Molecular mechanism underlying the thermal stability and pH-induced unfolding of CHABII

The 37-residue alpha/beta protein CHABII was previously demonstrated to undergo a gradual pH-induced unfolding. It has been shown that even at pH 4.0 CHABII still retained a highly native-like secondary structure and tertiary topology although its tight side-chain packing was severely disrupted, typical of the molten globule state. Here, we have expressed and refolded the recombinant proteins of CHABII and its mutant Phe21]-CHABII, and subsequently conducted extensive CD and NMR characterizations. The results indicated: (1) replacement of His21 by Phe in Phe21]-CHABII eliminated the pH-induced unfolding from pH 6.5 to 4.0, indicating that His21 was responsible for the observed pH-induced unfolding of CHABII. Further examinations revealed that although the pH-induced unfolding of CHABII was also triggered by the protonation of the His residue as previously uncovered for apomyoglobin, their molecular mechanisms are different. (2) Monitoring the pH-induced unfolding by 1H-15N HSQC spectroscopy allowed us to visualize the gradual development of the CHABII molten globule. At pH 4.0, the HSQC spectrum of CHABII was poorly dispersed with dispersions of approximately 1 ppm over proton dimension and 10 ppm over 15N dimension, characteristic of severely or even "completely unfolded" proteins. One the other hand, unambiguous assignments of the NOESY spectra of CHABII led to the identification of the persistent medium and long-range NOEs at pH 4.0, which define a highly native-like secondary structure and tertiary packing. This implies that the degree of the native-like topology might be underestimated in the previous characterization of partially folded and even completely unfolded proteins. (3) Replacement of His21 by Phe with higher side-chain hydrophobicity only caused a minor structural rearrangement but considerably enhanced the packing interaction of the hydrophobic core, as evident from a dramatic increase in NOE contacts in Phe21]-CHABII. The enhancement led to an increase of the thermal stability of Phe21]-CHABII by approximately 17 deg. C.