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The transition state for integral membrane protein folding.

ABSTRACT: Biology relies on the precise self-assembly of its molecular components. Generic principles of protein folding have emerged from extensive studies on small, water-soluble proteins, but it is unclear how these ideas are translated into more complex situations. In particular, the one-third of cellular proteins that reside in biological membranes will not fold like water-soluble proteins because membrane proteins need to expose, not hide, their hydrophobic surfaces. Here, we apply the powerful protein engineering method of Phi-value analysis to investigate the folding transition state of the alpha-helical membrane protein, bacteriorhodopsin, from a partially unfolded state. Our results imply that much of helix B of the seven-transmembrane helical protein is structured in the transition state with single-point alanine mutations in helix B giving Phi values >0.8. However, residues Y43 and T46 give lower Phi values of 0.3 and 0.5, respectively, suggesting a possible reduction in native structure in this region of the helix. Destabilizing mutations also increase the activation energy of folding, which is accompanied by an apparent movement of the transition state toward the partially unfolded state. This apparent transition state movement is most likely due to destabilization of the structured, unfolded state. These results contrast with the Hammond effect seen for several water-soluble proteins in which destabilizing mutations cause the transition state to move toward, and become closer in energy to, the folded state. We thus introduce a classic folding analysis method to membrane proteins, providing critical insight into the folding transition state.

SUBMITTER: Curnow P

PROVIDER: S-EPMC2630066 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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The transition state for integral membrane protein folding.

Curnow Paul P Booth Paula J PJ

Proceedings of the National Academy of Sciences of the United States of America 20090113 3

Biology relies on the precise self-assembly of its molecular components. Generic principles of protein folding have emerged from extensive studies on small, water-soluble proteins, but it is unclear how these ideas are translated into more complex situations. In particular, the one-third of cellular proteins that reside in biological membranes will not fold like water-soluble proteins because membrane proteins need to expose, not hide, their hydrophobic surfaces. Here, we apply the powerful prot ...[more]

PMID: 19141633

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Project description:Characterizing the conformations of protein in the transition state ensemble (TSE) is important for studying protein folding. A promising approach pioneered by Vendruscolo et al. [Nature (London) 409, 641 (2001)] to study TSE is to generate conformations that satisfy all constraints imposed by the experimentally measured φ values that provide information about the native likeness of the transition states. Faísca et al. [J. Chem. Phys. 129, 095108 (2008)] generated conformations of TSE based on the criterion that, starting from a TS conformation, the probabilities of folding and unfolding are about equal through Markov Chain Monte Carlo (MCMC) simulations. In this study, we use the technique of constrained sequential Monte Carlo method [Lin et al., J. Chem. Phys. 129, 094101 (2008); Zhang et al. Proteins 66, 61 (2007)] to generate TSE conformations of acylphosphatase of 98 residues that satisfy the φ-value constraints, as well as the criterion that each conformation has a folding probability of 0.5 by Monte Carlo simulations. We adopt a two stage process and first generate 5000 contact maps satisfying the φ-value constraints. Each contact map is then used to generate 1000 properly weighted conformations. After clustering similar conformations, we obtain a set of properly weighted samples of 4185 candidate clusters. Representative conformation of each of these cluster is then selected and 50 runs of Markov chain Monte Carlo (MCMC) simulation are carried using a regrowth move set. We then select a subset of 1501 conformations that have equal probabilities to fold and to unfold as the set of TSE. These 1501 samples characterize well the distribution of transition state ensemble conformations of acylphosphatase. Compared with previous studies, our approach can access much wider conformational space and can objectively generate conformations that satisfy the φ-value constraints and the criterion of 0.5 folding probability without bias. In contrast to previous studies, our results show that transition state conformations are very diverse and are far from nativelike when measured in cartesian root-mean-square deviation (cRMSD): the average cRMSD between TSE conformations and the native structure is 9.4 Å for this short protein, instead of 6 Å reported in previous studies. In addition, we found that the average fraction of native contacts in the TSE is 0.37, with enrichment in native-like β-sheets and a shortage of long range contacts, suggesting such contacts form at a later stage of folding. We further calculate the first passage time of folding of TSE conformations through calculation of physical time associated with the regrowth moves in MCMC simulation through mapping such moves to a Markovian state model, whose transition time was obtained by Langevin dynamics simulations. Our results indicate that despite the large structural diversity of the TSE, they are characterized by similar folding time. Our approach is general and can be used to study TSE in other macromolecules.

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The transition state for integral membrane protein folding.

Publications

The transition state for integral membrane protein folding.

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