Solution NMR structures of the major coat protein of filamentous bacteriophage M13 solubilized in Dodecyl Phosphocholine micelles, 25 lowest energy structures

Solution structure of the M13 major coat protein in detergent micelles: A basis for a model of phage assembly involving specific residues

The major coat protein (gVIIIp) of bacteriophage M13 solubilized in sodium dodecyl sulfate (SDS) detergent micelles was used as a model system to study this protein in the lipid-bound form. In order to probe the position of gVIIIp relative to the SDS micelles, stearate was added, spin-labeled at the 5- or 16-position with a doxyl group containing a stable nitroxide radical. The average position of the spin-labels in the micelles was derived from the line broadening of the resonances in the 13C spectrum of SDS. Subsequently, we derived a model of the relative position of gVIIIp in the SDS micelle from the effect of the spin-labels on the gVIIIp resonances, monitored via 1H-15N HSQC and TOCSY experiments. The results are consistent with the structure of gVIIIp having two helical strands. One strand is a long hydrophobic helix that spans the micelle, and the other is a shorter amphipathic helix on the surface of the micelle. These results are in good agreement with the structure of gVIIIp in membranes proposed by McDonnell et al. on the basis of solid state NMR data [McDonnell, P. A., Shon, K., Kim, Y., & Opella, S. J. (1993) J. Mol. Biol. 233, 447-463]. This study indicates that high-resolution NMR on this membrane protein, solubilized in detergent micelles, is a very suitable technique for mimicking these proteins in their natural environment. Furthermore, the data indicate that the structure of the micelle near the C-terminus of the major coat protein is distorted.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry 1994 Nov 8;33(44):12990-7
Location of M13 coat protein in sodium dodecyl sulfate micelles as determined by
NMR. Papavoine CH, Konings RN, Hilbers CW, van de Ven FJ

Location of M13 coat protein in sodium dodecyl sulfate micelles as determined by NMR.

The membrane-bound form of the major coat protein (gVIIIp) of bacteriophage M13
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 alpha-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. Comparison of the data of gVIIIp in DodPCho
micelles with those of gVIIIp in sodium dodecyl sulfate (SDS) micelles [Van de
Ven, F. J. M., van Os, J. W. M., Aelen, J. M. A., Wymenga, S. S., Remerowski,
M. L., Konings, R. N. H. & Hilbers, C. W. (1993) Biochemistry 32, 8322-8328;
Papavoine, C. H. M., Konings, R. N. H., Hilbers, C. W. & Van de Ven, F. J. M.
(1994) Biochemistry 33, 12,990-12,997] reveals that the structures of the
protein in the two detergent micelles are very similar. They differ only in the
arrangement of the detergent molecules around the protein. For gVIIIp 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.

Eur J Biochem 1995 Sep 1;232(2):490-500
NMR studies of the major coat protein of bacteriophage M13. Structural
information of gVIIIp in dodecylphosphocholine micelles. Papavoine CH, Aelen
JM, Konings RN, Hilbers CW, Van de Ven FJ

NMR studies of the major coat protein of bacteriophage M13. Structural information of gVIIIp in dodecylphosphocholine micelles.

The backbone dynamics of the major coat protein (gVIIIp) of the filamentous bacteriophage M13, solubilized in detergent micelles, have been studied using 15N nuclear magnetic resonance spectroscopy at three frequencies. Motional parameters and overall and internal correlation times were derived with the model-free approach. It was also checked whether these parameters had to be modified due to anisotropic motion of the protein/micelle complex. Reduced spectral density mapping was used to calculate the spectral densities at J(O), J(omegaN), and [J(omegaH)]. The spectral densities were interpreted by mapping a linear or scaled linear combination of two Lorentzians onto a J(O)-J(omega) plot. The major coat protein of bacteriophage M13 consists of two alpha-helices, one of which is hydrophobic and located within the micelle, while the other is amphipathic and located on the surface of the micelle. Our results indicate that the motion of the hydrophobic helix is restricted such that it corresponds to the overall tumbling of the protein/micelle complex. The interpretation of the relaxation data of the amphipathic helix by means of the model-free approach and the reduced spectral density mapping indicate that in addition to the overall motion all residues in this helix are subject to motion on the fast nanosecond and picosecond time scales. The motions of the vectors in the low nanosecond range are characterized by similar values of the spectral densities and correlation times and represent the motion of the amphipathic helix on and away from the surface of the micelle. The relaxation data of the residues in the hinge region connecting the helices show that there is an abrupt change from highly restricted to less restricted motion. Both the C-terminal and N-terminal residues are very mobile.

Biochemistry 1997 Apr 1;36(13):4015-26
Backbone dynamics of the major coat protein of bacteriophage M13 in detergent
micelles by 15N nuclear magnetic resonance relaxation measurements using the
model-free approach and reduced spectral density mapping. Papavoine CH,
Remerowski ML, Horstink LM, Konings RN, Hilbers CW, van de Ven FJ

Backbone dynamics of the major coat protein of bacteriophage M13 in detergent micelles by 15N nuclear magnetic resonance relaxation measurements using the model-free approach and reduced spectral density mapping.

The major coat protein (gVIIIp) of bacteriophage M13 complexed with SDS detergent micelles was used as a model system to study the lipid-bound conformation of the protein. Conditions were found that allowed the recording of good quality of NMR spectra. By making extensive use of three-dimensional heteronuclear (13C, 15N) NMR, we obtained a complete set of resonance assignments for 1HN, 1H alpha, 1H beta, 13C alpha, CO, and 15N and partially assigned the rest of the 1H spectrum. Analysis of NOE and chemical shift data reveals that gVIIIp is composed of two alpha-helical domains, one ranging from Pro-6 to Glu20 and the other ranging from Tyr-24 all the way to the C-terminus Ser-50. In contrast to the results reported by Henry and Sykes [Henry, G.D., & Sykes, B.D. (1992) Biochemistry 31, 5285-5297], at a high SDS to protein ratio the protein appears to be monomeric.

Biochemistry 1993 Aug 17;32(32):8322-8
Assignment of 1H, 15N, and backbone 13C resonances in detergent-solubilized M13
coat protein via multinuclear multidimensional NMR: a model for the coat
protein monomer. van de Ven FJ, van Os JW, Aelen JM, Wymenga SS, Remerowski ML,
Konings RN, Hilbers CW

Assignment of 1H, 15N, and backbone 13C resonances in detergent-solubilized M13 coat protein via multinuclear multidimensional NMR: a model for the coat protein monomer.

The membrane bound form of bacteriophage fd coat protein has a long hydrophobic membrane spanning helix and a shorter amphipathic helix in the plane of the bilayer. Residues near the N and C termini and in the turn connecting the two helices are mobile. The locations and orientations of the helical secondary structure elements and the protein backbone dynamics were characterized by combining results from multidimensional solution NMR experiments on protein samples in micelles and high resolution solid-state NMR experiments on protein samples in oriented and unoriented lipid bilayers. The coat protein is a monomer in micelles. The secondary structure of the membrane bound form of fd coat protein is very similar to that of the structural form found in the virus particles, since it is nearly all alpha helix. However, the membrane bound form of the protein differs from the structural form of the protein in virus particles in the arrangement of the secondary structure, since the membrane bound form of the protein has two distinct helical domains oriented perpendicular to each other and the structural form of the protein in the virus particles has a nearly continuous helix aligned approximately along the filament axis. In addition, there are substantial differences in the dynamics of residues in the bend between the two helices and near the C terminus, since they are mobile in the membrane bound form of the protein and not in the virus particles. Residues 1 to 5 at the N terminus are highly mobile and unstructured in both the membrane bound and structural forms of the coat protein.

J Mol Biol 1993 Oct 5;233(3):447-63
fd coat protein structure in membrane environments.
McDonnell PA, Shon K, Kim Y, Opella SJ

fd coat protein structure in membrane environments.

Couplings not reported are not resolved
due to overlap. Residue 06 is a proline
residue.
Data collected at both 400 and 500 MHz

Fd Major Coat Protein In Sds Micelles, Nmr, 20 Structures

Model-Building Studies Of Inovirus: Genetic Variations On A Geometric Theme

Molecular Models And Structural Comparisons Of Native And Mutant Class I Filamentous Bacteriophages Ff (Fd, F1, M13), If1 And I

Nmr Structure Of The Fd Bacteriophage Pviii Coat Protein In Lipid Bilayer Membranes

Solution Nmr Structures Of The Major Coat Protein Of Filamentous Bacteriophage M13 Solubilized In Dodecylphosphocholine Micelle

Solution Nmr Structures Of The Major Coat Protein Of Filamentous Bacteriophage M13 Solubilized In Sodium Dodecyl Sulphate Micel

Capsid Model Of M13 Bacteriophage Virus From Magic-angle Spinning Nmr And Rosetta Modeling

structural protein [Enterobacteria phage M13]

unnamed protein product [Enterobacteria phage f1]

gene VIII protein, partial [Enterobacteria phage f1]

major coat protein B [Enterobacteria phage f1]

gene-8 protein, g8p=major coat protein [bacteriophage fd, Peptide, 50 aa]

RecName: Full=Capsid protein G8P; AltName: Full=Coat protein B; AltName: Full=Gene 8 protein; Short=G8P; AltName: Full=Major co

RecName: Full=Capsid protein G8P; AltName: Full=Coat protein B; AltName: Full=Gene 8 protein; Short=G8P; AltName: Full=M13 proc

RESTRAINED MOLECULAR DYNAMICS, SIMULATED ANNEALING, THIS VERSION OF X-PLOR WAS EXTENDED FOR FLOATING CHIRALITY

The sample was in dry form added
to dry dodecyl phosphocholine.
To this H2O or D2O was added. This
sample was then vortexed and pH adjusted.
The end volume was 500 uL.

Analysis of the spectra to obtain assignments and structural information