A theoretical study of rotational diffusion models for rod-shaped viruses. The influence of motion on 31P nuclear magnetic resonance lineshapes and transversal relaxation.

Information about the interaction between nucleic acids and coat proteins in intact virus particles may be obtained by studying the restricted backbone dynamics of the incapsulated nucleic acids using 31P nuclear magnetic resonance (NMR) spectroscopy. In this article, simulations are carried out to investigate how reorientation of a rod-shaped virus particle as a whole and isolated nucleic acid motions within the virion influence the 31P NMR lineshape and transversal relaxation dominated by the phosphorus chemical shift anisotropy. Two opposite cases are considered on a theoretical level. First, isotropic rotational diffusion is used as a model for mobile nucleic acids that are loosely or partially bound to the protein coat. The effect of this type of diffusion on lineshape and transversal relaxation is calculated by solving the stochastic Liouville equation by an expansion in spherical functions. Next, uniaxial rotational diffusion is assumed to represent the mobility of phosphorus in a virion that rotates as a rigid rod about its length axis. This type of diffusion is approximated by an exchange process among discrete sites. As turns out from these simulations, the amplitude and the frequency of the motion can only be unequivocally determined from experimental data by a combined analysis of the lineshape and the transversal relaxation. In the fast motional region both the isotropic and the uniaxial diffusion model predict the same transversal relaxation as the Redfield theory. For very slow motion, transversal relaxation resembles the nonexponential relaxation as observed for water molecules undergoing translational diffusion in a magnetic field gradient. In this frequency region T2e is inversely proportional to the cube root of the diffusion coefficient. In addition to the isotropic and uniaxial diffusion models, a third model is presented, in which fast restricted nucleic acid backbone motions dominating the lineshape are superimposed on a slow rotation of the virion about its length axis, dominating transversal relaxation. In an accompanying article the models are applied to the 31P NMR results obtained for bacteriophage M13 and tobacco mosaic virus.     

Dynamics in a protein-lipid complex: nuclear magnetic resonance measurements on the headgroup of cardiolipin when bound to cytochrome c.

Deuterium and phosphorus nuclear magnetic resonance (NMR) has been used to investigate the dynamics of slow motional processes induced in bilayer cardiolipin upon binding with cytochrome c. 31P NMR line shapes suggest that protein binding induces less restricted, isotropic-like motions in the lipid phosphates within the ms time scale of this measurement. However, these motions impart rapid transverse relaxation to methylene deuterons adjacent to the phosphate in the lipid headgroup and so did not feature strongly in the NMR line shapes recorded from these nuclei by using the quadrupolar echo. Nonetheless, motional characteristics of the headgroup deuterons were accessible to a dynamic NMR approach using the Carr-Purcell-Meiboom-Gill multiple-pulse experiment. Compared to the well-studied case of deuterons in fatty acyl chains of bilayer phosphatidylcholine, the motions determining the 2H spin transverse relaxation in the headgroup of bilayer cardiolipin were much faster, having a lower limit in the 5-10 kHz range. On binding with cytochrome c, the T2 effecting motions in the cardiolipin headgroup became faster still, with rates comparable to the residual quadrupolar coupling frequency of the headgroup deuterons (approximately 25 kHz) and so coincided with the time scale for recording the quadrupolar echo (approximately 40 microseconds). It is concluded that the headgroup of cardiolipin does not exclusively report localized dynamic information but is particularly sensitive to collective motions occurring throughout the bilayer molecules. Although the rates of collective modes of motion may be dependent on the lipid type in pure lipid bilayers, these low-frequency fluctuations appear to occupy a similar dynamic range in a variety of lipid-protein systems, including the natural membranes.     

An on/off resonance rotating frame relaxation experiment to monitor millisecond to microsecond timescale dynamics

Rotating frame relaxation experiments in proteins are used to study slow motions on the microsecond to millisecond timescale. An on/off resonance rotating frame relaxation experiment (R(1)(rho)) has been developed that incorporates adiabatic rotations into a R(1)(rho)-R(1) constant relaxation time experiment with weak radio frequency field strengths in order to effectively lock the magnetization over a wide range of (15)N frequencies. The new pulse sequence allows the measurement of a wide range of chemical exchange timescales on the order of 1.0 to 0.05 ms over an asymmetric bandwidth from +1.7omega(l) to -0.5omega(l) in a single experiment. A total bandwidth of +/-l.7omega(l) is obtained by performing the experiment a second time with a reversed adiabatic rotation.     

Nuclear magnetic resonance evidence for retention of a lamellar membrane phase with curvature in the presence of large quantities of the HIV fusion peptide

The HIV fusion peptide (HFP) is a biologically relevant model system to understand virus/host cell fusion. ²H and ³¹P NMR spectroscopies were applied to probe the structure and motion of membranes with bound HFP and with a lipid headgroup and cholesterol composition comparable to that of membranes of host cells of HIV. The lamellar phase was retained for a variety of highly fusogenic HFP constructs as well as a non-fusogenic HFP construct and for the influenza virus fusion peptide. The lamellar phase is therefore a reasonable structure for modeling the location of HFP in lipid/cholesterol dispersions. Relative to no HFP, membrane dispersions with HFP had faster ³¹P transverse relaxation and faster transverse relaxation of acyl chain ²H nuclei closest to the lipid headgroups. Relative to no HFP, mechanically aligned membrane samples with HFP had broader ³¹P signals with a larger fraction of unoriented membrane. The relaxation and aligned sample data are consistent with bilayer curvature induced by the HFP which may be related to its fusion catalytic function. In some contrast to the subtle effects of HFP on a host-cell-like membrane composition, an isotropic phase was observed in dispersions rich in phosphatidylethanolamine lipids and with bound HFP.     

Structure of metal-nucleotide complexes bound to creatine kinase: 31P NMR measurements using Mn(II) and Co(II)

The structures of metal-nucleotide complexes bound to rabbit muscle creatine kinase have been studied by making measurements of paramagnetic effects of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), on the spin-relaxation rates of the 31P nuclei of ATP and ADP in these complexes. The experiments were performed on enzyme-bound complexes, thereby limiting the contributions to the observed relaxation rate to two exchanging complexes (with and without the cation). Measurements were made as a function of temperature in the range 5-35 degrees C and at three 31P NMR frequencies, 81, 121.5, and 190.2 MHz, in order to determine the effect of exchange on the observed relaxation rates. The relaxation rates in E X MnADP and E X MnATP are independent of frequency, and their temperature variation yields activation energies (delta E) in the range 5-8 kcal/mol; in the transition-state analogue complex E X MnADP X NO3- X Cre (Cre is creatine), delta E is increased to 17.3 kcal/mol. These results demonstrate that the relaxation rates in the Mn(II) complexes are exchange limited and are incapable of providing structural data. It is shown further that use of line-width measurements to estimate the lifetime of the paramagnetic complex leads to incorrect results. The relaxation rates in E X CoADP and E X CoATP exhibit frequency dependence and delta E values in the range 1-3 kcal/mol; i.e., these rates depend on the Co(II)-31P distances, whereas those in the E X CoADP X NO3- X Cre complex have delta E approximately 18 kcal/mol and are significantly contributed by exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical and molecular exchange effects on T2 relaxation of living tissues: a pulse spacing dependence study

Contrast in magnetic resonance imaging depends principally on the longitudinal relaxation (R1) and the transverse relaxation rate (R2) of the observed nuclei, most often the protons. The spin-spin relaxation rate (R2) is the result of several mechanisms. The dependence of the interpulse delay of the Carr-Purcell-Meiboom-Gill sequence on the transverse relaxation rate of the water was studied in rat organs in vitro. It gives an insight into the exchange mechanisms involved. The increase of the interpulse delay from 0.2 ms to 5 ms gives an R2 increase of 23, 15, 3, and 2 s-1 for the heart, the liver, the spleen and the brain, respectively. These increases are compared to the R2 increases obtained in 17O-enriched water, amino acid and albumin solutions atomic exchange takes place. The concentration of these materials in organs cannot explain the R2 increase of the organs with the interpulse delay. Water exchange between intra and extracellular compartments is proposed to explain the R2 increase with interpulse delays in organs like the heart and the liver.     

Phosphorus-31 relaxation rate studies of Mn 2+-alkaline phosphatase

Phosphorus-31 NMR relaxation rates for the ternary complex of manganese-alkaline phosphatase-phosphate have been measured and their temperature dependence studied. The exchange of phosphate into the complex is exchange limited with respect to the transverse relaxation rate but is fast with respect to longitudinal relaxation. The data show that the observed phosphate relaxation is an outer-sphere effect. The activation energy for phosphate exchange is E_a = 8 Kcal/mole as determined from the temperature dependence of the line width of the phosphorus resonance.     

31P and 1H NMR studies of the structure of enzyme-bound substrate complexes of lobster muscle arginine kinase: relaxation measurements with Mn(II) and Co(II)

The paramagnetic effects of Mn(II) and Co(II) on the spin-lattice relaxation rates of 31P nuclei of ATP and ADP and of Mn(II) on the spin-lattice relaxation rate of the delta protons of arginine bound to arginine kinase from lobster tail muscle have been measured. Temperature variation of 31P relaxation rates in E.MnADP and E.MnATP yields activation energies (delta E) in the range 6-10 kcal/mol. Thus, the 31P relaxation rates in these complexes are exchange limited and cannot provide structural information. However, the relaxation rates in E.CoADP and E.CoATP exhibit frequency dependence and delta E values in the range 1-2 kcal/mol; i.e., these rates depend upon 31P-Co(II) distances. These distances were calculated to be in the range 3.2-4.5 A, appropriate for direct coordination between Co(II) and the phosphoryl groups. The paramagnetic effect of Mn(II) on the 1H spin-lattice relaxation rate of the delta protons of arginine in the E.MnADP.Arg complex was also measured at three frequencies (viz., 200, 300, and 470 MHz). These 1H experiments were performed in the presence of sufficient excess of arginine to be observable over the protein background but with MnADP exclusively in the enzyme-bound form so that the enhancement in the relaxation rates of the delta protons of arginine arises entirely from the enzyme-bound complex. Both the observed frequency dependence of these rates and the delta E less than or equal to 1.0 +/- 0.3 kcal/mol indicate that this rate depends on the 1H-Mn(II) distances.(ABSTRACT TRUNCATED AT 250 WORDS)     

31P NMR studies of the structure of cation-nucleotide complexes bound to porcine muscle adenylate kinase

The paramagnetic effects on the spin-relaxation rates of 31P nuclei in complexes of porcine muscle adenylate kinase with ATP, GTP, GDP, and AMP were measured in the presence of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), to examine the structures of the enzyme-bound complexes. Experiments were performed exclusively on enzyme-bound complexes to limit contributions to observed relaxation rates to two exchanging complexes (with and without cation). Measurements were made at three frequencies, 81, 121.5, and 190.2 MHz, and as a function of temperature in the range 5-30 degrees C to determine the effect of exchange on the observed relaxation rates. Relaxation rates in the E.MnATP, E.MnGTP, and E.MnGDP complexes were shown to be exchange-limited and therefore without structural information. Relaxation rates for the complexes E.CoATP, E.CoGTP, and E.CoGDP were shown to depend on Co(II)-31P distances. Inability to precisely estimate spectral densities arising from electronic relaxation of Co(II) restricts calculations of Co(II)-31P distances in these complexes to upper and lower limits. At the center of these limits, the Co(II)-31P distances of beta-P and gamma-P in E.CoATP and E.CoGTP, and of beta-P (E.CoGDP), are in the range 3.1-3.5 A appropriate for the first coordination sphere. For all these complexes, the corresponding distance for alpha-P is appreciably larger in the range 3.9-4.5 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative comparison of errors in 15N transverse relaxation rates measured using various CPMG phasing schemes

Nitrogen-15 Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation experiment are widely used to characterize protein backbone dynamics and chemical exchange parameters. Although an accurate value of the transverse relaxation rate, R(2), is needed for accurate characterization of dynamics, the uncertainty in the R(2) value depends on the experimental settings and the details of the data analysis itself. Here, we present an analysis of the impact of CPMG pulse phase alternation on the accuracy of the (15)N CPMG R(2). Our simulations show that R(2) can be obtained accurately for a relatively wide spectral width, either using the conventional phase cycle or using phase alternation when the r.f. pulse power is accurately calibrated. However, when the r.f. pulse is miscalibrated, the conventional CPMG experiment exhibits more significant uncertainties in R(2) caused by the off-resonance effect than does the phase alternation experiment. Our experiments show that this effect becomes manifest under the circumstance that the systematic error exceeds that arising from experimental noise. Furthermore, our results provide the means to estimate practical parameter settings that yield accurate values of (15)N transverse relaxation rates in the both CPMG experiments.     

Studying base pair open-close kinetics of tRNALeu by TROSY-based proton exchange NMR spectroscopy

The millisecond conformational flexibility is functionally important for nucleic acids and can be studied through probing the base pair open-close kinetics by proton exchange nuclear magnetic resonance (NMR) spectroscopy. Here, the traditional imino proton exchange NMR experiments were modified with transverse relaxation optimized spectroscopy and were applied to accurately measure imino proton exchange rates of all base pairs in Escherichia coli tRNA^Leu (CAG), and their dependence on magnesium ion concentration. Finally, we correlated millisecond conformational flexibility with aminoacylation of tRNA^Leu and proposed that the flexibility of the acceptor stem and the core region might contribute to aminoacylation of tRNA^Leu.     

Separating the contributions to 15N transverse relaxation in a fibronectin type III domain

In proteins, dynamic mobility is an important feature of structure, stability, and biomolecular recognition. Uniquely sensitive to motion throughout the milli- to picosecond range, rates of transverse relaxation, R2, are commonly obtained for the characterization of chemical exchange, and the construction of motional models that attempt to separate overall and internal mobility. We have performed an in-depth study of transverse relaxation rates of backbone 15N nuclei in TNfn31–90, the third fibronectin type III domain from human tenascin. By combining the results of spin-echo (CPMG) and off-resonance T1 experiments, we present R2 rates at effective field strengths of 2 to 40 krad/s, obtaining a full spectrum of 16 independent R2 data points for most residues. Collecting such a large number of replicate measurements provides insight into intrinsic uncertainties. The median standard deviation in R2 for non-exchanging residues is 0.31, indicating that isolated measurements may not be sufficiently accurate for a precise interpretation of motional models. Chemical exchange events on a timescale of 570 s were observed in a cluster of residues at the C terminus. Rates of exchange for five other residues were faster than the sampled range of frequencies and could not be determined. Averaged 'exchange free' transverse relaxation rates, R20, were used to calculate the diffusion tensor for rotational motion. Despite a highly asymmetric moment of inertia, the narrow angular dispersion of N-H vectors within the sandwich proves insufficient to define deviations from isotropic rotation. Loop residues provide exclusive evidence for axially symmetric diffusion (Dpar/Dper=1.55).     

Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy

We present the protocol for the measurement and analysis of dark-state exchange saturation transfer (DEST), a novel solution NMR method for characterizing, at atomic resolution, the interaction between an NMR-'visible' free species and an NMR-'invisible' species transiently bound to a very high-molecular-weight (>1 MDa) macromolecular entity. The reduced rate of reorientational motion in the bound state that precludes characterization by traditional NMR methods permits the observation of DEST. (15)N-DEST profiles are measured on a sample comprising the dark state in exchange with an NMR-visible species; in addition, the difference (ΔR(2)) in (15)N transverse relaxation rates between this sample and a control sample comprising only the NMR-visible species is also obtained. The (15)N-DEST and ΔR(2) data for all residues are then fitted simultaneously to the McConnell equations for various exchange models describing the residue-specific dynamics in the bound state(s) and the interconversion rate constants. Although the length of the experiments depends strongly on sample conditions, approximately 1 week of NMR spectrometer time was sufficient for full characterization of samples of amyloid-β (Aβ) at concentrations of ~100 μM.     

Triple Quantum Decoherence under Multiple Refocusing: Slow Correlated Chemical Shift Modulations of C′ and N Nuclei in Proteins

A new experiment allows the identification of residues that feature slow conformational exchange in macromolecules. Rotations about dihedral angles that are slower than the global correlation time tau(c) cause a modulation of the isotropic chemical shifts of the nuclei. If these fluctuations are correlated they induce a differential line broadening between three-spin single-quantum and triple-quantum coherences involving three nuclei such as the carbonyl C', the neighbouring amide nitrogen N and the amide proton H(N) belonging to a pair of consecutive amino acids. A cross-correlated relaxation rate R (CS/CS)(C'N) can be determined that corresponds to the sum of the isotropic and anisotropic contributions to the chemical shift modulations of the carbonyl carbon and nitrogen nuclei. Only the isotropic contributions depend on the pulse repetition rate of a multiple-refocusing sequence. An attenuation of the relaxation rate with increasing pulse repetition rate can therefore be attributed to slow motions. The asparagine N25 residue of ubiquitin, located in the first alpha-helix, is shown to feature significant slow conformational exchange.     

Phosphorus-31 studies on lecithin in ethanol solutions

³¹P relaxation times of lecithin in ethanol solutions have been measured in dependence on temperature and water concentration. Trial calculations have been carried out on the assumption of a 2-site exchange model. The results suggest first, the relaxation behaviour is determined by various motional and exchange processes; second, at 29 MHz the dipole-dipole interaction between ³¹P and protons provides the dominant contribution; third, in general we are not concerned with the case of “extreme narrowing”. Moreover, there are no negligible intermolecular contributions to relaxation.     

Dynamics of the phosphate group in phospholipid bilayers. A 31P angular dependent nuclear spin relaxation time study.

To understand 31P relaxation processes and hence molecular dynamics in the phospholipid multilayer it is important to measure the dependence of the 31P spin-lattice relaxation time on as many variables as the physical system allows. Such measurements of the 31P spin-lattice relaxation rate have been reported both as a function of Larmor frequency and temperature for egg phosphatidylcholine liposomes (Milburn, M.P., and K.R. Jeffrey. 1987. Biophys. J. 52:791-799). In principle, the spin-lattice relaxation rate in an anisotropic environment such as a bilayer will be a function of the angle between the bilayer normal and the magnetic field. However, the measurement of this angular dependence has not been possible because the rapid (on the time-scale of the spin-lattice relaxation rate) diffusion of the lipid molecules over the curved surface of the liposome average this dependence (Milburn, M.P., and K.R. Jeffrey. 1987. Biophys. J. 52:791-799; Brown, M.F., and J.H. Davis. 1981. Chem. Phys. Lett. 79:431-435). This paper reports the results of the measurement of the 31P spin-lattice relaxation rate as a function of this angle, beta', (the angle between the bilayer normal and the external magnetic field) using samples oriented between glass plates. These measurements were made at high field (145.7 MHz) where the spin-lattice relaxation processes are dominated by the chemical shielding interaction (Milburn, M.P., and K.R. Jeffrey. 1987. Biophys. J. 52:791-799). A model of molecular motion that includes a fast axially symmetric rotation of the phosphate group (tau i approximately 10(-9) s) and a wobble of the head group tilt with respect to this rotation axis has been used to describe both the angular dependence of the spin-lattice relaxation and the spectral anisotropy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Off-resonance rotating frame spin-lattice NMR relaxation studies of phosphorus metabolite rotational diffusion in bovine lens homogenates

The rotational diffusion behavior of phosphorus metabolites present in calf lens cortical and nuclear homogenates was investigated by the NMR technique of 31P off-resonance rotating frame spin-lattice relaxation as a means of assessing the occurrence and extent of phosphorus metabolite-lens protein interactions. 31P NMR spectra of calf lens homogenates were obtained at 10 and 18 degrees C (below and above the cold cataract phase transition temperature, respectively) at 7.05 T. Effective rotational correlation times (tau 0,eff) for the major phosphorus metabolites present in cortical and nuclear bovine calf lens homogenates were derived from nonlinear least-squares analysis of R vs omega e (spectral intensity ratio vs precessional frequency about the effective field) data with the assumption of isotropic reorientational motion. Intramolecular dipole-dipole (1H-31P, 31P-31P), chemical shift anisotropy (CSA), and solvent (water) translational intermolecular dipole-dipole (1H-31P) relaxation contributions were assumed in the analyses. In those cases where the limiting value of the spectral intensity ratio failed to reach unity at large offset frequency, a modified formalism incorporating chemical exchange mediated saturation transfer between two sites was used. Values of tau 0,eff for phosphorus metabolites present in the cortex varied from a low of ca. 2 ns [L-alpha-glycero-phosphocholine (GPC)] to a high of 12 ns (alpha-ATP) at 10 degrees C, whereas at 18 degrees C the range was from ca. 1 to 9 ns. For the nucleus the tau 0,eff values ranged from ca. 3 ns (GPC) to 41 ns (Pi) at 10 degrees C; at 18 degrees C the corresponding values ranged from 4 to 39 ns. For PME (phosphomonoester; in lens the predominant metabolite is L-alpha-glycerol phosphate) at 18 degrees C evidence was obtained for binding and subsequent exchange with solid like protein domains. The diversity in tau 0,eff values for lenticular phosphorus metabolites is suggestive of differential binding to more slowly tumbling macromolecular species, most likely lens crystallin proteins. Corresponding measurement of tau 0,eff values for the mobile protein fraction present in calf lens cortical and nuclear homogenates at 10 and 18 degrees C, by 13C off-resonance rotating frame spin-lattice relaxation, provided average macromolecular correlation times that were assumed to represent the bound metabolite state. A fast-exchange model (on the T1 time scale), between free and bound forms, was employed in the analysis of the metabolite R vs omega e curves to yield the     

Evaluation of <Superscript>15</Superscript>N-detected H–N correlation experiments on increasingly large RNAs

Recently, ¹⁵N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a ¹⁵N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three ¹⁵N-detected H–N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established ¹⁵N-detected BEST-TROSY experiment, which proves to be the most sensitive ¹⁵N-detected H–N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the ¹⁵N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the ¹H- than in the ¹⁵N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that ¹⁵N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs.     

NMR in cancer: VIII. Phosphorus-31 as a nuclear probe for malignant tumors

Spin-lattice relaxation times (T1) for 31P were determined in normal and malignant tissues by a saturation technique employing a 90 degree -tau-90 degrees pulse sequence. Results for five normal tissues from rat were (in seconds): 2.33 +/- .14 for liver; 2.19 +/- .05 for muscle; 1.13 +/- .05 for brain; 1.43 +/- .15 fro kidney; and 1.97 +/- .12 for intestine. Results for two rat malignancies, Novikoff hepatoma and Walker sarcoma, were 5.98 +/- .57 and 5.38 +/- .68, respectively, and for Crocker sarcoma of mouse, 5.19 +/- 1.42. No individual measurement of malignant tissue overlapped any of the normal measurements; probabilities of insignificance ranged from .029 for Crocker sarcoma to .000184 for Novikoff hepatoma. The data call attention to another nucleus of potential value for NMR detection of internal malignancies in humans. Also suggested, because of the strategic placement of the 31P nucleus in the nucleic acid molecule, is a possible new probe for exploring the mechanism of carcinogenesis.