Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large ΔC_p's

Abstract

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8-anilino-1-naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274-329 K. At 298 K, values of ΔG°, ΔC_p, and C_m were 3.1 ± 0.2 kcal mol^-1, 5.9 ± 0.8 kcal mol^-1 K^-1 (15.9 cal (mol-residue)^-1 K^-1), and 0.8 M, respectively, at pH3.0 and 14.5 ± 0.4 kcal mol^-1, 8.3 ± 0.7 kcalmo^l-1 K^-1 (22.4kcal (mol-residue^-1 K^-1), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of ΔG° and ΔC_p similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of ΔC_p per mol-residue for the molten globule is comparable to corresponding values of ΔCp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of ΔC_p per mol-residue for the unfolding of the native state is among the highest currently known for any protein.