Magic angle spinning carbon-13 NMR of tobacco mosaic virus. An application of the high-resolution solid-state NMR spectroscopy to very large biological systems.

Magic angle spinning 13C NMR was used to study tobacco mosaic virus (TMV) in solution. Well-resolved 13C NMR spectra were obtained, in which several carbon resonances of amino acids of the TMV coat protein subunits that are not observable by conventional high-resolution NMR spectroscopy can be designed. RNA resonance were absent, however, in the magic angle spinning 13C NMR spectra. Since three different binding sites are available for each nucleotide of the RNA, this is probably due to a line broadening caused by distributions of isotropic chemical shift values. In 13C-enriched TM 13C-13C dipolar interactions also gave rise to line broadening. By suitable pulse techniques that discriminate carbon resonances on the basis of their T1 and T1 rho values, it was possible to select particular groups of carbon nuclei with characteristic motional properties. Magic angle spinning 13C NMR spectra obtained with these pulse techniques are extremely well resolved.     

A solid-state 31P-NMR investigation of the allosteric transition in glycogen phosphorylase b.

The catalytic role of the cofactor phosphate moiety at the active site of glycogen phosphorylase has been the subject of many investigations including solution-state high-resolution 31P-NMR studies. In this study the pyridoxal phosphate moiety in both the inactive and active forms of microcrystalline phosphorylase b has been investigated by high-resolution 31P magic-angle spinning NMR. The symmetry of the shielding tensor in model compounds at varying degrees of ionization is investigated and the results indicate a marked difference between the dianionic and monoanionic model compounds. Consequently the observed similarity in the principal tensor components describing the shielding tensor of the phosphorus nuclei present at the active site of both the R- and T-state conformations suggests that there is no change in ionization site upon activation in contrast to suggestions based upon isotropic shifts. Since previous relaxation measurements have pointed to the need to consider motional influences in such systems, several plausible models are considered. Subject to the assumption of congruency between the principal axis system describing the shielding interaction and molecular frame determined by the molecular symmetry axes, we conclude that the phosphate cofactor is dianionic in both forms.     

Sodium-23 NMR relaxation times in nucleated red blood cells and suspensions of nuclei.

The relaxation behavior of intracellular 23Na in suspensions of chicken erythrocytes and of their nuclei was investigated. The transverse magnetization was found to decay biexponentially. The average relaxation rates for the nucleated chicken erythrocytes are considerably shorter than the average relaxation rates obtained for dog and human nonnucleated red blood cells. Of particular significance is the twofold decrease in the short component of T2. Calculations based on the measured 23Na NMR relaxation rates in suspensions of nuclei indicate that most of the difference between the relaxation rates in the mammalian as compared to the chicken erythrocytes, can be accounted for by the contribution of the nuclei in the latter.     

High-resolution proton and carbon-13 NMR of membranes: why sonicate?

We have obtained high-field (11.7-T) proton and carbon-13 Fourier transform (FT) nuclear magnetic resonance (NMR) spectra of egg lecithin and egg lecithin-cholesterol (1:1) multibilayers, using "magic-angle" sample spinning (MASS) techniques, and sonicated egg lecithin and egg lecithin-cholesterol (1:1) vesicles, using conventional FT NMR methods. Resolution of the proton and carbon-13 MASS NMR spectra of the pure egg lecithin samples is essentially identical with that of sonicated samples, but spectra of the unsonicated lipid, using MASS, can be obtained very much faster than with the more dilute, sonicated systems. With the 1:1 lecithin-cholesterol systems, proton MASS NMR spectra are virtually identical with conventional FT spectra of sonicated samples, while with 13C NMR, we demonstrate that most 13C nuclei in the cholesterol moiety can be monitored, even though these same nuclei are essentially invisible, i.e., are severely broadened, in the corresponding sonicated systems. In addition, 13C MASS NMR, spectra can again be recorded much faster than with sonicated samples, due to concentration effects. Taken together, these results strongly suggest there will seldom be need in the future to resort to ultrasonic disruption of lipid bilayer membranes in order to obtain high-resolution proton or carbon-13 NMR spectra.     

High-resolution, high-pressure NMR studies of proteins.

Advanced high-resolution NMR spectroscopy, including two-dimensional NMR techniques, combined with high pressure capability, represents a powerful new tool in the study of proteins. This contribution is organized in the following way. First, the specialized instrumentation needed for high-pressure NMR experiments is discussed, with specific emphasis on the design features and performance characteristics of a high-sensitivity, high-resolution, variable-temperature NMR probe operating at 500 MHz and at pressures of up to 500 MPa. An overview of several recent studies using 1D and 2D high-resolution, high-pressure NMR spectroscopy to investigate the pressure-induced reversible unfolding and pressure-assisted cold denaturation of lysozyme, ribonuclease A, and ubiquitin is presented. Specifically, the relationship between the residual secondary structure of pressure-assisted, cold-denatured states and the structure of early folding intermediates is discussed.     

High-resolution cytometry of FISH dots in interphase cell nuclei.

BACKGROUND: Flow cytometry (FCM) and laser scanning cytometry (LSCM) provide indispensable tools for measuring large number of cells with low resolution. Confocal microscopy, on the other hand, is used for measuring small number of cells with high resolution. In this paper, we present a reasonable compromise between the two extremes. METHODS: We have developed a completely automated, high-resolution system (high-resolution cytometer, HRCM) capable of analyzing microscope slides with FISH-stained interphase nuclei in two dimensions as well as in three dimensions using a fully motorized epi-fluorescence microscope and a cooled digital CCD camera fully controlled by a high-performance computer which performs both acquisition and related on-line image analysis. The images of different dyes are acquired sequentially using highly specific filters and superimposed in computer memory. For each nucleus and each hybridization dot, user-selected attributes (such as position, size, intensity, etc.) are computed off-line using another processor or computer connected with a network. RESULTS: Using HRCM, it is possible to analyze multi-color preparations including UV-excited dyes as well as repeatedly hybridized preparations reacquiring individual nuclei. The speed of the acquisition and analysis is about 50 nuclei per minute in two dimensions and 1 nucleus per minute in three dimensions, but depends on the density of nuclei on the slide; the precision of the lateral and axial measurements is approximately 100 nm. CONCLUSIONS: Thus, using overnight acquisition, quantities comparable to those of FCM or LSCM measurements can be analyzed with an accuracy comparable to confocal microscopy. HRCM is suitable for a number of clinical and scientific tasks: routine diagnostics, follow-up of therapy, studies of chromatin structure, and many other different aspects of cell research.     

Structure and metabolism of mammalian liver glycogen monitored by carbon-13 nuclear magnetic resonance

Natural-abundance 13C NMR signals from glycogen are observable in situ within the perfused livers of rats. The nuclear magnetic relaxation properties (T1, T2, eta + 1) of glycogen were measured for glycogen in situ and in vitro and were found to be identical. All of the carbon nuclei in glycogen contribute to the high-resolution NMR spectrum, in spite of glycogen's very large molecular weight. The metabolism of glycogen in situ in the perfused rat liver was followed by 13C NMR. Stimulation of the fed rat liver by physiological glucagon levels led to rapid glycogenolysis. Perfusion of the liver with [1-13C]glucose led to net glycolysis, with concomitant scrambling of the label from C1 to C6 due to triosephosphate isomerase activity.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignment of human guanylate kinase

Human guanylate kinase (hGMPK) is a critical enzyme that, in addition to phosphorylating its physiological substrate (d)GMP, catalyzes the second phosphorylation step in the conversion of anti-viral and anti-cancer nucleoside analogs to their corresponding active nucleoside analog triphosphates. Until now, a high-resolution structure of hGMPK is unavailable and thus, we studied free hGMPK by NMR and assigned the chemical shift resonances of backbone and side chain ¹H, ¹³C, and ¹⁵N nuclei as a first step towards the enzyme’s structural and mechanistic analysis with atomic resolution.     

STD-NMR: application to transient interactions between biomolecules—a quantitative approach

Saturation transfer difference NMR (STD NMR) spectroscopy is one of the most powerful NMR techniques for detection and characterization of transient (fast) receptor–ligand interactions in solution. By observing the signals of a small molecule (ligand) with spectroscopic properties suitable for high-resolution studies, irrespective of receptor size, STD NMR enables quantitative structural and affinity information to be obtained about the molecular recognition process under study. Approximately one decade after its introduction, the technique has reached maturity, and is highly robust and useful. The objective of this article is to review the current status of this powerful technique, with particular emphasis on quantitative applications, within the framework of the (bio-)chemistry of molecular recognition.     

A 31P NMR study of mitochondrial inorganic phosphate visibility: effects of Ca2+, Mn2+, and the pH gradient.

The effects of external pH, temperature, and Ca2+ and Mn2+ concentrations on the compartmentation and NMR visibility of inorganic phosphate (Pi) were studied in isolated rat liver mitochondria respiring on succinate and glutamate. Mitochondrial matrix Pi is totally visible by NMR at 8 degrees C and at low external concentrations of Pi. However, when the external Pi concentration is increased above 7 mM, the pH gradient decreases, the amount of matrix Pi increases, and the fraction not observed by NMR increases. Raising the temperature to 25 degrees C also decreases the pH gradient and the Pi fraction observed by NMR. At physiologically relevant concentrations, Ca2+ and Mn2+ do not seem to play a major role in matrix Pi NMR invisibility. For Ca2+ concentrations above 30 nmol/mg of protein, formation of insoluble complexes will cause loss of Pi signal intensity. For Mn2+ concentrations above 2 nmol/mg of protein, the Pi peak can be broadened sufficiently to preclude detection of a high-resolution signal. The results indicate that mitochondrial matrix Pi should be mostly observable up to 25 degrees C by high-resolution NMR. While the exact nature of the NMR-invisible phosphate in perfused or in vivo liver is yet to be determined, better success at detecting and resolving both Pi pools by NMR is indicated at high field, low temperature, and optimized pulsing conditions.     

The use of solid-state NMR and X-ray crystallography as complementary tools for studying molecular recognition

Solid-state NMR is rapidly becoming available as a routine technique for studying the structure of crystalline or noncrystalline solids. This technique has an advantage over crystallography in that single crystals are not necessary, but it has the disadvantage that the information obtained does not produce a direct picture of the molecule and its environment. On the other hand, solid-state NMR can be done on mixtures, and it gives information about phase distribution in a manner similar to that of X-ray powder pattern analysis.Crystallographic effects such as polymorphism, multiple molecules per asymmetric unit, disorder and salvation can frequently be detected using NMR. Sometimes molecular point group symmetry can also be deduced based on the number of independent nuclei that are detected. The NMR method is sensitive to changes in the electronic structure of a molecule as sensed by the nuclei, and the effects are measured as changes in the isotropic chemical shift of individual nuclei.In this paper, we will give examples of the combined use of X-ray crystallography and ¹³CP/MAS (cross polarization/magic angle spinning) NMR for studying hostguest materials and cocrystals. We have learned how to use NMR to tell us about keto/enol composition in the solid state, to detect the presence of trapped solvent molecules, to detect hydrogen-bond formation and to evaluate molecular conformation and unusual packing pattern effects. We will also present a brief background of the ¹³CP/MAS NMR technique and three case studies in which solid-state NMR and X-ray crystallography are used together to understand materials' structures and properties     

High-resolution electron cryomicroscopy of macromolecular assemblies

Electron cryomicroscopy is a high-resolution imaging technique that is particularly appropriate for the structural determination of large macromolecular assemblies, which are difficult to study by X-ray crystallography or NMR spectroscopy. For some biological molecules that form two-dimensional crystals, the application of electron cryomicroscopy and image reconstruction can help elucidate structures at atomic resolution. In instances where crystals cannot be formed, atomic-resolution information can be obtained by combining high-resolution structures of individual components determined by X-ray crystallography or NMR with image-derived reconstructions at moderate resolution. This can provide unique and crucial information on the mechanisms of these complexes. Finally, image reconstructions can be used to augment X-ray studies by providing initial models that facilitate phasing of crystals of large macromolecular machines such as ribosomes and viruses.     

NMR studies on fully hydrated membrane proteins, with emphasis on bacteriorhodopsin as a typical and prototype membrane protein

The 3D structures or dynamic feature of fully hydrated membrane proteins are very important at ambient temperature, in relation to understanding their biological activities, although their data, especially from the flexible portions such as surface regions, are unavailable from X-ray diffraction or cryoelectron microscope at low temperature. In contrast, high-resolution solid-state NMR spectroscopy has proved to be a very convenient alternative means to be able to reveal their dynamic structures. To clarify this problem, we describe here how we are able to reveal such structures and dynamic features, based on intrinsic probes from high-resolution solid-state NMR studies on bacteriorhodopsin (bR) as a typical membrane protein in 2D crystal, regenerated preparation in lipid bilayer and detergents. It turned out that their dynamic features are substantially altered upon their environments where bR is present. We further review NMR applications to study structure and dynamics of a variety of membrane proteins, including sensory rhodopsin, rhodopsin, photoreaction centers, diacylglycerol kinases, etc.     

NMR studies on fully hydrated membrane proteins, with emphasis on bacteriorhodopsin as a typical and prototype membrane protein

The 3D structures or dynamic feature of fully hydrated membrane proteins are very important at ambient temperature, in relation to understanding their biological activities, although their data, especially from the flexible portions such as surface regions, are unavailable from X-ray diffraction or cryoelectron microscope at low temperature. In contrast, high-resolution solid-state NMR spectroscopy has proved to be a very convenient alternative means to be able to reveal their dynamic structures. To clarify this problem, we describe here how we are able to reveal such structures and dynamic features, based on intrinsic probes from high-resolution solid-state NMR studies on bacteriorhodopsin (bR) as a typical membrane protein in 2D crystal, regenerated preparation in lipid bilayer and detergents. It turned out that their dynamic features are substantially altered upon their environments where bR is present. We further review NMR applications to study structure and dynamics of a variety of membrane proteins, including sensory rhodopsin, rhodopsin, photoreaction centers, diacylglycerol kinases, etc.     

Lipid molecular motion and enzyme activity in sarcoplasmic reticulum membrane.

In biochemically active sarcoplasmic reticulum vesicles (SR) the physical state of the membrane lipids was studied by high angle x-ray diffraction and proton nuclear magnetic resonance (NMR) at 220 MHz, and related to thermal effects observed in SR functional parameters. It is shown by high angle x-ray diffraction that even at temperatures as low as 1 degree C nearly all the SR lipid hydrocarbon chains are in a disordered conformation and only a very small part (less than 3%) are in rigid crystalline order. Consistent with this observation, the NMR data indicate that the majority of SR phospholipid molecules are in a state of restricted anisotropic motion having no apparent crystalline order at temperatures as low as 5 degrees C. At this temperature most of the resonance signal is contained in a broad feature-less line of 700-Hz half-width. On the other hand, as the temperature is raised, high-resolution NMR signals, representing groups with highly isotropic motion, begin to grow in intensity. It is estimated that by 35 degrees C 90-100% of the phosphatidylcholine N-methyl protons and 35% of the hydrocarbon-chain protons give high-resolution signals. Concurrent studies on functional parameters reveal thermal effects giving rise to nonlinear Arrhenius plots for the rates of calcium transport and calcium activated ATPase. The thermal effects observed on functional parameters and on the character of phospholipid molecular motion exhibit a parallel behavior, suggesting a relationship between enzyme activity and the physical state of the membrane lipids.     

Conformational fluctuations of proteins revealed by variable pressure NMR

With the high-resolution variable-pressure NMR spectroscopy, one can study conformational fluctuations of proteins in a much wider conformational space than hitherto explored by NMR and other spectroscopic techniques. This is because a protein in solution generally exists as a dynamic mixture of conformers mutually differing in partial molar volume, and pressure can select the population of a conformer according to its relative volume. In this review, we describe how variable-pressure NMR can be used to probe conformational fluctuations of proteins in a wide conformational space from the folded to the fully unfolded structures, with actual examples. Furthermore, the newly emerging technique "NMR snapshots" expresses amply fluctuating protein structures as changes in atomic coordinates. Finally, the concept of conformational fluctuation is extended to include intermolecular association leading to amyloidosis.     

The structural and topological analysis of membrane-associated polypeptides by oriented solid-state NMR spectroscopy: established concepts and novel developments

Solid-state NMR spectroscopy is a powerful technique for the investigation of membrane-associated peptides and proteins as well as their interactions with lipids, and a variety of conceptually different approaches have been developed for their study. The technique is unique in allowing for the high-resolution investigation of liquid disordered lipid bilayers representing well the characteristics of natural membranes. Whereas magic angle solid-state NMR spectroscopy follows approaches that are related to those developed for solution NMR spectroscopy the use of static uniaxially oriented samples results in angular constraints which also provide information for the detailed analysis of polypeptide structures. This review introduces this latter concept theoretically and provides a number of examples. Furthermore, ongoing developments combining solid-state NMR spectroscopy with information from solution NMR spectroscopy and molecular modelling as well as exploratory studies using dynamic nuclear polarization solid-state NMR will be presented.     

X-ray and 13C solid-state NMR studies of N-benzoyl-phenylalanine.

A crystalline sample of N-benzoyl-DL-phenylalanine 1 and a polycrystalline sample of N-benzoyl-L-phenylalanine 2 were studied using 13C high-resolution solid-state NMR spectroscopy. The X-ray structure of the DL form was established. Sample 1 crystallizes in a monoclinic form with a P21/c space group, a=11.338(1) A, b=9.185(1) A, c=14.096(2) A, beta=107.53(3) degrees, V=1400(3) A3, Z=4 and R=0.053. The principal elements of the 13C chemical shift tensors deltaii for 1 and 2, selectively 13C (99%) labeled at the carboxyl groups were calculated. On the basis of 13C (delta)ii analysis the hydrogen bonding pattern for sample 2 was deduced. Enriched samples were used to establish the intermolecular distance between chemically equivalent nuclei for 1 and spatial proximity in heterogeneous domain for 2, employing the ODESSA pulse sequence. The consistence of the complementary approach covering X-ray data, analysis of the 13C (delta)ii parameters and ODESSA results is revealed.     

NMR and water relaxation of floral honey samples from wild and domesticated bees

The aqueous solutions of Bashkir floral honey of wild and domesticated bees were studied with high-resolution ¹H and ¹³C NMR and nuclear magnetic relaxation. NMR was shown to provide only qualitative data on the composition of the studied honey samples. Data on the composition of the minor components (amino acids), as well as the mobility of water protons in honey, indicate that the distinctions between honey from wild and domesticated bees are due to both the honey composition and the difference in the interactions of components with one another and with water.     

A polynomial-time algorithm for de novo protein backbone structure determination from nuclear magnetic resonance data.

We describe an efficient algorithm for protein backbone structure determination from solution Nuclear Magnetic Resonance (NMR) data. A key feature of our algorithm is that it finds the conformation and orientation of secondary structure elements as well as the global fold in polynomial time. This is the first polynomial-time algorithm for de novo high-resolution biomacromolecular structure determination using experimentally recorded data from either NMR spectroscopy or X-ray crystallography. Previous algorithmic formulations of this problem focused on using local distance restraints from NMR (e.g., nuclear Overhauser effect [NOE] restraints) to determine protein structure. This approach has been shown to be NP-hard, essentially due to the local nature of the constraints. In practice, approaches such as molecular dynamics and simulated annealing, which lack both combinatorial precision and guarantees on running time and solution quality, are used routinely for structure determination. We show that residual dipolar coupling (RDC) data, which gives global restraints on the orientation of internuclear bond vectors, can be used in conjunction with very sparse NOE data to obtain a polynomial-time algorithm for structure determination. Furthermore, an implementation of our algorithm has been applied to six different real biological NMR data sets recorded for three proteins. Our algorithm is combinatorially precise, polynomialtime, and uses much less NMR data to produce results that are as good or better than previous approaches in terms of accuracy of the computed structure as well as running time.