Somberg Noah H, Sučec Iva, Hong Mei
Department of Chemistry, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139, United States.
J Am Chem Soc. 2025 Jul 30;147(30):26534-26545. doi: 10.1021/jacs.5c06493. Epub 2025 Jul 19.
Solid-state NMR studies of ligand binding sites in proteins traditionally require assignment of the observed resonances to the amino acid sequence. This sequential assignment is time-consuming and constitutes a major bottleneck in protein solid-state NMR. To determine ligand binding sites in proteins whose structures are already known, experimentally measured protein-ligand distances can be analyzed much more rapidly if sequential assignment can be bypassed. Here we present an assignment-free NMR approach for determining ligand binding sites in proteins. We measure 2D C-C resolved C-F rotational-echo double-resonance (REDOR) spectra that probe protein-ligand proximities and assign the peaks in the 2D spectra to residue types based on the well-known characteristic chemical shifts of amino acids. We simulate the measured REDOR dephasing using a second-moment approximation with an empirically calibrated scaling factor that accounts for experimental imperfections. This efficient REDOR simulation is combined with simulated annealing to rapidly search for ligand positions that agree with the type-assigned REDOR dephasing. We demonstrate this approach on the model protein GB1 and show that the position of a single fluorine can be determined accurately. We then apply this technique to the bacterial transporter EmrE and show that the location of a tetra-fluorinated ligand is within the range of positions found by this assignment-free REDOR approach. This technique should accelerate studies of ligand binding sites in membrane proteins, amyloids, and large protein complexes, and should also be applicable to dynamic nuclear polarization experiments at cryogenic temperature where broad lines may prohibit sequential assignment.
传统上，蛋白质中配体结合位点的固态核磁共振研究需要将观察到的共振峰与氨基酸序列进行归属。这种序列归属很耗时，并且构成了蛋白质固态核磁共振的主要瓶颈。对于结构已知的蛋白质，若能绕过序列归属，实验测量的蛋白质-配体距离就能更快地进行分析。在此，我们提出一种无需归属的核磁共振方法来确定蛋白质中的配体结合位点。我们测量二维碳-碳分辨的碳-氟旋转回波双共振（REDOR）光谱，该光谱可探测蛋白质-配体的接近程度，并根据氨基酸众所周知的特征化学位移将二维光谱中的峰归属到残基类型。我们使用二阶矩近似并结合经验校准的比例因子来模拟测量的REDOR去相位，该比例因子考虑了实验缺陷。这种高效的REDOR模拟与模拟退火相结合，以快速搜索与类型归属的REDOR去相位相符的配体位置。我们在模型蛋白GB1上展示了这种方法，并表明可以准确确定单个氟的位置。然后我们将此技术应用于细菌转运蛋白EmrE，并表明四氟化配体的位置在通过这种无需归属的REDOR方法找到的位置范围内。该技术应能加速对膜蛋白、淀粉样蛋白和大型蛋白质复合物中配体结合位点的研究，并且也应适用于低温下的动态核极化实验，在该实验中宽谱线可能会阻碍序列归属。
