Protein conformational changes in the bacteriorhodopsin photocycle.

Area of Science:

• Structural Biology
• Biophysics
• Protein Dynamics

Background:

• Bacteriorhodopsin is a light-driven proton pump crucial for energy transduction.
• Understanding its photocycle and conformational changes is key to elucidating its mechanism.
• Mutations can trap specific photocycle intermediates, offering insights into structural dynamics.

Purpose of the Study:

• To comprehensively analyze conformational changes in wild-type and mutant bacteriorhodopsin during its photocycle using electron crystallography.
• To determine the structural basis for kinetic defects in various bacteriorhodopsin mutants.
• To elucidate the relationship between structural changes and photocycle intermediates.

Main Methods:

• Electron crystallography of flash-illuminated wild-type and mutant bacteriorhodopsin crystals.
• Trapping of photocycle intermediates by rapid freezing.
• Construction of projection difference Fourier maps at 3.5 Å resolution.

Main Results:

• A significant protein conformational change occurs within 1 ms after light activation in wild-type bacteriorhodopsin.
• Structural differences between M and N intermediates are small in the same protein but vary significantly across different mutants.
• Mutations can induce partial or full conformational changes even in the dark.

Conclusions:

• Observed structural changes in mutants with long-lived intermediates represent variations of a single fundamental conformational change.
• The photocycle involves two main protein conformations, approximating early (K, L, M1) and late (M2, N, O) intermediates.
• This conformational change is critical for switching Schiff base proton accessibility during proton pumping.