FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein.
A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants A7C, A9C, N12C, S13C, Q15C, A16C, S17C, and A18C in the N-terminal domain of this protein were produced and specifically labeled with the fluorescence probe AEDANS. The energy transfer data from the natural Trp-26 to AEDANS were analyzed assuming a two-helix protein model. Furthermore, the polarity Stokes shift of the AEDANS fluorescence maxima is taken into account. As a result the orientation and tilt of the N-terminal protein domain with respect to the bilayer interface were obtained, showing for the first time, to our knowledge, an overall alpha-helical protein conformation from amino acid residues 12-46, close to the protein conformation in the intact phage.
Nazarov
Petr V
PV
Laboratory of Biophysics, Wageningen University, Wageningen, The Netherlands.
Koehorst
Rob B M
RB
Vos
Werner L
WL
Apanasovich
Vladimir V
VV
Hemminga
Marcus A
MA
D017104:Bacteriophage M13 / Q000737:chemistry*; D036022:Capsid Proteins / Q000737:chemistry*; D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D008051:Lipid Bilayers / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D017434:Protein Structure, Tertiary
D036022:Capsid Proteins; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008565:Membrane Proteins; D009282:Naphthalenesulfonates; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; C048876:1,5-AEDANS; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; C017251:1,2-oleoylphosphatidylcholine; D003545:Cysteine