Structure of the DNA binding cleft of the gene 5 protein from bacteriophage fd.

The structure of the gene 5 DNA unwinding protein from bacteriophage fd has been solved to 2.3-A resolution by X-ray diffraction techniques. The molecule contains an extensive cleft region that we have identified as the DNA binding site on the basis of the residues that comprise its surface. The interior of the groove has a rather large number of basic amino acid residues that serve to draw the polynucleotide backbone into the cleft. Arrayed along the external edges of the groove are a number of aromatic amino acid side groups that are in position to stack upon the bases of the DNA and fix it in place. The structure and binding mechanism as we visualize it appear to be fully consistent with evidence provided by physical-chemical studies of the protein in solution.     

Filamentous phage studied by magic-angle spinning NMR: resonance assignment and secondary structure of the coat protein in Pf1

Assignments are presented for resonances in the magic-angle spinning solid-state NMR spectra of the major coat protein subunit of the filamentous bacteriophage Pf1. NMR spectra were collected on uniformly 13C and 15N isotopically enriched, polyethylene glycol precipitated samples of fully infectious and hydrated phage. Site-specific assignments were achieved for 231 of the 251 labeled atoms (92%) of the 46-residue-long coat protein, including 136 of the 138 backbone atoms, by means of two- and three-dimensional 15N and 13C correlation experiments. A single chemical shift was observed for the vast majority of atoms, suggesting a single conformation for the 7300 subunits in the 36 MDa virion in its high-temperature form. On the other hand, multiple chemical shifts were observed for the Calpha, Cbeta, and Cgamma atoms of T5 in the helix terminus and the Calpha and Cbeta atoms of M42 in the DNA interaction domain. The chemical shifts of the backbone atoms indicate that the coat protein conformation involves a 40-residue continuous alpha-helix extending from residue 6 to the C-terminus.     

NMR and protein dynamics

NMR techniques can give insight into a wide variety of motional events that occur in proteins over a range of timescales. In the first section of this article an overview of the results of dynamics studies, using NMR methods, on both small globular and larger multi-domain proteins is presented including the findings from investigations of non-native partly folded states. The second section of the article then concentrates on two topics where NMR can give residue specific quantitative data, namely coupling constant measurements and relaxation studies, including comparisons of these NMR data with results from crystallographic studies and theoretical molecular dynamics simulations. Finally the possible functional significance of the experimentally observed motions in proteins is discussed. © 1996 John Wiley & Sons, Inc.     

Liquid crystal properties of a self-assembling viral coat protein

Monomers of the fd bacteriophage major coat protein, g8p, interact to form a helical bent-core structure surrounding the viral deoxyribonucleic acid (DNA). It was hypothesised that g8p has mesophasic characteristics guiding viral coat assembly. Atomic force and scanning electron microscopy reveal that g8p monomers organise to form films that wrap around a central axis to form birefringent fibres. These fibres resemble the filament structure of bent-core thermotropic B7 liquid crystals. These results offer insight into the g8p self-assembly mechanism that results in the formation of new viral particles in vivo.     

Strategies for measurements of pseudocontact shifts in protein NMR spectroscopy.

Paramagnetic metal ions bound to proteins generate a dipolar field that can be accurately probed by pseudocontact shifts (PCS) displayed by the protein's nuclear spins. PCS are highly useful for determining the coordinates of individual spins in the molecule and for rapid structure determinations of entire protein-protein and protein-ligand complexes. However, PCS measurements require reliable resonance assignments for the molecule in its paramagnetic state and in a diamagnetic reference state. This article discusses different approaches for pairwise resonance assignments, with emphasis on a strategy which exploits chemical exchange between the two states.     

Assessing the stability of assembled filamentous phage coat protein P8

Assembly of viral protein coats is crucial to the protection of internal genetic cargo and is necessary for proper infection. Understanding the conditions for maintaining these supramolecular assemblies is of value for engineering-effective virus-based materials and related technologies. In this study, we examine the stability of the filamentous bacteriophage, fd-tet, in a variety of solvent and temperature conditions. On the basis of these results, we advise amenable reaction environments for modification of fd-tet. In particular, assessment of the temperature stability indicates that practical use of these viruses as reaction substrates is best performed at moderate temperatures, since loss in infectivity was found to occur within only 1-h incubation over 37°C. In addition, these findings reveal additional loss of infectivity after exposure to conditions near pH 4.5 which may be attributable to changes in the effective charge of the p8 major coat protein.     

Measuring protein concentrations by NMR spectroscopy

In applications of NMR to biological macromolecules in solution, the concentration of the NMR sample is an important parameter describing the sample and providing information for the selection and planning of experiments. Although concentrations can be measured directly by NMR spectroscopy, other methods are usually preferred to measure the concentration of macromolecules in NMR samples. The reasons are the difficulties in the correlation of the sample of interest with the signal intensity representing a known concentration. This correlation is usually obtained by adding to the sample a reference compound with known concentration and comparing the integral over resolved resonance lines of the molecules with known and unknown concentrations. For solutions of biological macromolecules it is very difficult to find a compound that does not interact with the macromolecules and has a resonance outside their spectral range. We introduce PULCON which is a method that correlates the absolute intensities of two spectra measured in different solution conditions. PULCON is easy to implement and apply on all NMR spectrometers; it does not need any special hardware or software. PULCON is very robust and at the same time delivers accurate concentrations of samples in the NMR tube. We demonstrate that PULCON has the potential to replace UV spectroscopy for concentration measurements of NMR samples.     

Random isotopolog libraries for protein perturbation studies. 13C NMR studies on lumazine protein of Photobacterium leiognathi

[graph: see text] Lumazine proteins of luminescent bacteria are paralogs of riboflavin synthase which are devoid of catalytic activity but bind the riboflavin synthase substrate, 6,7-dimethyl-8-ribityllumazine, with high affinity and are believed to serve as optical transponders for bioluminescence emission. Lumazine protein of Photobacterium leiognathi was expressed in a recombinant Escherichia coli host and was reconstituted with mixtures (random libraries) of 13C-labeled isotopologs of 6,7-dimethyl-8-ribityllumazine or riboflavin that had been prepared by biotransformation of [U-(13)C6]-, [1-(13)C1]-, [2-(13)C1]-, and [3-(13)C1]glucose. 13C NMR analysis of the protein/ligand complexes afforded the assignments of the 13C NMR chemical shifts for all carbon atoms of the protein-bound ligands by isotopolog abundance editing. The carbon atoms of the ribityl groups of both ligands studied were shifted up to 6 ppm upon binding to the protein. Chemical shift modulation of the side chain and chromophore carbon atoms due to protein/ligand interaction is discussed on the basis of the sequence similarity between lumazine protein and riboflavin synthase.     

Screening of protein kinases by ATP-STD NMR spectroscopy

ATP-STD NMR takes advantage of Mg2+ binding to ATP to adjust the ATP affinity for protein kinases permitting a wide range of Ki's to be determined for ATP competitive ligands. Substituting Mn2+ for Mg2+ creates a paramagnetic probe (MnATP) from which the proximity of non-ATP competitive ligands can be inferred. Internal standards and references are used to reduce false positives due to protein or compound degradation. Use of the natural ATP ligand confers active site-specificity that is not available a priori from other ligand binding experiments.     

Automated protein structure determination from NMR spectra

Fully automated structure determination of proteins in solution (FLYA) yields, without human intervention, three-dimensional protein structures starting from a set of multidimensional NMR spectra. Integrating existing and new software, automated peak picking over all spectra is followed by peak list filtering, the generation of an ensemble of initial chemical shift assignments, the determination of consensus chemical shift assignments for all (1)H, (13)C, and (15)N nuclei, the assignment of NOESY cross-peaks, the generation of distance restraints, and the calculation of the three-dimensional structure by torsion angle dynamics. The resulting, preliminary structure serves as additional input to the second stage of the procedure, in which a new ensemble of chemical shift assignments and a refined structure are calculated. The three-dimensional structures of three 12-16 kDa proteins computed with the FLYA algorithm coincided closely with the conventionally determined structures. Deviations were below 0.95 A for the backbone atom positions, excluding the flexible chain termini. 96-97% of all backbone and side-chain chemical shifts in the structured regions were assigned to the correct residues. The purely computational FLYA method is suitable for substituting all manual spectra analysis and thus overcomes a main efficiency limitation of the NMR method for protein structure determination.     

Dipolar waves as NMR maps of protein structure

The anisotropy of nuclear spin interactions results in a unique mapping of structure to the resonance frequencies and split tings observed in NMR spectra, however, the determination of molecular structure from experimentally measured spectral parameters is complicated by angular ambiguities resulting from the symmetry properties of dipole-dipole and chemical shift interactions. This issue can be addressed through the periodicity inherent in secondary structure elements, which can be used as an index of topology. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels to map protein structure in NMR spectra of both highly and weakly aligned samples. Dipolar waves describe the periodic wavelike variations of the magnitudes of the static heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings (RDCs), in solution NMR spectra. The corresponding properties of the RDCs in solution NMR spectra of weakly aligned helices represent a convergence of solid-state and solution NMR approaches to structure determination.     

Redirecting the coat protein of a spherical virus to assemble into tubular nanostructures

We take advantage of the self-assembly properties of the coat protein of a spherical virus to form uniform tubular nanostructures. Critical to redirecting assembly are the weak interprotein association energy inherent to virus assembly and the relatively rigid nature of the double-stranded DNA scaffold at their core.     

The study of Turnip Mosaic virus coat protein by surface enhanced Raman spectroscopy

Using Turnip Mosaic virus (TuMV) coat protein as material, the secondary structure has been studied by both normal Raman spectroscopy (NRS) and surface enhanced Raman spectroscopy (SERS). The NRS of TuMV coat protein under certain conditions showed the α-helix, β-sheet and random coil structure. The CSSC comformations are trans—gauche—gauche and gauche—gauche—gauche. The SERS spectrum of TuMV coat protein under certain conditions reveals the α-helix structure. By studying SERS at different adsorbing times, we have observed the amide III vibration of α-helix, β-sheet and random coil structure. The CSSC conformations drawn from the SERS spectra are trans—gauche—gauche and trans—gauche—trans. Besides the amide I, amide III and CSSC bands, the CαCN band, aromatic amino acid bands and some other bands can also be seen in the SERS spectra.     

Comparative study of structure and properties of nucleoproteides synthesized using plant virus coat protein

The self-assembly of virus-like artificial particles from the coat protein of a helical virus (potato virus X) and nucleic acids (RNA and DNA) is studied. The structure and properties of the particles are investigated by transmission electron microscopy, atomic force microscopy, and enzymatic analysis.     

Efficient modeling of symmetric protein aggregates from NMR data

An efficient approach to determine the structures of symmetric protein aggregates from liquid and solid-state NMR data is presented. Any symmetry can be used (cyclic or dihedral point symmetries, helical symmetries, crystallographic symmetries). Because the starting point is the random structure of the monomer, the knowledge of the 3D structure of the monomer is not required.