Use of the membrane-impermeable guanidinating reagent 2-S-( sup 14 C)thiuroniumethanesulfonate to demonstrate the orientation of light-harvesting proteins in Rhodobacter sphaeroides

Journal Article · Tue Jul 03 04:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00478a009· OSTI ID:6253926

Hundle, B S; Richards, W R ^[1]

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1. Simon Fraser Univ., Burnaby, British Columbia (Canada)

The radiolabeled guanidinating reagent 2-S-({sup 14}C)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. 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. The results have also demonstrated the effectiveness of the labeling reagent for future membrane-protein orientation studies.

• OSTI ID: 6253926
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:26; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
AMINES
AROMATICS
ATP-ASE
AZAARENES
BACTERIA
CARBON 14 COMPOUNDS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CHROMATOGRAPHY
CROSS-LINKING
ENZYMES
ESTERS
GUANINE
HETEROCYCLIC COMPOUNDS
HYDROLASES
HYDROXY COMPOUNDS
LABELLED COMPOUNDS
MEMBRANE PROTEINS
MEMBRANES
MICROORGANISMS
MOLECULAR STRUCTURE
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PERMEABILITY
PHOSPHOHYDROLASES
PHYTOCHROMES
PIGMENTS
POLYMERIZATION
PROTEINS
PURINES
SEPARATION PROCESSES
SULFONIC ACID ESTERS
THIN-LAYER CHROMATOGRAPHY