Accessibility and environment probing using cysteine residues introduced along the putative transmembrane domain of the major coat protein of bacteriophage M13.
The major coat protein of the filamentous bacteriophage M13 is located in the inner membrane of host cell Escherichia coli prior to assembly into virions. To identify the transmembrane domain of the coat protein, we have introduced unique cysteine residues along the putative transmembrane domain at position 25, 31, 33, 36, 38, 46, 47, 49, or 50. The mutant major coat protein was solubilized by membrane-mimicking detergents or reconstituted into mixed bilayers of phospholipids. Information about the environmental polarity was deduced from the wavelength of maximum emission, using N-[[(iodoacetyl)-amino)ethyl]-1-sulfonaphthylamine (IAEDANS) attached to the SH groups of the cysteines as a fluorescent probe. Additional information was obtained by determining the accessibility of AEDANS for the fluorescence quencher molecules acrylamide and 5-doxylstearic acid, and the reactivity of the cysteine's sulfhydryl group toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Our data suggest transmembrane boundaries close to residue 25 and 46, with residue 25 inside the hydrophobic part of the membrane in very close proximity to the membrane-water interface and residue 46 located at the membrane-water interface. Domains of the mutant coat protein which are packed or coated by cholate molecules and various other detergents [except for sodium dodecyl sulfate (SDS)] are at least similarly packed by phospholipid molecules in bilayers. SDS is a good solubilizing detergent but badly mimics the typical nature of a membrane structure. The overall results are interpreted with respect to the established conformation of the coat protein and its membrane anchoring mechanism.
Spruijt
R B
RB
Department of Molecular Physics, Wageningen Agricultural University, The Netherlands.
Wolfs
C J
CJ
Verver
J W
JW
Hemminga
M A
MA
D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000737:chemistry*; D036022:Capsid Proteins; D003497:Cyclic N-Oxides / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D015335:Molecular Probes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000235:genetics
D000178:Acrylamides; D036022:Capsid Proteins; D003497:Cyclic N-Oxides; D004268:DNA-Binding Proteins; D015335:Molecular Probes; D009282:Naphthalenesulfonates; D013438:Sulfhydryl Compounds; D014760:Viral Fusion Proteins; D020106:Acrylamide; C005072:5-doxylstearic acid; C048876:1,5-AEDANS; D003545:Cysteine