Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization.

An experimental strategy has been developed to increase the efficiency of dynamic nuclear polarization (DNP) in solid-state NMR studies. The method makes assignments simpler, faster, and more reliable via sequential correlations of both side-chain and Cα resonances. The approach is particularly suited to complex biomolecules and systems with significant chemical-shift degeneracy. It was designed to overcome the spectral congestion and line broadening that occur due to sample freezing at the cryogenic temperatures required for DNP. Nonuniform sampling (NUS) is incorporated to achieve time-efficient collection of multidimensional data. Additionally, fast (25 kHz) magic-angle spinning (MAS) provides optimal sensitivity and resolution. Data collected in <1 wk produced a virtually complete de novo assignment of the coat protein of Pf1 virus. The peak positions and linewidths for samples near 100 K are perturbed relative to those near 273 K. These temperature-induced perturbations are strongly correlated with hydration surfaces.

Polyelectrolyte properties of filamentous biopolymers and their consequences in biological fluids.

Anionic polyelectrolyte filaments are common in biological cells. DNA, RNA, the cytoskeletal filaments F-actin, microtubules, and intermediate filaments, and polysaccharides such as hyaluronan that form the pericellular matrix all have large net negative charge densities distributed over their surfaces. Several filamentous viruses with diameters and stiffnesses similar to those of cytoskeletal polymers also have similar negative charge densities. Extracellular protein filaments such collagen, fibrin and elastin, in contrast, have notably smaller charge densities and do not behave as highly charged polyelectrolytes in solution. This review summarizes data that demonstrate generic counterion-mediated effects on four structurally unrelated biopolymers of similar charge density: F-actin, vimentin, Pf1 virus, and DNA, and explores the possible biological and pathophysiological consequences of the polyelectrolyte properties of biological filaments.

Pf1 bacteriophage hydration by magic angle spinning solid-state NMR.

High resolution two- and three-dimensional heteronuclear correlation spectroscopy ((1)H-(13)C, (1)H-(15)N, and (1)H-(13)C-(13)C HETCOR) has provided a detailed characterization of the internal and external hydration water of the Pf1 virion. This long and slender virion (2000 nm × 7 nm) contains highly stretched DNA within a capsid of small protein subunits, each only 46 amino acid residues. HETCOR cross-peaks have been unambiguously assigned to 25 amino acids, including most external residues 1-21 as well as residues 39-40 and 43-46 deep inside the virion. In addition, the deoxyribose rings of the DNA near the virion axis are in contact with water. The sets of cross-peaks to the DNA and to all 25 amino acid residues were from the same hydration water (1)H resonance; some of the assigned residues do not have exchangeable side-chain protons. A mapping of the contacts onto structural models indicates the presence of water "tunnels" through a highly hydrophobic region of the capsid. The present results significantly extend and modify results from a lower resolution study, and yield a comprehensive hydration surface map of Pf1. In addition, the internal water could be distinguished from external hydration water by means of paramagnetic relaxation enhancement. The internal water population may serve as a conveniently localized magnetization reservoir for structural studies.

Partial alignment and measurement of residual dipolar couplings of proteins under high hydrostatic pressure.

High-pressure NMR spectroscopy has emerged as a complementary approach for investigating various structural and thermodynamic properties of macromolecules. Noticeably absent from the array of experimental restraints that have been employed to characterize protein structures at high hydrostatic pressure is the residual dipolar coupling, which requires the partial alignment of the macromolecule of interest. Here we examine five alignment media that are commonly used at ambient pressure for this purpose. We find that the spontaneous alignment of Pf1 phage, d(GpG) and a C12E5/n-hexnanol mixture in a magnetic field is preserved under high hydrostatic pressure. However, DMPC/DHPC bicelles and collagen gel are found to be unsuitable. Evidence is presented to demonstrate that pressure-induced structural changes can be identified using the residual dipolar coupling.

Very large residual dipolar couplings from deuterated ubiquitin.

Main-chain (1)H(N)-(15)N residual dipolar couplings (RDCs) ranging from approximately -200 to 200 Hz have been measured for ubiquitin under strong alignment conditions in Pf1 phage. This represents a ten-fold increase in the degree of alignment over the typical weakly aligned samples. The measurements are made possible by extensive proton-dilution of the sample, achieved by deuteration of the protein with partial back-substitution of labile protons from 25 % H(2)O / 75 % D(2)O buffer. The spectral quality is further improved by application of deuterium decoupling. Since standard experiments using fixed-delay INEPT elements cannot accommodate a broad range of couplings, the measurements were conducted using J-resolved and J-modulated versions of the HSQC and TROSY sequences. Due to unusually large variations in dipolar couplings, the trosy (sharp) and anti-trosy (broad) signals are often found to be interchanged in the TROSY spectra. To distinguish between the two, we have relied on their respective (15)N linewidths. This strategy ultimately allowed us to determine the signs of RDCs. The fitting of the measured RDC values to the crystallographic coordinates of ubiquitin yields the quality factor Q = 0.16, which confirms the perturbation-free character of the Pf1 alignment. Our results demonstrate that RDC data can be successfully acquired not only in dilute liquid crystals, but also in more concentrated ones. As a general rule, the increase in liquid crystal concentration improves the stability of alignment media and makes them more tolerant to variations in sample conditions. The technical ability to measure RDCs under moderately strong alignment conditions may open the door for development of alternative alignment media, including new types of media that mimic biologically relevant systems.

Intersubunit hydrophobic interactions in Pf1 filamentous phage.

Magic angle spinning solid-state NMR has been used to study the structural changes in the Pf1 filamentous bacteriophage, which occur near 10 °C. Comparisons of NMR spectra recorded above and below 10 °C reveal reversible perturbations in many NMR chemical shifts, most of which are assigned to atoms of hydrophobic side chains of the 46-residue subunit. The changes mainly involve groups located in patches on the interfaces between neighboring capsid subunits. The observations show that the transition adjusts the hydrophobic interfaces between fairly rigid subunits. The low temperature form has been generally more amenable to structure determination; spin diffusion experiments on this form revealed unambiguous contacts between side chains of neighboring subunits. These contacts are important constraints for structure modeling.

Chemical shifts for the unusual DNA structure in Pf1 bacteriophage from dynamic-nuclear-polarization-enhanced solid-state NMR spectroscopy.

Solid-state NMR spectra, including dynamic nuclear polarization enhanced 400 MHz spectra acquired at 100 K, as well as non-DNP spectra at a variety of field strengths and at temperatures in the range 213-243 K, have allowed the assignment of the (13)C and (15)N resonances of the unusual DNA structure in the Pf1 virion. The (13)C chemical shifts of C3' and C5', considered to be key reporters of deoxyribose conformation, fall near or beyond the edges of their respective ranges in available databases. The (13)C and (15)N chemical shifts of the DNA bases have above-average values for AC4, AC5, CC5, TC2, and TC5, and below average values for AC8, GC8, and GN2, pointing to an absence of Watson-Crick hydrogen bonding, yet the presence of some type of aromatic ring interaction. Crosspeaks between Tyr40 of the coat protein and several DNA atoms suggest that Tyr40 is involved in this ring interaction. In addition, these crosspeak resonances and several deoxyribose resonances are multiply split, presumably through the effects of ordered but differing interactions between capsid protein subunits and each type of nucleotide in each of the two DNA strands. Overall, these observations characterize and support the DNA model proposed by Liu and Day and refined by Tsuboi et al., which calls for the most highly stretched and twisted naturally occurring DNA yet encountered.

Consensus structure of Pf1 filamentous bacteriophage from X-ray fibre diffraction and solid-state NMR.

Filamentous bacteriophages (filamentous bacterial viruses or Inovirus) are simple and well-characterised macromolecular assemblies that are widely used in molecular biology and biophysics, both as paradigms for studying basic biological questions and as practical tools in areas as diverse as immunology and solid-state physics. The strains fd, M13 and f1 are virtually identical filamentous phages that infect bacteria expressing F-pili, and are sometimes grouped as the Ff phages. For historical reasons fd has often been used for structural studies, but M13 and f1 are more often used for biological experiments. Many other strains have been identified that are genetically quite distinct from Ff and yet have a similar molecular structure and life cycle. One of these, Pf1, gives the highest resolution X-ray fibre diffraction patterns known for filamentous bacteriophage. These diffraction patterns have been used in the past to derive a molecular model for the structure of the phage. Solid-state NMR experiments have been used in separate studies to derive a significantly different model of Pf1. Here we combine previously published X-ray fibre diffraction data and solid-state NMR data to give a consensus structure model for Pf1 filamentous bacteriophage, and we discuss the implications of this model for assembly of the phage at the bacterial membrane.

Conformational dynamics of an intact virus: order parameters for the coat protein of Pf1 bacteriophage.

This study has examined the atomic-level dynamics of the protein in the capsid of filamentous phage Pf1. This capsid consists of approximately 7,300 small subunits of only 46 aa in a helical array around a highly extended, circular single-stranded DNA molecule of 7,349 nt. Measurements were made of site-specific, solid-state NMR order parameters, S, the values which are dimensionless quantities between 0 (mobile) and 1 (static) that characterize the amplitudes of molecular bond angular motions that are faster than microseconds. It was found that the protein subunit backbone is very static, and of particular interest, it appears to be static at residues glycine 15 and glutamine 16 where it had been previously thought to be mobile. In contrast to the backbone, several side chains display large-amplitude angular motions. Side chains on the virion exterior that interact with solvent are highly mobile, but surprisingly, the side chains of residues arginine 44 and lysine 45 near the DNA deep in the interior of the virion are also highly mobile. The large-amplitude dynamic motion of these positively charged side chains in their interactions with the DNA were not previously expected. The results reveal a highly dynamic aspect of a DNA-protein interface within a virus.

A resonance assignment method for oriented-sample solid-state NMR of proteins.

A general sequential assignment strategy for uniformly (15)N-labeled uniaxially aligned membrane proteins is proposed. Mismatched Hartmann-Hahn magnetization transfer is employed to establish proton-mediated correlations among the neighboring (15)N backbone spins. Magnetically aligned Pf1 phage coat protein was used to illustrate the method. Exchanged and nonexchanged separated local field spectra were acquired and overlaid to distinguish the cross-peaks from the main peaks. Most of the original assignments from the literature were confirmed without selectively labeled samples. This method is applicable to proteins with arbitrary topology and will find use in assigning solid-state NMR spectra of oriented membrane proteins for their subsequent structure determination.

1H detection of heteronuclear dipolar oscillations with water suppression in single crystal peptide and oriented protein samples.

Oriented sample solid-state NMR is a complementary approach to protein structure determination with the distinct advantage that it can be applied to supramolecular assemblies, such as viruses and membrane proteins, under near-native conditions, which generally include high levels of hydration as found in living systems. Thus, in order to perform 1H detected versions of multi-dimensional experiments water suppression techniques must be integrated into the pulse sequences. For example, 1H-windowed detection of 1H-15N dipolar couplings enable multi-dimensional NMR experiments to be performed. Here we show that the addition of a solvent suppression pulse during the z-filter interval greatly improves the sensitivity of the experiments by suppressing the 1H signals from water present. This is demonstrated here with a crystal sample submerged in water and then extended to proteins. The combination of solvent-suppressed 1H detected PISEMO and the use of a strip shield-solenoid coil probe configuration provides a two-fold sensitivity enhancement in both the crystal sample and Pf1 coat protein sample compared to the 15N direct detection method. Here we also examine protein NMR line-widths and sensitivity enhancements in the context of window detected separated local field experiments for protein samples.

De novo NMR pulse sequence design using Monte-Carlo optimization techniques.

Separated Local Field (SLF) experiments have been routinely used for measuring 1H-15N heteronuclear dipolar couplings in oriented-sample solid-state NMR for structure determination of proteins. In the on-going pursuit of designing better-performing SLF pulse sequences (e.g. by increasing the number of subdwells, and varying the rf amplitudes and phases), analytical treatment of the relevant average Hamiltonian terms may become cumbersome and/or nearly impossible. Numerical simulations of NMR experiments using GPU processors can be employed to rapidly calculate spectra for moderately sized spin systems, which permit an efficient numeric optimization of pulse sequences by the Monte Carlo Simulated Annealing protocol. In this work, a computational strategy was developed to find the optimal phases and timings that substantially improve the 1H-15N dipolar linewidths over a broad range of dipolar couplings as compared to SAMPI4. More than 100 pulse sequences were developed de novo and tested on an N-acetyl Leucine crystal. Seventeen distinct pulse sequences were shown to produce sharper mean linewidths than SAMPI4. Overall, these pulse sequences have more variable parameters (involving non-quadrature phases) and do not involve symmetry between the odd and even dwells, which would likely preclude their rigorous analytical treatment. The top performing pulse sequence, termed ROULETTE-1, has 18% sharper mean linewidths than SAMPI4 when run on an N-acetyl Leucine crystal. This sequence was also shown to be robust over a broad range of 1H carrier frequencies and various crystal orientations. The performance of such an optimized pulse sequence was also illustrated on 15N Leucine-labeled Pf1 coat protein reconstituted in magnetically aligned bicelles. For the optimized pulse sequence the mean peak width was 14% sharper than SAMPI4, which in turn yielded a better signal to noise ratio, 20:1 vs. 17:1. This method is potentially extendable to de novo development of a variety of NMR experiments.

Optimal excitation of (23)Na nuclear spins in the presence of residual quadrupolar coupling and quadrupolar relaxation.

Optimal control theory is applied for designing pulse sequences to optimally excite a spin-3/2 system with residual quadrupolar coupling in the presence of quadrupolar relaxation. A homogeneous form of the master equation is constructed to simulate the dynamics of the spin system, and a general optimization procedure with a homogeneous form of the equation of motion is described. The optimized pulses are tested with (23)Na NMR, and their performance is compared with that of pulses optimized in the absence of relaxation.

Effects of deuteration on solid-state NMR spectra of single peptide crystals and oriented protein samples.

Extensive deuteration can be used to simplify NMR spectra by "diluting" and minimizing the effects of the abundant 1H nuclei. In solution-state NMR and magic angle spinning solid-state NMR of proteins, perdeuteration has been widely applied and its effects are well understood. Oriented sample solid-state NMR of proteins, however, is at a much earlier stage of development. In spite of the promise of the approach, the effects of sample deuteration are largely unknown. Here we map out the effects of perdeuteration on solid-state NMR spectra of aligned samples by closely examining differences in results obtained on fully protiated and perdeuterated samples, where all of the carbon sites have either 1H or 2H bonded to them, respectively. The 2H and 15N labeled samples are back-exchanged in 1H2O solution so that the amide 15N sites have a bonded 1H. Line-widths in the 15N chemical shift, 1H chemical shift, and 1H-15N dipolar coupling frequency dimensions were compared for peptide single crystals as well as membrane proteins aligned along with the phospholipids in bilayers with their normals perpendicular to the direction of the magnetic field. Remarkably, line-width differences were not found between fully protiated and perdeuterated samples. However, in the absence of effective 1H-1H homonuclear decoupling, the line-widths in the 1H-15N heteronuclear dipolar coupling frequency dimension were greatly narrowed in the perdeuterated samples. In proton-driven spin diffusion (PDSD) experiments, no effects of perdeuteration were observed. In contrast, in mismatched Hartmann-Hahn experiments, perdeuteration enhances cross-peak intensities by allowing more efficient spin-exchange with less polarization transfer back to the carbon-bound 1H. Here we show that in oriented sample solid-state NMR, the effects of perdeuteration can be exploited in experiments where 1H-1H homonuclear decoupling cannot be applied. These data also provide evidence for the possible contribution of direct 15N-15N dilute-spin mixing mechanism in proton-driven spin diffusion experiments.

Liquid crystalline phase of G-tetrad DNA for NMR study of detergent-solubilized proteins.

The liquid crystalline phase consisting of the potassium salt of the dinucleotide d(GpG) is compatible with detergents commonly used for solubilizing membrane proteins, including dodecylphosphocholine, the lysolipid 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, and small bicelles consisting of dihexanoyl phosphatidylcholine and dimyristoyl phosphatidylcholine. The chiral nematic liquid crystalline phase of d(GpG) consists of long columns of stacked G-tetrad structures and carry a net negative charge. For water-soluble systems, the protein alignment induced by d(GpG) is very similar to that observed for liquid crystalline Pf1 bacteriophage, but of opposite sign. Alignment of the detergent-solubilized fusion domain of hemagglutinin is demonstrated to be homogeneous and stable, resulting in high quality NMR spectra suitable for the measurement of residual dipolar couplings.

Solid-state NMR spectroscopy of a membrane protein in biphenyl phospholipid bicelles with the bilayer normal parallel to the magnetic field.

Bicelles composed of the long-chain biphenyl phospholipid TBBPC (1-tetradecanoyl-2-(4-(4-biphenyl)butanoyl)-sn-glycero-3-PC) and the short-chain phospholipid DHPC align with their bilayer normals parallel to the direction of the magnetic field. In contrast, in typical bicelles the long-chain phospholipid is DMPC or DPPC, and the bilayers align with their normals perpendicular to the field. Samples of the membrane-bound form of the major coat protein of Pf1 bacteriophage in TBBPC bicelles are stable for several months, align magnetically over a wide range of temperatures, and yield well-resolved solid-state NMR spectra similar to those obtained from samples aligned mechanically on glass plates or in DMPC bicelle samples "flipped" with lanthanide ions so that their bilayer normals are parallel to the field. The order parameter of the TBBPC bicelle sample decreases from approximately 0.9 to 0.8 upon increasing the temperature from 20 degrees C to 60 degrees C. Since the frequency spans of the chemical shift and dipolar coupling interactions are twice as large as those obtained from proteins in DMPC bicelles without lanthanide ions, TBBPC bicelles provide an opportunity for structural studies with higher spectral resolution of the metal-binding membrane proteins without the risk of chemical or spectroscopic interference from the added lanthanide ions. In addition, the large temperature range of these samples is advantageous for the studies of membrane proteins that are unstable at elevated temperatures and for experiments requiring measurements as a function of temperature.

Automated assignment of NMR spectra of macroscopically oriented proteins using simulated annealing.

An automated technique for the sequential assignment of NMR backbone resonances of oriented protein samples has been developed and tested based on 15N-15N homonuclear exchange and spin-exchanged separated local-field spectra. By treating the experimental spectral intensity as a pseudopotential, the Monte-Carlo Simulated Annealing algorithm has been employed to seek lowest-energy assignment solutions over a large sampling space where direct enumeration would be unfeasible. The determined sequential assignments have been scored based on the positions of the crosspeaks resulting from the possible orders for the main peaks. This approach is versatile in terms of the parameters that can be specified to achieve the best-fit result. At a minimum the algorithm requires a continuous segment of the main-peak chemical shifts obtained from a uniformly labeled sample and a spin-exchanged experimental spectrum represented as a 2D matrix array. With selective labeling experiments, groups of chemical shifts corresponding to specific locations in the protein backbone can be fixed, thereby decreasing the sampling space. The output from the program consists of a list of top-score main peak assignments, which can be subjected to further scoring criteria until a consensus solution is found. The algorithm has first been tested on a synthetic spectrum with randomly generated chemical shifts and dipolar couplings for the main peaks. The original assignments have been successfully recovered for as many as 100 main peaks when residue-type information was used even in the presence of substantial spectral peak overlap. The algorithm was then applied to assigning two sets of experimental spectra to recover and confirm the previously established assignments in an automated fashion. For the 20-residue transmembrane domain of Pf1 coat protein reconstituted in magnetically aligned bicelles, the original assignment by Park et al. (2010) was recovered by the automated algorithm with additional input from 5 selectively labeled amino acid spectra. The second case considered was the 46 residue Pf1 bacteriophage from Thiriot et al. (2005) and Knox et al. (2010), of which 38 residues were fit. Automated fitting resulted in several possible assignments but not exactly the original assignment. By using a post-fitting filtering procedure based on the number of missed cross peaks and Pf1 helical structure, a consensus spectroscopic assignment is proposed covering 84% of the original assignment. While the automated assignment works best in spectra with well-resolved crosspeaks, it also tolerates substantial spectral crowding to yield reasonable assignments in the cases where ambiguity and degeneracy of possible assignment solutions are inevitable.

Quadrupole sensitive pulse for signal filtering.

A longstanding problem in quadrupolar NMR of semi-solids is the selection of signals originating from ordered nuclei, i.e. those that experience a non-vanishing quadrupolar coupling. Established techniques, such as for example multiple-quantum filters are not adequate in situations when the radio frequency power is on the order of the quadrupolar coupling or the quadrupolar relaxation rates, such as may be the case on an MRI scanner, or in ex situ applications. In this manuscript we show a new method for the selective excitation of ordered spin-3/2 nuclei, which produces the desired results when the radio frequency power is approximately equal or smaller than quadrupolar frequency. Using a combination of simulations and experiments with (23)Na in NaCl solution, Pf1-solutions, and bovine patellar cartilage samples we further show how the value of the quadrupolar frequency and global features of a quadrupolar coupling distribution can be extracted from these experiments.

Recoupling of residual dipolar couplings in single-domain polymer-stabilized liquid crystals undergoing magic-angle spinning.

Measurement of dipolar couplings, chemical shift anisotropies, and quadrupole couplings in oriented media such as liquid crystals are of great importance for extraction of structural parameters in biological macromolecules. Here, we introduce a new technique, SAD-REDOR, that consists of recoupling heteronuclear dipolar couplings in molecules dissolved in a single-domain liquid crystal or other oriented medium through the combined use of magic-angle spinning and rotor-synchronized radiofrequency pulses. This application of the REDOR pulse sequence to oriented media offers several advantages such as selectivity over the type of coupling recovered and tunable scaling of the interaction. The effectiveness of the technique is demonstrated both theoretically and experimentally, using the recently developed polyacrylamide-stabilized Pf1 phage medium and 15N-labeled benzamide as the aligned molecule.

Capture and manipulation of magnetically aligned Pf1 with an aqueous polymer gel.

The magnetic alignment of the Pseudomonas bacteriophage Pf1 is captured indefinitely in a gel of the aqueous triblock copolymer Pluronic F-127. In addition to preserving high-resolution liquids NMR spectra for dissolved solutes, the gel prevents the reorientation of the phage allowing mechanical manipulation of the angle between the axis of the phage alignment and the static magnetic field. The residual 2H quadrupolar couplings for several solutes dissolved in this material as a function of the angle Theta between the non-spinning sample tube and the static magnetic field are consistent with the value of P(2)(cosTheta)=(3cos(2)Theta-1)/2. The variable-angle correlation spectrum for these solutes is shown to separate residual quadrupolar effects from isotropic chemical shifts. Finally, the compatibility of Pluronic F-127 with NMR studies of aqueous biological macromolecules is demonstrated in a measurement of residual dipolar couplings in an 15N-labeled nucleic acid.