Ultrafast Hydrogen Exchange Reveals Specific Structural Events during the Initial Stages of Folding of Cytochrome c

被引:29
|
作者
Fazelinia, Hossein [1 ]
Xu, Ming [1 ]
Cheng, Hong [1 ]
Roder, Heinrich [1 ,2 ]
机构
[1] Fox Chase Canc Ctr, Philadelphia, PA 19111 USA
[2] Univ Penn, Philadelphia, PA 19104 USA
关键词
MICROSECOND TIME-SCALE; AMIDE PROTON-EXCHANGE; RAPID MIXING METHODS; X-RAY-SCATTERING; HYDROPHOBIC COLLAPSE; RIBONUCLEASE-A; H/D EXCHANGE; SPEED LIMIT; PROTEIN; INTERMEDIATE;
D O I
10.1021/ja410437d
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Many proteins undergo a sharp decrease in chain dimensions during early stages of folding, prior to the rate-limiting step in folding. However, it remains unclear whether compact states are the result of specific folding events or a general hydrophobic collapse of the poly peptide chain driven by the change in solvent conditions. To address this fundamental question, we extended the temporal resolution of NMR-detected H/D exchange labeling experiments into the microsecond regime by adopting a microfluidics approach. By observing the competition between H/D exchange and folding as a function of labeling pH, coupled with direct measurement of exchange rates in the unfolded state, we were able to monitor hydrogen-bond formation for over 50 individual backbone NH groups within the initial 140 microseconds of folding of horse cytochrome c. Clusters of solvent-shielded amide protons were observed in two alpha-helical segments in the C-terminal half of the protein, while the N-terminal helix remained largely unstructured, suggesting that proximity in the primary structure is a major factor in promoting helix formation and association at early stages of folding, while the entropically more costly long-range contacts between the N- and C-terminal helices are established only during later stages. Our findings clearly indicate that the initial chain condensation in cytochrome c is driven by specific interactions among a subset of alpha-helical segments rather than a general hydrophobic collapse.
引用
收藏
页码:733 / 740
页数:8
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