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    The Role of Electrostatic Interactions in Folding of β-Proteins
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    Department of Chemistry, Emory University, Atlanta, Georgia 30322, United States
    *[email protected]
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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 2016, 138, 4, 1456–1464
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    https://doi.org/10.1021/jacs.5b13201
    Published January 10, 2016
    Copyright © 2016 American Chemical Society
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    Abstract

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    Atomic-level molecular dynamic simulations are capable of fully folding structurally diverse proteins; however, they are limited in their ability to accurately represent electrostatic interactions. Here we have experimentally tested the role of charged residues on stability and folding kinetics of one of the most widely simulated β-proteins, the WW domain. The folding of wild type Pin1 WW domain, which has two positively charged residues in the first turn, was compared to the fast folding mutant FiP35 Pin1, which introduces a negative charge into the first turn. A combination of FTIR spectroscopy and laser-induced temperature-jump coupled with infrared spectroscopy was used to probe changes in the amide I region. The relaxation dynamics of the peptide backbone, β-sheets and β-turns, and negatively charged aspartic acid side chain of FiP35 were measured independently by probing the corresponding bands assigned in the amide I region. Folding is initiated in the turns and the β-sheets form last. While the global folding mechanism is in good agreement with simulation predictions, we observe changes in the protonation state of aspartic acid during folding that have not been captured by simulation methods. The protonation state of aspartic acid is coupled to protein folding; the apparent pKa of aspartic acid in the folded protein is 6.4. The dynamics of the aspartic acid follow the dynamics of the intermediate phase, supporting assignment of this phase to formation of the first hairpin. These results demonstrate the importance of electrostatic interactions in turn stability and formation of extended β-sheet structures.

    Copyright © 2016 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.5b13201.

    • Temperature dependent FTIR and difference spectra of pH 7 Pin1 and pH 5.8 and pH 7 FiP35 WW Domains, SVD analysis of pH 7 FiP35 FTIR data, pH dependent fluorescence and CD data of FiP35, representative IR T-jump relaxation kinetics of FiP35 at low temperature, second derivative of FBP28 at 5 °C, and a complete table of relaxation kinetics for pH 7 and pH 5.8 at 1613, 1636, 1680, and 1710 cm–1. (PDF)

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    Cite this: J. Am. Chem. Soc. 2016, 138, 4, 1456–1464
    Click to copy citationCitation copied!
    https://doi.org/10.1021/jacs.5b13201
    Published January 10, 2016
    Copyright © 2016 American Chemical Society
    Request reuse permissions

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