Relaxation Enhancement Effects Research Articles

Dynamical properties of solid and hydrated collagen: Insight from nuclear magnetic resonance relaxometry.

1H spin-lattice Nuclear Magnetic Resonance relaxometry experiments have been performed for collagen and collagen-based artificial tissues in the frequency range of 10 kHz-20MHz. The studies were performed for non-hydrated and hydrated materials. The relaxation data have been interpreted as including relaxation contributions originating from 1H-1H and 1H-14N dipole-dipole interactions, the latter leading to Quadrupole Relaxation Enhancement effects. The 1H-1H relaxation contributions have been decomposed into terms associated with dynamical processes on different time scales. A comparison of the parameters for the non-hydrated and hydrated systems has shown that hydration leads to a decrease in the dipolar relaxation constants without significantly affecting the dynamical processes. In the next step, the relaxation data for the hydrated systems were interpreted in terms of a model assuming two-dimensional translational diffusion of water molecules in the vicinity of the macromolecular surfaces and a sub-diffusive motion leading to a power law of the frequency dependencies of the relaxation rates. It was found that the water diffusion process is slowed down by at least two orders of magnitude compared to bulk water diffusion. The frequency dependencies of the relaxation rates in hydrated tissues and hydrated collagen are characterized by different power laws (ωH-β, where ωH denotes the 1H resonance frequency): the first of about 0.4 and the second close to unity.

Activatable 19F MRI probe for detection of α-amylase activity

Abstract α-Amylase is an endo-type glycoside hydrolase and has drawn attention as a biomarker of various diseases. However, current detection methods rely on colorimetric and fluorometric approaches, which limits imaging in deep tissues. In this research, we developed an activatable probe, Tfb-G6-Gd, for detecting α-amylase using 19F MRI. In the presence of α-amylase, the 19F MRI signal of Tfb-G6-Gd increased by attenuation of paramagnetic relaxation enhancement effect. This probe showed a switching ability depending on α-amylase activity with high specificity.

Hydrazone Molecular Switches with Paramagnetic Center as 19F Magnetic Resonance Imaging Relaxation Enhancement Agents for pH Imaging.

The design and synthesis of hydrazone-based switches with a CF3 reporting group for 19F pH imaging using relaxation rate changes were described. A paramagnetic center was introduced into the hydrazone molecular switch scaffold by substitution of an ethyl functional group with a paramagnetic complex. The mechanism of activation relies on a gradual increase in T1 and T2 magnetic resonance imaging (MRI) relaxation times as pH decreases due to E/Z isomerization, which results in a change in the distance between fluorine atoms and the paramagnetic center. Among the three possible variants of the ligand, the meta isomer was found to offer the highest potential changes in relaxation rates due to the significant paramagnetic relaxation enhancement (PRE) effect and a stable position of the 19F signal, allowing for the tracking of a single narrow 19F resonance for imaging purposes. The selection of the most suitable Gd(III) paramagnetic ion for complexation was conducted by theoretical calculations based on the Bloch-Redfield-Wangsness (BRW) theory, taking into account the electron-nucleus dipole-dipole and Curie interactions only. The results were verified experimentally, confirming the accuracy of theoretical predictions, good solubility, and stability of the agents in water and the reversible transition between E and Z-H+ isomers. The results demonstrate the potential of this approach for pH imaging using relaxation rate changes instead of chemical shift.

PH-dependent aggregation of the Pmel17 repeat domain characterized by NMR.

In studying amyloid-forming proteins, it is imperative to determine the monomer conformational dynamics that precede fibrillation. This study utilizes NMR and the paramagnetic relaxation enhancement (PRE) effect to observe monomer properties of the repeat domain (RPT) from a human functional amyloid, premelanosomal protein (Pmel17). RPT is generated through Pmel17 post-translational processing during melanosome maturation. The melanosome is an acidic organelle within specialized cells where melanin biogenesis occurs. At low melanosomal pH, RPT self-assembles into amyloid fibrils, functioning as a scaffold for melanin deposition. Here, we report dynamics of the short (sRPT) isoform, which has been demonstrated to be a fibrillation nucleator. NMR experiments were performed to determine conformational differences in sRPT by comparing aggregation-prone vs. non-aggregating solution conditions at pH ranges from 4 to 6, respectively. We observed significant chemical shift perturbations localized to residues near the important singular tryptophan residue, demonstrating that the local chemical environment of the amyloid core region is sensitive to changes in pH even in the monomer form. Next, we introduced several cysteine point mutations in order to covalently attach PRE ligands to sRPT for observation of intramolecular interactions. Long-range PRE effects indicate potential contacts to residues on opposite ends of sRPT. These PRE effects might be an indication of initial molecular events to facilitate intermolecular interactions, which can go on to trigger fibrillation. These results also hint at a transient conformation that inhibits fibrillation. Thus, by raising or lowering solution pH, the relative population of this state would be modulated. Taken together, these results show that sRPT monomers adopt a conformation inconsistent with fully random coil at neutral pH and undergo conformational changes at lower pH values. These observations highlight the tight regulatory mechanisms that can affect fibrillation activity of proteins like RPT.

Nanoplatforms with synergistic redox cycles and rich defects for activatable image-guided tumor-specific therapy

<h2>Summary</h2> Multifunctional nanoplatforms for the early diagnosis and treatment of deep-seated tumors with high specificity are particularly desirable but face many challenges. Herein, nanoplatforms containing copper/manganese chalcogenide nanoflowers (CMC NFs) and ¹⁹F probes have been developed, which exhibit enhanced therapeutic efficiency and activatable ¹⁹F magnetic resonance imaging (MRI) capability within a tumor microenvironment (TME). CMC NFs containing synergistic redox pairs (Cu^ox/Cu^red, Mn^ox/Mn^red) and rich defects acted as integrated cascade enzyme mimics to effectively destroy the redox homeostasis of tumors via generation of reactive oxygen species (ROS) and GSH depletion. In addition, the ¹⁹F MRI signal could be switched on by attenuation of the paramagnetic relaxation enhancement (PRE) effect under TME conditions, affording a TME-activatable ¹⁹F MRI-guided tumor-specific chemodynamic therapeutic. These nanoplatforms could simultaneously boost therapeutic efficacy and regulate signal output, exhibiting significant potential for <i>in vivo</i> drug tracking and deep-seated tumor diagnosis and treatment.

Paramagnetic relaxation enhancement NMR as a tool to probe guest binding and exchange in metallohosts

Paramagnetic metallohost systems can bind guest molecules and find application as biomimetic catalysts. Due to the presence of the paramagnetic metal center, rigorous characterization of these systems by NMR spectroscopy can be very difficult. We report here that metallohost-guest systems can be studied by using the paramagnetic relaxation enhancement (PRE) effect. Manganese(III) porphyrin cage compounds are shown through their PRE to thread and bind viologen guests, including a polymeric one. The binding constants and dethreading activation parameters are lower than those of the metal-free porphyrin cage compounds, which is proposed to be a result of charge repulsion of the trivalent metal center and dicationic viologen guest. The threading rate of the manganese(III) porphyrin cage onto the polymer is more than 10 times faster than that of the non-metallated one, which is ascribed to initial binding of the cage to the polymer chain prior to threading, and to an entron effect.

Magnetic resonance spectroscopic imaging of downfield proton resonances in the human brain at 3 T.

To develop an MRSI technique capable of mapping downfield proton resonances in the human brain. A spectral-spatial excitation and frequency-selective refocusing scheme, in combination with 2D phase encoding, was developed for mapping of downfield resonances without any perturbation of the water magnetization. An alternative scheme using spectral-spatial refocusing was also investigated for simultaneous detection of both downfield and upfield resonances. The method was tested in 5 healthy human volunteers. Downfield metabolite maps with a nominal spatial resolution of 1.5 cm3 were recorded at 3 T in a scan time of 12 minutes. Cramer-Rao lower bounds for nine different downfield peaks were 20% or less over a single supraventricular slice. Downfield spectral profiles were similar to those in the literature recorded previously using single-voxel localization methods. The same approach was also used for upfield MRSI, and simultaneous upfield and downfield acquisitions. The developed MRSI pulse sequence was shown to be an efficient way of rapidly mapping downfield resonances in the human brain at 3 T, maximizing sensitivity through the relaxation enhancement effect. Because the MRSI approach is efficient in terms of data collection and can be readily implemented at short TE, somewhat higher spatial resolution can be achieved than has been reported in previous single-voxel downfield MRS studies. With this approach, nine downfield resonances could be mapped in a single slice for the first time using MRSI at 3 T.

Successive Free-Radical C(sp 2 )–C(sp 2 ) Coupling Reactions to Form Graphene

Successive Free-Radical C(sp ² )–C(sp ² ) Coupling Reactions to Form Graphene

Development of Off-On Switching 19F MRI Probes for Cathepsin K Activity Detection

Abstract Cathepsin K is a protease expressed in osteoclasts that degrades bone tissue, such as type I collagen fibers. Overexpression of cathepsin K is involved in osteoporosis, rheumatoid arthritis, and bone metastasis. Therefore, detecting cathepsin K activity is important for understanding the mechanism of these diseases and developing new drugs. However, current chemical probes cannot be employed for the detection of cathepsin K activity in animal deep-tissue. In this study, we developed novel 19F magnetic resonance imaging (MRI) probes (FLAME-(Gd-X), X = Acp, Deg, Deg2) to detect cathepsin K. In FLAME-(Gd-X), the Gd3+ complex was modified on the surface of perfluorocarbon-encapsulated silica nanoparticles through cathepsin K substrate and three different hydrophobic/hydrophilic linkers. The 19F NMR signal intensities of these probes were suppressed by the paramagnetic relaxation enhancement (PRE) effect of the Gd3+ complexes. The 19F MRI signal intensities of FLAME-Gd-Acp and FLAME-Gd-Deg specifically increased with the substrate cleavage by cathepsin K. The 19F MRI probes based on the PRE effect can be applied to the in vivo detection of cathepsin K activity.

Towards applying NMR relaxometry as a diagnostic tool for bone and soft tissue sarcomas: a pilot study

This work explores what Fast Field-Cycling Nuclear Magnetic Resonance (FFC-NMR) relaxometry brings for the study of sarcoma to guide future in vivo analyses of patients. We present the results of an ex vivo pilot study involving 10 cases of biopsy-proven sarcoma and we propose a quantitative method to analyse 1H NMR relaxation dispersion profiles based on a model-free approach describing the main dynamical processes in the tissues and assessing the amplitude of the Quadrupole Relaxation Enhancement effects due to 14N. This approach showed five distinct groups of dispersion profiles indicating five discrete categories of sarcoma, with differences attributable to microstructure and rigidity. Data from tissues surrounding sarcomas indicated very significant variations with the proximity to tumour, which may be attributed to varying water content but also to tissue remodelling processes due to the sarcoma. This pilot study illustrates the potential of FFC relaxometry for the detection and characterisation of sarcoma.

Fluorinated ZnFeIII Hollow Metal-Organic Framework as a 19F NMR Probe for Highly Sensitive and Selective Detection of Hydrogen Sulfide.

Hydrogen sulfide (H2S) is considered as a highly toxic environmental pollutant and an important signal transmitter in physiological processes, and the selective and reliable detection of H2S is of great concern and remains challenging. Herein, we report a smart sensitive “off–on” 19F NMR sensor for H2S by partially introducing a fluorinated ligand to construct a hollow dual metal–organic framework (MOF) nanosystem, F-ZnFeIII hMOF, in which the fluorinated ligand acts as the 19F signal source but is initially quenched due to the strong paramagnetic relaxation enhancement (PRE) effect from neighboring Fe3+ nodes. Upon exposure to sulfide ions, reduction of Fe3+ to Fe2+ is specifically triggered, which attenuates PRE efficiency, thus turning on the 19F NMR signal. The unique hollow MOF architecture benefits the mobility of 19F atoms, thereby improving the response sensitivity. Meanwhile, the desirable H2S-sorption feature and appropriate redox potential of Fe3+/Fe2+ account for the favorable selectivity. The increase in the 19F signal is linear with the concentration of sulfide in the range of 20 to 150 μM with a detection limit of 2.8 μM. The probe is well demonstrated by analyzing H2S in complex matrixes such as biological and foodstuff samples.

Structure of a Protein-RNA Complex by Solid-State NMR Spectroscopy.

Solid‐state NMR (ssNMR) is applicable to high molecular‐weight (MW) protein assemblies in a non‐amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein–RNA complexes (RNP) solely from ssNMR data. Instead of using low‐sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical‐shift‐perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein–RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.

Structure of a Protein–RNA Complex by Solid‐State NMR Spectroscopy

AbstractSolid‐state NMR (ssNMR) is applicable to high molecular‐weight (MW) protein assemblies in a non‐amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein–RNA complexes (RNP) solely from ssNMR data. Instead of using low‐sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical‐shift‐perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein–RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.

Dynamics of Solid Proteins by Means of Nuclear Magnetic Resonance Relaxometry.

1H Nuclear magnetic resonance (NMR) relaxometry was exploited to investigate the dynamics of solid proteins. The relaxation experiments were performed at 37 °C over a broad frequency range, from approximately 10 kHz to 40 MHz. Two relaxation contributions to the overall 1H spin–lattice relaxation were revealed; they were associated with 1H–1H and 1H–14N magnetic dipole–dipole interactions, respectively. The 1H–1H relaxation contribution was interpreted in terms of three dynamical processes occurring on timescales of 10−6 s, 10−7 s, and 10−8 s, respectively. The 1H–14N relaxation contribution shows quadrupole relaxation enhancement effects. A thorough analysis of the data was performed revealing similarities in the protein dynamics, despite their different structures. Among several parameters characterizing the protein dynamics and structure (e.g., electric field gradient tensor at the position of 14N nuclei), the orientation of the 1H–14N dipole–dipole axis, with respect to the principal axis system of the electric field gradient, was determined, showing that, for lysozyme, it was considerably different than for the other proteins. Moreover, the validity range of a closed form expression describing the 1H–14N relaxation contribution was determined by a comparison with a general approach based on the stochastic Liouville equation.

Detecting acid phosphatase enzymatic activity with phenol as a chemical exchange saturation transfer magnetic resonance imaging contrast agent (PhenolCEST MRI)

Phenol contains an exchangeable hydroxyl proton resonant at 4.8 ppm from the resonance frequency of water in the 1H nuclear magnetic resonance (1H NMR) spectrum, enabling itself to be detected at sub-mM concentration by either chemical exchange saturation transfer magnetic resonance imaging (CEST MRI) or exchange-based T2 relaxation enhancement (T2ex) effect under acidic and basic conditions, respectively. We recently investigated the T2ex effects of phenol and its derivatives, but the CEST characteristics of phenols are unknown in detail, and no study on using the natural CEST MRI effects of phenol for detecting enzymatic activity has been conducted. Herein, on the basis of the inherent CEST MR property of phenol, namely phenolCEST, we developed the first MRI approach to detect acid phosphatase (AcP) enzymatic activity. Upon the activity of AcP at pH = 5.0, non-CEST-detectable enzyme substrate phenyl phosphate was converted to CEST-detectable phenol, providing a simple way to quantify AcP activity directly without the need for a second signalling probe. We showed the application of this phenolCEST biosensor for measuring AcP activity in both enzyme solutions and cell lysates of prostate cells. This work opens a door for the utilization of phenolCEST MRI technique in sensor design and development.

Multi-quantum quadrupole relaxation enhancement effects in 209Bi compounds.

1H spin-lattice nuclear magnetic resonance relaxation experiments have been performed for triphenylbismuth dichloride (C18H15BiCl2) and phenylbismuth dichloride (C6H5BiCl2) in powder. The frequency range of 20-128 MHz has been covered. Due to 1H-209Bi dipole-dipole interactions, a rich set of pronounced Quadrupole Relaxation Enhancement (QRE) peaks (quadrupole peaks) has been observed. The QRE patterns for both compounds have been explained in terms of single- and double-quantum transitions of the participating nuclei. The analysis has revealed a complex, quantum-mechanical mechanism of the QRE effects. The mechanism goes far beyond the simple explanation of the existence of three quadrupole peaks for 14N reported in literature. The analysis has been supported by nuclear quadrupole resonance results that independently provided the 209Bi quadrupole parameters (amplitude of the quadrupole coupling constant and asymmetry parameter).

A Physical Modeling Approach to Determine Protein Structures from Paramagnetic NMR Measurements

Paramagnetic NMR approaches to protein structure determination can provide critical long-range information that may be difficult to obtain using standard NOE measurements, particularly for larger proteins. The paramagnetic relaxation enhancement (PRE) effect can be used to infer distances between residues and site-specifically engineered paramagnetic spin labels. These measurements are both sparse, due to the cost of making spin-labeled mutants, and ambiguous, due to the inherent flexibility of the spin-label. Here, we demonstrate the application of MELD (Modeling Employing Limited Data), a Bayesian approach combing sparse and ambiguous data with physical modeling, to the structure determination of a Calmodulin-peptide complex. Ten single-cysteine mutants were labeled with the common MTSL spin label, and PRE measurements were taken. Our approach can identify dominant conformations within 4 Å RMSD of the reference crystal structure using an implicit solvent model, while simulations without PRE data yield no near-native conformations. Inclusion of an explicit solvent significantly improves the model in comparison to the reference.

Determining Protein Structures by Iterating between Computation and Experiment

An increasing variety of experimental techniques for protein structure determination rely on the site-specific incorporation of probes. Examples include NMR using the paramagnetic relaxation enhancement (PRE) effect, fluorescence using FRET and related techniques, and site-directed chemical cross-linking quantified by mass-spectrometry. In each case, the probe locations must be carefully chosen, which poses a major challenge, as some probe locations are highly informative, whereas others yield little useful information. In the absence of structural knowledge, how does one efficiently choose probe locations? We present a method called Rational Probe Mutagenesis (RPM) that relies on iteration between computation and experiment to rapidly determine protein structures. RPM is based on the idea of reducing the “width” of the conformational ensemble as rapidly as possible. An ensemble is generated using whatever data is currently available. This ensemble is then analyzed to find the experiment that maximally discriminates between possible structures. The resulting experimental data is then used to generate a new updated ensemble and the process repeated. We demonstrate RPM on several simple model systems and a challenging real-world solid-state NMR dataset, where we show that RPM can choose highly informative experiments, leading to potential time and cost savings by avoiding redundant or uninformative experiments.

Quadrupole relaxation enhancement and polarisation transfer in DMSO solution of [Bi(NO3)3(H2O)3]*18-crown-6 in solid state

ABSTRACT1H spin-lattice relaxation studies have been performed for pure [Bi(NO3)3(H2O)3]*18-crown-6 in powder and its solution in dimethyl sulfoxide (DMSO). The experiments have been carried out in the frequency range of 10 kHz–30 MHz and the temperature range of 240–277 K; at 277 K the solution is already frozen. The 1H relaxation of pure [Bi(NO3)3(H2O)3]*18-crown-6 has been interpreted in terms of three dynamical processes. Quadrupole Relaxation Enhancement effects have been observed in the frozen DMSO solution of [Bi(NO3)3(H2O)3]*18-crown-6. The specific mechanisms of the 1H spin-lattice relaxation enhancement have been discussed distinguishing between effects caused by time independent (residual) and fluctuating 1H-209Bi dipole-dipole interactions.

1H spin–lattice relaxation in water solution of 209Bi counterparts of Gd3+contrast agents

ABSTRACT1H spin–lattice relaxation studies of water solutions of Bismuth-ethylenediamine-tetraacetic acid (Bi-EDTA), Bismuth-ethylenediamine-tetrakis(methylenephosphonic) acid (Bi-EDTP), Bismuth-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (Bi-DOTA), Bismuth-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonic acid) (Bi-DOTP) and Bismuth-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (Bi-DO3A) have been performed in order to compare Quadrupole Relaxation Enhancement (QRE) effects with Paramagnetic Relaxation Enhancement (PRE) from the perspective of exploiting the first one as a novel contrast mechanism for Magnetic Resonance Imaging (MRI). The selected compounds can be considered as 209Bi counterparts of Gd3+ complexes. The relaxation experiments have been performed in a broad frequency range of 5 kHz–30 MHz. The relaxation contribution associated with QRE has been extracted from the data and compared with PRE. Similarities and differences between the two effects have been discussed.