Linking function to global and local dynamics in an elevator-type transporter

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 GltPh. 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.

Automated summary

This study used single-molecule fluorescence microscopy to compare the timing of conformational transitions and substrate uptake in the transporter GltPh, finding that the movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous. Mutations that increase elevator transitions and reduce substrate affinity can diminish transport rate heterogeneities and boost transport efficiency, suggesting potential gain-of-function properties that impact transport rates.