Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 118, Issue 49, Pages -Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2025520118
Keywords
single-molecule FRET; hydrogen deuterium exchange mass spectrometry; conformational dynamics; rate-limiting step; glutamate transporter
Categories
Funding
- National Institute of Neurological Disorders and Stroke (NINDS) [R37NS085318, R01NS111767]
- American Heart Association (AHA) Fellowship [19PRE34380215, 7R01GM098859]
- European Union's Horizon 2020 research and innovation program under the Maire Sklodowska-Curie Grant [MEMDYN 660083]
- Wellcome Trust [109854/Z/15/Z]
- Leverhulme Trust [RPG-2019-178]
- Wellcome Trust [109854/Z/15/Z] Funding Source: Wellcome Trust
Ask authors/readers for more resources
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.
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available