Conservative mutation Met8 -> Leu affects the folding process and structural stability of squash trypsin inhibitor CMTI-I

Protein molecules can accommodate a large number of mutations without notic eable effects on their stability and folding kinetics. On the other hand, s ome mutations can have quite strong effects on protein conformational prope rties. Such mutations either destabilize secondary structures, e.g., alpha- helices, are incompatible with dose packing of protein hydrophobic cores, o r lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met 8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rear rangement of a hydrophobic cluster formed by side chains of residues 8, Ile 6, and Leu17 that leads to partial breaking of a hydrogen bond formed by th e amide group of Leu17 with water and to a reduction of a hydrophobic surfa ce buried within the cluster. The mutation perturbs also the protein foldin g, In aerobic conditions the reduced wild-type protein folds effectively in to its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is t hat conservative mutations of hydrophobic residues can destabilize a protei n structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobi c clusters formed by such residues can lead to local changes in protein hyd ration; and consequently, can affect considerably protein stability and fol ding process.