Single-molecule transport kinetics of a glutamate transporter homolog shows static disorder.

Science Advances
Didar CiftciOlga Boudker

Abstract

Kinetic properties of membrane transporters are typically poorly defined because high-resolution functional assays analogous to single-channel recordings are lacking. Here, we measure single-molecule transport kinetics of a glutamate transporter homolog from Pyrococcus horikoshii, GltPh, using fluorescently labeled periplasmic amino acid binding protein as a fluorescence resonance energy transfer-based sensor. We show that individual transporters can function at rates varying by at least two orders of magnitude that persist for multiple turnovers. A gain-of-function mutant shows increased population of the fast-acting transporters, leading to a 10-fold increase in the mean transport rate. These findings, which are broadly consistent with earlier single-molecule measurements of GltPh conformational dynamics, suggest that GltPh transport is defined by kinetically distinct populations that exhibit long-lasting "molecular memory."

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Methods Mentioned

BETA
fluorescence
FRET
electron microscopy
protein folding
size exclusion chromatography
electrophoresis

Software Mentioned

GraphPad
Spartan
MATLAB script
Prism

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