Study of the gradual protein unfolding by NMR spectroscopy
Jianxing Song, Zheng Wei

Study of the gradual protein unfolding by NMR spectroscopy



Jianxing Song and Zheng Wei

Departments of Biochemistry and Biological Sciences
National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260



Protein folding problem is a great challenge in molecular biology and many issues associated with the folding mechanism still remain unsolved and controversial. It has been extensively thought that small single-domain proteins fold and unfold via a two-state mechanism. However, several years ago, our characterization of the pH-induced unfolding of a 37-residue CHABII first indicated that a continuum of equilibrium intermediates existed even between the native and molten-globule states of the protein (1-2). Therefore, NMR spectroscopy was further used to gain atomic-resolution details as well as the molecular mechanism of the gradual unfolding of CHABII (3). The results led to the following findings:
1) In contrast to the common belief, at least for CHABII, the disruption of the tight side-chain packing could be a gradual process.
2) At pH 4.0, CHABII formed the smallest molten globule identified so far, with a highly native-like secondary structure and tertiary topology, but a severely-disrupted side-chain packing.
3) The gradual unfolding of CHABII was triggered by the progressive protonation of His21 but its mechanism is different from that previously uncovered for apomyoglobin.
4) Replacement of His21 by Phe significantly enhanced the tertiary packing as evident from a significant increase in NOE number. This enhancement of the packing resulted in an increase in the thermal stability by 17 ºC.
Currently, we are investigating the relationship between the gradual unfolding and NMR backbone and sidechain dynamics on the different time scales.

References:

1. Song J, Gilquin B, Jamin N, Drakopoulou E, Guenneugues M, Dauplais M, Vita C, Menez A. (1997) NMR solution structure of a two-disulfide derivative of charybdotoxin: Biochemistry. 36, 3760-6

2. Song J, Jamin N, Gilquin B, Vita C, Menez A. (1996) A gradual disruption of tight side-chain packing: 2D 1H-NMR characterization of acid-induced unfolding of CHABII Nat Struct Biol. 6, 129-34.

3. Wei Z & Song J. (2005) Molecular mechanism underlying the thermal stability and pH-induced unfolding of CHABII J Mol Biol. 348, 205-18.