In the last several years, our group has studied the folding behavior of small single domain proteins, including cyt c, Rd-apocyt b562, barnase, PDZ domain, and FAT domain. We found that these proteins fold through partially unfolded intermediates that exist after the rate-limiting step. We called them """"""""hidden intermediates"""""""" since they can not be detected in conventional kinetic experiments. Further, we have developed a native-state hydrogen exchange-directed protein engineering method for populating the intermediates and determined the first high-resolution structures of the intermediates by multi-dimensional NMR methods. Recently, we have extended our studies to include multi-domain proteins such as T4 lysozyme and a redesigned protein by coupling protein A B-domain with Rd-apocyt b562. The results obtained from these studies provide strong support for the hypothesis that the kinetic principle of protein folding is the step-wise folding of cooperative structure units (foldons) (see pictures in the Gallery). We also provided theoretical arguments on why proteins should fold in a step-wise manner and why the current funnel-like energy landscape view is inadequate to describe the folding behavior of proteins, i.e., desolvation during folding leads to energy barrier on the energy landscape, random search, and coopertive formation of partially unfolded intermediates.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
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Zhou, Zheng; Feng, Hanqiao; Ghirlando, Rodolfo et al. (2008) The high-resolution NMR structure of the early folding intermediate of the Thermus thermophilus ribonuclease H. J Mol Biol 384:531-9
Tu, Chao; Tan, Yu Hong; Shaw, Gary et al. (2008) Impact of low-frequency hotspot mutation R282Q on the structure of p53 DNA-binding domain as revealed by crystallography at 1.54 angstroms resolution. Acta Crystallogr D Biol Crystallogr 64:471-7
Kato, Hidenori; Vu, Ngoc Diep; Feng, Hanqiao et al. (2007) The folding pathway of T4 lysozyme: an on-pathway hidden folding intermediate. J Mol Biol 365:881-91
Kato, Hidenori; Feng, Hanqiao; Bai, Yawen (2007) The folding pathway of T4 lysozyme: the high-resolution structure and folding of a hidden intermediate. J Mol Biol 365:870-80
Bai, Yawen (2006) Protein folding pathways studied by pulsed- and native-state hydrogen exchange. Chem Rev 106:1757-68
Korzhnev, Dmitry M; Bezsonova, Irina; Evanics, Ferenc et al. (2006) Probing the transition state ensemble of a protein folding reaction by pressure-dependent NMR relaxation dispersion. J Am Chem Soc 128:5262-9
Ai, Xuanjun; Zhou, Zheng; Bai, Yawen et al. (2006) 15N NMR spin relaxation dispersion study of the molecular crowding effects on protein folding under native conditions. J Am Chem Soc 128:3916-7
Bai, Yawen (2006) Energy barriers, cooperativity, and hidden intermediates in the folding of small proteins. Biochem Biophys Res Commun 340:976-83
Choy, Wing-Yiu; Zhou, Zheng; Bai, Yawen et al. (2005) An 15N NMR spin relaxation dispersion study of the folding of a pair of engineered mutants of apocytochrome b562. J Am Chem Soc 127:5066-72
Zhou, Zheng; Feng, Hanqiao; Zhou, Hongyi et al. (2005) Design and folding of a multidomain protein. Biochemistry 44:12107-12

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