Chemically crosslinked protein dimers: stability and denaturation effects.
Nine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; 1,6-bismaleimidohexane (BMH), 1,3-dibromo-2-propanol (DBP), and the chemical warfare agent, mustard gas (bis(2-chloroethyl)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCl) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two-state model of unfolding. The monofunctional reagent, epsilon-maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unfolded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fitted to a three-state thermodynamic model of two successive transitions in which the individual subunits cooperatively unfold. These two unfolding transitions were very different from the unfolding of the monomeric 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 contrast to the GuHCl denaturations, analysis of this data fit a two-state model well, but with greatly elevated van't Hoff enthalpies in many cases. However, clear correlations between the thermal and GuHCl denaturations exist, and the differences in thermal unfolding can be rationalized by postulating interactions of the denatured crosslinked proteins.
Byrne
M P
MP
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701-1201, USA.
Stites
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D003432:Cross-Linking Reagents; D004355:Drug Stability; D019791:Guanidine; D006146:Guanidines; D046911:Macromolecular Substances; D008301:Maleimides; D008836:Micrococcal Nuclease / Q000737:chemistry*; D008958:Models, Molecular; D009151:Mustard Gas; D020005:Propanols; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
D003432:Cross-Linking Reagents; D006146:Guanidines; D046911:Macromolecular Substances; D008301:Maleimides; D020005:Propanols; C075594:1,6-bismaleimidohexane; C098109:1,3-dibromo-2-propanol; D008836:Micrococcal Nuclease; D019791:Guanidine; D009151:Mustard Gas