Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency—yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter Glt_Ph. We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.

转运体循环通过大的结构变化以跨生物膜转运分子。这些变化和功能之间的时间关系，以及决定其速率的分子特性，决定了运输效率——但大多数情况下仍然未知。使用单分子荧光显微镜，我们比较了电梯型转运蛋白 Glt _{Ph中构象转变和底物摄取的时间}. 我们表明，载有底物的运输域跨膜的电梯状运动和底物释放在动力学上是不均匀的，单个分子之间的速率变化数量级。增加电梯转换频率和降低底物亲和力的突变减少了运输速率的异质性并提高了运输效率。氢氘交换与质谱联用揭示了底物结合位点周围二级结构的不稳定，这表明增加的局部动力学会导致更快的全局构象变化速率，并赋予设定传输速率的功能增益特性。