CHEMICALLY CROSS-LINKED PROTEIN DIMERS - STABILITY AND DENATURATION EFFECTS

Nine single substitution cysteine mutants of staphylococcal nuclease ( nuclease) were preferentially crosslinked at the introduced cysteine r esidues using three different bifunctional crosslinking reagents; 1,6- bismaleimidohexane (BMH), 1,3-dibromo-2-propanol (DBP), and the chemic al warfare agent, mustard gas (bis(2-chloroethyl)sulfide; mustard). BM H and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as spe cific. Guanidine hydrochloride (GuHCl) denat urations of the resulting dimeric proteins exhibited biphasic unfoldin g behavior that did not fit the two-state model of unfolding. The mono functional reagent, epsilon-maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unf olded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fit ted to a three-state thermodynamic model of two successive transitions in which the individual subunits cooperatively unfold. These two unfo lding transitions were very different from the unfolding of the monome ric protein. These differences in unfolding behavior can be attributed in large part to changes in the denatured state. In addition to GuHCl titrations, the crosslinked dimers were also thermally unfolded. In c ontrast to the GuHCl denaturations, analysis of this data fit a two-st ate model well, but with greatly elevated van't Hoff enthalpies in man y cases. However, clear correlations between the thermal and GuHCl den aturations exist, and the differences in thermal unfolding can be rati onalized by postulating interactions of the denatured crosslinked prot eins.