The folding kinetics of Rd-apocytochrome b562 is two-state, but native-state hydrogen exchange experiments show that there are discrete partially unfolded (PUF) structures in equilibrium with the native state. These PUF structures are called hidden intermediates because they are not detected in kinetic experiments and they exist after the rate-limiting step. Structures of the mimics of hidden intermediates of Rd-apocytochrome b562 are resolved by NMR. Based upon their relative stability and structural features, the folding mechanism was proposed to follow a specific pathway (unfolded → rate-limiting transition state → PUF1 → PUF2 → native). Investigating the roles of equilibrium PUF structures in folding kinetics and their interrelationship not only deepens our understanding of the details of folding mechanism but also provides guides in protein design and prevention of misfolding. We performed molecular dynamics simulations starting from a hidden intermediate and the native state of Rd-apocytochrome b562 in explicit solvent, for a total of 37.18 μs mainly with Anton. We validated our simulations by detailed comparison with experimental data and other computations. We have verified that we sampled the post rate-limiting transition state region only. Markov state model was used to analyze the simulation results. We replace the specific pathway model with a network model. Transition-path theory was employed to calculate the net effective flux from the most unfolded state towards the most folded state in the network. The proposed sequential folding pathway via PUF1 then more stable, more native-like PUF2 is one of the routes in our network, but it is not dominant. The dominant path visits PUF2 without going through PUF1. There is also a route from PUF1 directly to the most folded state in the network without visiting PUF2. Our results indicate that the PUF states are not necessarily sequential in the folding. The major routes predicted in our network are testable by future experiments such as single molecule experiment.
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7 October 2015
Research Article|
October 01 2015
Network representation of conformational transitions between hidden intermediates of Rd-apocytochrome b562
Mojie Duan;
Mojie Duan
1Gustaf H. Carlson School of Chemistry and Biochemistry,
Clark University
, 950 Main Street, Worcester, Massachusetts 01610, USA
2Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics,
Chinese Academy of Sciences
, Wuhan 430071, China
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Hanzhong Liu;
Hanzhong Liu
a)
1Gustaf H. Carlson School of Chemistry and Biochemistry,
Clark University
, 950 Main Street, Worcester, Massachusetts 01610, USA
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Minghai Li;
Minghai Li
c)
1Gustaf H. Carlson School of Chemistry and Biochemistry,
Clark University
, 950 Main Street, Worcester, Massachusetts 01610, USA
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Shuanghong Huo
Shuanghong Huo
b)
1Gustaf H. Carlson School of Chemistry and Biochemistry,
Clark University
, 950 Main Street, Worcester, Massachusetts 01610, USA
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a)
M. Duan and H. Liu contributed equally to this work.
b)
Authors to whom correspondence should be addressed. Electronic addresses: shuo@clarku.edu and mjduan@wipm.ac.cn
c)
Present address: Physics Department, Northeastern University, Boston, Massachusetts 02115, USA.
J. Chem. Phys. 143, 135101 (2015)
Article history
Received:
May 30 2015
Accepted:
September 14 2015
Citation
Mojie Duan, Hanzhong Liu, Minghai Li, Shuanghong Huo; Network representation of conformational transitions between hidden intermediates of Rd-apocytochrome b562. J. Chem. Phys. 7 October 2015; 143 (13): 135101. https://doi.org/10.1063/1.4931921
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