A new formalism for the simultaneous determination of the membrane embedment and aggregation of membrane proteins is developed. This method is based on steady-state Förster (or fluorescence) resonance energy transfer (FRET) experiments on site-directed fluorescence labeled proteins in combination with global data analysis utilizing simulation-based fitting. The simulation of FRET was validated by a comparison with a known analytical solution for energy transfer in idealized membrane systems. The applicability of the simulation-based fitting approach was verified on simulated FRET data and then applied to determine the structural properties of the well-known major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants Y24C, G38C, and T46C of this protein were produced and specifically labeled with the fluorescence label AEDANS. The energy transfer data from the natural tryptophan at position 26, which is used as a donor, to AEDANS were analyzed assuming a helix model for the transmembrane domain of the protein. As a result of the FRET data analysis, the topology and bilayer embedment of this domain were quantitatively characterized. The resulting tilt of the transmembrane helix of the protein is 18 +/- 2 degrees. The tryptophan is located at a distance of 8.5 +/- 0.5 A from the membrane center. No specific aggregation of the protein was found. The methodology developed here is not limited to M13 major coat protein and can be used in principle to study the bilayer embedment of any small protein with a single transmembrane domain.
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| http://dx.doi.org/10.1529/biophysj.106.082867 | DOI Listing |
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