Backbone chemical shift assignments for the Toho-1 β-lactamase (263 amino acids, 28.9 kDa) are reported based on triple resonance solution-state NMR experiments performed on a uniformly ²H,¹³C,¹⁵N-labeled sample. These assignments allow for subsequent site-specific characterization at the chemical, structural, and dynamical levels. At the chemical level, titration with the non-β-lactam β-lactamase inhibitor avibactam is found to give chemical shift perturbations indicative of tight covalent binding that allow for mapping of the inhibitor binding site. At the structural level, protein secondary structure is predicted based on the backbone chemical shifts and protein residue sequence using TALOS-N and found to agree well with structural characterization from X-ray crystallography. At the dynamical level, model-free analysis of ¹⁵N relaxation data at a single field of 16.4 T reveals well-ordered structures for the ligand-free and avibactam-bound enzymes with generalized order parameters of ~ 0.85. Complementary relaxation dispersion experiments indicate that there is an escalation in motions on the millisecond timescale in the vicinity of the active site upon substrate binding. The combination of high rigidity on short timescales and active site flexibility on longer timescales is consistent with hypotheses for achieving both high catalytic efficiency and broad substrate specificity: the induced active site dynamics allows variously sized substrates to be accommodated and increases the probability that the optimal conformation for catalysis will be sampled.

Toho-1 β-内酰胺酶（263 个氨基酸，28.9 kDa）的主链化学位移归属是基于在均匀² H、 ¹³ C、 ¹⁵ N 标记的样品上进行的三重共振溶液态 NMR 实验报告的。这些分配允许随后在化学、结构和动力学水平上进行特定位点的表征。在化学水平上，发现用非β-内酰胺β-内酰胺酶抑制剂阿维巴坦进行滴定会产生化学位移扰动，表明紧密的共价结合，从而可以绘制抑制剂结合位点的图谱。在结构水平上，使用 TALOS-N 根据主链化学位移和蛋白质残基序列预测蛋白质二级结构，并发现与 X 射线晶体学的结构表征非常吻合。在动力学水平上，对 16.4 T 单一场的¹⁵ N 弛豫数据进行无模型分析，揭示了无配体和阿维巴坦结合酶的良好有序结构，其广义有序参数约为 0.85。互补弛豫分散实验表明，在底物结合时，活性位点附近的运动在毫秒时间尺度上有所升级。短时间尺度上的高刚性和较长时间尺度上的活性位点灵活性的组合与实现高催化效率和广泛的底物特异性的假设是一致的：诱导的活性位点动力学允许适应不同尺寸的底物并增加最佳构象的可能性将对催化进行采样。