Kinetic Mechanisms of Fast Glutamate Sensing by Fluorescent Protein Probes.

ABSTRACT: We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding for studying the kinetic mechanism of glutamate binding. iGluSnFR variants termed iGlu_h, iGlu_m, and iGlu_l cover a broad range of K_d-s (5.8 μM and 2.1 and 50 mM, respectively), and a novel fluorescently labeled indicator, Fl-GluBP, has a K_d of 9.7 μM. The fluorescence response kinetics of all the probes are consistent with a two-step mechanism involving ligand binding and isomerization either of the apo or the ligand-bound binding protein. Although the previously characterized ultrafast indicators iGlu_u and iGlu_f

SUBMITTER: Coates C 

PROVIDER: S-EPMC6950763 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature