Membrane-bound conformation of M13 major coat protein: a structure validation through FRET-derived constraints.
M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein has a single tryptophan residue that is used as a donor in fluorescence (or Förster) resonance energy transfer (FRET) experiments, using AEDANS-labeled cysteines as acceptors. Based on FRET-derived constraints, a straight alpha-helix is proposed as the membrane-bound conformation of the coat protein. Different models were tested to represent the molecular conformations of the donor and acceptor moieties. The best model was used to make a quantitative comparison of the FRET data to the structures of M13 coat protein and related coat proteins in the Protein Data Bank. This shows that the membrane-bound conformation of the coat protein is similar to the structure of the coat protein in the bacteriophage that was obtained from x-ray diffraction. Coat protein embedded in stacked, oriented bilayers and in micelles turns out to be strongly affected by the environmental stress of these membrane-mimicking environments. Our findings emphasize the need to study membrane proteins in a suitable environment, such as in fully hydrated unilamellar vesicles. Although larger proteins than M13 major coat protein may be able to handle environmental stress in a different way, any membrane protein with water exposed parts in the C or N termini and hydrophilic loop regions should be treated with care.
Vos
Werner L
WL
Laboratory of Biophysics, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.
Koehorst
Rob B M
RB
Spruijt
Ruud B
RB
Hemminga
Marcus A
MA
D036022:Capsid Proteins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D030562:Databases, Protein; D003902:Detergents / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008051:Lipid Bilayers / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D015233:Models, Statistical; D008968:Molecular Conformation; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014364:Tryptophan / Q000737:chemistry; D014961:X-Ray Diffraction
D036022:Capsid Proteins; D003902:Detergents; D008051:Lipid Bilayers; D008823:Micelles; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; C025668:coat protein, Bacteriophage M13; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D014364:Tryptophan; C017251:1,2-oleoylphosphatidylcholine; D003545:Cysteine