Membrane-bound Conformation of M13 Major Coat Protein

Abstract

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.One of the most challenging problems in structural biology of the 21st century is the unraveling of the structure and function of membrane proteins. For the water-soluble proteins, x-ray diffraction and high field solution NMR spectroscopy are the most suitable tools for structure determination, but for membrane proteins that need to be embedded in an amphipathic environment, there is not yet a well defined strategy for obtaining a protein structure (1). Among the spectroscopic methods, site-directed spectroscopic approaches are becoming increasingly important as alternative tools for structure determination of membrane proteins. These techniques are based on site-directed mutagenesis in combination with specific labeling and provide detailed information about the local environment of the labeled sites and membrane embedment (2-5).Herein is described a further enhancement of the site-directed approach using site-directed labeling to determine distances within a membrane protein from fluorescence (or Förster) resonance energy transfer (FRET). 2FRET is based on a dipolar interaction in which energy is transferred from one chromophore, the donor, to another chromophore, the acceptor (6). In the present work, we applied FRET to determine the membrane-bound structure of the bacteriophage M13 major coat protein. M13 is a small protein, composed of 50 amino acid residues. The protein can adopt a stable conformation in the phage particle stabilized by prot...

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“…There are, however, other more extended structures that deviate from such a structure. This model suggests a much more flexible protein structure, reflecting a more dynamical detergent system (Vos et al 2005, 2009). …”

“…The inner cylinder indicates the viral DNA. b Structure and membrane embedding of M13 coat protein in fully hydrated vesicles of 18:1PC (and mixed phospholipid systems with C 18 acyl chains), based upon recent site-directed labelling spectroscopy (Koehorst et al 2004; Nazarov et al 2007; Stopar et al 2006b; Vos et al 2005, 2007). The protein is effectively anchored with the C-terminal domain at the membrane–water interface by three “snorkelling” lysines (Lys40, Lys43, and Lys44) and two “anti-snorkelling” phenylalanines (Phe42 and Phe45).…”

“…Although convincing, the model suffered from the fact that the coat protein needed to be reconstituted in oriented lipid bilayers that distort, primarily, the topology of the N terminus, forcing it to bend on the lipid bilayer. These protein–lipid systems do not leave enough space between the bilayers, which perturbs the topology of the N-terminal protein domain (Vos et al 2005, 2009). Solution NMR spectroscopy for the protein reconstituted in lipid bilayers is not possible.…”

“…There are, however, strong experimental arguments against orientation of the transmembrane helix parallel to the lipid bilayer normal (Koehorst et al 2004). Experimental data from both NMR (Glaubitz et al 2000; Marassi and Opella 2003; Opella et al 2008) and fluorescence spectroscopy (Koehorst et al 2004; Nazarov et al 2006; Vos et al 2005, 2007) suggest a tilted orientation of the protein. Taking tilt into consideration, one should probably abandon the notion of a perfect L-shaped protein in a lipid bilayer.…”

“…Using the QuikChange site-directed mutagenesis procedure (Stratagene, La Jolla, CA, USA) unique cysteine residues at different positions along the primary sequence of the major coat protein were introduced (Spruijt et al 2004). Such a mutagenesis approach is basically unlimited and at the moment 40 out of the 50 amino acid residues of the major coat protein have been substituted by cysteines (Koehorst et al 2004; Nazarov et al 2007; Spruijt et al 2004; Stopar et al 2006b; Vos et al 2005, 2007). Replacement of essential amino acid residues, principally aromatic and charged amino acid residues, was avoided, as this could give rise to anomalous results with regard to the behaviour of the protein in membranes.…”