Structural studies on transmembrane proteins. 1. Model study using bacteriorhodopsin mutants containing single...

Area of Science:

• Biochemistry
• Structural Biology
• Membrane Protein Research

Background:

• Polytopic transmembrane proteins are crucial for cellular functions.
• Understanding their structure is vital for drug development and biological insights.
• Bacteriorhodopsin serves as a model system for studying transmembrane protein structure and function.

Purpose of the Study:

• To develop novel methods for structural studies of polytopic transmembrane proteins.
• To investigate the topographic domain and membrane helix orientation of bacteriorhodopsin using cysteine mutants.

Main Methods:

• Preparation of four bacteriorhodopsin mutants with single cysteine residues at specific sites.
• Regeneration of the chromophore and proton pumping assays in detergent micelles.
• Reconstitution into asolectin vesicles for reactivity studies with iodoacetic acid.
• Derivatization of sulfhydryl groups in denatured states and assessment of protein folding.

Main Results:

• Mutants Gly-72-Cys and Ser-169-Cys in looped regions showed reactive sulfhydryl groups in vesicles.
• Mutants Thr-90-Cys and Leu-92-Cys in helix C had unreactive sulfhydryl groups in vesicles.
• Derivatized mutants Gly-72-Cys and Ser-169-Cys folded normally; Thr-90-Cys and Leu-92-Cys showed impaired folding.
• Bulky labeling of Leu-92-Cys further impaired folding, indicating steric sensitivity.

Conclusions:

• Cysteine accessibility in bacteriorhodopsin mutants correlates with their location (looped vs. membrane-embedded).
• The folding and reactivity of these mutants provide information on topographic domain and helix orientation.
• This approach offers a valuable tool for structural characterization of transmembrane proteins.