Membrane-Anchoring Interactions of M13 Major Coat Protein

• Published in: Biochemistry (Easton) Vol. 40; no. 30; pp. 8815 - 8820
• Main Authors: Meijer, Alexander B, Spruijt, Ruud B, Wolfs, Cor J. A. M, Hemminga, Marcus A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 31.07.2001
• Subjects: Amino Acid Sequence; Bacteriophage M13 - genetics; Bacteriophage M13 - metabolism; Biofysica; Biophysics; Capsid - chemistry; Capsid - genetics; Capsid - metabolism; Capsid Proteins; Cysteine - genetics; Dimyristoylphosphatidylcholine - metabolism; Electron Spin Resonance Spectroscopy; EPS; Fluorescent Dyes - metabolism; Laboratorium voor Biofysica; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Naphthalenesulfonates - metabolism; Phosphatidylcholines - metabolism; Spin Labels; Tryptophan - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi002956s

The response to hydrophobic mismatch of membrane-bound M13 major coat protein is measured using site-directed fluorescence and ESR spectroscopy. For this purpose, we investigate the membrane-anchoring interactions of M13 coat protein in model systems consisting of phosphatidylcholine bilayers that vary in hydrophobic thickness. Mutant coat proteins are prepared with an AEDANS-labeled single cysteine residue in the hinge region of the protein or at the C-terminal side of the transmembrane helix. In addition, the fluorescence of the tryptophan residue is studied as a monitor for the N-terminal side of the transmembrane helix. The fluorescence results show that the hinge region and C-terminal side of the transmembrane helix hardly respond to hydrophobic mismatch. In contrast, the N-terminal side of the helical transmembrane domain shifts to a more apolar environment, when the hydrophobic thickness is increased. The apparent strong membrane-anchoring interactions of the C-terminus are confirmed using a mutant that contains a longer transmembrane domain. As a result of this mutation, the tryptophan residue at the N-terminal side of the helical domain clearly shifts to a more polar environment, whereas the labeled position 46 at the C-terminal side is not affected. The phenylalanines in the C-terminal part of the protein play an important role in these apparent strong anchoring interactions. This is demonstrated with a mutant in which both phenylalanines are replaced by alanine residues. The phenylalanine residues in the C-terminus affect the location in the membrane of the entire transmembrane domain of the protein.