Stepwise unfolding of a multi-tryptophan protein MPT63 with immunoglobulin-like fold: detection of zone-wise perturbation during guanidine hydrochloride-induced unfolding using phosphorescence spectroscopy

Abstract

A combination of circular dichroism, steady-state and time-resolved fluorescence, and low temperature phosphorescence spectroscopy has been used to study guanidine hydrochloride (Gdn)-induced stepwise unfolding of MPT63, a small protein with immunoglobulin-like fold. A simple, rapid and inexpensive low temperature (77 K) phosphorescence (LTP) study of MPT63, having four Trp residues, in a suitable aqueous cryogenic solvent clearly indicates that the most exposed Trp 82 and the deeply buried Trp 129 exhibit a greater probability of being unfolded in comparison to the partially exposed Trp 48 during Gdn-induced unfolding. The partially exposed Trp 48 practically conserves its environment up to 1.6 M Gdn concentrations. The excitation wavelength-dependent LTP and lifetime of the triplet state further support the contention. Tyrosine residues which are silent in free MPT63 start exhibiting emission when the concentration of Gdn reaches 1.6 M. The overall solvent exposure of the Trp residues in the fully unfolded state of MPT63 is observed to be less than that observed for a Trp residue in a tripeptide and a heptapeptide in a 40% ethylene glycol matrix. The specific detection of perturbation of the environment of individual Trp residues during gradual unfolding has been achieved without using any Trp substituted mutant. This is possible since the wild-type MPT63 exhibits unusual optical resolution of all four Trp residues by LTP due to the special architecture of MPT63, where the four Trp residues are well distributed in its tertiary structure.