Cooperativity of a Protein Folding Reaction Probed at Multiple Chain Positions by Real-Time 2D NMR Spectroscopy
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- 作者：Clemens Steegborn ; Henriette Schneider-Hassloff ; Markus Zeeb ; and Jochen Balbach
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：July 11, 2000
- 年：2000
- 卷：39
- 期：27
- 页码：7910 - 7919
- 全文大小：193K
- 年卷期：v.39,no.27(July 11, 2000)
- ISSN：1520-4995
文摘
The refolding reaction of S54G/P55N ribonuclease T1 is a two-step process, where fastformation of a partly folded intermediate is followed by the slow reaction to the native state, limited bya trans 
cis isomerization of Pro39. The hydrodynamic radius of this kinetic folding intermediate wasdetermined by real-time diffusion NMR spectroscopy. Its folding to the native state was monitored by aseries of 128 very fast 2D 15N-HMQC spectra, to observe the kinetics of 66 individual backbone amideprobes. We find that the intermediate is as compact as the native protein with many native chemicalshifts. All 66 analyzed amide probes follow the rate-limiting prolyl isomerization, which indicates thatthis cooperative refolding reaction is fully synchronized. The stability of the folding intermediate wasdetermined from the protection factors of 45 amide protons derived from a competition between refoldingand H/D exchange. The intermediate has already gained 40% of the Gibbs free energy of refolding withmany protected amides in not-yet-native regions.
cis isomerization of Pro39. The hydrodynamic radius of this kinetic folding intermediate wasdetermined by real-time diffusion NMR spectroscopy. Its folding to the native state was monitored by aseries of 128 very fast 2D 15N-HMQC spectra, to observe the kinetics of 66 individual backbone amideprobes. We find that the intermediate is as compact as the native protein with many native chemicalshifts. All 66 analyzed amide probes follow the rate-limiting prolyl isomerization, which indicates thatthis cooperative refolding reaction is fully synchronized. The stability of the folding intermediate wasdetermined from the protection factors of 45 amide protons derived from a competition between refoldingand H/D exchange. The intermediate has already gained 40% of the Gibbs free energy of refolding withmany protected amides in not-yet-native regions.