in thermal stability between the wild-type and mutated protein were observed. Therefore, we infer that the E152 residue is important in regulating the beta-lactam hydrolysing properties of NDM-5.