The time-zero HSQC method improves the linear free energy relationship of a polypeptide chain through the accurate measurement of residue-specific equilibrium constants

• Published in: Journal of biomolecular NMR 2022-06, Vol.76 (3), p.87-94
• Main Authors: Hayashi, Seiichiro, Kohda, Daisuke
• Format: Article
• Language: English
• Subjects: Amino acids; Aqueous solutions; Bias; Biochemistry; Biological and Medical Physics; Biological properties; Biophysics; Chains; Chemical equilibrium; Exchanging; Free energy; Linkages; NMR; Nuclear magnetic resonance; Physicochemical properties; Physics; Physics and Astronomy; Polypeptides; Protein folding; Rate constants; Residues; Ring structures; Spectroscopy; Spectroscopy/Spectrometry; Sulfides
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1007/s10858-022-00396-y

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 R 1 and R 2 relaxation rates of 1 H 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.