Adams Zoë C, Olson Erika J, Lopez-Silva Tania L, Lian Zhengwen, Kim Audrey Y, Holcomb Matthew, Zimmermann Jörg, Adhikary Ramkrishna, Dawson Philip E
Department of Chemistry, The Scripps Research Institute 10550 North Torrey Pines Road La Jolla California 92037 USA
Chemical Biology Laboratory, National Cancer Institute, National Institutes of Health Frederick MD 21702 USA.
Chem Sci. 2022 Aug 16;13(34):10020-10028. doi: 10.1039/d1sc06562a. eCollection 2022 Aug 31.
The characterization of self-assembling molecules presents significant experimental challenges, especially when associated with phase separation or precipitation. Transparent window infrared (IR) spectroscopy leverages site-specific probes that absorb in the "transparent window" region of the biomolecular IR spectrum. Carbon-deuterium (C-D) bonds are especially compelling transparent window probes since they are non-perturbative, can be readily introduced site selectively into peptides and proteins, and their stretch frequencies are sensitive to changes in the local molecular environment. Importantly, IR spectroscopy can be applied to a wide range of molecular samples regardless of solubility or physical state, making it an ideal technique for addressing the solubility challenges presented by self-assembling molecules. Here, we present the first continuous observation of transparent window probes following stopped-flow initiation. To demonstrate utility in a self-assembling system, we selected the MAX1 peptide hydrogel, a biocompatible material that has significant promise for use in drug delivery and medical applications. C-D labeled valine was synthetically introduced into five distinct positions of the twenty-residue MAX1 β-hairpin peptide. Consistent with current structural models, steady-state IR absorption frequencies and linewidths of C-D bonds at all labeled positions indicate that these side chains occupy a hydrophobic region of the hydrogel and that the motion of side chains located in the middle of the hairpin is more restricted than those located on the hairpin ends. Following a rapid change in ionic strength to initiate self-assembly, the peptide absorption spectra were monitored as function of time, allowing determination of site-specific time constants. We find that within the experimental resolution, MAX1 self-assembly occurs as a cooperative process. These studies suggest that stopped-flow transparent window FTIR can be extended to other time-resolved applications, such as protein folding and enzyme kinetics.
自组装分子的表征面临着重大的实验挑战，尤其是当与相分离或沉淀相关时。透明窗红外（IR）光谱利用在生物分子红外光谱的“透明窗”区域吸收的位点特异性探针。碳-氘（C-D）键是特别有吸引力的透明窗探针，因为它们是非扰动性的，可以很容易地位点选择性地引入到肽和蛋白质中，并且它们的伸缩频率对局部分子环境的变化敏感。重要的是，红外光谱可以应用于广泛的分子样品，而不管其溶解性或物理状态如何，这使其成为解决自组装分子所带来的溶解性挑战的理想技术。在这里，我们展示了在停流启动后对透明窗探针的首次连续观察。为了证明在自组装系统中的实用性，我们选择了MAX1肽水凝胶，一种在药物递送和医学应用中有很大应用前景的生物相容性材料。将C-D标记的缬氨酸合成引入到二十残基MAX1β-发夹肽的五个不同位置。与当前的结构模型一致，所有标记位置的C-D键的稳态红外吸收频率和线宽表明这些侧链占据水凝胶的疏水区域，并且位于发夹中间的侧链的运动比位于发夹末端的侧链更受限制。在离子强度快速变化以启动自组装后，监测肽吸收光谱随时间的变化，从而确定位点特异性时间常数。我们发现在实验分辨率范围内，MAX1自组装是一个协同过程。这些研究表明，停流透明窗傅里叶变换红外光谱可以扩展到其他时间分辨应用，如蛋白质折叠和酶动力学。
