Structural and kinetic mapping of side-chain exposure onto the protein energy landscape

被引:16
作者
Bernstein, Rachel [2 ,3 ]
Schmidt, Kierstin L. [1 ,4 ]
Harbury, Pehr B. [1 ]
Marqusee, Susan [3 ,5 ]
机构
[1] Stanford Univ, Dept Biochem, Sch Med, Stanford, CA 94305 USA
[2] Univ Calif Berkeley, Dept Chem, Berkeley, CA 94720 USA
[3] Univ Calif Berkeley, Inst Quantitat Biosci QB3, Berkeley, CA 94720 USA
[4] Stanford Univ, Dept Chem, Stanford, CA 94305 USA
[5] Univ Calif Berkeley, Dept Mol & Cell Biol, Berkeley, CA 94720 USA
基金
美国国家卫生研究院;
关键词
intermediate; thiol exchange; STATE HYDROGEN-EXCHANGE; THIOL-DISULFIDE EXCHANGE; PARTIALLY FOLDED STATE; COLI RIBONUCLEASE-H; CONFORMATIONAL FLUCTUATIONS; FOLDING INTERMEDIATE; MASS-SPECTROMETRY; MOLTEN GLOBULE; HUMAN LYSOZYME; CYTOCHROME-C;
D O I
10.1073/pnas.1103629108
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Identification and characterization of structural fluctuations that occur under native conditions is crucial for understanding protein folding and function, but such fluctuations are often rare and transient, making them difficult to study. Native-state hydrogen exchange (NSHX) has been a powerful tool for identifying such rarely populated conformations, but it generally reveals no information about the placement of these species along the folding reaction coordinate or the barriers separating them from the folded state and provides little insight into side-chain packing. To complement such studies, we have performed native-state alkyl-proton exchange, a method analogous to NSHX that monitors cysteine modification rather than backbone amide exchange, to examine the folding landscape of Escherichia coli ribonuclease H, a protein well characterized by hydrogen exchange. We have chosen experimental conditions such that the rate-limiting barrier acts as a kinetic partition: residues that become exposed only upon crossing the unfolding barrier are modified in the EX1 regime (alkylation rates report on the rate of unfolding), while those exposed on the native side of the barrier are modified predominantly in the EX2 regime (alkylation rates report on equilibrium populations). This kinetic partitioning allows for identification and placement of partially unfolded forms along the reaction coordinate. Using this approach we detect previously unidentified, rarely populated conformations residing on the native side of the barrier and identify side chains that are modified only upon crossing the unfolding barrier. Thus, in a single experiment under native conditions, both sides of the rate-limiting barrier are investigated.
引用
收藏
页码:10532 / 10537
页数:6
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