Microsecond exchange of internal water molecules in bacteriorhodopsin

Area of Science:

• Biophysics
• Structural Biology
• Protein Dynamics

Background:

• Bacteriorhodopsin (BR) utilizes internal water molecules in its proton-conducting pathway for proton translocation.
• Understanding the exchange rate of these water molecules is critical for elucidating BR's mechanism.
• Conformational fluctuations in BR influence internal water exchange, providing insights into protein dynamics.

Purpose of the Study:

• To determine the exchange rate of internal water molecules in bacteriorhodopsin (BR) with bulk water.
• To investigate the role of water exchange kinetics in the proton translocation mechanism of BR.
• To correlate water exchange dynamics with protein conformational substates.

Main Methods:

• Multinuclear magnetic relaxation dispersion (MRD) studies using 1H, 2H, and 17O isotopes.
• Analysis of water residence times in BR using 2H and 17O MRD data.
• Determination of the rotational correlation time of detergent-solubilized BR.

Main Results:

• At least seven water molecules in BR exhibit residence times between 0.1-10 microseconds at 277 K.
• Five of these water molecules have residence times in the more restricted range of 0.1-0.5 microseconds.
• Most internal water molecules in BR exchange on a timescale shorter than the rate-limiting step of the photocycle.

Conclusions:

• The rapid exchange of internal water molecules in BR is a key feature of its proton translocation mechanism.
• Water exchange kinetics provide insights into the protein's conformational dynamics and accessibility of functional substates.
• Detergent-solubilized BR is stable and suitable for biophysical studies, with a determined rotational correlation time consistent with a monomeric complex.