NMR Studies of the Major Coat Protein of Bacteriophage M13

Abstract

The membrane-bound form of the major coat protein (gV11Ip) of bacteriophage M I 3 has been studied using nuclear magnetic resonance spectroscopy. As membrane mimetics, we used dodecylphosphocholine (DodPCho) detergent micelles to solubilize the protein. We were able to nearly completely assign all resonances of the protein solubilized in DodPCho micelles by using both homonuclear and heteronuclear multidimensional experiments. Based on the patterns of the nuclear Overhauser enhancements and the chemical shifts of the resonances, we deduced the secondary structure of the protein. Additional structural information was obtained from amide proton exchange data and J-coupling constants. The protein consists of two a-helices which are connected by a hinge region around residue 21. From spin-label experiments, the location of the protein relative to the DodPCho micelles was determined. One, hydrophobic, helix spans the micelle, and another, amphipathic, helix, is located beneath the surface of the micelle. For gVTTTp in SDS micelles, we found a micellar structure which is distorted near the C-terminus of the protein; whereas for DodPCho micelles, both distorted and regular elliptical micelles occur. This distortion is probably due to the interaction of the positively charged lysine side chains with the negatively charged head group of the detergent molecules.Keywords: major coat protein; bacteriophage M I 3 ; NMR; dodecylphosphocholine; structural analysis.Our knowledge of biological processes involving membranebound proteins is limited in comparison with those involving cytoplasmic proteins. This is due, in large part, to the lack of structural information caused by difficulties in applying the methods of X-ray diffraction and NMR to membrane-bound proteins. Membrane-associated proteins are more difficult to crystallize than globular proteins. Multidimensional high-resolution NMR methods, which work well in the elucidation of the structures of small water-soluble proteins (Wiithrich, 1986) are less well-suited for studying large, slowly reorienting complexes, like membrane-bound proteins. To obtain structural information for these proteins with these techniques, model systems, mostly in the form of detergent micelles, are used as membrane mimetics. In high-resolution NMR, glucagon (Braun et al., 1981) and melittin (Brown et al., 1982) were the initial model systems Correspondence to F. J. M. Van de Ven,

Studies of the Structure of the N‐Terminal Domain from the Y4 Receptor—a G Protein‐Coupled Receptor—and its Interaction with Hormones from the NPY Family

“…The proximity of protons of the N‐terminal domain to the micelle surface was probed with the aid of micelle‐integrating spin‐labels. The paramagnetic moiety of 5‐doxyl stearic acid has been shown to reside in the headgroup region 32. Consistently with the assumption that structuring of the N‐terminal segment is induced by binding to the micelle, signals from the amide moieties within that segment experienced the largest signal reduction (see Figure S10).…”

Using O 2 to probe membrane immersion depth by 19 F NMR

“…By comparison, in an oriented bicelle medium consisting of a 25% (wt͞wt) dispersion of TFOM and DMPC͞DHPC ϭ 3, differential downfield shifts resulting from addition of Dy 3ϩ EDTA could be seen for position 3, confirming our assignment and the notion that position 3 lies closest to the membrane-water interface. Membrane-soluble and water-soluble ESR spin labels have been used widely in NMR studies of membrane proteins and membrane peptides, primarily in detergent micelles to determine surface exposure and immersion depth (39)(40)(41)(42)(43)(44). In general, these experiments involve the addition of spin-labeled probes, in which the nitroxide or paramagnetic species is located (on a lipid or fatty acid chain) at a particular depth in the micelle.…”

Structure and dynamics of the M13 coat signal sequence in membranes by multidimensional high-resolution and solid-state NMR spectroscopy

“…14 This technique was used to determine the structures of several membrane polypeptides, including hormones, toxins, antibiotics, and viral coat proteins, in the presence of SDS or DPC micelles, respectively. [15][16][17] By contrast, polypeptides associated with phospholipid bilayers exhibit slow correlation times, and the NMR spectra of these samples are characterized by the anisotropy of nuclear interactions. The spectral line shapes of solid-state NMR spectra therefore exhibit the orientational distributions of nuclear interactions and can be used to characterize the structure, orientation, dynamics, and interactions of bilayer-associated polypeptides.…”