The Conformational Switches of a Bacterial Light-Driven Sodium Pump Characterized by Time-Resolved Resonance Raman...

Area of Science:

• Biophysics
• Structural Biology
• Spectroscopy

Background:

• Krokinobacter eikastus rhodopsin 2 (KR2) is a microbial light-driven ion pump.
• It transports Na+ out of the cell, coupled to its retinal chromophore's photocycle.
• The chromophore is attached via a Schiff base linkage.

Purpose of the Study:

• To characterize the operational switches of KR2 controlling Na+ uptake and release.
• To investigate the photocycle intermediates and kinetics using time-resolved spectroscopy.

Main Methods:

• Time-resolved pump-probe resonance Raman (TR RR) spectroscopy.
• Spectroscopic investigation of the parent state using different excitation lines.
• Analysis of chromophore structures and kinetics of photocycle intermediates.

Main Results:

• Two substates were identified with differing Asp116 counterion and water positions relative to the Schiff base.
• One substate corresponds to the active configuration for Na+ binding.
• Two redshifted intermediates (O1 and O2) were identified within milliseconds, featuring protonated 13-cis and all-trans configurations, respectively.

Conclusions:

• The identified substates and intermediates provide insight into KR2's Na+ transport mechanism.
• Chromophore isomerization in intermediates O1 and O2 is identified as the switch for Na+ release.
• TR RR spectroscopy is advantageous for determining chromophore structures and kinetics of photocycle intermediates.