Assignment of amide proton and nitrogen-15 NMR resonances in detergent-solubilized M13 coat protein: a model for the coat protein dimer

Abstract

The major coat protein of the filamentous coliphage M13 is a 50-residue integral membrane protein. Detergent-solubilized M13 coat protein is a promising candidate for structure determination by nuclear magnetic resonance methods as the protein can be prepared in large quantities and the protein-containing micelle is reasonably small. Under the conditions of our experiments, SDS-bound coat protein exists as a dimer with an apparent molecular weight of 27,000. Broad lines and poor resolution in the 1H spectrum have led us to adopt an 15N-directed approach, in which the coat protein was labeled both uniformly with 15N and selectively with [alpha-15N]alanine, -glycine, -valine, -leucine, -isoleucine, phenylalanine, -lysine, -tyrosine, and -methionine. Nitrogen resonances were assigned as far as possible using carboxypeptidase digestion, double-labeling, and an independent knowledge of the amide proton exchange rates determined from neighboring assigned 13C-labeled carbonyl carbons. 1H/15N heteronuclear multiple quantum coherence (HMQC) spectroscopy of both uniform and site-selectively-labeled proteins subsequently correlated amide nitrogen with amide proton chemical shifts, and the assignments were completed sequentially from homonuclear NOESY and HMQC-NOESY spectra. The most slowly exchanging amide protons were shown to occur in a continuous stretch extending from methionine-28 to phenylalanine-42. This sequence includes most of the resonances of the hydrophobic core, although it is shifted toward the C-terminal end of the protein. Strong NH to NH (i,i+1) nuclear Overhauser enhancements are a feature of the coat protein, which appears to be largely helical. Between 20 and 25 residues give rise to 2 juxtaposed resonances which can be seen clearly in the HMQC spectrum of uniform 15N-labeled coat protein. These residues are concentrated in a region extending from the beginning of the membrane-spanning sequence through to the disordered region near the C-terminus. We propose that dodecyl sulfate-bound M13 coat protein consists of two independent domains, an N-terminal helix which is in a state of moderately fast dynamic flux and a long, stable, C-terminal membrane-spanning helix, which undergoes extensive interactions with a second monomer. Amide 1H chemical shifts are consistent with this picture; in addition, a marked periodicity is observed at the C-terminal end of the molecule.

FT-IR spectroscopy of the major coat protein of M13 and Pf1 in the phage and reconstituted into phospholipid systems

“…The approach presented here provides a new approach that shows that the C-and N-terminal parts of these coat proteins contain a large amount of secondary structure that resembles a discontinuous a-helix, indicating that these parts probably arising from an increased amount of molecular motion. This concept is in agreement with conclusions arising from NMR (Shon et al, 1991;Henry & Sykes, 1992; Van de Ven et al, 1993) and FT-IR studies (Thiaudiere et al, 1993).…”

“…As a result of the high amount of a-helical structure observed, it was suggested that the coat protein when embedded in lipid bilayers is predominantly in an a-helical conformation. A more detailed structure of M l3 coat protein in a lipidic environment follows from high-resolution NMR studies of the protein solubilized in sodium dodecyl sulfate (Henry & Sykes, 1992;Van de Ven et al, 1993). From this work it follows that micellar-bound M13 coat protein consists of two a-helical domains linked by a short region of uncertain conformation.…”

Complete resolution of the solid-state NMR spectrum of a uniformly 15 N-labeled membrane protein in phospholipid bilayers

“…Newly synthesized copies of the protein are stored in the membrane of infected Escherichia coli bacteria prior to incorporation into virus particles as they are extruded through the membrane. The structure of the membrane bound form of this protein has been determined in micelles by solution NMR spectroscopy (29)(30)(31)(32). It has an amphipathic helix that lies in the plane of the membrane (residues 7-16), and a longer hydrophobic membrane spanning helix (residues 27-44).…”

NMR Studies of the Major Coat Protein of Bacteriophage M13

“…It has been reported as being a dimer with a large fraction of a-helix and P-sheet (Nozaki et al, 1978;Datema et al, 1987) and a helical dimer (Henry and Sykes, 1992), but also as an almost completely a-helical monomer (McDonnell et al, 1993). It has been shown that these differences are brought about by the isolation and purification methods used for the preparation of the samples (Hemminga et al, 1992), and by sample conditions (e.g.…”

“…The major coat protein has a long history within the NMR field but it was not until 1992 that all backbone NH and 15N resonances were assigned (Henry and Sykes, 1992). This was followed by the identification of the "Ca, ' T O , 'Ha and sidechain 'H resonances using multidimensional multinuclear NMR (Van de Ven et al, 1993).…”