Amide Signals Research Articles

Mitochondrial oxidative phosphorylation capacity in skeletal muscle measured by ultrafast Z-spectroscopy (UFZ) MRI at 3T.

To investigate the feasibility of rapid CEST MRI acquisition for evaluating oxidative phosphorylation (OXPHOS) in human skeletal muscle at 3T, utilizing ultrafast Z-spectroscopy (UFZ) combined with MRI and the Polynomial and Lorentzian line-shape Fitting (PLOF) technique. UFZ MRI on muscle was evaluated with turbo spin echo (TSE) and 3D EPI readouts. Five healthy subjects performed in-magnet plantar flexion exercise (PFE) and subsequent changes of amide, PCr, and partial PCr mixed Cr (Cr+) CEST dynamic signals post-exercise were enabled by PLOF fitting. PCr/Cr CEST signal was further refined through pH correction by using the ratios between PCr/Cr and amide signals, named PCAR/CAR, respectively. UFZ MRI with TSE readout significantly reduces acquisition time, achieving a temporal resolution of <50 s for collecting high-resolution Z-spectra. Following PFE, the recovery/decay times (τ) for both PCr and Cr in the gastrocnemius muscle of the calf were notably longer when determined using PCr/Cr CEST compared to those after pH correction with amideCEST, namely = 87.1 ± 15.8 s and = 98.1 ± 20.4 s versus = 32.9 ± 19.7 s and = 43.0 ± 13.0 s, respectively. obtained via 31P MRS ( = 50.3 ± 6.2 s) closely resemble those obtained from pH-corrected PCr/Cr CEST signals. The outcomes suggest potential of UFZ MRI as a robust tool for non-invasive assessment of mitochondrial function in skeletal muscles. pH correction is critical for the reliable OXPHOS measurement by CEST.

Multi-pool chemical exchange saturation transfer MRI in glioma grading, molecular subtyping and evaluating tumor proliferation.

To evaluate the performance of multi-pool Chemical exchange saturation transfer (CEST) MRI in prediction of glioma grade, isocitrate dehydrogenase (IDH) mutation, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) loss and Ki-67 labeling index (LI), based on the fifth edition of the World Health Organization classification of central nervous system tumors (WHO CNS5). 95 patients with adult-type diffuse gliomas were analyzed. The amide, direct water saturation (DS), nuclear Overhauser enhancement (NOE), semi-solid magnetization transfer (MT) and amine signals were derived using Lorentzian fitting, and asymmetry-based amide proton transfer-weighted (APTwasym) signal was calculated. The mean value of tumor region was measured and intergroup differences were estimated using student-t test. The receiver operating curve (ROC) and area under the curve (AUC) analysis were used to evaluate the diagnostic performance of signals and their combinations. Spearman correlation analysis was performed to evaluate tumor proliferation. The amide and DS signals were significantly higher in high-grade gliomas compared to low-grade gliomas, as well as in IDH-wildtype gliomas compared to IDH-mutant gliomas (all p < 0.001). The DS, MT and amine signals showed significantly differences between ATRX loss and retention in grade 2/3 IDH-mutant gliomas (all p < 0.05). The combination of signals showed the highest AUC in prediction of grade (0.857), IDH mutation (0.814) and ATRX loss (0.769). Additionally, the amide and DS signals were positively correlated with Ki-67 LI (both p < 0.001). Multi-pool CEST MRI demonstrated good potential to predict glioma grade, IDH mutation, ATRX loss and Ki-67 LI.

Pitfalls in measurements of R1 relaxation rates of protein backbone 15N nuclei.

The dynamics of the backbone and side-chains of protein are routinely studied by interpreting experimentally determined 15N spin relaxation rates. R1(15N), the longitudinal relaxation rate, reports on fast motions and encodes, together with the transverse relaxation R2, structural information about the shape of the molecule and the orientation of the amide bond vectors in the internal diffusion frame. Determining error-free 15N longitudinal relaxation rates remains a challenge for small, disordered, and medium-sized proteins. Here, we show that mono-exponential fitting is sufficient, with no statistical preference for bi-exponential fitting up to 800MHz. A detailed comparison of the TROSY and HSQC techniques at medium and high fields showed no statistically significant differences. The least error-prone DD/CSA interference removal technique is the selective inversion of amide signals while avoiding water resonance. The exchange of amide with solvent deuterons appears to affect the rate R1 of solvent-exposed amides in all fields tested and in each DD/CSA interference removal technique in a statistically significant manner. In summary, the most accurate R1(15N) rates in proteins are achieved by selective amide inversion, without the addition of D2O. Importantly, at high magnetic fields stronger than 800MHz, when non-mono-exponential decay is involved, it is advisable to consider elimination of the shortest delays (typically up to 0.32s) or bi-exponential fitting.

Dual contrast CEST MRI for pH-weighted imaging in stroke.

pH enhanced (pHenh ) CEST imaging combines the pH sensitivity from amide and guanidino signals, but the saturation parameters have not been optimized. We propose pHdual as a variant of pHenh that suppresses background signal variations, while enhancing pH sensitivity and potential for imaging ischemic brain injury of stroke. Simulation and in vivo rodent stroke experiments of pHenh MRI were performed with varied RF saturation powers for both amide and guanidino protons to optimize the contrast between lesion/normal tissues, while simultaneously minimizing signal variations across different types of normal tissues. In acute stroke, contrast and volume ratio measured by pHdual imaging were compared with an amide-CEST approach, and perfusion and diffusion MRI. Simulation experiments indicated that amide and guanidino CEST signals exhibit unique sensitivities across different pH ranges, with pHenh producing greater sensitivity over a broader pH regime. The pHenh data of rodent stroke brain demonstrated that the lesion/normal tissue contrast was maximized for an RF saturation power pair of 0.5 μT at 2.0 ppm and 1.0 μT at 3.6 ppm, whereas an optimal contrast-to-variation ratio (CVR) was obtained with a 0.7 μT saturation at 2.0 ppm and 0.8 μT at 3.6 ppm. In acute stroke, CVR optimized pHenh (i.e., pHdual ) achieved a higher sensitivity than the three-point amide-CEST approach, and distinct patterns of lesion tissue compared to diffusion and perfusion MRI. pHdual MRI improves the sensitivity of pH-weighted imaging and will be a valuable tool for assessing tissue viability in stroke.

Dynamic Structure of Organic Compounds in Solution According to NMR Data and Quantum Mechanical Calculations: IV. Benzamide

To study the structure and dynamics of nitrogen-containing compounds, NMR parameters with directly involved nitrogen can provide valuable structure information. However, this information can only be obtained using15N-enriched compounds due to low natural abundance of 15N and extremely short relaxation of 14N nuclei. For synthesis of these compounds from 15N-ammonium salts, 15N-enriched benzamides often used as intermediates. In the present work, we study the dynamic structure of benzamide caused by two independent factors: hindered internal rotation of the NH2 group around the C(O)-N bond and the amide group as a whole relative to the benzene ring. This deeper knowledge of mechanism and parameters of these processes in amides is important for meaningful interpretation and prediction of the biological activity of aromatic amides in living systems, the strength and conformation of its supramolecular complexes with lanthanide and actinide ions. Double enriched [2H5, 15N]benzamide was synthesized to avoid unwanted superposition of intense aromatic multiplet on the amide signals in the 1H NMR spectra. In the 1H spectrum of this compound observed only intense signals of amide protons, which allowed accurate quantitative characterization of the parameters of the dynamic processes under study. The experimental data obtained is in good agreement with our results of simulation by quantum molecular dynamics techniques.

Comparison of model-free Lorentzian and spinlock model-based fittings in quantitative CEST imaging ofacutestroke.

CEST MRI detects complex tissue changes following acute stroke. Our study aimed to test if spinlock model-based fitting of the quasi-steady-state (QUASS)-reconstructed equilibrium CEST MRI improves the determination of multi-pool signal changes over the commonly-used model-free Lorentzian fitting in acute stroke. Multiple three-pool CEST Z-spectra were simulated using Bloch-McConnell equations for a range of T1 , relaxation delay, and saturation times. The multi-pool CEST signals were solved from the simulated Z-spectra to test the accuracy of routine Lorentzian (model-free) and spinlock (model-based) fittings without and with QUASS reconstruction. In addition, multiparametric MRI scans were obtained in rat models of acute stroke, including relaxation, diffusion, and CEST Z-spectrum. Finally, we compared model-free and model-based per-pixel CEST quantification in vivo. The spinlock model-based fitting of QUASS CEST MRI provided a nearly T1 -independent determination of multi-pool CEST signals, advantageous over the fittings of apparent CEST MRI (model-free and model-based). In vivo data also demonstrated that the spinlock model-based QUASS fitting captured significantly different changes in semisolid magnetization transfer (-0.9 ± 0.8 vs. 0.3 ± 0.8%), amide (-1.1 ± 0.4 vs. -0.5 ± 0.2%), and guanidyl (1.0 ± 0.4 vs. 0.7 ± 0.3%) signals over the model-free Lorentzian analysis. Our study demonstrated that spinlock model-based fitting of QUASS CEST MRI improved the determination of the underlying tissue changes following acute stroke, promising further clinical translation of quantitative CEST imaging.

Investigation of the influence of pH on the properties and morphology of gelatin hydrogels

AbstractThe behavior of gelatin hydrogels is influenced by the charges located on the amino acid side chains throughout the gelatin molecules. The presence and distribution of ionisable side chains influences the surface activity of gelatin and ultimately determines the material properties. Herein, we report the influence of pH on mechanical properties as studied by texture analysis supported by data from polarimetry, zeta potential, pH titrations and NMR experiments. When adjusted to more extreme pH values (pH 2 and 12), softer gelatin blocks were observed. However, at pH values close to the isoelectric point (pH 5–10), the material is firmer. This behavior is related to the helical content. At pH 2 and pH 12 the surface of the gelatin carries a net charge, positive and negative, respectively, that inhibits the formation of tight helices and lowers the physical crosslink network density. Chemical shift perturbations were observed for the acidic amino acids glutamic and aspartic acid, under acidic pH, where their peaks shifted to higher ppm. Intense amide signals were observed at acidic pH but diminished with increasing pH. This was due to an increase in the rate of chemical exchange between the solvent and peptide amide protons as the pH increases.

CEST2022 - mapping multi-pool CEST signal changes in an animal model of brain tumor with quasi-steady-state reconstruction-empowered CEST quantification

Chemical exchange saturated transfer (CEST) MRI has biomarker potential to assess tissue microenvironment in brain tumors. Multi-pool Lorentzian or spinlock models provides useful insights into the CEST contrast mechanism. However, T1 contribution to the complex overlapping effects of brain tumors is difficult under the non-equilibrium state. Therefore, this study evaluated T1 contributions on multi-pool parameters with quasi-steady-state (QUASS) algorithm reconstructed equilibrium data. MRI scans were performed in rat brain tumor models, including relaxation, diffusion, and CEST imaging. A pixel-wise seven-pool spinlock-model was employed to fit QUASS reconstructed CEST Z-spectra and evaluated the magnetization transfer (MT), amide, amine, guanidyl, and nuclear-overhauled effect (NOE) signals in tumor and normal tissues. In addition, T1 was estimated from the spinlock-model fitting and compared with measured T1. We observed tumor had a statistically significant increase in the amide signal (p < 0.001) and decreases in the MT and NOE signals (p < 0.001). On the other hand, the differences in amine and guanidyl between the tumor and contralateral normal regions were not statistically significant. The differences between measured and estimated T1 values were 8% in the normal tissue and 4% in the tumor. Furthermore, the isolated MT signal strongly correlated with R1 (r = 0.96, P < 0.001). In summary, we successfully unraveled multi-factorial effects in the CEST signal using spinlock-model fitting and QUASS method and demonstrated the effect of T1 relaxation on MT and NOE.

The exchange rate of creatine CEST in mouse brain.

To estimate the exchange rate of creatine (Cr) CEST and to evaluate the pH sensitivity of guanidinium (Guan) CEST in the mouse brain. Polynomial and Lorentzian line-shape fitting (PLOF) were implemented to extract the amine, amide, and Guan CEST signals from the brain Z-spectrum at 11.7T. Wild-type (WT) and knockout mice with the guanidinoacetate N-methyltransferase deficiency (GAMT-/- ) that have low Cr and phosphocreatine (PCr) concentrations in the brain were used to extract the CrCEST signal. To quantify the CrCEST exchange rate, a two-step Bloch-McConnell (BM) fitting was used to fit the CrCEST line-shape, B1 -dependent CrCEST, and the pH response with different B1 values. The pH in the brain cells was altered by hypercapnia to measure the pH sensitivity of GuanCEST. Comparison between the Z-spectra of WT and GAMT-/- mice suggest that the CrCEST is between 20% and 25% of the GuanCEST in the Z-spectrum at 1.95 ppm between B1 = 0.8 and 2 μT. The CrCEST exchange rate was found to be around 240-480 s-1 in the mouse brain, which is significantly lower than that in solutions (∼1000 s-1 ). The hypercapnia study on the mouse brain revealed that CrCEST at B1 = 2 μT and amineCEST at B1 = 0.8 μT are highly sensitive to pH change in the WT mouse brain. The in vivo CrCEST exchange rate is slow, and the acquisition parameters for the CrCEST should be adjusted accordingly. CrCEST is the major contribution to the opposite pH-dependence of GuanCEST signal under different conditions of B1 in the brain.

Amide Spectral Fingerprints are Hydrogen Bonding-Mediated.

The origin of the peculiar amide spectral features of proteins in aqueous solution is investigated, by exploiting a combined theoretical and experimental approach to study UV Resonance Raman (RR) spectra of peptide molecular models, namely N-acetylglycine-N-methylamide (NAGMA) and N-acetylalanine-N-methylamide (NALMA). UVRR spectra are recorded by tuning Synchrotron Radiation at several excitation wavelengths and modeled by using a recently developed multiscale protocol based on a polarizable QM/MM approach. Thanks to the unparalleled agreement between theory and experiment, we demonstrate that specific hydrogen bond interactions, which dominate hydration dynamics around these solutes, play a crucial role in the selective enhancement of amide signals. These results further argue the capability of vibrational spectroscopy methods as valuable tools for refined structural analysis of peptides and proteins in aqueous solution.

Observation of Arginine Side-Chain Motions Coupled to the Global Conformational Exchange Process in Deubiquitinase A

Coupled motions have been demonstrated to be functionally important in a number of enzymes. Noncovalent side-chain interactions play essential roles in coordinating the motions across different structural elements in a protein. However, most of the dynamic studies of proteins are focused on backbone amides or methyl groups in the side chains and little is known about the polar and charged side chains. We have previously characterized the conformational dynamics of deubiquitinase A (DUBA), an isopeptidase, on the microsecond-to-millisecond (μs–ms) time scales with the amide 1H Carr–Purcell–Meiboom–Gill (CPMG) experiment. We detected a global conformational exchange process on a time scale of approximately 200 μs, which involves most of the structural elements in DUBA, including the active site and the substrate binding interface. Here, we extend our previous study on backbone amides to the arginine side-chain Nε–Hε groups using a modified 1H CPMG pulse sequence that can efficiently detect both backbone amide and arginine side-chain Nε–Hε signals in a single experiment. We found that the side chains of three arginines display motions on the same time scale as the backbone amides. Mutations of two of the three arginines to alanines result in a decrease in enzyme activity. One of these two arginines is located in a loop involved in substrate binding. This loop is not visible in the backbone amide-detected experiments due to excess line broadening induced by motions on the μs–ms time scales. These results clearly demonstrate that the motions of some arginine side chains are coupled to the global conformational exchange process and provide an additional probe for motions in a functionally important loop that did not yield visible backbone amide signals, suggesting the value of side-chain experiments on DUBA. The modified 1H CPMG pulse sequence allows the simultaneous characterization of backbone and arginine side-chain dynamics without any increase in data acquisition time and can be applied to the dynamic studies of any protein that displays measurable amide 1H relaxation dispersion.

Mapping intracellular pH in tumors using amide and guanidyl CEST-MRI at 9.4 T.

In principle, non-invasive mapping of the intracellular pH (pHi ) in vivo is possible using endogenous chemical exchange saturation transfer (CEST)-MRI of the amide and guanidyl signals. However, the application for cancer imaging is still impeded, as current state-of-the-art approaches do not allow for simultaneous compensation of concomitant effects that vary within tumors. In this study, we present a novel method for absolute pHi mapping using endogenous CEST-MRI, which simultaneously compensates for concentration changes, superimposing CEST signals, magnetization transfer contrast, and spillover dilution. Compensation of the concomitant effects was achieved by a ratiometric approach (i.e. the ratio of one CEST signal at different B1 ) in combination with the relaxation-compensated inverse magnetization transfer ratio MTRRex and a separate first-order polynomial-Lorentzian fit of the amide and guanidyl signals at 9.4 T. Calibration of pH values was accomplished using in vivo-like model suspensions from porcine brain lysates. Applicability of the presented method in vivo was demonstrated in n = 19 tumor-bearing mice. In porcine brain lysates, measurement of pH was feasible over a broad range of physiologically relevant pH values of 6.2 to 8.0, while being independent of changes in concentration. A median pHi of approximately 7.2 was found in the lesions of 19 tumor-bearing mice. The presented method enables non-invasive mapping of absolute pHi values in tumors using CEST-MRI, which was so far prevented by concomitant effects. Consequently, pre-clinical studies on pHi changes in tumors are possible allowing the assessment of pHi in vivo as a biomarker for cancer diagnosis or treatment monitoring.

Large Multidomain Protein NMR: HIV-1 Reverse Transcriptase Precursor in Solution.

NMR studies of large proteins, over 100 kDa, in solution are technically challenging and, therefore, of considerable interest in the biophysics field. The challenge arises because the molecular tumbling of a protein in solution considerably slows as molecular mass increases, reducing the ability to detect resonances. In fact, the typical 1H-13C or 1H-15N correlation spectrum of a large protein, using a 13C- or 15N-uniformly labeled protein, shows severe line-broadening and signal overlap. Selective isotope labeling of methyl groups is a useful strategy to reduce these issues, however, the reduction in the number of signals that goes hand-in-hand with such a strategy is, in turn, disadvantageous for characterizing the overall features of the protein. When domain motion exists in large proteins, the domain motion differently affects backbone amide signals and methyl groups. Thus, the use of multiple NMR probes, such as 1H, 19F, 13C, and 15N, is ideal to gain overall structural or dynamical information for large proteins. We discuss the utility of observing different NMR nuclei when characterizing a large protein, namely, the 66 kDa multi-domain HIV-1 reverse transcriptase that forms a homodimer in solution. Importantly, we present a biophysical approach, complemented by biochemical assays, to understand not only the homodimer, p66/p66, but also the conformational changes that contribute to its maturation to a heterodimer, p66/p51, upon HIV-1 protease cleavage.

Amide signal intensities may be reduced in the motor cortex and the corticospinal tract of ALS patients.

The aim of the study is to assess amide concentration changes in ALS patients compared with healthy controls by using quantitative amide proton transfer (APT) and multiparameter magnetic resonance imaging, and testing its correlation with clinical scores. Sixteen ALS patients and sixteen healthy controls were recruited as part of the Canadian ALS Neuroimaging Consortium, and multimodal magnetic resonance imaging was performed at 3T, including APT and diffusion imaging. Lorentz fitting was used to quantify the amide effect. Clinical disability was evaluated using the revised ALS functional rating scale (ALSFRS-R), and its correlation with image characteristics was assessed. The diagnostic performance of different imaging parameters was evaluated with receiver operating characteristic analysis. Our results showed that the amide peak was significantly different between the motor cortex and other gray matter territories within the brain of ALS patients (p < 0.001). Compared with controls, amide signal intensities in ALS were significantly reduced in the motor cortex (p < 0.001) and corticospinal tract (p = 0.046), while abnormalities were not detected using routine imaging methods. There was no significant correlation between amide and ALSFRS-R score. The diagnostic accuracy of the amide peak was superior to that of diffusion imaging. This study demonstrated changes of amide signal intensities in the motor cortex and corticospinal tract of ALS patients. • The neurodegenerative disease amyotrophic lateral sclerosis (ALS) has a lack of objective imaging indicators for diagnosis and assessment. • Analysis of amide proton transfer imaging revealed changes in the motor cortex and corticospinal tract of ALS patients that were not visible on standard magnetic resonance imaging. • The diagnostic accuracy of the amide peak was superior to that of diffusion imaging.

Characterization of Membrane Protein-Lipid Interactions in Unfolded OmpX with Enhanced Time Resolution by Hyperpolarized NMR.

Proton nuclear spins of dodecyl phosphocholine molecules below the critical micelle concentration are hyperpolarized by using dissolution dynamic nuclear polarization (D-DNP). NMR signal enhancements of 1210±400 and 1610±550 are obtained at 9.4 T, for choline methyls in the head group of the lipid and for the tail-end methyl group, respectively. This polarization is transferred to the unfolded protein through the nuclear Overhauser effect, after dilution to a final denaturant concentration of 0.8 M urea. As a result, the amide and aromatic side-chain signals of the protein are increased up to sixfold. Selective inversion pulses applied either on the head-group or tail-group of the lipid are used to identify the source of the transferred polarization. The normalized cross-relaxation rates of σN,tail =-1.8±0.1 s-1 M-1 and σN,head =-0.5±0.3 s-1 M-1 are obtained, showing a larger polarization transfer from the tail groups. These cross-relaxation rates are determined at a low urea concentration, which constitutes refolding conditions for the protein. The sensitivity enhancement by D-DNP permits to access these conditions with a measurement time on the order of seconds, and may further open the possibility to investigate structural changes in membrane proteins during folding.

Recent developments in isotope-aided NMR methods for supramolecular protein complexes –SAIL aromatic TROSY

BackgroundThe structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals. MethodsThe 82 kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra. ResultsThe MSG proteins, selectively labeled with either [δ1,ε1,ε3,η2]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously. ConclusionsWe demonstrated that the aromatic ring 13CH pairs without directly bonded 13C and adjacent 1H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available. General significanceThe method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology.

The pH sensitivity of APT-CEST using phosphorus spectroscopy as a reference method.

The pH value is a potential physiological marker for clinical diagnosis as it is altered in pathologies such as tumors. While intracellular pH can be measured noninvasively via phosphorus spectroscopy (31 P MRSI), Amide Proton Transfer-Chemical Exchange Saturation Transfer (APT-CEST) MRI has been suggested as an alternative method for pH quantification. To assess the suitability of APT-CEST contrast for pH quantification, two approaches (magnetization transfer ratio asymmetry [MTRasym ] and Lorentzian difference analysis [LDA]) for analyzing the Z-spectrum have been correlated with pH values obtained by 31 P MRSI. Fourteen patients with glioblastoma and 12 healthy controls were included. In contrast to MTRasym , the LDA is modeling the direct water saturation and the semi-solid magnetization transfer, allowing a separate evaluation of the aliphatic nuclear Overhauser effect and the APT-CEST. The results of our study show that the pH values obtained by 31 P MRSI correspond well with both methods describing the APT-CEST contrast. Two-sample t-test showed significant differences in MTRasym , LDA and pH obtained by 31 P MRSI for regions of interest in glioblastoma, contralateral control areas and normal appearing white matter (P<0.001). A slightly improved correlation between the amide signal and pH was found after performing LDA (r=0.78) compared with MTRasym (r=0.70). While both methods can be used to monitor pH changes, the LDA approach appears to be better suited.

Towards complete polypeptide backbone NH assignment via combinatorial labeling.

Combinatorial selective isotope labeling is a valuable tool to facilitate polypeptide backbone resonance assignment in cases of low sensitivity or extensive chemical shift degeneracy. It involves recording of 15N-HSQC and 2D HN-projections of triple-resonance spectra on a limited set of samples containing different combinations of labeled and unlabeled amino acid types. Using labeling schemes in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in 15N or 13C isotopes - individually as well as simultaneously - usually yields abundant amino-acid type information of consecutive residues i and i - 1. Although this results in a large number of anchor points that can be used in the sequential assignment process, for most amide signals the exact positioning of the corresponding residue the polypeptide sequence still relies on matching intra- and interresidual 13C chemical shifts obtained from 3D spectra. An obvious way to obtain more sequence-specific assignments directly with combinatorial labeling would be to increase the number of samples. This is, however, undesirable because of increased sample preparation efforts and costs. Irrespective of the number of samples, unambiguous assignments cannot be accomplished for i - 1/i pairs that are not unique in the sequence. Here we show that the ambiguity for non-unique pairs can be resolved by including information about the labeling state of residues i + 1 and i - 2. Application to a 35-residue peptide resulted in complete assignments of all detectable signals in the 15N HSQC which, due to its repetitive sequence and 13C chemical shift degeneracies, was difficult to achieve by other means. For a medium-sized protein (165 residues, rotational correlation time 8.2 ns) the improved protocol allowed the extent of backbone amide assignment to be expanded to 88% solely using a suite of 2D 1H-15N correlated spectra.

Optimization and repeatability of multipool chemical exchange saturation transfer MRI of the prostate at 3.0 T.

BackgroundChemical exchange saturation transfer (CEST) can potentially support cancer imaging with metabolically derived information. Multiparametric prostate MRI has improved diagnosis but may benefit from additional information to reduce the need for biopsies.PurposeTo optimize an acquisition and postprocessing protocol for 3.0 T multipool CEST analysis of prostate data and evaluate the repeatability of the technique.Study TypeProspective.SubjectsFive healthy volunteers (age range: 24–47 years; median age: 28 years) underwent two sessions (interval range: 7–27 days; median interval: 20 days) and two biopsy‐proven prostate cancer patients were evaluated once. Patient 1 (71 years) had a Gleason 3 + 4 transition zone (TZ) tumor and patient 2 (55 years) had a Gleason 4 + 3 peripheral zone (PZ) tumor.Field Strength3.0 T. Sequences run: T2‐weighted turbo‐spin‐echo (TSE); diffusion‐weighted imaging; CEST; WASABI (for B0 determination).AssessmentSaturation, readout, and fit‐model parameters were optimized to maximize in vivo amide and nuclear Overhauser effect (NOE) signals. Repeatability (intrasession and intersession) was evaluated in healthy volunteers. Subsequently, preliminary evaluation of signal differences was made in patients. Regions of interest were drawn by two post‐FRCR board‐certified readers, both with over 5 years of experience in multiparametric prostate MRI.Statistical TestsRepeatability was assessed using Bland–Altman analysis, coefficient of variation (CV), and 95% limits of agreement (LOA). Statistical significance of CEST contrast was calculated using a nonparametric Mann–Whitney U‐test.ResultsThe optimized saturation scheme was found to be 60 sinc‐Gaussian pulses with 40 msec pulse duration, at 50% duty‐cycle with continuous‐wave pulse equivalent B1 power (B1CWPE) of 0.92 μT. The magnetization transfer (MT) contribution to the fit‐model was centered at –1.27 ppm. Intersession coefficients of variation (CVs) of the amide, NOE, and magnetization transfer (MT) and asymmetric magnetization transfer ratio (MTRasym) signals of 25%, 23%, 18%, and 200%, respectively, were observed. Fit‐metric and MTRasym CVs agreed between readers to within 4 and 10 percentage points, respectively.Data ConclusionSignal differences of 0.03–0.10 (17–43%) detectable depending upon pool, with MT the most repeatable (signal difference of 17–22% detectable). Level of Evidence: 2 Technical Efficacy: Stage 2J. Magn. Reson. Imaging 2019;50:1238–1250.

Perspective: next generation isotope-aided methods for protein NMR spectroscopy

In this perspective, we describe our efforts to innovate the current isotope-aided NMR methodology to investigate biologically important large proteins and protein complexes, for which only limited structural information could be obtained by conventional NMR approaches. At the present time, it is widely believed that only backbone amide and methyl signals are amenable for investigating such difficult targets. Therefore, our primary mission is to disseminate our novel knowledge within the biological NMR community; specifically, that any type of NMR signals other than methyl and amide groups can be obtained, even for quite large proteins, by optimizing the transverse relaxation properties by isotope labeling methods. The idea of “TROSY by isotope labeling” has been cultivated through our endeavors aiming to improve the original stereo-array isotope labeling (SAIL) method (Kainosho et al., Nature 440:52–57, 2006). The SAIL TROSY methods subsequently culminated in the successful observations of individual NMR signals for the side-chain aliphatic and aromatic 13CH groups in large proteins, as exemplified by the 82 kDa single domain protein, malate synthase G. Meanwhile, the expected role of NMR spectroscopy in the emerging integrative structural biology has been rapidly shifting, from structure determination to the acquisition of biologically relevant structural dynamics, which are poorly accessible by X-ray crystallography or cryo-electron microscopy. Therefore, the newly accessible NMR probes, in addition to the methyl and amide signals, will open up a new horizon for investigating difficult protein targets, such as membrane proteins and supramolecular complexes, by NMR spectroscopy. We briefly introduce our latest results, showing that the protons attached to 12C-atoms give profoundly narrow 1H-NMR signals even for large proteins, by isolating them from the other protons using the selective deuteration. The direct 1H observation methods exhibit the highest sensitivities, as compared to heteronuclear multidimensional spectroscopy, in which the 1H-signals are acquired via the spin-coupled 13C- and/or 15N-nuclei. Although the selective deuteration method was launched a half century ago, as the first milestone in the following prosperous history of isotope-aided NMR methods, our results strongly imply that the low-dimensional 1H-direct observation NMR methods should be revitalized in the coming era, featuring ultrahigh-field spectrometers beyond 1 GHz.