Flip-back Pulse Research Articles

A facile approach for estimating radio-frequency field strength of low-receptivity nuclei

Radio-frequency (RF) field calibration is essential in NMR spectroscopy. A common practice is to collect a nutation curve by varying the pulse length in a direct single-pulse excitation experiment or in a cross-polarization magic-angle spinning with a flip-back pulse experiment. From the null points on this curve, one can calculate the RF field strength. Nevertheless, the practical implementation is not always straightforward or can even be unrealizable, especially for low-receptivity nuclei owing to their associated low sensitivity. Several researchers used an approach that involves utilizing other nuclei with more sensitivity but nearly identical Larmor frequencies to that of the nucleus of interest. However, such an approach has not been a common practice so far. In this work, we have systematically revisited this approach using 3.2 mm rotors on different sets of nuclei covering a Larmor frequency range up to 80 MHz. The effect of solid- and solution-states on RF field strength measurements has been investigated. The detection of each set of nuclei is then carried out with a resonant circuit in the NMR probe consisting of identical coils and capacitors. Our methodology is illustrated by recording 135/137Ba NMR spectra of BaTiO3 without prior 135/137Ba RF field calibration.

Broadband Cross-Polarization to Half-Integer Quadrupolar Nuclei: Wideline Static NMR Spectroscopy.

Cross-polarization (CP) is a technique commonly used for the signal enhancement of NMR spectra; however, applications to quadrupolar nuclei have heretofore been limited due to a number of problems, including poor spin-locking efficiency, inconvenient relaxation times, and reduced CP efficiencies over broad spectral bandwidths─this is unfortunate, since they constitute 73% of NMR-active nuclei in the periodic table. The Broadband Adiabatic Inversion CP (BRAIN-CP) pulse sequence has proven useful for the signal enhancement of wideline and ultra-wideline (i.e., 250 kHz to several MHz in breadth) powder patterns arising from stationary samples; however, a comprehensive investigation of its application to half-integer quadrupolar nuclei (HIQN) is currently lacking. Herein, we present theoretical and experimental considerations for applying BRAIN-CP to acquire central-transition (CT, +1/2 ↔ -1/2) powder patterns of HIQN. Consideration is given to parameters crucial to the success of the experiment, such as the Hartmann-Hahn (HH) matching conditions and the phase modulation of the contact pulse. Modifications to the BRAIN-CP sequence such as flip-back (FB) pulses and ramped contact pulses applied to the 1H spins are used for the reduction of experimental times and increased CP bandwidth capabilities, respectively. Spectra for a series of quadrupolar nuclei with broad CT powder patterns, including 35Cl (S = 3/2), 55Mn (S = 5/2), 59Co (S = 7/2), and 93Nb (S = 9/2), are acquired via direct excitation (CPMG and WCPMG) and indirect excitation (CP/CPMG and BRAIN-CP) methods. We demonstrate that proper implementation of the sequence can enable 1H-S broadband CP over a bandwidth of 1 MHz, which to the best of our knowledge is the largest CP bandwidth reported to date. Finally, we establish the basic principles necessary for simplified optimization and execution of the BRAIN-CP pulse sequence for a wide range of HIQNs.

Axial T2-DRIVE MRI myelography is highly accurate in diagnosing preganglionic traumatic brachial plexus injuries: why pseudomeningoceles should not be used as a primary diagnostic sign

To evaluate the accuracy of brachial plexus magnetic resonance imaging (MRI) utilising the three-dimensional (3D)-T2-turbo spin echo (TSE) with 90° flip-back pulse ("DRIVE") myelography in detecting nerve root avulsions in patients with traumatic brachial plexus injuries. A prospective study of 24 patients planned for brachial plexus reconstructive surgery following trauma from April 2019 to October 2021. Preoperative 1.5 T MRI of the brachial plexus was performed utilising axial T2-DRIVE, looking for signs of avulsions (absent dural rootlets, pauci-rootlet appearance and thickened rootlets; the presence of pseudomeningoceles was noted only as an ancillary sign). Comparison against the reference standard of extra-dural brachial plexus exploration was performed. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated. Eighteen patients had at least one root avulsion at surgery, five showed isolated post-ganglionic injuries, and one had normal brachial plexus exploration. Thirty-nine avulsed roots were found at surgery (out of 108 explored in 24 patients). Preoperative MRI identified the specific avulsed roots accurately in each patient. Two false-positive diagnoses of C5 and C6 avulsions were made in one patient. On MRI, absence of the rootlets was seen in 73.2% (n=30) of avulsions, pauci-rootlet appearance in 24.4% (n=10) and thickening of the rootlets in 2.4% (n=1). Pseudomeningoceles were found only in 68.3% (n=28) of avulsions. The overall sensitivity, specificity, PPV, NPV, and accuracy of MRI were 100%, 97.1%, 95.1%, 100% and 98.1%, respectively. 3D-T2-DRIVE is highly accurate in evaluating pre-ganglionic traumatic brachial plexus injuries. Pseudomeningoceles can be considered an ancillary feature of avulsion given the clarity of rootlet visualisation by this sequence.

Sensitivity enhancement in 2D Double Cross Polarization experiments under fast MAS by recycling unused protons

We demonstrate sensitivity enhancement via recycling of proton magnetization in 2D Double Cross Polarization (Double CP) experiments performed on fully protonated and uniformly labeled (13C, 15N) samples at a magic angle spinning rate of 60 kHz. Unused proton magnetization is preserved during t1 evolution either by locking it with CW irradiation or by employing rotor-synchronized pi pulses. A flip-back pulse together with a modified second CP block preserves unused proton magnetization resulting in enhanced sensitivity. We have achieved sensitivity enhancements of 15–20% and 25–28% in 1H–13C and 1H–15N 2D Double CP experiments respectively. At shorter recycle delays (∼0.25T1), relative sensitivity enhancements of 40–45% and 55% were obtained in 1H–13C and 1H–15N 2D Double CP experiments respectively. An analysis of the sensitivity enhancements and theoretical estimation of lineshapes in indirect dimension in the presence of proton recycling is provided.

Maximizing the sensitivity in 13C cross-polarization magic-angle-spinning solid-state NMR measurements with flip-back pulses

The flip-back pulse combined with cross-polarization magic-angle spinning (CPMAS-FB) has been widely used to reduce the optimal repetition delay, resulting in enhancing sensitivity per unit time. Despite its common use in samples with long 1H T1 relaxation time, the experimental setup of the repetition delay in CPMAS-FB is not obvious. In this article, a simple model is used to derive the optimal repetition delay and expected sensitivity gain through the 1H T1 relaxation time and signal decay during 1H spinlock.

Forcing the 'lazy' protons to work.

The combination of cross-polarization (CP) with flip-back (FB) pulse has enabled in NMR the enhancement of 13C sensitivity and the decrease of the recycling delay at both moderate and fast magic-angle spinning (MAS) frequencies. However, only continuous-wave (CW) decoupling is presently compatible with FB-pulse (FB-CW), and depending on the CW radio-frequency (rf) field, either an insignificant sensitivity gain or an acquisition time-dependent gain and a low 13C resolution are obtained. In this study, we propose a new FB-pulse method in which radio frequency-driven recoupling (RFDR) is used as the 1H-13C decoupling scheme to overcome these drawbacks. The performances of FB-RFDR in terms of decoupling efficiency and sensitivity gain are tested on both natural abundance (NA) and uniformly 13C-15N labeled l-histidine·HCl·H2O (Hist) samples at a MAS frequency of νR = 70 kHz. The results show the superiority of RFDR over the CW decoupling with respect to these criteria. Importantly, they reveal that the sensitivity gain offered by FB-RFDR is nearly independent of the decoupling/acquisition duration. The application of FB-RFDR on NA-Hist and sucrose yields a sensitivity gain between 60 and 100% compared to conventional FB-CW and CPMAS-SPINAL experiments. Moreover, we compare the 13C sensitivities of NA-Hist obtained by our 1D FB-RFDR method and 2D 1H-{13C} double-CP acquisition. Both methods provide similar 13C sensitivity and are complementary. Indeed, the 2D method has the advantage of also providing the 1H-13C spatial proximities, but its sensitivity for quaternary carbons is limited; whereas our 1D FB-RFDR method is more independent of the type of carbon, and can provide a 13C 1D spectrum in a shorter experimental time. We also test the feasibility of FB-RFDR at a moderate frequency of νR = 20 kHz, but the experimental results demonstrate a poor resolution as well as a negligible sensitivity gain.

Recovery of bulk proton magnetization and sensitivity enhancement in ultrafast magic-angle spinning solid-state NMR.

The sensitivity of solid-state NMR experiments is limited by the proton magnetization recovery delay and by the duty cycle of the instrument. Ultrafast magic-angle spinning (MAS) can improve the duty cycle by employing experiments with low-power radio frequency (RF) irradiation which reduce RF heating. On the other hand, schemes to reduce the magnetization recovery delay have been proposed for low MAS rates, but the enhancements rely on selective transfers where the bulk of the (1)H magnetization pool does not contribute to the transfer. We demonstrate here that significant sensitivity enhancements for selective and broadband experiments are obtained at ultrafast MAS by preservation and recovery of bulk (1)H magnetization. We used [(13)C, (15)N]-labeled glutamine as a model compound, spinning in a 1.3 mm rotor at a MAS frequency of 65 kHz. Using low-power (1)H RF (13.4 kHz), we obtain efficient (1)H spin locking and (1)H-(13)C decoupling at ultrafast MAS. As a result, large amounts of (1)H magnetization, from 35% to 42% of the initial polarization, are preserved after cross-polarization and decoupling. Restoring this magnetization to the longitudinal axis using a flip-back pulse leads to an enhancement of the sensitivity, an increase ranging from 14% to 21% in the maximal achievable sensitivity regime and from 24% to 50% in the fast pulsing regime, and to a shortening of the optimal recycling delay to 68% of its original duration. The analysis of the recovery and sensitivity curves reveals that the sensitivity gains do not rely on a selective transfer where few protons contribute but rather on careful conservation of bulk (1)H magnetization. This makes our method compatible with broadband experiments and uniformly labeled materials, in contrast to the enhancement schemes proposed for low MAS. We tested seven different cross-polarization schemes and determined that recovery of bulk (1)H magnetization is a general method for sensitivity enhancement. The physical insight gained about the behavior of proton magnetization sharing under spin lock will be helpful to break further sensitivity boundaries, when even higher external magnetic fields and faster spinning rates are employed.

Multi-echo length and offset VARied saturation (MeLOVARS) method for improved CEST imaging.

The aim of this study was to develop a technique for rapid collection of chemical exchange saturation transfer images with the saturation varied to modulate signal loss transfer and enhance contrast. Multi-echo Length and Offset VARied Saturation (MeLOVARS) divides the saturation pulse of length Tsat into N = 3-8 submodules, each consisting of a saturation pulse with length of Tsat /N (∼0.3-1 s), one or more low flip-angle gradient-echo readout(s) and a flip back pulse. This results in N readouts with increasing saturation time from Tsat /N to Tsat without extra scan time. For phantoms, eight images with Tsat incremented every 0.5 s from 0.5-4 s were collected simultaneously using MeLOVARS, which allows rapid determination of exchange rates for agent protons. For live mice bearing glioblastomas, the Z-spectra for five different Tsat values from 0.5 to 2.5 s were acquired in a time normally used for one Tsat . With the additional Tsat -dependence information, LOVARS phase maps were produced with a more clearly defined tumor boundary and an estimated 4.3-fold enhanced contrast-to-noise ratio (CNR). We also show that enhancing CNR is achievable by simply averaging the collected images or transforming them using the principal component analysis. MeLOVARS enables collection of multiple saturation-time-weighted images without extra time, producing a LOVARS phase map with increased CNR.

Advanced new relaxation filter-selective signal excitation methods for (13)C solid-state nuclear magnetic resonance.

We have developed new relaxation filter-selective signal excitation (RFS) methods for (13)C solid-state NMR, which enable extraction of the spectrum of a target component from a mixture of several components. These methods are based on the equalization of proton relaxation time in a single domain via rapid intraproton spin diffusion and the difference in proton relaxation time of individual components in the mixture. We recently reported two types of RFS methods using proton spin-lattice relaxation time in the rotating frame ((1)H T1rho) in (13)C solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, to increase the availability of RFS methods, we focus on proton spin-lattice relaxation time ((1)H T1). Introduction of simple pulse sequences to one-dimensional experiments reduced data acquisition time and increased flexibility, and led to the development of two new types of RFS methods using (1)H T1. We then demonstrated these methods by selectively exciting the (13)C signals of target components in a commercially available drug and a number of physical mixtures, and we showed them to be applicable to the quantitative analysis of individual components in these solid mixtures with an experimental duration of 1.5 to about 10 h. The practicality and versatility of these four RFS methods were increased by combining two or more of them, or by using a flip-back pulse, which is an effective means of shortening experimental duration. These RFS methods are suitable for use in a broad range of fields.

Optimization of rapid acquisition with relaxation enhancement (RARE) pulse sequence parameters for ¹⁹F-MRI studies.

To optimize signal-to-noise ratio (SNR) in fast spin echo (rapid acquisition with relaxation enhancement [RARE]) sequences and to improve sensitivity in ¹⁹F magnetic resonance imaging (MRI) on a 7T preclinical MRI system, based on a previous experimental evaluation of T₁ and T₂ actual relaxation times. Relative SNR changes were theoretically calculated at given relaxation times (T₁, T₂) and mapped in RARE parameter space (TR, number of echoes, flip back pulse), at fixed acquisition times. T₁ and T₂ of KPF₆ phantom samples (solution, agar mixtures, ex vivo perfused brain) were measured and experimental SNR values were compared with simulations, at optimal and suboptimal RARE parameter values. The optimized setting largely depended on T₁, T₂ times and the use of flip back pulse improved SNR up to 30% in case of low T₁/T₂ ratios. Relaxation times in different conditions showed negligible changes in T₁ (below 14%) and more evident changes in T₂ (-95% from water solution to ex vivo brain). Experimental data confirmed theoretical forecasts, within an error margin always below 4.1% at SNR losses of ~20% and below 8.8% at SNR losses of ~40%. The optimized settings permitted a detection threshold at a concentration of 0.5 mM, corresponding to 6.22 × 10¹⁶ fluorine atoms per voxel. Optimal settings according to measured relaxation times can significantly improve the sensitivity threshold in ¹⁹F MRI studies. They were provided in a wide range of (T₁, T₂) values and experimentally validated showing good agreement.

Water proton spin saturation affects measured protein backbone 15N spin relaxation rates

Protein backbone 15N NMR spin relaxation rates are useful in characterizing the protein dynamics and structures. To observe the protein nuclear-spin resonances a pulse sequence has to include a water suppression scheme. There are two commonly employed methods, saturating or dephasing the water spins with pulse field gradients and keeping them unperturbed with flip-back pulses. Here different water suppression methods were incorporated into pulse sequences to measure 15N longitudinal T 1 and transversal rotating-frame T 1 ρ spin relaxation. Unexpectedly the 15N T 1 relaxation time constants varied significantly with the choice of water suppression method. For a 25-kDa Escherichia coli. glutamine binding protein (GlnBP) the T 1 values acquired with the pulse sequence containing a water dephasing gradient are on average 20% longer than the ones obtained using a pulse sequence containing the water flip-back pulse. In contrast the two T 1 ρ data sets are correlated without an apparent offset. The average T 1 difference was reduced to 12% when the experimental recycle delay was doubled, while the average T 1 values from the flip-back measurements were nearly unchanged. Analysis of spectral signal to noise ratios ( s/ n) showed the apparent slower 15N relaxation obtained with the water dephasing experiment originated from the differences in 1H N recovery for each relaxation time point. This in turn offset signal reduction from 15N relaxation decay. The artifact becomes noticeable when the measured 15N relaxation time constant is comparable to recycle delay, e.g., the 15N T 1 of medium to large proteins. The 15N relaxation rates measured with either water suppression schemes yield reasonable fits to the structure. However, data from the saturated scheme results in significantly lower Model-Free order parameters (〈 S 2〉 = 0.81) than the non-saturated ones (〈 S 2〉 = 0.88), indicating such order parameters may be previously underestimated.

Flip-back, an old trick to face highly contrasted relaxation times: Application in the characterization of pharmaceutical mixtures by CPMAS NMR

The 13C– 1H CPMAS with flip-back pulse NMR experiment is revisited in view of applications to pharmaceutical mixtures. The analysis of the kinetics of relaxation and CP transfer with and without the flip-back pulse shows that a significant gain in 13C signal can be expected (thus in experimental time) from the flip-back pulse for protons with long T 1. The gain is of the order of T 1 of the protons expressed in seconds. The experiment is applied on samples with highly contrasted spin-lattice relaxation times T 1 for protons, situation encountered in pharmaceutical mixtures. The application of the flip-back increases significantly the relative signal intensity of the component with the longer T 1, making this component detectable even after using short recycle delays. Therefore, this CPMAS with flip-back experiment could be used routinely to get 13C CPMAS NMR spectra of mixtures in constant experimental time and signal-to-noise ratio without the need for optimization of the recycle delays, and for whatever may be the degree of crystallinity of the active principal ingredient (API) and/or excipients.

Modulation of the Structure, Catalytic Activity, and Fidelity of African Swine Fever Virus DNA Polymerase X by a Reversible Disulfide Switch

African swine fever virus polymerase X (pol X) is the smallest DNA polymerase known (174 amino acids), and its tertiary structure resembles the C-terminal half of prototypical X-family pol beta, which includes a catalytic dNTP-binding site (palm domain) and a finger domain. This structural similarity and the presence of viral genes coding for other base excision repair proteins suggest that pol X functions in a manner similar to pol beta, but inconsistencies concerning pol X catalysis have been reported. We examined the structural and functional properties of two forms of pol X using spectroscopic and kinetic analysis. Using (1)H-(15)N correlated NMR, we unambiguously demonstrated the slow interconversion of pol X between a reduced (pol X(red)) and an oxidized form (pol X(ox)), confirmed by mass spectrometry. Steady-state kinetic analysis revealed that pol X(ox), with a disulfide bond between Cys-81 and Cys-86, has approximately 10-fold lower fidelity than pol X(red) during dNTP insertion opposite a template G. The disulfide linkage is located between two beta-strands in the palm domain, near the putative dNTP-binding site. Structural alignment of pol X with a pol beta ternary structure suggests that the disulfide switch may modulate fidelity by altering the ability of the palm domain to align and stabilize the primer terminus and catalytic metal ion for deprotonation of the 3'-OH group and subsequent phosphoryl transfer. Thus, DNA polymerase fidelity is altered by the redox state of the enzyme and its related conformational changes.

8013 ORAL Disclosing/Informing the diagnosis of cancer to Turkish People and their close relatives: An Opinion Survey

In experiments performed on protonated proteins at high fields, 80% of the NMR spectrometer time is spent waiting for the 1H atoms to recover their polarization after recording the free induction decay. Selective excitation of a fraction of the protons in a large molecule has previously been shown to lead to faster longitudinal relaxation for the selected protons [K. Pervushin, B. Vögeli, A. Eletsky, Longitudinal 1H relaxation optimization in TROSY NMR spectroscopy, J. Am. Chem. Soc. 124 (2002) 12898–12902; P. Schanda, B. Brutscher, Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds, J. Am. Chem. Soc. 127 (2005) 8014–8015; H.S. Attreya, T. Szyperski, G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment, Proc. Natl. Acad. Sci. USA 101 (2004) 9642–9647]. The pool of non-selected protons acts as a “thermal bath” and spin-diffusion processes (“flip-flop” transitions) channel the excess energy from the excited pool to the non-selected protons in regions of the molecule where other relaxation processes can dissipate the excess energy. We present here a sensitivity enhanced HSQC sequence (COST-HSQC), based on one selective E-BURP pulse, which can be used on protonated 15N enriched proteins (with or without 13C isotopic enrichment). This experiment is compared to a gradient sensitivity enhanced HSQC with a water flip-back pulse (the water flip-back pulse quenches the spin diffusion between 1HN and 1Hα spins). This experiment is shown to have significant advantages in some circumstances. Some observed limitations, namely sample overheating with short recovery delays and complex longitudinal relaxation behaviour are discussed and analysed.

Unambiguous Determination of the Ionization State of a Glycoside Hydrolase Active Site Lysine by 1H−15N Heteronuclear Correlation Spectroscopy

We have investigated the lysine side chain amines in the 34 kDa catalytic domain from Cellulomonas fimi beta-(1,4)-glycosidase Cex (or CfXyn10A) using 1H-detected 15N heteronuclear correlation NMR spectroscopy. Signals from the 1Hzeta ( approximately 8 ppm) and 15Nzeta ( approximately 35 ppm) of Lys302 in the unmodified enzyme and Lys47 in a trapped cellobiosyl-enzyme intermediate were detected in a 1H-15N HMQC spectrum (pH 6.5 and 30 degrees C). The amine of Lys302 forms a buried ion pair, and that of Lys47 is hydrogen bonded to the cellobioside. Both lysines are positively charged, as unambiguously demonstrated by the splitting of their 15Nzeta signals into quartets (|1JNH| approximately 75 Hz) in a 1H-15N HSQC spectrum recorded without 1H decoupling during 15N evolution. Qualitative insights into the dynamic properties of these lysines are also provided by the deviations of their quartet intensity ratios from that of approximately 3:1:1:3 expected for a highly mobile amine. On the basis of the observed ratios of approximately 1:1:1:1 for the quartet of Lys302 and approximately 0.5:1:1:0.5 for Lys47, the amine of the latter active site residue is most rigidly positioned. Signals from at least 8 and 10 additional positively charged, mobile amines in Cex were observed at 10 degrees C and pH 6.5 and 5.6, respectively. By using conditions of reduced temperature, slightly acidic pH, and low general base concentrations, as well as water flipback pulses to minimize the effects of hydrogen exchange, 1H-15N correlation experiments provide a sensitive route to directly investigate the charge states and dynamic properties of the N-terminal and side chain amines in proteins and protein complexes.

Cooling overall spin temperature: Protein NMR experiments optimized for longitudinal relaxation effects

In experiments performed on protonated proteins at high fields, 80% of the NMR spectrometer time is spent waiting for the 1H atoms to recover their polarization after recording the free induction decay. Selective excitation of a fraction of the protons in a large molecule has previously been shown to lead to faster longitudinal relaxation for the selected protons [K. Pervushin, B. Vögeli, A. Eletsky, Longitudinal 1H relaxation optimization in TROSY NMR spectroscopy, J. Am. Chem. Soc. 124 (2002) 12898–12902; P. Schanda, B. Brutscher, Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds, J. Am. Chem. Soc. 127 (2005) 8014–8015; H.S. Attreya, T. Szyperski, G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment, Proc. Natl. Acad. Sci. USA 101 (2004) 9642–9647]. The pool of non-selected protons acts as a “thermal bath” and spin-diffusion processes (“flip-flop” transitions) channel the excess energy from the excited pool to the non-selected protons in regions of the molecule where other relaxation processes can dissipate the excess energy. We present here a sensitivity enhanced HSQC sequence (COST-HSQC), based on one selective E-BURP pulse, which can be used on protonated 15N enriched proteins (with or without 13C isotopic enrichment). This experiment is compared to a gradient sensitivity enhanced HSQC with a water flip-back pulse (the water flip-back pulse quenches the spin diffusion between 1H N and 1H α spins). This experiment is shown to have significant advantages in some circumstances. Some observed limitations, namely sample overheating with short recovery delays and complex longitudinal relaxation behaviour are discussed and analysed.

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

In large molecular structures, the magnetization of all hydrogen atoms in the solute is strongly coupled to the water magnetization through chemical exchange between solvent water and labile protons of macromolecular components, and through dipole-dipole interactions and the associated "spin diffusion" due to slow molecular tumbling. In NMR experiments with such systems, the extent of the water polarization is thus of utmost importance. This paper presents a formalism that describes the propagation of the water polarization during the course of different NMR experiments, and then compares the results of model calculations for optimized water polarization with experimental data. It thus demonstrates that NMR spectra of large molecular structures can be improved with the use of paramagnetic spin relaxation agents which selectively enhance the relaxation of water protons, so that a substantial gain in signal-to-noise can be achieved. The presently proposed use of a relaxation agent can also replace the water flip-back pulses when working with structures larger than about 30 kDa. This may be a valid alternative in situations where flip-back pulses are difficult to introduce into the overall experimental scheme, or where they would interfere with other requirements of the NMR experiment.

Structural and Dynamics Studies of the D54A Mutant of Human T Cell Leukemia Virus-1 Capsid Protein

The human T cell leukemia virus and the human immunodeficiency virus share a highly conserved, predominantly helical two-domain mature capsid (CA) protein structure with an N-terminal beta-hairpin. Despite overall structural similarity, differences exist in the backbone dynamic properties of the CA N-terminal domain. Since studies with other retroviruses suggest that the beta-hairpin is critical for formation of a CA-CA interface, we investigated the functional role of the human T cell leukemia virus beta-hairpin by disrupting the salt bridge between Pro(1) and Asp(54) that stabilizes the beta-hairpin. NMR (15)N relaxation data were used to characterize the backbone dynamics of the D54A mutant in the context of the N-terminal domains, compared with the wild-type counterpart. Moreover, the effect of the mutation on proteolytic processing and release of virus-like particles (VLPs) from human cells in culture was determined. Conformational and dynamic changes resulting from the mutation were detected by NMR spectroscopy. The mutation also altered the conformation of mature CA in cells and VLPs, as reflected by differential antibody recognition of the wild-type and mutated CA proteins. In contrast, the mutation did not detectably affect antibody recognition of the CA protein precursor or release of VLPs assembled by the precursor, consistent with the fact that the hairpin cannot form in the precursor molecule. The particle morphology and size were not detectably affected. The results indicate that the beta-hairpin contributes to the overall structure of the mature CA protein and suggest that differences in the backbone dynamics of the beta-hairpin contribute to mature CA structure, possibly introducing flexibility into interface formation during proteolytic maturation.

Hyperpolarized 13C MR angiography using trueFISP.

A (13)C-enriched water-soluble compound (bis-1,1-(hydroxymethyl)-1-(13)C-cyclopropane-D(8)), with a (13)C-concentration of approximately 200 mM, was hyperpolarized to approximately 15% using dynamic nuclear polarization, and then used as a contrast medium (CM) for contrast-enhanced magnetic resonance angiography (CE-MRA). The long relaxation times (in vitro: T(1) approximately 82 s, T(2) approximately 18 s; in vivo: T(1) approximately 38 s, T(2) approximately 1.3 s) are ideal for steady-state free precession (SSFP) imaging with a true fast imaging and steady precession (trueFISP) pulse sequence. It was shown both theoretically and experimentally that the optimal flip angle was 180 degrees. CE-MRA was performed in four anesthetized live rats after intravenous injection of 3 ml CM. The angiograms covered the thoracic/abdominal region in two of the animals, and the head-neck region in the other two. Fifteen consecutive images were acquired in each experiment, with a flip-back pulse at the end of each image acquisition. In the angiograms, the vena cava (SNR approximately 240), aorta, renal arteries, carotid arteries (SNR approximately 75), jugular veins, and several other vessels were visible. The SNR in the cardiac region was 500. Magnetization was preserved from one image acquisition to the next using the flip-back technique (SNR(cardiac) approximately 10 in the 15th image).

Solution NMR techniques for large molecular and supramolecular structures.

Transverse relaxation-optimized spectroscopy (TROSY) or generation of heteronuclear multiple quantum coherences during the frequency labeling period and TROSY during the acquisition period have been combined either with cross-correlated relaxation-induced polarization transfer (CRIPT) or cross-correlated relaxation-enhanced polarization transfer (CRINEPT) to obtain two-dimensional (2D) solution NMR correlation spectra of (15)N,(2)H-labeled homo-oligomeric macromolecules with molecular weights from 110 to 800 kDa. With the experimental conditions used, the line widths of the TROSY-components of the (1)H- and (15)N-signals were of the order of 60 Hz at 400 kDa, whereas, for structures of size 800 kDa, the line widths were about 75 Hz for (15)N and 110 Hz for (1)H. This paper describes the experimental schemes used and details of their setup for individual measurements. The performance of NMR experiments with large structures depends critically on the choice of the polarization transfer times, the relaxation delays between subsequent recordings, and the water-handling routines. Optimal transfer times for 2D [(15)N,(1)H]-CRIPT-TROSY experiments in H(2)O solutions were found to be 6 ms for a molecular weight of approximately 200 kDa, 2.8 ms for 400 kDa, and 1.4 ms for 800 kDa. These data validate theoretical predictions of inverse proportionality between optimal transfer time and size of the structure. The proton longitudinal relaxation times in H(2)O solution were found to be of the order of 0.8 s for structure sizes around 200 kDa, 0.4 s at 400 kDa, and 0.3 s at 800 kDa, which enabled the use of recycle times below 1 s. Since improper water handling results in severe signal loss, the water resonance was kept along the z-axis during the entire duration of the experiments by adjusting each water flip-back pulse individually.