Exploring the Gating Mechanism of the Human Copper Transporter, hCtr1, Using EPR Spectroscopy

Biomolecules, Journal Year: 2025, Volume and Issue: 15(1), P. 127 - 127

Published: Jan. 14, 2025

Ctr1 is a membrane-spanning homotrimer that facilitates copper uptake in eukaryotic cells with high affinity. While structural details of the transmembrane domain human have been elucidated using X-ray crystallography and cryo-EM, transfer mechanisms conformational changes control gating mechanism remain poorly understood. The role extracellular N-terminal domains particularly unclear due to absence high-resolution structure full-length hCtr1 protein limited biochemical biophysical characterization transporter solution cell. In this study, we employed distance electron paramagnetic resonance investigate hCtr1, both vitro cells, as function Cu(I) binding. Our results demonstrate at specific concentrations, chains move closer lumen facilitate transfer. Additionally, while these concentrations intracellular part penetrating lumen, suggesting ball-and-chain mechanism. Moreover, phenomenon was observed for reconstituted micelles native cell membranes. However, measured values were slightly different, membrane’s characteristics therefore its lipid composition also impact even regulate hCtr1.

Language: Английский

Efficient Orthogonal Spin Labeling of Proteins via Aldehyde Cyclization for Pulsed Dipolar EPR Distance Measurements

Journal of the American Chemical Society, Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 28, 2024

Pulsed dipolar electron paramagnetic resonance (PD-EPR) measurement is a powerful technique for characterizing the interactions and conformational changes of biomolecules. The extraction these distance restraints from PD-EPR experiments relies on manipulation spin–spin pairs. orthogonal spin labeling approach offers unique advantages by providing multiple distances between different Here, we report an efficient based exploiting cyclization 1,2-aminothiol moiety in protein (e.g., N-terminal cysteine) with aldehyde group label thiol substitution (or addition) reaction label. We demonstrated that this method enables high accuracy precision constraints through single sample. This was applied to characterize oligomeric state trigger factor (TF) Escherichia coli, important chaperone, solution cell lysates measurements Contrary popular belief, TF exists mainly monomeric not as dimer lysate.

Language: Английский