Structures of Glt Ph ( 7, 22 ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓– 30), and archaeal and mammalian homologs ( 31 ⇓ ⇓ ⇓ ⇓ ⇓– 37), show that the transporters assemble into homotrimers via scaffold domains. Glt Ph is an extensively studied archaeal aspartate transporter that is homologous to human excitatory amino acid transporters (EAATs). Here, we combine these approaches to link changes in local protein dynamics to the larger-scale conformational transitions and substrate transport in wild-type (WT) and gain-of-function mutants of Glt Ph.

Hydrogen–deuterium exchange followed by mass spectrometry (HDX-MS) has been used to pinpoint local changes in structural dynamics in diverse biological systems ( 19 ⇓– 21). Single-molecule Forster resonance energy transfer (smFRET)-based total internal reflection fluorescence (TIRF) microscopy ( 2 ⇓– 4) has been used to monitor the dynamics of the OFS to IFS transitions ( 5 ⇓ ⇓– 8) and single-transporter activity ( 9) in the elevator-type transporter Glt Ph and other transporters ( 10 ⇓ ⇓ ⇓ ⇓ ⇓ ⇓ ⇓– 18). Transport efficiency depends on the rates of these rearrangements, but linking function and structural dynamics has presented methodological challenges. In each of these so-called outward- and inward-facing states (OFS and IFS, respectively), further isomerizations accompany substrate binding and release. They undergo concerted conformational changes, allowing alternate exposure of their substrate-binding sites to external and internal solutions ( 1). Transporters are integral membrane proteins that move solutes across lipid bilayers.

hydrogen deuterium exchange mass spectrometry.

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. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. 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.

Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter Glt Ph. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency-yet remain mostly unknown. Transporters cycle through large structural changes to translocate molecules across biological membranes.

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