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Biophys J. 2016 Dec 6;111(11):2368-2376. doi: 10.1016/j.bpj.2016.08.027.

High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape.

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Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York.
Department of Biophysics, Johns Hopkins University, Baltimore, Maryland.
Unité de Virologie Structurale, Centre National de la Recherche Scientifique UMR 3569, Institut Pasteur, Paris, France.
Centre de Biochimie Structurale, Centre National de la Recherche Scientifique UMR 5048, Institut National de la Santé et de la Recherche Médicale, Université de Montpellier, France.
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.
Department of Physics, Rensselaer Polytechnic Institute, Troy, New York.
Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York. Electronic address:


A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific 1H-15N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms.

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