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 -helical protein conformation from amino acid residues 12¿46, close to the protein conformation in the intact phage.
| Main Authors: | , , , , |
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| Format: | Article/Letter to editor biblioteca |
| Language: | English |
| Subjects: | bacteriophage m13, conformation, dynamics, energy-transfer, fluorescence, micelles, modulation, peptides, spectroscopy, transmembrane domain, |
| Online Access: | https://research.wur.nl/en/publications/fret-study-of-membrane-proteins-determination-of-the-tilt-and-ori |
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