Measurements Of Transverse Relaxation Rates Research Articles

Precision measurements of relaxation rates of degenerate 1H transitions in methyl groups of small proteins

An NMR experiment is designed for accurate and robust measurement of transverse relaxation rates of degenerate 1H transitions in selectively 13CH3-labeled, deuterated small proteins. The measurement is based on the use of acute (<90°) angle 1H radio-frequency pulses and relies on selection of the slow- and fast-relaxing components of methyl magnetization following the relaxation period in separate experiments. The R2 decay series recorded with selection of the fast-relaxing components serves as a useful complement to the R2 series acquired with selection of the slow-relaxing part, and permits the extension of the range of relative contributions of the fast- and slow-relaxing parts to apparent signal decay. The approach is experimentally verified on 13CH3 methyl groups of the ILV-{13CH3}-labeled protein ubiquitin at 10 °C and 25 °C. The obtained methyl 1H relaxation rates are in remarkably good agreement with the values obtained from well-established NMR techniques.

In-Situ Relaxation Rate Measurement in Magnetic Modulated Atomic Magnetometers

Traditional practice of suppressing low-frequency noise in atomic magnetometers is mainly through the application of magnetic field modulation, while magnetic fields of this kind introduce extra transverse relaxation, which results in a decrease of sensitivity. In this study, a novel method of measuring relaxation rate induced by spin-destruction, wall collisions and modulation magnetic fields is proposed. The optical pumping model is studied both in optical-thin and optical-thick vapor cell through the analysis of total relaxation, thus the optical pumping rate can be separated from total relaxation by analyzing the optical depth. Furthermore, based on the analysis of optical pumping rate and relaxation rate, the polarization of the electron ensemble was evaluated. A compact magnetometer with single-beam configuration is developed for the proof of concept, and the experimental results show good agreements with theoretical calculation. Our method provides a simple and feasible approach to in-situ measurement of transverse relaxation rate and optical pumping rate with physical structure of the device untouched, which is especially advantageous for designing and optimizing compact atomic magnetometers.

The measurement of relaxation rates of degenerate 1H transitions in methyl groups of proteins using acute angle radiofrequency pulses

A simple NMR experiment for the measurement of transverse relaxation rates of degenerate 1H spins in 13CH3 methyl groups of deuterated proteins, is described. The experiment relies on the use of acute-angle 1H radio-frequency pulses, whereby a series of methyl 1H R2 decays is acquired with different values of the 1H pulse flip-angle, which modulates the relative contributions of the slow- and fast-relaxing components of 1H magnetization during the relaxation delay. These are subsequently analyzed simultaneously to extract the transverse relaxation rates of both components (RS and RF), along with the rate of cross-relaxation between these components, δ. The dipolar 1H–1H cross-correlated relaxation rate η = (RF – RS)/2 and the cross-relaxation rate δ, extracted from such acute-angle-pulse R2 decay series are compared with those derived from well-established methodology that uses relaxation-violated methyl 1H coherence transfer (so-called ‘forbidden’ experiments). Good agreement is achieved for the rates η derived from the two techniques, while slightly more accurate values of δ are obtained from analysis of acute-angle-pulse R2 series. Recording of acute-angle-pulse R2 series represents an attractive alternative to existing methodologies for quantifying methyl 1H relaxation and dynamics of the methyl three-fold symmetry axis in selectively [13CH3]-methyl-labeled, deuterated proteins – particularly so for very high-molecular-weight proteins, where the measurements of RF rates or the build-up of methyl 1H magnetization in relaxation-violated coherence transfer experiments, are problematic due to low sensitivity.

MagA expression attenuates iron export activity in undifferentiated multipotent P19 cells.

Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport protein from magnetotactic bacteria, is a useful gene-based magnetic resonance contrast agent. Hemagglutinin-tagged MagA was stably expressed in undifferentiated embryonic mouse teratocarcinoma, multipotent P19 cells to provide a suitable model for tracking these cells during differentiation. Western blot and immunocytochemistry confirmed the expression and membrane localization of MagA in P19 cells. Surprisingly, elemental iron analysis using inductively-coupled plasma mass spectrometry revealed significant iron uptake in both parental and MagA-expressing P19 cells, cultured in the presence of iron-supplemented medium. Withdrawal of this extracellular iron supplement revealed unexpected iron export activity in P19 cells, which MagA expression attenuated. The influence of iron supplementation on parental and MagA-expressing cells was not reflected by longitudinal relaxation rates. Measurement of transverse relaxation rates (R2* and R2) reflected changes in total cellular iron content but did not clearly distinguish MagA-expressing cells from the parental cell type, despite significant differences in the uptake and retention of total cellular iron. Unlike other cell types, the reversible component R2′ (R2* ‒ R2) provided only a moderately strong correlation to amount of cellular iron, normalized to amount of protein. This is the first report to characterize MagA expression in a previously unrecognized iron exporting cell type. The interplay between contrast gene expression and systemic iron metabolism substantiates the potential for diverting cellular iron toward the formation of a novel iron compartment, however rudimentary when using a single magnetotactic bacterial gene expression system like magA. Since relatively few mammalian cells export iron, the P19 cell line provides a tractable model of ferroportin activity, suitable for magnetic resonance analysis of key iron-handling activities and their influence on gene-based MRI contrast.

Impact of two-bond 15N-15N scalar couplings on 15N transverse relaxation measurements for arginine side chains of proteins.

NMR relaxation of arginine (Arg) 15Nε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal 15N-15N scalar couplings on measurements of transverse relaxation rates (R 2 ) for Arg side-chain 15Nε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal 15N-15N couplings ( 2 J NN ) of the 15Nε nuclei and found that the magnitudes of the 2 J NN coupling constants were virtually uniform with an average of 1.2Hz. Our simulations, assuming ideal 180° rotations for all 15N nuclei, suggested that the two 2 J NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr-Purcell-Meiboom-Gill (CPMG) scheme for Arg 15Nε R 2 measurements. However, our experimental data show that the expected modulation via two 2 J NN couplings vanishes during the 15N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831-836, 2006). This effect allows for accurate measurements of R 2 relaxation rates for Arg side-chain 15Nε nuclei despite the presence of two 2 J NN couplings. Although the so-called recoupling conditions may cause overestimate of R 2 rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings.

Cross-Correlated Relaxation of Dipolar Coupling and Chemical-Shift Anisotropy in Magic-Angle Spinning R1ρ NMR Measurements: Application to Protein Backbone Dynamics Measurements.

Transverse relaxation rate measurements in magic-angle spinning solid-state nuclear magnetic resonance provide information about molecular motions occurring on nanosecond-to-millisecond (ns-ms) time scales. The measurement of heteronuclear ((13)C, (15)N) relaxation rate constants in the presence of a spin-lock radiofrequency field (R1ρ relaxation) provides access to such motions, and an increasing number of studies involving R1ρ relaxation in proteins have been reported. However, two factors that influence the observed relaxation rate constants have so far been neglected, namely, (1) the role of CSA/dipolar cross-correlated relaxation (CCR) and (2) the impact of fast proton spin flips (i.e., proton spin diffusion and relaxation). We show that CSA/D CCR in R1ρ experiments is measurable and that the CCR rate constant depends on ns-ms motions; it can thus provide insight into dynamics. We find that proton spin diffusion attenuates this CCR due to its decoupling effect on the doublet components. For measurements of dynamics, the use of R1ρ rate constants has practical advantages over the use of CCR rates, and this article reveals factors that have so far been disregarded and which are important for accurate measurements and interpretation.

Evaluation of a multiple spin- and gradient-echo (SAGE) EPI acquisition with SENSE acceleration: Applications for perfusion imaging in and outside the brain

Perfusion-based changes in MR signal intensity can occur in response to the introduction of exogenous contrast agents and endogenous tissue properties (e.g. blood oxygenation). MR measurements aimed at capturing these changes often implement single-shot echo planar imaging (ssEPI). In recent years ssEPI readouts have been combined with parallel imaging (PI) to allow fast dynamic multi-slice imaging as well as the incorporation of multiple echoes. A multiple spin- and gradient-echo (SAGE) EPI acquisition has recently been developed to allow measurement of transverse relaxation rate (R2 and R2⁎) changes in dynamic susceptibility contrast (DSC)-MRI experiments in the brain. With SAGE EPI, the use of PI can influence image quality, temporal resolution, and achievable echo times. The effect of PI on dynamic SAGE measurements, however, has not been evaluated. In this work, a SAGE EPI acquisition utilizing SENSE PI and partial Fourier (PF) acceleration was developed and evaluated. Voxel-wise measures of R2 and R2⁎ in healthy brain were compared using SAGE EPI and conventional non-EPI multiple echo acquisitions with varying SENSE and PF acceleration. A conservative SENSE factor of 2 with PF factor of 0.73 was found to provide accurate measures of R2 and R2⁎ in white (WM) (rR2=[0.55–0.79], rR2⁎=[0.47–0.71]) and gray (GM) matter (rR2=[0.26–0.59], rR2⁎=[0.39–0.74]) across subjects. The combined use of SENSE and PF allowed the first dynamic SAGE EPI measurements in muscle, with a SENSE factor of 3 and PF factor of 0.6 providing reliable relaxation rate estimates when compared to multi-echo methods. Application of the optimized SAGE protocol in DSC-MRI of high-grade glioma patients provided T1 leakage-corrected estimates of CBV and CBF as well as mean vessel diameter (mVD) and simultaneous measures of DCE-MRI parameters Ktrans and ve. Likewise, application of SAGE in a muscle reperfusion model allowed dynamic measures of R2′, a parameter that has been shown to correlate with muscle oxy-hemoglobin saturation.

The effect of magnetically induced linear aggregates on proton transverse relaxation rates of aqueous suspensions of polymer coated magnetic nanoparticles

It has been recently reported that for some suspensions of magnetic nanoparticles the transverse proton relaxation rate, R(2), is dependent on the time that the sample is exposed to an applied magnetic field. This time dependence has been linked to the formation of linear aggregates or chains in an applied magnetic field via numerical modeling. It is widely known that chain formation occurs in more concentrated ferrofluids systems and that this has an affect on the ferrofluid properties. In this work we examine the relationships between colloidal stability, the formation of these linear structures, and changes observed in the proton transverse relaxation rate of aqueous suspensions of magnetic particles. A series of iron oxide nanoparticles with varying stabilizing ligand brush lengths were synthesized. These systems were characterized with dynamic light scattering, transmission electron microscopy, dark-field optical microscopy, and proton transverse relaxation rate measurements. The dark field optical microscopy and R(2) measurements were made in similar magnetic fields over the same time scale so as to correlate the reduction of the transverse relaxivity with the formation of linear aggregates. Our results indicate that varying the ligand length has a direct effect on the colloidal arrangement of the system in a magnetic field, producing differences in the rate and size of chain formation, and hence systematic changes in transverse relaxation rates over time. With increasing ligand brush length, attractive inter-particle interactions are reduced, which results in slower aggregate formation and shorter linear aggregate length. These results have implications for the stabilization, characterization and potentially the toxicity of magnetic nanoparticle systems used in biomedical applications.

Intrinsic Dynamics of DNA–Polymer Complexes: A Mechanism for DNA Release

The transfer of genetic material into cells using nonviral vectors offers unique potential for therapeutics; however, the efficacy of delivery depends upon a poorly understood, multistep pathway, limiting the prospects for successful gene delivery. Mechanistic insight into DNA association and release has been hampered by a lack of atomic resolution structural and dynamic information for DNA-polymer complexes (polyplexes). Here, we report a dendrimer-based polyplex system containing poly(ethyleneglycol) (PEG) arms that is suitable for atomic-level characterization by solution NMR spectroscopy. NMR chemical shift, line width, and proton transverse relaxation rate measurements reveal that free and dendrimer-bound polyplex DNA exchange rapidly relative to the NMR time scale (<millisecond). The dendrimers retain a high degree of mobility in the polyplex, whereas the DNA shows restrained mobility, suggesting that the polyplex is a highly dynamic complex with a rapidly exchanging dendrimer atmosphere around a more rigid DNA framework.

A convenient method for the measurements of transverse relaxation rates in homonuclear scalar coupled spin systems

A new solution-state NMR method is proposed to determine apparent transverse NMR relaxation rates in both weakly and strongly scalar coupled two-spin systems.

Distribution of cardiac iron measured by magnetic resonance imaging (MRI)‐R*2

To assess regional iron distribution by magnetic resonance imaging (MRI)-R(2)* within the heart of patients with β-thalassemia major (TM) and other iron overload diseases. Breathhold electrocardiogram (ECG)-gated MRI (1.5 T) of the heart was used for the measurement of transverse relaxation rates R(2)* in 32 patients (11-79 years). In a mid-papillary short-axis slice divided into septal, anterior, lateral, and posterior quadrants, R(2)* was analyzed from region of interest (ROI)-based signal intensities from 12 echo times (TE = 1.3-26 msec). Typical boundary effects were evaluated in detail. The segmentation of the cardiac wall resulted in highly significant correlations of R(2)* between septal and all other quadrants. In the patient group with R(2)* < 50 s(-1) (normal), all quadrants show higher normalized median rates (126%-174%) than the septum (P < 10(-4)), while this was relatively smaller in the group with septal R(2)* > 50 s(-1). Typical boundary effects on segmental R(2)* from blood, lung tissue, epicardial fat, and hepatic iron could not be easily separated from segmental iron distribution. The measurement of MRI-R2* in the interventricular septum is the least affected method by boundary effects to detect patients with iron overload at risk of developing heart failure.

The optimal MR acquisition strategy for exponential decay constants estimation

Estimating the relaxation constant of an exponentially decaying signal from experimental MR data is fundamental in diffusion tensor imaging, fractional anisotropy mapping, measurements of transverse relaxation rates and contrast agent uptake. The precision of such measurements depends on the choice of acquisition parameters made at the design stage of the experiments. In this report, chi(2) fitting of multipoint data is used to demonstrate that the most efficient acquisition strategy is a two-point scheme. We also conjecture that the smallest coefficient of variation of the decay constant achievable in any N-point experiment is 3.6 times larger than that in the image intensity obtained by averaging N acquisitions with minimal exponential weighting.

Improved transverse relaxation rate measurement techniques for the assessment of hepatic and myocardial iron content

To develop and validate an optimized respiratory-gated, gradient-echo sampling of free induction decay and echo (GESFIDE) pulse sequence for the simultaneous measurement of R2, R2*, and R2' in the liver or heart. Fifteen subjects (12 thalassemia patients and three normal volunteers) were scanned using an optimized navigator-gated GESFIDE pulse sequence for the measurement of R2, R2*, and R2' in the liver and heart. For imaging the myocardium, dark-blood preparation was used to suppress the blood signal to improve accuracy. The results were compared with those obtained from breath-held GESFIDE and multi-gradient-echo (GRE) scans. Good agreement between breath-held and navigator-gated scans was found for R2, R2*, and R2' values in the liver (slopes = 0.97-0.99, r = 0.997-0.998, P < 0.0001) and for R2* in the heart (slope = 1.02, r = 0.85, P < 0.0001). Both R2* and R2' were closely correlated to R2 in the liver, with correlation factors of 0.998 and 0.994, respectively, but weaker correlations were observed in the heart (r = 0.72 for R2* vs. R2 and r = 0.51 for R2' vs. R2). The improved sequence enables free-breathing measurements of transverse relaxation rates of the myocardium and liver. The method precludes the need for multiple breath-held scans and possible misregistration issues, and may prove most beneficial for imaging young children and patients who may have difficulty with prolonged or repeated breath-holds.

Study of the behaviour of amino acids in aqueous solution by time‐domain NMR and high‐resolution NMR

The study of protein hydration by time-domain NMR is complicated by the great number of interactions involved, resulting from the presence of several amino acids and the possible modifications produced by the various structures. Moreover, a good comprehension of the molecular interactions of the simple amino acids in solution is essential to elucidate the mechanism of the biological functions of proteins. Measurements of transverse relaxation rates of the protons of water (R(2) = 1/T(2)) in aqueous solutions of amino acids such as L-glycine, L-asparagine, L-arginine and L-tryptophan were carried out in order to study the effects of chemical exchange and molecular diffusion on the amplitude of R(2). The values of R(2) measured by the Carr-Purcell-Meiboom-Gill (CPMG) sequence were studied while varying the solution pH and the parameters of the CPMG sequence. The dependence of R(2) on pH and tau (inter-pulse delay between the first and the second pulses of the CPMG sequence) is interpreted in terms of chemical exchange between the protons of water and those of the labile amino acid groups. This interpretation was confirmed by the analysis of the proton spectra acquired using a 300 MHz NMR spectrometer.

Detection limits for ferrimagnetic particle concentrations using magnetic resonance imaging based proton transverse relaxation rate measurements

A clinical magnetic resonance imaging (MRI) system was used to measure proton transverse relaxation rates (R2) in agar gels with varying concentrations of ferrimagnetic iron oxide nanoparticles in a field strength of 1.5 T. The nanoparticles were prepared by coprecipitation of ferric and ferrous ions in the presence of either dextran or polyvinyl alcohol. The method of preparation resulted in loosely packed clusters (dextran) or branched chains (polyvinyl alcohol) of particles containing of the order of 600 and 400 particles, respectively. For both methods of particle preparation, concentrations of ferrimagnetic iron in agar gel less than 0.01 mg ml−1 had no measurable effect on the value of R2 for the gel. The results indicate that MRI-based R2 measurements using 1.5 T clinical scanners are not quite sensitive enough to detect the very low concentrations of nanoparticulate biogenic magnetite reported in human brain tissue.

Multiple-quantum J-resolved NMR spectroscopy (MQ-JRES): measurement of multiple-quantum relaxation rates and relative signs of spin coupling constants.

The technique of multiple-quantum J-resolved NMR spectroscopy (MQ-JRES) is introduced and applied to the spin system SI(3)-M (such as in the example given here, the (13)CH(3)-(12)CH in alanine). The SI(3) spin system was excited to its highest quantum state (8S(y)I(x)I(y)I(y)), which consists of four coherences: quadruple quantum of (3I + S), double quantum of (3I - S), double quantum of (I + S), and zero quantum of (I - S). In the MQ spectrum generated from the projection onto the F(1) dimension, the resonances of the different multiple-quantum coherences are resolved by their coupling constants to the remote spin (M). The absorptive lineshapes in both F(1) and F(2) dimensions enable accurate measurements of transverse relaxation rates, and both amplitude and relative signs of the long-range coupling constants are to be derived from either frequency or time domain data. The selective detection of MQ-JRES spectra of the individual MQ coherences using either phase cycling or pulsed field gradients is presented.

Estimating the time scale of chemical exchange of proteins from measurements of transverse relaxation rates in solution.

Chemical (conformational) exchange on the ms--microsecond time scale is reliably identified by the observation of transverse relaxation rates, Rex, that depend upon the strength of the effective field (omega 1eff = gamma B1eff) used in spin lock or CPMG experiments. In order to determine if the exchange correlation time, tau ex, is the fast or slow limit, measurements of (i) signal line shape and (ii) temperature dependence of Rex have been commonly used in studies of stable, small molecules. However, these approaches are often not applicable to proteins, because sample stability and solubility, respectively, limit the temperature range and signal sensitivity of experiments. Herein we use a complex, but general, two-site exchange equation to show when the simple fast exchange equations for Rex are good approximations, in the case of proteins. We then present a simple empirical equation that approximately predicts Rex in all exchange regimes, and explains these results in a clear, straightforward manner. Finally we show how one can reliably determine whether tau ex is in the fast or slow exchange limit.

Water Mobility and Crystallization Action of Lactose‐water Systems by Oxygen‐17 and Carbon‐13 NMR

ABSTRACTSystems ranging in concentration from 10‐60g lactose/100g water were investigated by fully proton decoupled 17O and fully proton decoupled 13C Nuclear Magnetic Resonance (NMR). The relation between 17O NMR transverse relaxation rate measurements and lactose concentration showed water mobility decreased linearly with increasing lactose concentration up to 25g lactose/100g water and decreased rapidly and nonlinearly above that level. Water mobility was used to monitor lactose crystallization in supersaturated solutions with time. The 13C NMR showed no significant differences in chemical shifts of carbon atom peaks in lactose molecules during crystallization. However, depending on the method of sample preparation, mutarotation could be observed.

Zn(II)–D-(−)penicillamine complexes in aqueous solution. Zn-67 nuclear magnetic resonance study

D–(−)-Penicillamine interactions with Zn(II) have been studied in aqueous solutions as a function of pH, penicillamine concentration, and temperature, using Zn-67 nuclear magnetic resonance. Longitudinal and transverse relaxation rate measurements show the presence in solution of complexes in fast exchange with the aquated Zn(II) cation, and belonging to the extreme narrowing regime. Using equilibrium constant values from the literature, the relaxation behaviour was modelled. Characteristic Zn-67 line width values for the two complexes Zn(Pen) (v = (6200 ± 500) Hz) and [Zn(Pen H)]+ (v = (6000 ± 1000) Hz) were found. Equality of the two values is in agreement with zinc chelation by the sulfhydryl and the amino groups.

Errors in measurements of transverse relaxation rates

Errors which may occur in the measurement of transverse relaxation rates in liquids by wideband multiple pulse techniques are described. Numerical analysis of Bloch equations is used to evaluate the effects of imperfect pulses, and results of pulse imperfections are demonstrated experimentally. Effects of field-frequency instability and of diffusion combined with external field gradients are discussed. Methods for avoiding several of these errors are described and applied to the measurement of T 2 for degassed benzene. It is experimentally demonstrated that if certain criteria are met, measurements of T 2 with accuracy of ±1–2% are feasible.