NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.     

Carbon-13 nuclear magnetic resonance studies of myocardial glycogen metabolism in live guinea pigs

Myocardial glycogen metabolism was studied in live guinea pigs by 13C NMR at 20.19 MHz. Open-chest surgery was used to expose the heart, which was then positioned within a solenoidal radio frequency coil for NMR measurements. The time course of myocardial glycogen synthesis during 1-h infusions of 0.5 g of D-[1-13C]glucose (and insulin) into the jugular vein was investigated. The possible turnover of the 13C-labeled glycogen was also studied in vivo by following the labeled glucose infusion with a similar infusion of unlabeled glucose. The degree of 13C enrichment of the C-1 glycogen carbons during these infusions was measured in heart extracts by 1H NMR at 360 MHz. High-quality proton-decoupled 13C NMR spectra of the labeled C-1 carbons of myocardial glycogen in vivo were obtained in 1 min of data accumulation. This time resolution allowed measurement of the time course of glycogenolysis of the 13C-labeled glycogen during anoxia by 13C NMR in vivo. With the solenoidal coil used for 13C NMR, the spin-lattice relaxation time of the labeled C-1 carbons of myocardial glycogen could be measured in vivo. For a comparison, spin-lattice relaxation times of heart glycogen were measured in vitro at 90.55 MHz. Natural abundance 13C NMR studies of the quantitative hydrolysis of extracted heart glycogen in vitro at 90.55 MHz showed that virtually all the carbons in heart glycogen contribute to the 13C NMR signals. The same result was obtained in 13C NMR studies of glycogen hydrolysis in excised guinea pig heart.     

Conformation and dynamic structure of poly(inosinic acid) in neutral aqueous solution by 1H-, 2H-, 13C-, and 31P-NMR spectroscopy

The conformation and dynamic structure of single-stranded poly(inosinic acid), poly(I), in aqueous solution at neutral pH have been investigated by nmr of four nuclei at different frequencies: ¹H (90 and 250 MHz), ²H (13.8 MHz), ¹³C (75.4 MHz), and ³¹P (36.4 and 111.6 MHz). Measurements of the proton-proton coupling constants and of the ¹H and ¹³C chemical shifts versus temperature show that the ribose is flexible and that base-base stacking is not very significant for concentrations varying from 0.04 to 0.10M in the monomer unit. On the other hand, the proton T₁ ratios between the sugar protons, T₁ (H₁′)/T₁ (H₃′), indicate a predominance of the anti orientation of the base around the glycosidic bond. The local motions of the ribose and the base were studied at different temperatures by measurements of nuclear Overhauser enhancement (NOE) of protonated carbons, the ratio of the proton relaxation times measured at two frequencies (90 and 250 MHz), and the deuterium quadrupolar transverse relaxation time T₂. For a given temperature between 22 and 62°C, the ¹³C-{¹H} NOE value is practically the same for seven protonated carbons (C₂, C₈, C_1′, C_2′, C_3′, C_4′, C_5′). This is also true for the T₁ ratio of the corresponding protons. Thus, the motion of the ribose–base unit can be considered as isotropic and characterized by a single correlation time, τ_c, for all protons and carbons. The τ_c values determined from either the ¹³C-{¹H} NOE or proton T₁ ratios, T₁(90 MHz)/T₁(250 MHz), and/or deuterium transverse relaxation time T₂ agree well. The molecular motion of the sugar-phosphate backbone (O-P-O) and the chemical-shift anisotropy (CSA) were deduced from T₁ (³¹P) and ³¹P-{¹H} NOE measurements at two frequencies. The CSA contribution to the phosphorus relaxation is about 12% at 36.4 MHz and 72% at 111.6 MHz, corresponding to a value of 118 ppm for the CSA (σ = σ∥ ? σ?). Activation energies of 2–6 kcal/mol for the motion of the ribose–base unit and the sugarphosphate backbone were evaluated from the proton and phosphorus relaxation data.     

Structure and dynamics of flour by solid state NMR: effects of hydration and wheat aging

The effects of accelerated aging of wheat seeds on structural and dynamic properties of dry and hydrated (ca 10 wt % H(2)O) flour at a molecular level were investigated by several high and low resolution solid-state NMR techniques. Identification and characterization of domains with different mobility was performed by (13)C direct excitation (DE) and cross-polarization (CP) magic angle spinning (MAS), as well as by (1)H static and MAS experiments. (1)H spin-lattice relaxation times (T(1) and T(1)(rho)) measurements were carried out to investigate molecular motions in different frequency ranges. Experimental data show that the main components of flour (starch and gluten proteins) are in a glassy phase, whereas the mobile fraction is constituted by lipids and, in hydrated samples, absorbed water. A lower proportion of rigid domains, as well as an increased dynamics of all flour components are observed after both seeds aging and flour hydration. Linear average dimensions between 20 and 200 A are estimated for water domains in hydrated samples.     

A pulsed N.M.R study of D2O bound to 1,2 dipalmitoyl phosphatidylcholine

Spin lattice relaxation times in both the lab and rotating frame, have been measured for deuterons (²H) in a number of unsonicated dispersions of 1,2 dipalmitoyl phosphatidylcholine in D₂O over a range of resonant frequencies from 13 MHz to 1 MHz for temperatures from ?20°C to 65°C.The proton (¹H) spin lattice relaxation time for the lecithin was measured for resonant frequencies of 8.5 MHz, and 40 MHz over a similar range of temperatures.The results agree with broadline measurements by Salsbury et al. [1], and for the liquid crystal phase are consistent with an anisotropic tumbling model of the water molecules bound to the lecithin headgroup. This tumbling occurs with correlation times of ≤10^?10 sec and ≈ 10^?6 sec about axes parallel to and perpendicular to the bisector of the D-O-D angle within a D₂O molecule, hydrogen bonded to the negatively charged phosphate headgroup.     

Pulsed nuclear magnetic resonance study of 17-O, 2-D, and 1-H of water in frog striated muscle.

Whole gastrocnemius muscles were incubated in Ringer's solution enriched with H2-17O; the paired contralateral gastrocnemius muscles were incubated in a similar solution enriched with deuterons, as well. Subsequently, the longitudinal relaxation times (T1) were measured 17-O, 2-D, and 1-H, both at 8.1 MHz and at 4.3 MHz. The results indicate that: (a) the absolute values of T1 characterizing the three nuclides are different in muscle and pure water. (b) the longitudinal relaxation rates of all three have an identical frequency dependence over the range studied, (c) the ratio (T1)2D/(T1)17ois the same in muscle water and pure water, while the ratio (T1)1H/(T1)17o is 2.1 times greater in pure water than it is in muscle water, and (d) 30-49 percent substitution of 2-D for 1-H has very little effect on the spin-lattice relaxation of tissue water protons. These data suggest that muscle water is in rapid exchange between a small fraction of immobilized molecules and a large fraction of free water. The results render unlikely the possibility that hypothetical ordering of muscle water significantly contributes to its longitudinal relaxation.     

Cross-correlation suppressed T1 and NOE experiments for protein side-chain 13CH2 groups

Relaxation measurements of side-chain ¹³CH₂-groups of uniformly ¹³C labeled human ubiquitin were performed at 600 MHz and 800 MHz magnetic field strength at 30°C. Dipole-dipole cross-correlated relaxation effects in T₁ experiments were suppressed by the combination of radio-frequency pulses and pulsed field gradients during the relaxation delay leading to monoexponential relaxation decays that allow a more accurate extraction of the ¹³C T₁ relaxation times. Heteronuclear ¹H-¹³C NOEs obtained by using different proton saturation schemes indicate that the influence of cross-correlation is small. The experimental T₁ and NOE data were interpreted in a model-free way in terms of a generalized order parameter and an internal correlation time.     

Dipolar NMR relaxation of nonprotonated aromatic carbons in proteins. Structural and dynamical effects.

The crystal structure and a 96-ps molecular dynamics simulation used to analyze structural and motional contributions to spin-lattice (T1) relaxation times of phenylalanine and tyrosine C gamma carbons of the pancreatic trypsin inhibitor. The H beta and H delta protons geminal to C gamma are calculated to account for approximately 80% of the dipolar relaxation for each residue. Experimental T1 values for the phenylalanine residues obtained at 25 MHz are observed to be 15-25% longer than estimates based on the rigid crystal structure. It is shown how an increase in T1 can be related to order parameters for the picosecond motional averaging of the important C,H dipolar interactions, and how these order parameters can be calculated from a protein molecular dynamics trajectory.     

The relationship between the transverse and longitudinal nuclear magnetic resonance relaxation rates of muscle water.

Whole frog sartorius and gastrocnemius muscles were incubated in Ringer's solutions, either unenriched or enriched with H2 17Oor 2D2O. Subsequently, the rates of transverse (1/T2) and of longitudinal (1/T1) nuclear magnetic relaxation were measured for 17O, 2D, and 1H at room temperature and at 8.1 MHz. The ratio (T1/T2) for 17O was measured to be approximately 1.5-2.0, close to the value roughly estimated from the Larmor frequency dependence of 1/T1 alone over the range 4.3-8.1 MHz. On the other hand (T1/T2) for 2D and 1H were both measured to lie in the range 9-11. Insofar as the entire 17O signal was detected, the data indicate the presence of an exchange mechanism between the major fraction of intracellular water and a minor fraction characterized by enhanced rates of relaxation. Possible molecular mechanisms are presented.     

The distribution of lipid attached spin probes in bilayers: application to membrane protein topology

The distribution of the lipid-attached doxyl electron paramagnetic resonance (EPR) spin label in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes has been studied by (1)H and (13)C magic angle spinning nuclear magnetic resonance relaxation measurements. The doxyl spin label was covalently attached to the 5th, 10th, and 16th carbons of the sn-2 stearic acid chain of a 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-sn-glycero-3-phosphocholine analog. Due to the unpaired electron of the spin label, (1)H and (13)C lipid relaxation rates are enhanced by paramagnetic relaxation. For all lipid segments the influence of paramagnetic relaxation is observed even at low probe concentrations. Paramagnetic relaxation rates provide a measure for the interaction strength between lipid segments and the doxyl group. Plotted along the membrane director a transverse distribution profile of the EPR probe is obtained. The chain-attached spin labels are broadly distributed in the membrane with a maximum at the approximate chain position of the probe. Both (1)H and (13)C relaxation measurements show these broad distributions of the doxyl group in the membrane indicating that (1)H spin diffusion does not influence the relaxation measurements. The broad distributions of the EPR label result from the high degree of mobility and structural heterogeneity in liquid-crystalline membranes. Knowing the distribution profiles of the EPR probes, their influence on relaxation behavior of membrane inserted peptide and protein segments can be studied by (13)C magic angle spinning nuclear magnetic resonance. As an example, the location of Ala residues positioned at three sites of the transmembrane WALP-16 peptide was investigated. All three doxyl-labeled phospholipid analogs induce paramagnetic relaxation of the respective Ala site. However, for well ordered secondary structures the strongest relaxation enhancement is observed for that doxyl group in the closest proximity to the respective Ala. Thus, this approach allows study of membrane insertion of protein segments with respect to the high molecular mobility in liquid-crystalline membranes.     

A single-quantum methyl <Superscript>13</Superscript>C-relaxation dispersion experiment with improved sensitivity

A pulse sequence is described for recording single-quantum (13)C-methyl relaxation dispersion profiles of (13)C-selectively labeled methyl groups in proteins that offers significant improvements in sensitivity relative to existing approaches where initial magnetization derives from (13)C polarization. Sensitivity gains in the new experiment are achieved by making use of polarization from (1)H spins and (1)H --> (13)C --> (1)H type magnetization transfers. Its utility has been established by applications involving three different protein systems ranging in molecular weight from 8 to 28 kDa, produced using a number of different selective labeling approaches. In all cases exchange parameters from both (13)C-->(1)H and (1)H --> (13)C --> (1)H classes of experiment are in good agreement, with gains in sensitivity of between 1.7 and 4-fold realized using the new scheme.     

Study of high molecular weight wheat glutenin subunit 1Dx5 by 13C and 1H solid-state NMR spectroscopy. I. Role of covalent crosslinking

This work describes a carbon and proton solid-state NMR study of the hydration of a high molecular weight wheat glutenin subunit, 1Dx5. The effect of the presence of disulfide bonds on the hydration behavior of the subunit is investigated by a comparison of the unalkylated and alkylated forms of the protein. Hydration induces partial plasticization of the protein so that some segments become more mobile than others. The 13C cross-polarization and magic-angle spinning (MAS) spectra of the samples in the dry state and at two hydration levels (approximately 40 and approximately 65% D2O) were used to monitor the protein fraction resisting plasticization (trains). Conversely, 13C single pulse excitation and 1H-MAS experiments were used to gain information on the more plasticized segments (loops). The molecular motion of the two protein dynamic populations was further characterized by 13C T1 and 1H T(1rho), T2, and T1 relaxation times. The results suggest that hydration leads to the formation of a network held by a cooperative action of hydrogen bonded glutamines and some hydrophobic interactions. The looser protein segments are suggested to be glycine- and glutamine-rich segments. The primary structure is therefore expected to significantly determine the proportion of trains and loops in the network. The presence of disulfide bonds was observed to promote easier plasticization of the protein and the formation of a more mobile network, probably involving a higher number of loops and/or larger loops.     

Hydration of Escherichia coli lipids. Deuterium T1 relaxation time studies of phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine

The hydration properties of Escherichia coli lipids (phosphatidylglycerol, phosphatidylethanolamine) and synthetic 1,2-dioleoyl-sn-glycero-3-phosphocholine in H2O/2H2O mixtures (9:1, v/v) were investigated with 2H-NMR. Comparison of the 2H2O spin lattice relaxation time (T1) as a function of the water content revealed a remarkable quantitative similarity of all three lipid-H2O systems. Two distinct hydration regions could be discerned in the T1 relaxation time profile. (1) A minimum of 11-16 water molecules was needed to form a primary hydration shell, characterized by an average relaxation time of T1 approximately equal to 90 ms. (2) Additional water was found to be in exchange with the primary hydration shell. The exchange process could be described in terms of a two-site exchange model, assuming rapid exchange between bulk water with T1 = 500 ms and hydration water with T1 = 80-120 ms. Analysis of the linewidth and the residual quadrupole splitting (at low water content) confirmed the size of the primary hydration layer. However, each lipid-water system exhibited a somewhat different linewidth behavior, and a detailed molecular interpretation appeared to be preposterous.     

An NMR database for simulations of membrane dynamics

Computational methods are powerful in capturing the results of experimental studies in terms of force fields that both explain and predict biological structures. Validation of molecular simulations requires comparison with experimental data to test and confirm computational predictions. Here we report a comprehensive database of NMR results for membrane phospholipids with interpretations intended to be accessible by non-NMR specialists. Experimental 13C-1H and 2H NMR segmental order parameters (S(CH) or S(CD)) and spin-lattice (Zeeman) relaxation times (T(1Z)) are summarized in convenient tabular form for various saturated, unsaturated, and biological membrane phospholipids. Segmental order parameters give direct information about bilayer structural properties, including the area per lipid and volumetric hydrocarbon thickness. In addition, relaxation rates provide complementary information about molecular dynamics. Particular attention is paid to the magnetic field dependence (frequency dispersion) of the NMR relaxation rates in terms of various simplified power laws. Model-free reduction of the T(1Z) studies in terms of a power-law formalism shows that the relaxation rates for saturated phosphatidylcholines follow a single frequency-dispersive trend within the MHz regime. We show how analytical models can guide the continued development of atomistic and coarse-grained force fields. Our interpretation suggests that lipid diffusion and collective order fluctuations are implicitly governed by the viscoelastic nature of the liquid-crystalline ensemble. Collective bilayer excitations are emergent over mesoscopic length scales that fall between the molecular and bilayer dimensions, and are important for lipid organization and lipid-protein interactions. Future conceptual advances and theoretical reductions will foster understanding of biomembrane structural dynamics through a synergy of NMR measurements and molecular simulations.     

Effect of solvent exchange on the supramolecular structure, the molecular mobility and the dissolution behavior of cellulose in LiCl/DMAc

We investigated the effect of solvent exchange on the supramolecular structure and the molecular mobility of the cellulose molecule to clarify the mechanism of the dissolution of cellulose in lithium chloride/N,N-dimethylacetamide (LiCl/DMAc). Among the celluloses that were solvent exchanged in different ways, the DMAc-treated celluloses dissolved most rapidly. Dissolution of the acetone-treated celluloses was much slower than the DMAc-treated ones, but considerably faster than the untreated one. Such differences in the dissolution behavior were well explained by the differences in the surface fractal dimension calculated from the small-angle X-ray scattering profiles and in the (1)H spin-lattice and spin-spin relaxation times estimated from the solid-state NMR spectroscopic measurements. Furthermore, it was suggested from the IR spectra and the (13)C spin-lattice relaxation times of cellulose that DMAc is adsorbed on the surface of cellulose even after vacuum-drying and affects the molecular mobility and hydrogen-bonding state of cellulose.     

The state of water in biological systems as studied by proton and deuterium relaxation.

Careful experiments on the measurement of the intensity of the deuterium NMR signal for 2-H2 O in muscle and in its distillate were performed, and they showed that all 2-H2 O muscle is "NMR visible". The spin-lattice relaxation time (T1) of the water protons in the muscle and liver of mice and in egg white has been studied at six frequencies ranging from 4.5 to 6.0 MHz over the temperature range of +37 to --70 degrees C. T1 values of deuterons in 2H2 O of gastrocnemius muscle and liver of mice have been measured at three frequencies (4.5, 9.21 and 15.35 MHz) over the temperature range of +37 to --20 degrees C. Calculations on T1 for both proton and deuteron have been made and compared with the experimental data. It is suggested that the reduction of the T1 values compared to pure water and the frequency dependence of T1 are due to water molecules in the hydration layer of the macromolecules, and that the bulk of water molecules in the biological tissues and egg white undergoes relaxation like ordinary liquid water.     

NMR relaxation of protein and water protons in diamagnetic hemoglobin solutions

We have measured T1 and T2 of protein and water protons in hemoglobin solutions using broad-line pulse techniques; selective excitation and detection methods enabled the intrinsic protein and water relaxation rates, as well as the spin-transfer rate between them, to be obtained at 5, 10, and 20 MHz. Water and protein T1 data were also obtained at 100 and 200 MHz for hemoglobin in H2O/D2O mixtures by using commercial Fourier-transform instruments. The T1 data conform to a simple model of two well-mixed spin systems with single intrinsic relaxation times and an average spin-transfer rate, with each phase recovering from a radio-frequency excitation with a biexponential time dependence. At low frequencies, protein T1 and T2 agree reasonably with a model of dipolar relaxation of an array of fixed protons tumbling in solution, explicitly calculating methyl and methylene relaxation and using a continuum approximation for the others. Differing values in H2O and D2O are mainly ascribed to solvent viscosity. For water-proton relaxation, T1, T2, and spin transfer were measured for H2O and HDO, which enabled a separation of inter-and intramolecular contributions to relaxation. Despite such detail, few firm conclusions could be reached about hydration water. But it seems clear that few long-lived hydration sites are needed to explain T1 and T2, and the spin-transfer value mandates fewer than five sites with a lifetime longer than 10(-8) s.     

Transfer RNA structure by carbon NMR: C2 of adenine, uracil and cytosine.

Fourier transform 13C NMR spectra of E. coli tRNA enriched on 13C in either position 2 of adenine (60 atom % 13C) or in position 2 of uracil (82%) and cytosine (63%) were taken at 25.16 MHz over the temperature range 10 degrees - 76 degrees. For C2 of adenine the peak as initially 5 ppm wide, but narrowed to 0.5 ppm as the molecule unfolded. C2 of uracil displayed behavior similar to that of adenine while the cytosine peak, initially relatively narrow at low temperature, sharpened less dramatically. Comparison of spectra at 26.16 MHz and 67.9 MHz showed that the peak widths for folded tRNA were determined largely by chemical shift non-equivalence. T2 T2 measurements suggested that intrinsic line widths of most cytosine C2 peaks were 4 Hz and 2-3 Hz for uracil. Adenine C2 with a directly bonded proton had resonances of about 40 Hz line width. T1 values were measured for C2 of adenine and the ribose carbons of tRNA. Consideration of dipolar relaxation and chemical shift anisotrophy led to a calculated rotational correlation time of 1.6 +/- 0.4 x 10(-8) sec for the adenines and 1.3 +/- 0.3 x 10(-8) sec for the ribose carbons.     

Modelling the dynamics of an antigenic peptide using NMR relaxation data.

The internal dynamics of a cyclic peptide which was designed to mimic an antigenic loop of the haemagglutinin, is studied through heteronuclear relaxation along the 13C alpha-1H alpha vectors and through homonuclear relaxation along the 1H alpha-1HN and 1H beta-1H beta' vectors. Order parameters are extracted from the longitudinal and cross-relaxation data. Molecular dynamics simulations are performed and the order parameters are calculated in different ways from the trajectories. The simulation, which is performed in vacuo, gives smaller order parameters (vector motions of larger amplitude) than the experimental results. However, the general features of the experimental order parameters are reproduced by the molecular dynamics simulation. The flexibility of the molecule can then be investigated from the results of the molecular dynamics. It shows that the mobility observed through the order parameters is due to motions in flanking regions, remote from the observed vectors.     

13C relaxation experiments for aromatic side chains employing longitudinal- and transverse-relaxation optimized NMR spectroscopy

Aromatic side chains are prevalent in protein binding sites, perform functional roles in enzymatic catalysis, and form an integral part of the hydrophobic core of proteins. Thus, it is of great interest to probe the conformational dynamics of aromatic side chains and its response to biologically relevant events. Indeed, measurements of (13)C relaxation rates in aromatic moieties have a long history in biomolecular NMR, primarily in the context of samples without isotope enrichment that avoid complications due to the strong coupling between neighboring (13)C spins present in uniformly enriched proteins. Recently established protocols for specific (13)C labeling of aromatic side chains enable measurement of (13)C relaxation that can be analyzed in a straightforward manner. Here we present longitudinal- and transverse-relaxation optimized pulse sequences for measuring R (1), R (2), and {(1)H}-(13)C NOE in specifically (13)C-labeled aromatic side chains. The optimized R (1) and R (2) experiments offer an increase in sensitivity of up to 35 % for medium-sized proteins, and increasingly greater gains are expected with increasing molecular weight and higher static magnetic field strengths. Our results highlight the importance of controlling the magnetizations of water and aliphatic protons during the relaxation period in order to obtain accurate relaxation rate measurements and achieve full sensitivity enhancement. We further demonstrate that potential complications due to residual two-bond (13)C-(13)C scalar couplings or dipolar interactions with neighboring (1)H spins do not significantly affect the experiments. The approach presented here should serve as a valuable complement to methods developed for other types of protein side chains.