Membrane-mediated assembly of filamentous bacteriophage Pf1 coat protein.

Filamentous bacteriophage Pf1 assembles by a membrane-mediated process during which the viral DNA is secreted through the membrane while being encapsulated by the major coat protein. Neutron diffraction studies showed that in the virus most of the coat protein consists of two alpha-helical segments arranged end-to-end with an intervening mobile surface loop. Nuclear magnetic resonance studies of the coat protein in the membrane-bound form have shown that the secondary structure is essentially identical to that in the intact virus. A comparison indicates that during membrane-mediated viral assembly, while the secondary structure of the coat protein is largely conserved, its tertiary structure changes substantially.

NMR studies of the structure and dynamics of membrane-bound bacteriophage Pf1 coat protein.

Filamentous bacteriophage coat protein undergoes a remarkable structural transition during the viral assembly process as it is transferred from the membrane environment of the cell, where it spans the phospholipid bilayer, to the newly extruded virus particles. Nuclear magnetic resonance (NMR) studies show the membrane-bound form of the 46-residue Pf1 coat protein to be surprisingly complex with five distinct regions. The secondary structure consists of a long hydrophobic helix (residues 19 to 42) that spans the bilayer and a short amphipathic helix (residues 6 to 13) parallel to the plane of the bilayer. The NH2-terminus (residues 1 to 5), the COOH-terminus (residues 43 to 46), and residues 14 to 18 connecting the two helices are mobile. By comparing the structure and dynamics of the membrane-bound coat protein with that of the viral form as determined by NMR and neutron diffraction, essential features of assembly process can be identified.

NMR structural studies of membrane proteins.

The three-dimensional structures of membrane proteins are essential for understanding their functions, interactions and architectures. Their requirement for lipids has hampered structure determination by conventional approaches. With optimized samples, it is possible to apply solution NMR methods to small membrane proteins in micelles; however, lipid bilayers are the definitive environment for membrane proteins and this requires solid-state NMR methods. Newly developed solid-state NMR experiments enable completely resolved spectra to be obtained from uniformly isotopically labeled membrane proteins in phospholipid lipid bilayers. The resulting operational constraints can be used for the determination of the structures of membrane proteins.

Application of Maximum Entropy reconstruction to PISEMA spectra.

Maximum Entropy reconstruction is applied to two-dimensional PISEMA spectra of stationary samples of peptide crystals and proteins in magnetically aligned virus particles and membrane bilayers. Improvements in signal-to-noise ratios were observed with minimal distortion of the spectra when Maximum Entropy reconstruction was applied to non-linearly sampled data in the indirect dimension. Maximum Entropy reconstruction was also applied in the direct dimension by selecting sub-sets of data from the free induction decays. Because the noise is uncorrelated in the spectra obtained by Maximum Entropy reconstruction of data with different non-linear sampling schedules, it is possible to improve the signal-to-noise ratios by co-addition of multiple spectra derived from one experimental data set. The combined application of Maximum Entropy to data in the indirect and direct dimensions has the potential to lead to substantial reductions in the total amount of experimental time required for acquisition of data in multidimensional NMR experiments.

The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor.

The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane alpha-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 A at its narrowest, to 8.6 A at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.

Selectively deuterated amino acid analogues. Synthesis, incorporation into proteins and NMR properties.

Selectively deuterated analogues of histidine, tyrosine, phenylalanine and tryptophan have been synthesized by chemical exchange. These analogues have been characterized by NMR spectrometry and used for growth of bacteria. Active lactose repressor protein has been isolated from cells grown on the deuterated amino acids, and denatured 1H and 2H NMR spectra have been determined for the protein.

NMR experiments on aligned samples of membrane proteins.

NMR methods can be used to determine the structures of membrane proteins. Lipids can be chosen so that protein-containing micelles, bicelles, or bilayers are available as samples. All three types of samples can be aligned weakly or strongly, depending on their rotational correlation time. Solution NMR methods can be used with weakly aligned micelle and small bicelle samples. Solid-state NMR methods can be used with mechanically aligned bilayer and magnetically aligned bicelle samples.

Protein structure by solid state nuclear magnetic resonance. Residues 40 to 45 of bacteriophage fd coat protein.

The three-dimensional structure of part of the coat protein in the filamentous bacteriophage fd is described by nuclear magnetic resonance (n.m.r.). Residues 40 to 45 are in a somewhat distorted alpha-helix. This n.m.r. approach for determining protein structure relies on the spectral manifestations of chemical shift and heteronuclear dipolar couplings in a symmetrical assembly of protein subunits oriented parallel to the applied magnetic field. The angles between individual peptide linkages and the filament axis of the virion constitute the basic source of structural information. These angles are directly related to x, y, z co-ordinates for describing the protein structure.

fd coat protein structure in membrane environments: structural dynamics of the loop between the hydrophobic trans-membrane helix and the amphipathic in-plane helix.

By performing multidimensional solution NMR experiments on micelle samples it was possible to determine the structure of the membrane-bound form of fd coat protein based on short-range distance and dihedral angle constraints using distance geometry and simulated annealing calculations. Its dynamics were described by 15N relaxation measurements (T1, T2, heteronuclear nuclear Overhauser enhancement (NOE)) fitted with the Lipari-Szabo model-free formalism adapted for the transmembrane and in-plane helices of a membrane protein. The overall correlation time of the protein in micelles was found to be approximately 9 ns, and the local motion of each backbone N-H vector was described by an order parameter and an effective correlation time. The 50 residue protein has an amphipathic alpha-helix (residues 7 to 16) and a hydrophobic alpha-helix (residues 27 to 44), which were found to be approximately perpendicular on the basis of NOEs in the residues that connect the two helices. The residues connecting the helices are of particular interest in membrane proteins, and in this case the loop consists of two turns. The relaxation data show the presence of an extra motion in the amphipathic alpha-helix on the nanosecond timescale and additional flexibility of several residues in the loop connecting the two helices.

Secondary structure of filamentous bacteriophage coat protein is preserved in lipid environments.

1H nuclear magnetic resonance experiments have shown that the amide hydrogens of residues 30 to 40 of bacteriophage Pf1 coat protein in micelles undergo very slow exchange with solvent deuterons. The amide 1H resonances from these residues were used to monitor the structural stability of the membrane-spanning helix of the coat protein during the transition of the coat protein from its structural form, in the virus particle, to the membrane-bound form, in micelles. The helix was found to remain folded on the 10(-3) second time-scale of the experiment, which indicates that no major disruption or rearrangement of the central part of the protein structure occurs during the process of coat protein solubilization by detergent. The results also suggest that a helical peptide can associate with lipids without reorganization of its secondary structure. However, a general model for the insertion of proteins into membranes cannot be established from these results, because the mechanism of the detergent solubilization process may differ somewhat from that of the membrane insertion process.

A simple protocol for identification of helical and mobile residues in membrane proteins.

A simple molecular dynamics (MD) simulations is protocol shown to predict whether a residue is in a structured alpha-helical segment or in a mobile loop or terminal segment of a membrane protein. The results are verified by comparisons with experimental NMR data. The protocol consists of performing several independent MD simulations on a polypeptide sequence of interest in a dielectric continuum with a relative permittivity epsilon = 2. The time histories of the individual angles between NH bond vectors at time 0 and time t later are calculated, and then Gaussian smoothing of typically 50 ps is applied. The smoothed data are subtracted from the original data to yield the short time fluctuations of the NH bond vectors, and then the rms deviations of the angles are calculated and compared to experimental NMR results. Pf1 coat protein and the c subunit of the E. coli F1F0 ATP synthase are used as examples of membrane proteins. The calculated NH bond rms fluctuations are in qualitative agreement with experimental NMR data in showing that each of these proteins has a mobile segment connecting two helices, as well as mobile N and C-terminal regions. This MD simulations protocol can demonstrate the presence of both the amphipathic and hydrophobic helices while hydropathy plots are able to detect only the hydrophobic helices present in membrane proteins.

31P nuclear magnetic resonance of the RNA in tobacco mosaic virus.

Solid state 31P n.m.r. data concerning the structure of the RNA in TMV are presented in light of the prior diffraction and model building results on this system (Stubbs et al., 1977; Stubbs & Stauffacher, 1981). The 31P chemical shift anisotropy powder pattern of a stationary, unoriented solution of TMV shows the RNA to be immobilized by the coat protein-RNA interactions, since the principal values (sigma 11 = 83, sigma 22 = 25, sigma 33 = -108 p.p.m. relative to external 85% H3PO4) are essentially the same as those of a static phosphodiester group. There are three peaks in the isotropic 31P n.m.r. spectrum obtained with magic angle sample spinning, indicating three distinct phosphate environments. There are three peaks in the 31P n.m.r. spectrum from an oriented TMV solution, indicating three distinct phosphate orientations.

fd coat protein structure in membrane environments.

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.

NMR structure of the principal neutralizing determinant of HIV-1 displayed in filamentous bacteriophage coat protein.

An NMR approach for structure determination of short peptides displayed on the surface of filamentous bacteriophage virions is demonstrated using the hexapeptide GPGRAF that constitutes the principal neutralizing determinant of HIV-1. This peptide was inserted near the N terminus of the major coat protein of bacteriophage fd. NMR studies of the recombinant protein solubilized in detergent micelles showed that the inserted peptide adopts a double bend S-shaped conformation that is similar to the antibody-bound structure determined by X-ray crystallography. This indicates that a peptide displayed on the bacteriophage coat protein has an enhanced propensity to adopt a conformation similar to that found in the native protein from which it is derived. This approach may be generally applicable to the structure determination of peptide epitopes and other small peptides.

Effects of temperature and Y21M mutation on conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd.

Solid-state NMR spectroscopy was used to analyze the conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd. Both one and two-dimensional solid-state NMR spectra of magnetically aligned samples of fd bacteriophage reveal that an increase in temperature and a single site substitution (Tyr21 to Met, Y21M) reduce the conformational heterogeneity observed throughout wild-type pVIII. The NMR results are consistent with previous studies indicating that conformational flexibility in the hinge-bend segment that links the amphipathic and hydrophobic helices in the membrane-bound form of the protein plays an essential role during phage assembly, which involves a major change in the tertiary, but not secondary, structure of the coat protein.

Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy.

Magainin 2 is a 23-residue peptide that forms an amphipathic alpha-helix in membrane environments. It functions as an antibiotic and is known to disrupt the electrochemical gradients across the cell membranes of many bacteria, fungi, and some tumor cells, although it does not lyse red blood cells. One- and two-dimensional solid-state 15N NMR spectra of specifically 15N-labeled magainin 2 in oriented bilayer samples show that the secondary structure of essentially the entire peptide is alpha-helix, immobilized by its interactions with the phospholipids, and oriented parallel to the membrane surface.

Structure of a malaria parasite antigenic determinant displayed on filamentous bacteriophage determined by NMR spectroscopy: implications for the structure of continuous peptide epitopes of proteins.

The NANP repeating sequence of the circumsporozoite protein of Plasmodium falciparum was displayed on the surface of fd filamentous bacteriophage as a 12-residue insert (NANP)(3) in the N-terminal region of the major coat protein (pVIII). The structure of the epitope determined by multidimensional solution NMR spectroscopy of the modified pVIII protein in lipid micelles was shown to be a twofold repeat of an extended and non-hydrogen-bonded loop based on the sequence NPNA, demonstrating that the repeating sequence is NPNA, not NANP. Further, high resolution solid-state NMR spectra of intact hybrid virions containing the modified pVIII proteins demonstrate that the peptides displayed on the surface of the virion adopt a single, stable conformation; this is consistent with their pronounced immunogenicity as well as their ability to mimic the antigenicity of their native parent proteins.

Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers.

The colicin E1 channel polypeptide was shown to be organized anisotropically in membranes by solid-state NMR analysis of samples of uniformly 15N-labeled protein in oriented planar phospholipid bilayers. The 190 residue C-terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid-state NMR spectroscopy in oriented membrane bilayers. The 15N-NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans-membrane and in-plane helical segments; (3) trans-membrane helices account for approximately 20-25% of the channel polypeptide, which is equivalent to 38-48 residues of the 190-residue polypeptide. The results of the two-dimensional PISEMA spectrum are interpreted in terms of a single trans-membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38-residue hydrophobic segment near the C-terminus of the colicin E1 channel polypeptide.

Lanthanide ions bind specifically to an added "EF-hand" and orient a membrane protein in micelles for solution NMR spectroscopy.

Twelve amino acid residues corresponding to an "EF-hand" calcium-binding site were added to the N-terminus of a protein, providing a specific lanthanide ion binding that weakly orients the protein in solution. A comparison of spectra of the protein with and without the EF-hand residues demonstrates that the structure of the native protein is not perturbed by this modification, since there are minimal chemical shift changes. With a lanthanide but not calcium bound to the EF-hand, the protein is weakly oriented by the magnetic field, since residual dipolar couplings can be measured. Since the signs and magnitudes of the couplings varied with the type of lanthanide, this demonstrated the ability to obtain multiple orientations of the protein in solution. The sample is a membrane protein in lipid micelles that disrupted the commonly employed bicelle and filamentous phage solutions; therefore, the addition of a specific metal binding site in the form of an EF-hand may provide a general approach to orienting proteins where the addition of external agents is problematic. An additional benefit is that the lanthanide ions perturb the protein resonances in ways that provide unique orientational and distance constraints.