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Non-random-coil behavior as a consequence of extensive PPII structure in the denatured state.

ABSTRACT: Unfolded proteins may contain a native or nonnative residual structure, which has important implications for the thermodynamics and kinetics of folding, as well as for misfolding and aggregation diseases. However, it has been universally accepted that residual structure should not affect the global size scaling of the denatured chain, which obeys the statistics of random coil polymers. Here we use a single-molecule optical technique--fluorescence correlation spectroscopy--to probe the denatured state of a set of repeat proteins containing an increasing number of identical domains, from 2 to 20. The availability of this set allows us to obtain the scaling law for the unfolded state of these proteins, which turns out to be unusually compact, strongly deviating from random coil statistics. The origin of this unexpected behavior is traced to the presence of an extensive nonnative polyproline II helical structure, which we localize to specific segments of the polypeptide chain. We show that the experimentally observed effects of polyproline II on the size scaling of the denatured state can be well-described by simple polymer models. Our findings suggest a hitherto unforeseen potential of nonnative structure to induce significant compaction of denatured proteins, significantly affecting folding pathways and kinetics.

SUBMITTER: Cortajarena AL

PROVIDER: S-EPMC2603145 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Non-random-coil behavior as a consequence of extensive PPII structure in the denatured state.

Cortajarena Aitziber L AL Lois Gregg G Sherman Eilon E O'Hern Corey S CS Regan Lynne L Haran Gilad G

Journal of molecular biology 20080711 1

Unfolded proteins may contain a native or nonnative residual structure, which has important implications for the thermodynamics and kinetics of folding, as well as for misfolding and aggregation diseases. However, it has been universally accepted that residual structure should not affect the global size scaling of the denatured chain, which obeys the statistics of random coil polymers. Here we use a single-molecule optical technique--fluorescence correlation spectroscopy--to probe the denatured ...[more]

PMID: 18644382

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Project description:Cold denaturation is a general property of globular proteins, and the process provides insight into the origins of the cooperativity of protein folding and the nature of partially folded states. Unfortunately, studies of protein cold denaturation have been hindered by the fact that the cold denatured state is normally difficult to access experimentally. Special conditions such as addition of high concentrations of denaturant, encapsulation into reverse micelles, the formation of emulsified solutions, high pressure, or extremes of pH have been applied, but these can perturb the unfolded state of proteins. The cold denatured state of the C-terminal domain of the ribosomal protein L9 can be populated under native-like conditions by taking advantage of a destabilizing point mutation which leads to cold denaturation at temperatures above 0 degrees C. This state is in slow exchange with the native state on the NMR time scale. Virtually complete backbone (15)N, (13)C, and (1)H as well as side-chain (13)C(beta) and (1)H(beta) chemical shift assignments were obtained for the cold denatured state at pH 5.7, 12 degrees C. Chemical shift analysis, backbone N-H residual dipolar couplings, amide proton NOEs, and R(2) relaxation rates all indicate that the cold denatured state of CTL9 (the C-terminal domain of the ribosomal protein L9) not only contains significant native-like secondary structure but also non-native structure. The regions corresponding to the two native alpha-helices show a strong tendency to populate helical Phi and Psi angles. The segment which connects alpha-helix 2 and beta-strand 2 (residues 107-124) in the native state exhibits a significant preference to form non-native helical structure in the cold denatured state. The structure observed in the cold denatured state of the I98A mutant is similar to that observed in the pH 3.8 unfolded state of wild type CTL9 at 25 degrees C, suggesting that it is a robust feature of the denatured state ensemble of this protein. The implications for protein folding and for studies of cold denatured states are discussed.

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Non-random-coil behavior as a consequence of extensive PPII structure in the denatured state.

Publications

Non-random-coil behavior as a consequence of extensive PPII structure in the denatured state.

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