期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：49
DOI：10.1073/pnas.2025520118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Structural snapshots of membrane transporters show that they cycle through several conformational states to bring substrates across the membrane. The rates of these molecular motions determine the activity of the transporters. In this work, we directly compare real-time single-molecule measurements of conformational changes and substrate transport rates in the model glutamate transporter homologue, Glt
Ph. We couple these experiments with hydrogen–deuterium exchange mass spectrometry measurements of local protein flexibility to directly link local and global conformational dynamics with function.
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.
