Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, United States.
J Am Chem Soc. 2022 Jun 8;144(22):9920-9925. doi: 10.1021/jacs.2c02704. Epub 2022 May 26.
Elucidation of the detailed mechanisms by which biological macromolecules undergo major structural conversions, such as folding, complex formation, and self-assembly, is a central concern of biophysical chemistry that will benefit from new experimental methods. We describe a simple technique for initiating a structural conversion process by a rapid decrease in the temperature of a solution, i.e., a rapid inverse temperature jump. By pumping solutions through copper capillary tubes that are thermally anchored to heated and cooled blocks, solution temperatures can be switched from 95 to 30 °C (or lower) in about 0.8 ms. For time-resolved solid-state nuclear magnetic resonance (ssNMR), solutions can then be frozen rapidly by spraying into cold isopentane after a variable structural evolution time τ. As an initial demonstration, we use this "inverse T-jump" technique to characterize the kinetics and mechanism by which the 26-residue peptide melittin converts from its primarily disordered, monomeric state at 95 °C to its α-helical, tetrameric state at 30 °C. One- and two-dimensional ssNMR spectra of frozen solutions with various values of τ, recorded at 25 K with signal enhancements from dynamic nuclear polarization, show that both helical secondary structure and intermolecular contacts develop on the same time scale of about 6 ms. The dependences on τ of both intraresidue crosspeak patterns and inter-residue crosspeak volumes in two-dimensional spectra can be fit with a unidirectional dimerization model, consistent with dimerization being the rate-limiting step for melittin tetramer formation.
阐明生物大分子经历主要结构转化（如折叠、复合物形成和自组装）的详细机制是生物物理化学的核心关注点，这将受益于新的实验方法。我们描述了一种通过快速降低溶液温度来引发结构转化过程的简单技术，即快速逆温度跃变。通过将溶液泵入热锚定在加热和冷却块上的铜毛细管中，可以在约 0.8 毫秒内将溶液温度从 95°C 降至 30°C（或更低）。对于时间分辨固态核磁共振（ssNMR），在经过可变的结构演化时间 τ 后，溶液可以通过喷雾到冷异戊烷中快速冷冻。作为初步演示，我们使用这种“逆 T 跳跃”技术来表征蜂肽 26 残基从 95°C 时主要无序的单体状态到 30°C 时的α螺旋四聚体状态的动力学和机制。在 25 K 下用动态核极化增强记录的具有各种 τ 值的冷冻溶液的一维和二维 ssNMR 谱表明，螺旋二级结构和分子间接触都在大约 6 毫秒的相同时间尺度上发展。二维谱中内残基交叉峰图案和间残基交叉峰体积对 τ 的依赖性可以用单向二聚化模型拟合，这与二聚化是蜂肽四聚体形成的限速步骤一致。
