Real-time identification of two substrate-binding intermediates for the light-driven sodium pump rhodopsin

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Membrane transport proteins facilitate solute movement across cell membranes.
• Conformational changes are essential for transporter function, switching substrate-binding site accessibility.
• Detecting transient intermediates in transport cycles is challenging.

Purpose of the Study:

• To identify and characterize short-lived intermediates in the transport cycle of Na⁺-pump rhodopsin.
• To elucidate the sequential formation and properties of Na⁺-binding intermediates.
• To understand the mechanism of unidirectional ion transport.

Main Methods:

• Time-resolved absorption spectroscopy triggered by short laser pulses.
• Analysis of absorption changes to detect transient intermediates.
• Investigating the effect of Na⁺ concentration on intermediate equilibria.

Main Results:

• Real-time identification of Na⁺-binding intermediates O1 and O2.
• O1 and O2 exhibit red-shifted absorption spectra and form transient equilibria.
• Na⁺ binding to O1 and O2 confirmed by shifts in equilibria with increasing Na⁺ concentration.
• O1 equilibrium suggests Na⁺ uptake on the cytoplasmic side; O2 equilibrium suggests Na⁺ release on the extracellular side.

Conclusions:

• The O1 to O2 transition involves an irreversible switch in binding site accessibility.
• This transition is a key mechanism governing unidirectional Na⁺ transport.
• The findings provide insights into the functional mechanism of Na⁺-pump rhodopsin.