Which of the following describes the function of hydrophobic photoaffinity labeling?
Mapping membrane-embedded regions benefits from probes restricted to hydrophobic phase. Hydrophobic photoaffinity labeling utilizes synthetic phospholipid analogues bearing photoactivatable moieties including aryl azide generating nitrene upon UV about three hundred fifty nanometers, benzophenone generating ketyl radical, diazirine generating carbene highly reactive inserting into C-H bonds within three to five angstroms. Probes partition spontaneously into acyl chain region due to hydrophobicity, concentration in bilayer interior high. Upon irradiation during brief pulse, covalent adducts form between probe and amino acid side chains in contact with lipid-facing surfaces corresponding to transmembrane alpha helices or beta strands. Extramembranous extracellular and cytoplasmic domains exposed to aqueous phase remain unlabeled because probe absent there. After labeling, proteins digested with trypsin chymotrypsin, labeled peptides isolated by chromatography, residues identified by mass spectrometry sequencing defining membrane boundaries. Water soluble photoaffinity reagents label opposite. Technique historically mapped bacteriorhodopsin seven helices and glycophorin helix before crystallography, confirming transmembrane region identification approach essential for topology modeling.
Ref: Brunner, Photolabeling and Hydrophobic Core Mapping, Annu Rev Biochem 1993.