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.

EXSY（交换光谱）NMR 提供了在慢速交换方案中处于两态交换中的多肽链的残基特异性平衡常数K和残基特异性动力学速率常数k 。在 log k与 log K图中发现的线性自由能关系 (LFER)被认为是蛋白质分子平滑折叠和构象变化的物理化学基础。为了准确测定热力学和动力学参数，测量偏差由 R ₁和 R ₂弛豫率^{1的状态特定差异引起}HSQC 和 EXSY 实验中的 H 和其他原子核必须最小化。在这里，我们展示了时间零 HSQC 采集方案 (HSQC0) 与 EXSY 的特殊分析方法 (Π 分析) 相结合是有效的。例如，我们将 HSQC0 + Π 方法应用于水溶液中 nukacin ISK-1 的二态交换。Nukacin ISK-1 是一种含有 27 个残基的羊毛硫抗生素肽，含有三个单硫键。由此产生的基于无偏差残基的 LFER 为 nukacin ISK-1 拓扑互变的过渡状态提供了有价值的见解。我们发现两个氨基酸残基在基于残基的 LFER 关系中是例外。我们推断这两个残基可以在过渡态采用特殊构象，允许一些侧链穿过由单硫键之一形成的环结构。在这种情况下，这两个残基是控制 nukacin ISK-1 的物理化学性质和生物活性的有用目标。