(618e) Solution Studies of Protein Folding Using a Site-Specific Probe of Cysteine Accessibility

Cysteine is a moderately hydrophobic amino acid that is under represented in most proteins, rendering it a useful probe of protein structure. In a reformulation of classical methods, we have developed a method to assess the accessibility of specific exposed and buried cysteines that provides a sensitive measure of the local structure of protein domains. The cysteine probes of choice are selectively reactive fluorophors, as they permit the extent of labeling to be interrogated using multiple measurement modalities including fluorescence microscopy, fluorescence of 1D and 2D protein gels, and spectroscopic analysis via HPLC. We have applied this method to study a muscular dystrophy relevant recombinant Ig domain from the Lamin A/C tail, using a 1-D gel system. By measuring the temperature dependant labeling of an endogenous cysteine, we calculate a change in the fractional accessibility that compares well with circular dichroism studies. A shortcoming of the gel based methodology is the inability to differentiate between the labeling of multiple cysteines in a given polypeptide. We address this by borrowing the proteomic approach of analyzing tryptic peptides via RP-HPLC. In this approach, proteins are labeled in their native state and then fragmented by trypsin. Labeled peptides of varying hydrophobicity are separated, and the extent of labeling quantified. The number of cysteines in a polypeptide that can be analyzed in this way is limited only by the ability of the HPLC to resolve the labeled tryptic fragments, providing a power site specific method for assessing local protein structure.