화학공학소재연구정보센터
Journal of Physical Chemistry B, Vol.122, No.49, 11083-11094, 2018
Effect of Mutations on the Global and Site-Specific Stability and Folding of an Elementary Protein Structural Motif
Understanding the folding mechanism of proteins requires detailed knowledge of the roles of individual amino acid residues in stabilization of specific elements and local segments of the native structure. Recently, we have utilized the combination of circular dichroism (CD) and site-specific C-13 isotopically edited infrared spectroscopy (IR) coupled with the Ising-like model for protein folding to map the thermal unfolding at the residue level of a de novo designed helix-turn-elix motif ata. Here we use the same methodology to study how the sequence of local thermal unfolding is affected by selected mutations introduced into the most and least stable parts of the motif. Seven different mutants of ata are screened to find substitutions with the most pronounced effects on the overall stability. Subsequently, thermal unfolding of two mutated alpha t alpha sequences is studied with site-specific resolution, using four distinct C-13 isotopologues of each. The data are analyzed with the Ising-like model, which builds on a previous parametrization for the original alpha t alpha sequence and tests different ways of incorporating the amino acid substitution. We show that for both more and less stable mutants only the adjustment of all interaction parameters of the model can yield a satisfactory fit to the experimental data. The stabilizing and destabilizing mutations result, respectively, in a similar increase and decrease of the stability of all probed local segments, irrespective of their position with respect to the mutation site. Consequently, the relative order of their unfolding remains essentially unchanged. These results underline the importance of the interconnectivity of the stabilizing interaction network and cooperativity of the protein structure, which is evident even in a small motif with apparently noncooperative, heterogeneous unfolding. Overall, our findings are consistent with the native structure being the dominant factor in determining the folding mechanism, regardless of the details of its overall or local thermodynamic stabilization.