Use of the membrane-impermeable guanidinating reagent 2-S-[14C]thiuroniumethanesulfonate to demonstrate the orientation of light-harvesting proteins in Rhodobacter sphaeroides.
The radiolabeled guanidinating reagent 2-S-[14C]thiuroniumethanesulfonate reacts with the epsilon-amino groups of accessible lysyl residues of membrane proteins under relatively mild labeling conditions, yielding labeled homoarginyl residues. Model studies have shown that the resulting homoarginyl residues do act as new cleavage sites for trypsin, but only at a very slow rate of hydrolysis. The reagent has been shown to be impermeable to the intracytoplasmic membranes of Rhodobacter sphaeroides: when cytoplasmic-side-out chromatophores were treated with the reagent, it reacted with all four of the light-harvesting proteins, all of which have one or more lysyl residues on the N-terminal sides of their hydrophobic regions. However, when periplasmic-side-out vesicles, prepared by cytochrome c affinity chromatography, were treated with the guanidinating reagent, three of the light-harvesting proteins (B850 alpha, B850 beta, and B870 beta) were not labeled. The only light-harvesting protein to be labeled (B870 alpha) was the only one of the four to have a lysyl residue on the C-terminal side of its hydrophobic region. Guanidinated B870 alpha polypeptides from both the cytoplasmic-side-out chromatophores and the periplasmic-side-out membrane vesicles were purified and digested with trypsin. The resulting peptide fragments were then separated by high-performance liquid chromatography and analyzed for radioactivity. The results have confirmed the asymmetric orientation of the light-harvesting proteins of R. sphaeroides, with their N-termini on the cytoplasmic side of the intracytoplasmic membrane. In the case of the B870 alpha subunit, the protein has been shown to be transmembrane with its C-terminus on the periplasmic side of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Hundle
B S
BS
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Richards
W R
WR
D000595:Amino Acid Sequence; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D007425:Intracellular Membranes / Q000737:chemistry*; D008565:Membrane Proteins / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism / Q000648:ultrastructure*; D011487:Protein Conformation; D012242:Rhodobacter sphaeroides / Q000378:metabolism* / Q000648:ultrastructure; D013890:Thiourea / Q000031:analogs & derivatives* / Q000378:metabolism
D008565:Membrane Proteins; D045322:Photosynthetic Reaction Center Complex Proteins; C065865:2-S-thiuronium ethanesulfonate; D013890:Thiourea; D005998:Glycine