Abstract
Directed stabilization of globular proteins via substitution of a minimal number of amino acid residues is one of the most complicated experimental tasks. This work summarizes our research on the effect of amino acid substitutions on the protein stability utilizing the outputs of the analysis of intrinsic disorder predisposition of target proteins. This allowed us to formulate the basis of one of the possible approaches to the stabilization of globular proteins. The idea is quite simple. To stabilize a protein as a whole, one needs to find its "weakest spot" and stabilize it, but the question is how this weak spot can be found in a query protein. Our approach is based on the utilization of the computational tools for the per-residue evaluation of intrinsic disorder predisposition to search for the "weakest spot" of a query protein (i.e., the region(s) with the highest local predisposition for intrinsic disorder). When such "weakest spot" is found, it can be stabilized through a limited number of point mutations by introducing order-promoting residues at hot spots, thereby increasing structural stability of a protein as a whole. Using this approach, we were able to obtain stable mutant forms of several globular proteins, such as Gαo, GFP, ribosome protein L1, and circular permutant of apical domain of GroEL.
Highlights
Elevating protein stability could seem quite a simple and trivial task at first glance, since the interactions and forces responsible for protein stability are well-known from the textbooks [1].Many examples of protein stabilization via introduction of additional disulfide bond(s) are widely described in the literature [2,3,4,5]
One might choose publications, where the authors stabilized proteins by introducing artificially engineered disulfide bonds. Via using programs such as PONDR® FIT, one can find parts of the polypeptide chain that are weakened and try to answer the question if there is really a correlation between the stabilization of the protein and the location of the mutation in the sites that are predicted as intrinsically disorder? it turned out that the literature data analysis would not help in finding such a correlation, since many factors influence the stability of proteins with artificially introduced disulfide bonds, such as the size of the protein, the specific features of its structure, as well as the design features of disulfide bonds
It is clear that the disulfide bonds within the hydrophobic core of the protein could lead to either destabilization of a protein or to protein stabilization, if it stably joints different structural parts of a protein
Summary
Many examples of protein stabilization via introduction of additional disulfide bond(s) are widely described in the literature [2,3,4,5]. For some proteins, stabilizing substitution can be found out by comparison of their amino acid sequences with their homologs from thermophilic organisms [9]. Detailed study of literature makes it clear that in many cases, the stabilizing substitutions of amino acid residues represent the results of long-term search and brute-force selection of various substitution variants, whereas in other cases it is a simple luck, a success apparently brought by chance, or a concourse of favorable circumstances. Currently there are no universal “receipts”, which can help an experimenter to elevate the stability of any globular protein by designing an amino
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