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, Marcus A. Hemminga*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

20 Citations (Scopus)

Abstract

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 α-helical protein conformation from amino acid residues 12-46, close to the protein conformation in the intact phage.

Original languageEnglish
Pages (from-to)1296-1305
Number of pages10
JournalBiophysical Journal
Volume92
Issue number4
DOIs
Publication statusPublished - Feb 2007
Externally publishedYes

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