To understand a protein's function and to design new proteins with new functions, it is essential to know the physical principles that control the structure, folding, stability, and dynamics of protein molecules. Our long-term research interest is to investigate these principles and use them to solve practical problems in basic medical research and development of protein drugs. Currently, we are studying the mechanism of protein folding using various tools including stopped-flow spectroscopy, NMR, hydrogen exchange, protein engineering and phage-display.In the past several years, we focused on studying the folding mechanism of small proteins including cytochrome C (104 amino acids), Rd-apocyt b562 (106 amino acids), and barnase (110 amino acids). These small proteins fold kinetically in an apparent two-state manner. However, we found that they have partially folded hidden intermediates after the rate-limiting transition states. Moreover, using a hydrogen exchange-directed protein engineering approach, we were able to populate the partially unfolded intermediate of Rd-apocyt b562 and determine its high-resolution structure. This is the first high-resolution structure of a folding intermediate. To our surprise, significant non-native hydrophobic interactions were found in the partially unfolded structure. These results contradict the so called """"""""New View"""""""" of protein folding that hypothesizes that small proteins fold through multiple pathways without going through intermediate states but provide strong evidence for the earlier hypothesis that intermediates are important for solving the large-scale conformation search problem: the Levinthal paradox. We have also developed a theoretical model to explain why different small proteins (less than 120 amino acids) fold with different rates, which span six orders of magnitude from microseconds to seconds. We found that the size (number of folded residues) of the transition state is dominantly controlled by the topological complexity of the native structure and correlates with the folding rate. We also developed a theoretical model to explain why small proteins fold through intermediates.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
United States
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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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