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J. Biol. Chem., Vol. 280, Issue 46, 38522-38527, November 18, 2005

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Membrane-bound Conformation of M13 Major Coat Protein

A STRUCTURE VALIDATION THROUGH FRET-DERIVED CONSTRAINTS^*

From the Laboratory of Biophysics, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands

M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein has a single tryptophan residue that is used as a donor in fluorescence (or Förster) resonance energy transfer (FRET) experiments, using AEDANS-labeled cysteines as acceptors. Based on FRET-derived constraints, a straight -helix is proposed as the membrane-bound conformation of the coat protein. Different models were tested to represent the molecular conformations of the donor and acceptor moieties. The best model was used to make a quantitative comparison of the FRET data to the structures of M13 coat protein and related coat proteins in the Protein Data Bank. This shows that the membrane-bound conformation of the coat protein is similar to the structure of the coat protein in the bacteriophage that was obtained from x-ray diffraction. Coat protein embedded in stacked, oriented bilayers and in micelles turns out to be strongly affected by the environmental stress of these membrane-mimicking environments. Our findings emphasize the need to study membrane proteins in a suitable environment, such as in fully hydrated unilamellar vesicles. Although larger proteins than M13 major coat protein may be able to handle environmental stress in a different way, any membrane protein with water exposed parts in the C or N termini and hydrophilic loop regions should be treated with care.

Received for publication, May 31, 2005 , and in revised form, July 28, 2005.

^* This work was funded from the European Commission under Contract QLG-CT-2000-01801. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Laboratory of Biophysics, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands. Tel.: 31-317-482044; Fax: 31-317-482725; E-mail: marcus.hemminga{at}wur.nl.

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