This manuscript presents an NMR strategy to investigate conformational differences in protein-inhibitor complexes, when the inhibitors tightly bind to a protein at sub-nanomolar dissociation constants and are highly analogous to each other. Using HIV-1 protease (PR), we previously evaluated amide chemical shift differences, ΔCSPs, of PR bound to darunavir (DRV) compared to PR bound to several DRV analogue inhibitors, to investigate subtle but significant long-distance conformation changes caused by the inhibitor's chemical moiety variation [Khan, S. N., Persons, J. D. Paulsen, J. L., Guerrero, M., Schiffer, C. A., Kurt-Yilmaz, N., and Ishima, R., Biochemistry, (2018), 57, 1652-1662]. However, ΔCSPs are not ideal for investigating subtle PR-inhibitor interface differences because intrinsic differences in the electron shielding of the inhibitors affect protein ΔCSPs. NMR relaxation is also not suitable as it is not sensitive enough to detect small conformational differences in rigid regions among similar PR-inhibitor complexes. Thus, to gain insight into conformational differences at the inhibitor-protein interface, we recorded 15N-half filtered NOESY spectra of PR bound to two highly analogous inhibitors and assessed NOEs between PR amide protons and inhibitor protons, between PR amide protons and hydroxyl side chains, and between PR amide protons and water protons. We also verified the PR amide-water NOEs using 2D water-NOE/ROE experiments. Differences in water-amide proton NOE peaks, possibly due to amide-protein hydrogen bonds, were observed between subunit A and subunit B, and between the DRV-bound form and an analogous inhibitor-bound form, which may contribute to remote conformational changes.

中文翻译：

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检测与溶液中高度类似抑制剂复合的蛋白质中构象差异的核磁共振策略

这份手稿提出了一种 NMR 策略来研究蛋白质-抑制剂复合物的构象差异，当抑制剂以亚纳摩尔解离常数与蛋白质紧密结合并且彼此高度相似时。使用 HIV-1 蛋白酶 (PR)，我们之前评估了与达芦那韦 (DRV) 结合的 PR 与结合几种 DRV 类似物抑制剂的 PR 的酰胺化学位移差异ΔCSP，以研究由抑制剂的化学部分变异 [Khan, SN, Persons, JD Paulsen, JL, Guerrero, M., Schiffer, CA, Kurt-Yilmaz, N. 和 Ishima, R., Biochemistry, (2018), 57, 1652-1662] . 然而，ΔCSP 不适合研究 PR 抑制剂界面的细微差异，因为抑制剂电子屏蔽的内在差异会影响蛋白质 ΔCSP。NMR 弛豫也不适合，因为它不够灵敏，无法检测相似 PR 抑制剂复合物之间刚性区域的微小构象差异。因此，为了深入了解抑制剂-蛋白质界面的构象差异，我们记录了 PR 与两种高度类似的抑制剂结合的 15N 半滤波 NOESY 谱，并评估了 PR 酰胺质子和抑制剂质子之间、PR 酰胺质子和羟基侧链之间的 NOE ，以及 PR 酰胺质子和水质子之间。我们还使用二维水-NOE/ROE 实验验证了 PR 酰胺-水 NOE。水-酰胺质子 NOE 峰的差异，可能是由于酰胺-蛋白质氢键，