An EPR study of the protein proaerolysin
Date
2002
Authors
McAlduff, Michael James
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Abstract
The purpose of this thesis was to investigate the protein proaerolysin and its active form aerolysin. The nitroxyl spin label (l-oxyl-2,2,5,5-tetramethylpyrrolidine-3-yl)methylmethanethiosulfonate was used to selectively label the protein at cysteines located throughout the protein. Four mutants were chosen because of their location in the protein. Variants 300C PA, 445C PA, 241C PA, and 242C PA were all labeled successfully with the spin label. Their EPR spectra were analyzed to determine what kind of motion was occurring in the aqueous phase. Various additives were used in the hope of identifying large changes in the EPR spectra and attributing them to some change in the structure of the protein. Trypsin, protease K, and SDS were all added to at least one of the mutants. Temperature studies were carried out on 445C PA + MTSL in the proaerolysin and aerolysin forms and correlation times were determined to be on the order of 10·11-10·12 seconds. Simulations were performed on the spin label to determine some physical parameters; these simulations were found to be a good basis for comparing spectra of labeled protein and free spin in solution.