We present the first demonstration of ligand-induced conformational changes in a biological molecule, a protein, by sum-frequency generation (SFG). Constructs of KRasG12D protein were prepared by selectively deuterating residues of a single amino acid type using isotope-labeled amino acids and cell-free protein synthesis. By attaching labeled protein to a supported bilayer membrane via a His-tag to Ni-NTA-bearing lipids, we ensured that single layers of ordered molecules were formed while preserving the protein’s native structure. Exceptionally large SFG amide I signals were produced in both labeled and unlabeled proteins, demonstrating a high degree of orientational order upon attachment to the bilayer. Deuterated protein also produced SFG signals in the CDx spectral region, which were not present in the unlabeled protein. The CDx signals were measured before and after binding a peptide inhibitor, KRpep-2d, revealing shifts in SFG intensity due to conformational changes at the labeled sites. In particular, peaks associated with CDx stretching vibrations for alanine, valine, and glycine changed substantially in amplitude upon inhibitor binding. By inspection of the crystal structure, these three residues are uniquely colocated on the protein surface in and near the nucleotide binding site, which is in allosteric communication with the site of peptide inhibitor binding, suggesting an approach to identify a ligand’s binding site. The technique offers a highly sensitive, nonperturbative method of mapping ligand-induced conformational changes and allosteric networks in biological molecules for studies of the relationship between structure and function and mechanisms of action in drug discovery.

中文翻译：

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通过和频生成检测配体诱导的蛋白质分子构象变化


我们首次通过和频产生 （SFG） 展示了生物分子（一种蛋白质）中配体诱导的构象变化。通过使用同位素标记的氨基酸和无细胞蛋白质合成选择性地氘化单一氨基酸类型的残基来制备 KRasG12D 蛋白的构建体。通过将标记的蛋白质通过 His 标签连接到承载 Ni-NTA 的脂质上，我们确保了在保留蛋白质天然结构的同时形成单层有序分子。在标记和未标记的蛋白质中均产生非常大的 SFG 酰胺 I 信号，在附着到双层时表现出高度的定向顺序。氘代蛋白还在 CDx 光谱区域产生 SFG 信号，而这些信号在未标记的蛋白质中不存在。在结合肽抑制剂 KRpep-2d 之前和之后测量 CDx 信号，揭示了由于标记位点的构象变化而导致的 SFG 强度变化。特别是，与丙氨酸、缬氨酸和甘氨酸的 CDx 拉伸振动相关的峰在抑制剂结合后振幅发生显著变化。通过检查晶体结构，这三个残基唯一地位于核苷酸结合位点内和附近的蛋白质表面，该位点与肽抑制剂结合位点进行变构通讯，提出了一种识别配体结合位点的方法。该技术提供了一种高度灵敏、无扰动的方法，用于绘制生物分子中配体诱导的构象变化和变构网络，用于研究药物发现中结构和功能之间的关系以及作用机制。