Internal protein motions, concentrated glycerol, and hydrogen exchange studied in myoglobin

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Abstract
The effect of concentrations of glycerol on H-exchange (HX) rates was studied by using [sperm whole] myoglobulin as a test protein. Concentrated glycerol has only a small slowing effect on the HX kinetics of freely exposed amides, studied in a small molecule model (acetamide). Larger effects occur in structured proteins. The effect of solvent glycerol on different parts of the HX curve of myoglobin was studied by use of selective kinetic labeling approach. Concentrated glycerol exerts an apparently reverse effect on protein H exchange; the faster exchanging surface protons are least affected, while the slower and slower amide NH is further slowed by larger and larger factors. These results seem inconsistent with solvent penetration models which generally visualize slower and slower protons as being placed, and undergoing exchange, farther and farther from the solvent-protein interface. The results are as expected for the local unfolding model for protein H exchange since concentrated glycerol stabilizes proteins against unfolding. In the local unfolding model, slower exchanging protoms are released by way of higher energy and therefore generally larger, unfolding reactions. Larger unfoldings must be more inhibited by the glycerol effect.