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FRET Study of Membrane Proteins: Determination of the Tilt and Orientation of the N-Terminal Domain of M13 Major Coat Protein

Petr V. Nazarov ^* , Rob B. M. Koehorst ^*, Werner L. Vos ^*, Vladimir V. Apanasovich  and Marcus A. Hemminga ^*

^* Laboratory of Biophysics, Wageningen University, Wageningen, The Netherlands; and Department of Systems Analysis, Faculty of Radio Physics, Belarusian State University, Minsk 220050, Belarus

Correspondence: Address reprint requests to Marcus A. Hemminga, Laboratory of Biophysics, Wageningen University, PO Box 8128, 6700 ET Wageningen, The Netherlands. Tel.: 31-317-482635 or 31-317-482044; Fax: 31-317-482725; E-mail: marcus.hemminga{at}wur.nl.

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.

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