Molecular mechanisms underlying the thermal stability and acid-induced unfolding of CHABII

Abstract

CHABII was previously demonstrated to undergo a gradual pH-induced unfolding. CHABII at pH 4.0 offers an attractive model for deeper understanding of the molten globule state. In the present study, the recombinant proteins of CHABII and its mutant [Phe21]-CHABII were expressed and refolded. Extensive CD, NMR characterizations showed that replacement of His21 by Phe in [Phe21]-CHABII eliminated the pH-induced unfolding from pH 6.5 to 4.0 and considerably enhanced the packing interaction of the hydrophobic core, increasing the thermal stability of [Phe21]-CHABII by ~17 degrees. For CHABII at pH 4.0, the 1H-15N HSQC spectrum was poorly-dispersed while the persistence of medium- and long-range NOEs indicated its highly native-like topology, strongly implying that the degree of the native-like topology might be significantly underestimated in the previous characterization of partially-folded and even a??completely-unfoldeda?? proteins.