Photoaffinity labeling of bacteriorhodopsin

Area of Science:

• Biochemistry
• Structural Biology
• Membrane Proteins

Background:

• Bacteriorhodopsin (bR) is a light-driven proton pump crucial for energy transduction.
• Understanding the precise structure of bR, particularly the retinal chromophore's location, is key to elucidating its mechanism.
• Previous studies have proposed models for bR's seven-helix structure.

Purpose of the Study:

• To determine the cross-linking sites of optically pure retinal analogues within bacterioopsin.
• To refine the structural model of bacteriorhodopsin by identifying specific amino acid residues interacting with the retinal chromophore.
• To establish the orientation of the retinal chromophore and its ionone ring within the membrane.

Main Methods:

• Incorporation of 14C-labeled, optically pure 3S- and 3R-(diazoacetoxy)-all-trans-retinals into bacterioopsin to form 3S- and 3R-diazo-bR analogues.
• UV irradiation (254 nm) to generate carbenes for cross-linking retinal to nearby amino acid residues.
• Cyanogen bromide fragmentation and analysis to identify cross-linked peptides and specific amino acid residues.

Main Results:

• The 3S- and 3R-diazo analogues cross-linked to specific cyanogen bromide fragments (CN 7/CN9 and CN 8/CN 9, respectively).
• Thr121 and Gly122 in fragment CN 7 were identified as cross-linked residues for the 3S-diazo analogue.
• These findings support specific assignments for the seven transmembrane helices of bacteriorhodopsin.
• The ionone ring of the chromophore is oriented towards the extracellular side of the membrane.

Conclusions:

• The study precisely mapped the retinal chromophore's attachment points within bacteriorhodopsin.
• The orientation of the retinal chromophore, with the ionone ring facing the extracellular side, was confirmed.
• This work provides critical data for refining models of bacteriorhodopsin structure and function.