Paramagnetic Relaxation Enhancement Research Articles

Highly Sensitive Detection of Caspase-3/7 Activity in Living Mice Using Enzyme-Responsive 19F MRI Nanoprobes.

Highly sensitive imaging of enzymatic activities in the deep tissues of living mammals provides useful information about their biological functions and for developing new drugs; however, such imaging is challenging. 19F magnetic resonance imaging (MRI) is suitable for noninvasive visualization of enzymatic activities without endogenous background signals. Although various enzyme-responsive 19F MRI probes have been developed, most cannot be used for in vivo imaging because of their low sensitivity. Recently, we developed unique nanoparticles, called FLAMEs, that are composed of a liquid perfluorocarbon core and a robust silica shell, and demonstrated their outstanding sensitivity in vivo. Here, we report a highly functionalized nanoprobe, FLAME-DEVD 2, with an OFF/ON 19F MRI switch for detecting caspase-3/7 activity based on the paramagnetic relaxation enhancement effect. To improve the cleavage efficiency of peptides by caspase-3, we designed a novel Gd3+ complex-conjugated peptide, DEVD X ( X = 1, 2), which is a substrate peptide sequence tandemly repeated X times, and demonstrated that DEVD 2 showed faster cleavage kinetics than DEVD 1. By incorporating this novel concept into a signal activation strategy, FLAME-DEVD 2 showed a high 19F MRI signal enhancement rate in response to caspase-3 activity. After intravenous injection of FLAME-DEVD 2 and an apoptosis-inducing reagent, caspase-3/7 activity in the spleen of a living mouse was successfully imaged by 19F MRI. This imaging platform shows great potential for highly sensitive detection of enzymatic activities in vivo.

Synthesis, Stability and Relaxivity of TEEPO-Met: An Organic Radical as a Potential Tumour Targeting Contrast Agent for Magnetic Resonance Imaging.

Cancer is a widespread and life-threatening disease and its early-stage diagnosis is vital. One of the most effective, non-invasive tools in medical diagnostics is magnetic resonance imaging (MRI) with the aid of contrast agents. Contrast agents that are currently in clinical use contain metals, causing some restrictions in their use. Also, these contrast agents are mainly non-specific without any tissue targeting capabilities. Subsequently, the interest has notably increased in the research of organic, metal-free contrast agents. This study presents a new, stable organic radical, TEEPO-Met, where a radical moiety 2,2,6,6-tetraethylpiperidinoxide (TEEPO) is attached to an amino acid, methionine (Met), as a potentially tumour-targeting moiety. We describe the synthesis, stability assessment with electron paramagnetic resonance (EPR) spectroscopy and relaxation enhancement abilities by an in vitro nuclear magnetic resonance (NMR) and phantom MRI studies of TEEPO-Met. The new compound proved to be stable notably longer than the average imaging time in conditions mimicking a biological matrix. Also, it significantly reduced the relaxation times of water, making it a promising candidate as a novel tumour targeting contrast agent for MRI.

Protein-solvent interfaces in human Y145Stop prion protein amyloid fibrils probed by paramagnetic solid-state NMR spectroscopy

The C-terminally truncated Y145Stop variant of prion protein (PrP23-144), which is associated with heritable PrP cerebral amyloid angiopathy in humans and also capable of triggering a transmissible prion disease in mice, serves as a useful in vitro model for investigating the molecular and structural basis of amyloid strains and cross-seeding specificities. Here, we determine the protein-solvent interfaces in human PrP23-144 amyloid fibrils generated from recombinant 13C,15N-enriched protein and incubated in aqueous solution containing paramagnetic Cu(II)-EDTA, by measuring residue-specific 15N longitudinal paramagnetic relaxation enhancements using two-dimensional magic-angle spinning solid-state NMR spectroscopy. To further probe the interactions of the amyloid core residues with solvent molecules we perform complementary measurements of amide hydrogen/deuterium exchange detected by solid-state NMR and solution NMR methods. The solvent accessibility data are evaluated in the context of the structural model for human PrP23-144 amyloid.

Theory and practice of using solvent paramagnetic relaxation enhancement to characterize protein conformational dynamics.

Paramagnetic relaxation enhancement (PRE) has been established as a powerful tool in NMR for investigating protein structure and dynamics. The PRE is usually measured with a paramagnetic probe covalently attached at a specific site of an otherwise diamagnetic protein. The present work provides the numerical formulation for probing protein structure and conformational dynamics based on the solvent PRE (sPRE) measurement, using two alternative approaches. An inert paramagnetic cosolute randomly collides with the protein, and the resulting sPRE manifests the relative solvent exposure of protein nuclei. To make the back-calculated sPRE values most consistent with the observed values, the protein structure is either refined against the sPRE, or an ensemble of conformers is selected from a pre-generated library using a Monte Carlo algorithm. The ensemble structure comprises either N conformers of equal occupancy, or two conformers with different relative populations. We demonstrate the sPRE method using GB1, a structurally rigid protein, and calmodulin, a protein comprising two domains and existing in open and closed states. The sPRE can be computed with a stand-alone program for rapid evaluation, or with the invocation of a module in the latest release of the structure calculation software Xplor-NIH. As a label-free method, the sPRE measurement can be readily integrated with other biophysical techniques. The current limitations of the sPRE method are also discussed, regarding accurate measurement and theoretical calculation, model selection and suitable timescale.

Measurement of Kinetics and Active Site Distances in Metalloenzymes Using Paramagnetic NMR with 13C Hyperpolarization.

Paramagnetic relaxation enhancement (PRE) conjoint with hyperpolarized NMR reveals structural information on the enzyme-product complex in an ongoing metalloenzyme-catalyzed reaction. Substrates of pseudouridine monophosphate glycosidase are hyperpolarized using the dynamic nuclear polarization (DNP) method. Time series of 13C NMR spectra are subsequently measured with the enzyme containing diamagnetic Mg2+ or paramagnetic Mn2+ ions in the active site. The differences of the signal evolution and line widths in the Mg2+ vs Mn2+ reactions are explained through PRE in the enzyme-bound product, which is in fast exchange with its free form. Here, a strong distance dependence of the paramagnetically enhanced relaxation rates enables the calculation of distances from product atoms to the metal center in the complexed structure. The same method can be used to add structural information to real-time characterizations of chemical processes involving compounds with naturally present or artificially introduced paramagnetic sites.

The Unusual Transmembrane Partition of the Hexameric Channel of the Hepatitis C Virus

The p7 protein of the hepatitis C virus (HCV) can oligomerize in membrane to form cation channels. Previous studies showed that the channel assembly in detergent micelles adopts a unique flower-shaped oligomer, but the unusual architecture also presented problems for understanding how this viroporin resides in the membrane. Moreover, the oligomeric state of p7 remains controversial, as both hexamer and heptamer have been proposed. Here we address the above issues using p7 reconstituted in bicelles that mimic a lipid bilayer. We found,using a recently developed oligomer-labeling method, that p7 forms hexamers in the bicelles. Solvent paramagnetic relaxation enhancement analyses showed that the bilayer thickness around the HCV ion channel is substantially smaller than expected, and thus a significant portion of the previously assigned membrane-embedded region is solvent exposed. Our study provides an effective approach for characterizing the transmembrane partition of small ion channels in near lipid bilayer environment.

Fast Magic-Angle-Spinning 19F Spin Exchange NMR for Determining Nanometer 19F-19F Distances in Proteins and Pharmaceutical Compounds.

Internuclear distances measured using NMR provide crucial constraints of three-dimensional structures but are often restricted to about 5 Å due to the weakness of nuclear-spin dipolar couplings. For studying macromolecular assemblies in biology and materials science, distance constraints beyond 1 nm will be extremely valuable. Here we present an extensive and quantitative analysis of the feasibility of 19F spin exchange NMR for precise and robust measurements of interatomic distances up to 1.6 nm at a magnetic field of 14.1 T, under 20-40 kHz magic-angle spinning (MAS). The measured distances are comparable to those achievable from paramagnetic relaxation enhancement but have higher precision, which is better than ±1 Å for short distances and ±2 Å for long distances. For 19F spins with the same isotropic chemical shift but different anisotropic chemical shifts, intermediate MAS frequencies of 15-25 kHz without 1H irradiation accelerate spin exchange. For spectrally resolved 19F-19F spin exchange, 1H-19F dipolar recoupling significantly speeds up 19F-19F spin exchange. On the basis of data from five fluorinated synthetic, pharmaceutical, and biological compounds, we obtained two general curves for spin exchange between CF groups and between CF3 and CF groups. These curves allow 19F-19F distances to be extracted from the measured spin exchange rates after taking into account 19F chemical shifts. These results demonstrate the robustness of 19F spin exchange NMR for distance measurements in a wide range of biological and chemical systems.

Investigating energy-based pool structure selection in the structure ensemble modeling with experimental distance constraints: The example from a multidomain protein Pub1.

The structural variations of multidomain proteins with flexible parts mediate many biological processes, and a structure ensemble can be determined by selecting a weighted combination of representative structures from a simulated structure pool, producing the best fit to experimental constraints such as interatomic distance. In this study, a hybrid structure-based and physics-based atomistic force field with an efficient sampling strategy is adopted to simulate a model di-domain protein against experimental paramagnetic relaxation enhancement (PRE) data that correspond to distance constraints. The molecular dynamics simulations produce a wide range of conformations depicted on a protein energy landscape. Subsequently, a conformational ensemble recovered with low-energy structures and the minimum-size restraint is identified in good agreement with experimental PRE rates, and the result is also supported by chemical shift perturbations and small-angle X-ray scattering data. It is illustrated that the regularizations of energy and ensemble-size prevent an arbitrary interpretation of protein conformations. Moreover, energy is found to serve as a critical control to refine the structure pool and prevent data overfitting, because the absence of energy regularization exposes ensemble construction to the noise from high-energy structures and causes a more ambiguous representation of protein conformations. Finally, we perform structure-ensemble optimizations with a topology-based structure pool, to enhance the understanding on the ensemble results from different sources of pool candidates.

Simultaneous detection of intra- and inter-molecular paramagnetic relaxation enhancements in protein complexes.

Paramagnetic relaxation enhancement (PRE) measurements constitute a powerful approach for detecting both permanent and transient protein-protein interactions. Typical PRE experiments require an intrinsic or engineered paramagnetic site on one of the two interacting partners; while a second, diamagnetic binding partner is labeled with stable isotopes (15N or 13C). Multiple paramagnetic labeled centers or reversed labeling schemes are often necessary to obtain sufficient distance restraints to model protein-protein complexes, making this approach time consuming and expensive. Here, we show a new strategy that combines a modified pulse sequence (1HN-Γ2-CCLS) with an asymmetric labeling scheme to enable the detection of both intra- and inter-molecular PREs simultaneously using only one sample preparation. We applied this strategy to the non-covalent dimer of ubiquitin. Our method confirmed the previously identified binding interface for the transient di-ubiquitin complex, and at the same time, unveiled the internal structural dynamics rearrangements of ubiquitin upon interaction. In addition to reducing the cost of sample preparation and speed up PRE measurements, by detecting the intra-molecular PRE this new strategy will make it possible to measure and calibrate inter-molecular distances more accurately for both symmetric and asymmetric protein-protein complexes.

Integrating NMR, SAXS, and Atomistic Simulations: Structure and Dynamics of a Two-Domain Protein

Multidomain proteins with two or more independently folded functional domains are prevalent in nature. Whereas most multidomain proteins are linked linearly in sequence, roughly one-tenth possess domain insertions where a guest domain is implanted into a loop of a host domain, such that the two domains are connected by a pair of interdomain linkers. Here, we characterized the influence of the interdomain linkers on the structure and dynamics of a domain-insertion protein in which the guest LysM domain is inserted into a central loop of the host CVNH domain. Expanding upon our previous crystallographic and NMR studies, we applied SAXS in combination with NMR paramagnetic relaxation enhancement to construct a structural model of the overall two-domain system. Although the two domains have no fixed relative orientation, certain orientations were found to be preferred over others. We also assessed the accuracies of molecular mechanics force fields in modeling the structure and dynamics of tethered multidomain proteins by integrating our experimental results with microsecond-scale atomistic molecular dynamics simulations. In particular, our evaluation of two different combinations of the latest force fields and water models revealed that both combinations accurately reproduce certain structural and dynamical properties, but are inaccurate for others. Overall, our study illustrates the value of integrating experimental NMR and SAXS studies with long timescale atomistic simulations for characterizing structural ensembles of flexibly linked multidomain systems.

Nuclear magnetic resonance-based determination of dioxygen binding sites in protein cavities.

The location and ligand accessibility of internal cavities in cysteine-free wild-type T4 lysozyme was investigated using O2 gas-pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O2 in solvent to 8.9 mM, O2 -induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C-terminal domain. To determine the number of O2 binding sites and their atomic coordinates from the 1/r6 distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid-search. Two O2 -accessible sites were identified in internal cavities: One site was consistent with the xenon-binding site in the protein in crystal, and the other site was established to be a novel ligand-binding site. MD simulations performed at 10 and 100 mM O2 revealed dioxygen ingress and egress as well as rotational and translational motions of O2 in the cavities. It is therefore suggested that conformational fluctuations within the ground-state ensemble transiently develop channels for O2 association with the internal protein cavities.

Synthesis and Characterization of a Magnetically Active 19F Molecular Beacon.

Gene expression is used extensively to describe cellular characteristics and behaviors; however, most methods of assessing gene expression are unsuitable for living samples, requiring destructive processes such as fixation or lysis. Recently, molecular beacons have become a viable tool for live-cell imaging of mRNA molecules in situ. Historically, beacon-mediated imaging has been limited to fluorescence-based approaches. We propose the design and synthesis of a novel molecular beacon for magnetic resonance detection of any desired target nucleotide sequence. The biologically compatible synthesis incorporates commonly used bioconjugation reactions in aqueous conditions and is accessible for laboratories without extensive synthesis capabilities. The resulting beacon uses fluorine (19F) as a reporter, which is broadened, or turned "off", via paramagnetic relaxation enhancement from a stabilized nitroxide radical spin label when the beacon is not bound to its nucleic acid target. Therefore, the 19F NMR signal of the beacon is quenched in its hairpin conformation when the spin label and the 19F substituent are held in proximity, but the signal is recovered upon beacon hybridization to its specific complementary nucleotide sequence by physical separation of the radical from the 19F reporter. This study establishes a path for magnetic resonance-based assessment of specific mRNA expression, providing new possibilities for applying molecular beacon technology in living systems.

The Use of 13C Direct-Detect NMR to Characterize Flexible and Disordered Proteins.

NMR spectroscopy remains the only experimental technique that provides (near) atomistic structural information for intrinsically disordered proteins (IDPs), but their sequence and structure characteristics still pose major challenges for high-resolution spectroscopy. Carbon-13 direct-detect NMR spectroscopy can overcome poor spectral dispersion and other difficulties associated with traditional 1H-detected NMR of nonaggregating disordered proteins. This chapter presents spectroscopic protocols suitable for complete characterization of IDPs that rely exclusively on 13C direct-detect experiments. The protocols described span initial characterization and chemical shift assignment; structure constraint through residual dipolar coupling and paramagnetic relaxation enhancement measurements; and assessment of intramolecular dynamics through 15N spin relaxation. The experiments described empower investigators to establish molecular mechanisms and structure-function relationships for IDPs and other proteins characterized by high internal flexibility.

Tilted, Uninterrupted, Monomeric HIV-1 gp41 Transmembrane Helix from Residual Dipolar Couplings.

Cryo-electron microscopy and X-ray crystallography have shown that the pre- and postfusion states of the HIV-1 gp41 viral coat protein, although very different from one another, each adopt C3 symmetric structures. A stable homotrimeric structure for the transmembrane domain (TM) also was modeled and supported by experimental data. For a C3 symmetric structure, alignment in an anisotropic medium must be axially symmetric, with the unique axis of the alignment tensor coinciding with the C3 axis. However, NMR residual dipolar couplings (RDCs) measured under three different alignment conditions were found to be incompatible with C3 symmetry. Subsequent measurements by paramagnetic relaxation enhancement, analytical ultracentrifugation, and DEER EPR, indicate that the transmembrane domain is monomeric. 15N NMR relaxation data and RDCs show that TM is highly ordered and uninterrupted for a total length of 32 residues, extending well into the membrane proximal external region.

Application of paramagnetic relaxation enhancements to accelerate the acquisition of 2D and 3D solid-state NMR spectra of oriented membrane proteins.

Oriented sample solid-state NMR (OS-ssNMR) spectroscopy is uniquely suited to determine membrane protein topology at the atomic resolution in liquid crystalline bilayers under physiological temperature. However, the inherent low sensitivity of this technique has hindered the throughput of multidimensional experiments necessary for resonance assignments and structure determination. In this work, we show that doping membrane protein bicelle preparations with paramagnetic ion chelated lipids and exploiting paramagnetic relaxation effects it is possible to accelerate the acquisition of both 2D and 3D multidimensional experiments with significant saving in time. We demonstrate the efficacy of this method for a small membrane protein, sarcolipin, reconstituted in DMPC/POPC/DHPC oriented bicelles. In particular, using Cu2+-DMPE-DTPA as a dopant, we observed a decrease of 1H T1 of sarcolipin by 2/3, allowing us to reduce the recycle delay up to 3 times. We anticipate that these new developments will enable the routine acquisition of multidimensional OS-ssNMR experiments.

Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis.

Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein-ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.

Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR.

Suitable membrane mimetics are crucial to the performance of structural and functional studies of membrane proteins. Phospholipid nanodiscs (formed when a membrane scaffold protein encircles a small portion of a lipid bilayer) have native-like membrane properties. These have been used for a variety of functional studies, but structural studies by high-resolution solution-state NMR spectroscopy of membrane proteins in commonly used nanodiscs of 10-nm diameter were limited by the high molecular weight of these particles, which caused unfavorably large NMR line widths. We have recently constructed truncated versions of the membrane scaffold protein, allowing the preparation of a range of stepwise-smaller nanodiscs (6- to 8-nm diameter) to overcome this limitation. Here, we present a protocol on the assembly of phospholipid nanodiscs of various sizes for structural studies of membrane proteins with solution-state NMR spectroscopy. We describe specific isotope-labeling schemes required for working with large membrane protein systems in nanodiscs, and provide guidelines on the setup of NMR non-uniform sampling (NUS) data acquisition and high-resolution NMR spectra reconstruction. We discuss critical points and pitfalls relating to optimization of nanodiscs for NMR spectroscopy and outline a strategy for the high-resolution structure determination and positioning of isotope-labeled membrane proteins in nanodiscs using nuclear Overhauser enhancement spectroscopy (NOESY) spectroscopy, residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs). Depending on the target protein of interest, nanodisc assembly and purification can be achieved within 12-24 h. Although the focus of this protocol is on protein NMR, these nanodiscs can also be used for (cryo-) electron microscopy (EM) and small-angle X-ray and neutron-scattering studies.

Stability and Water Accessibility of the Trimeric Membrane Anchors of the HIV-1 Envelope Spikes.

HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. Recent studies showed that its transmembrane domain (TMD) forms a trimer in lipid bilayer whose structure has several peculiar features that remain difficult to explain. One is the presence of an arginine R696 in the middle of the TM helix. Additionally, the N- and C-terminal halves of the TM helix form trimeric cores of opposite nature (hydrophobic and hydrophilic, respectively). Here we determined the membrane partition and solvent accessibility of the TMD in bicelles that mimic a lipid bilayer. Solvent paramagnetic relaxation enhancement analysis showed that the R696 is indeed positioned close to the center of the bilayer, but, surprisingly, can exchange rapidly with water as indicated by hydrogen-deuterium exchange measurements. The solvent accessibility of R696 is likely mediated by the hydrophilic core, which also showed fast water exchange. In contrast, the N-terminal hydrophobic core showed extremely slow solvent exchange, suggesting the trimer formed by this region is extraordinarily stable. Our data explain how R696 is accommodated in the middle of the membrane while reporting the overall stability of the Env TMD trimer in lipid bilayer.

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

The human chemokine interleukin-8 (IL-8; CXCL8) is a key mediator of innate immune and inflammatory responses. This small, soluble protein triggers a host of biological effects upon binding and activating CXCR1, a G protein-coupled receptor, located in the cell membrane of neutrophils. Here, we describe 1H-detected magic angle spinning solid-state NMR studies of monomeric IL-8 (1-66) bound to full-length and truncated constructs of CXCR1 in phospholipid bilayers under physiological conditions. Cross-polarization experiments demonstrate that most backbone amide sites of IL-8 (1-66) are immobilized and that their chemical shifts are perturbed upon binding to CXCR1, demonstrating that the dynamics and environments of chemokine residues are affected by interactions with the chemokine receptor. Comparisons of spectra of IL-8 (1-66) bound to full-length CXCR1 (1-350) and to N-terminal truncated construct NT-CXCR1 (39-350) identify specific chemokine residues involved in interactions with binding sites associated with N-terminal residues (binding site-I) and extracellular loop and helical residues (binding site-II) of the receptor. Intermolecular paramagnetic relaxation enhancement broadening of IL-8 (1-66) signals results from interactions of the chemokine with CXCR1 (1-350) containing Mn2+ chelated to an unnatural amino acid assists in the characterization of the receptor-bound form of the chemokine.

Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy.

We demonstrate rapid quantitative measurements of site-resolved paramagnetic relaxation enhancements (PREs), which are a source of valuable structural restraints corresponding to electron-nucleus distances in the ∼10-20 Å regime, in solid-state nuclear magnetic resonance (NMR) spectra of proteins containing covalent Cu2+-binding tags. Specifically, using protein GB1 K28C-EDTA-Cu2+ mutant as a model, we show the determination of backbone amide 15N longitudinal and 1H transverse PREs within a few hours of experiment time based on proton-detected 2D or 3D correlation spectra recorded with magic-angle spinning frequencies ≥ ∼ 60 kHz for samples containing ∼10-50 nanomoles of 2H,13C,15N-labeled protein back-exchanged in H2O. Additionally, we show that the electron relaxation time for the Cu2+ center, needed to convert PREs into distances, can be estimated directly from the experimental data. Altogether, these results are important for establishing solid-state NMR based on paramagnetic-tagging as a routine tool for structure determination of natively diamagnetic proteins.