Abstract
EXSY (exchange spectroscopy) NMR provides the residue-specific equilibrium constants, K, and residue-specific kinetic rate constants, k, of a polypeptide chain in a two-state exchange in the slow exchange regime. A linear free energy relationship (LFER) discovered in a log k versus log K plot is considered to be a physicochemical basis for smooth folding and conformational changes of protein molecules. For accurate determination of the thermodynamic and kinetic parameters, the measurement bias arising from state-specific differences in the R1 and R2 relaxation rates of 1H and other nuclei in HSQC and EXSY experiments must be minimized. Here, we showed that the time-zero HSQC acquisition scheme (HSQC0) is effective for this purpose, in combination with a special analytical method (Π analysis) for EXSY. As an example, we applied the HSQC0 + Π method to the two-state exchange of nukacin ISK-1 in an aqueous solution. Nukacin ISK-1 is a 27-residue lantibiotic peptide containing three mono-sulfide linkages. The resultant bias-free residue-based LFER provided valuable insights into the transition state of the topological interconversion of nukacin ISK-1. We found that two amino acid residues were exceptions in the residue-based LFER relationship. We inferred that the two residues could adopt special conformations in the transition state, to allow the threading of some side chains through a ring structure formed by one of the mono-sulfide linkages. In this context, the two residues are a useful target for the manipulation of the physicochemical properties and biological activities of nukacin ISK-1.
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All data generated or analyzed during this study are included in this published article and the supplementary information files. The source data underlying Figs. 2b, 3 and 5, and Supplementary Figs. S1 and S3 are provided as supplementary Excel files, which contain executable equations for the HSQC0 and Π analyses.
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Acknowledgements
We thank Drs. Kenji Sonomoto and Takeshi Zendo (Kyushu University, Japan) for advice on the production of nukacin ISK-1. We also thank Dr. Daisuke Fujinami (University of Shizuoka, Japan) for productive discussions. This work was partly performed in the Medical Research Center Initiative for High Depth Omics of the Medical Institute of Bioregulation, Kyushu University.
Funding
This work was supported by the Japan Society for the Promotion of Science (JSPS, Japan) KAKENHI Grant Number JP21H02448 and the Mitsubishi Foundation (Japan) Research Grants in the Natural Sciences Grant Number 202110017 to D.K.
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SH conducted material preparation and NMR measurements, SH and DK contributed to the NMR data analyses, and DK wrote the manuscript.
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Hayashi, S., Kohda, D. The time-zero HSQC method improves the linear free energy relationship of a polypeptide chain through the accurate measurement of residue-specific equilibrium constants. J Biomol NMR 76, 87–94 (2022). https://doi.org/10.1007/s10858-022-00396-y
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DOI: https://doi.org/10.1007/s10858-022-00396-y