Graduate Thesis Or Dissertation
 

Alkylation kinetics of the human retinoid X receptor α using cysteine as a local probe

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  • The unfolding of human retinoid X receptor α in the presence of the denaturant guanidine HCl was studied using stopped-flow absorbance spectroscopy. The protein's four native cysteine residues were mutated to obtain four single-cysteine mutants, each with the other three cysteine residues mutated to alanine. The thiols of these hRXRα mutants were alkylated with DTNB, Ellman's reagent, and the appearance of the product TNB was followed over time with a spectrophotometer at 412 nm. Alkylation of the amino acid L-cysteine by DTNB was also studied in order to provide a model system for the thiol-DTNB reaction. The kinetic analysis of the observed rates of reaction of the protein's cysteine residues with DTNB suggests that each thiol is in equilibrium between an open and a closed state, DTNB forms intermediates with both the open and closed forms of the thiol, but only the open-DTNB intermediate is alkylated. The energetic parameters obtained for the unfolding by GuHCl are well separated for each thiol, suggesting that the observed events represent local instead of global unfolding. Previous investigation of the global unfolding of hRXRα by Harder et al. [Harder2004] further supports the present hypothesis, as the midpoint of the observed unfolding events take place before the midpoint of global unfolding of the protein. Circular dichroism and tryptophan fluorescence emission were used to determine structural differences between the mutants and the wild-type.
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