| Abstract
| - A simple theoretical model for increasing the protein stability by adequately redesigning thedistribution of charged residues on the surface of the native protein was tested experimentally. Using themolecule of ubiquitin as a model system, we predicted possible amino acid substitutions on the surfaceof this protein which would lead to an increase in its stability. Experimental validation for this predictionwas achieved by measuring the stabilities of single-site-substituted ubiquitin variants using urea-inducedunfolding monitored by far-UV CD spectroscopy. We show that the generated variants of ubiquitin areindeed more stable than the wild-type protein, in qualitative agreement with the theoretical prediction. Asa positive control, theoretical predictions for destabilizing amino acid substitutions on the surface of theubiquitin molecule were considered as well. These predictions were also tested experimentally usingcorrespondingly designed variants of ubiquitin. We found that these variants are less stable than the wild-type protein, again in agreement with the theoretical prediction. These observations provide guidelinesfor rational design of more stable proteins and suggest a possible mechanism of structural stability ofproteins from thermophilic organisms.
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