| Autor: |
Lai JK; Department of Chemistry , University of Wyoming , Laramie , Wyoming 82071 , United States., Kubelka GS; Department of Chemistry , University of Wyoming , Laramie , Wyoming 82071 , United States., Kubelka J; Department of Chemistry , University of Wyoming , Laramie , Wyoming 82071 , United States. |
| Jazyk: |
angličtina |
| Zdroj: |
The journal of physical chemistry. B [J Phys Chem B] 2018 Dec 13; Vol. 122 (49), pp. 11083-11094. Date of Electronic Publication: 2018 Jul 24. |
| DOI: |
10.1021/acs.jpcb.8b05280 |
| Abstrakt: |
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 13 C 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-helix motif αtα. 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 αtα are screened to find substitutions with the most pronounced effects on the overall stability. Subsequently, thermal unfolding of two mutated αtα sequences is studied with site-specific resolution, using four distinct 13 C isotopologues of each. The data are analyzed with the Ising-like model, which builds on a previous parametrization for the original αtα 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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
|
