Exploring Regions of Conformational Space Occupied by Two-Domain Proteins

The presence of heterogeneity in the interdomain arrangement of several biomolecules is required for their function. Here we present a method to obtain crucial clues to distinguish between different kinds of protein conformational distributions based on experimental NMR data. The method explores subregions of the conformational space and provides both upper and lower bounds of probability for the system to be in each subregion

Solid-State NMR Crystallography through Paramagnetic Restraints

Pseudocontact shifts (PCSs) measured by solid-state NMR spectroscopy (SS-NMR) on microcrystalline powders of a paramagnetic metalloprotein permit NMR crystallography. Along with other restraints for SS-NMR experiments, the protein molecular structure as well as the correct crystal packing are obtained

Solid-State NMR Crystallography through Paramagnetic Restraints

Pseudocontact shifts (PCSs) measured by solid-state NMR spectroscopy (SS-NMR) on microcrystalline powders of a paramagnetic metalloprotein permit NMR crystallography. Along with other restraints for SS-NMR experiments, the protein molecular structure as well as the correct crystal packing are obtained

Effect of Magnetic Coupling on Water Proton Relaxivity in a Series of Transition Metal Gd^III Complexes

A fundamental challenge in the design of bioresponsive (or bioactivated) Gd^III-based magnetic resonance (MR) imaging probes is the considerable background signal present in the “preactivated” state that arises from outer-sphere relaxation processes. When sufficient concentrations of a bioresponsive agent are present (i.e., a detectable signal in the image), the inner- and outer-sphere contributions to <i>r</i>₁ may be misinterpreted to conclude that the agent has been activated, when it has not. Of the several parameters that determine the observed MR signal of an agent, only the electron relaxation time (<i>T</i>_1e) impacts both the inner- and outer-sphere relaxation. Therefore, strategies to minimize this background signal must be developed to create a near zero-background (or truly “off” state) of the agent. Here, we demonstrate that intramolecular magnetic exchange coupling when Gd^III is coupled to a paramagnetic transition metal provides a means to overcome the contribution of second- and outer-sphere contributions to the observed relaxivity. We have prepared a series of complexes with the general formula LMLn­(μ-O₂CCH₃)­(O₂CCH₃)₂ (M = Co, Cu, Zn). Solid-state magnetic susceptibility measurements reveal significant magnetic coupling between Gd^III and the transition metal ion. Nuclear magnetic relaxation dispersion (NMRD) analysis confirms that the observed differences in relaxivity are associated with the modulation of <i>T</i>_1e at Gd^III. These results clearly demonstrate that magnetic exchange coupling between Gd^III and a transition metal ion can provide a significant decrease in <i>T</i>_1e (and therefore the relaxivity of Gd^III). This design strategy is being exploited to prepare new generations of <i>preclinical</i> bioresponsive MR imaging probes with near zero-background

Large Protein Assemblies for High-Relaxivity Contrast Agents: The Case of Gadolinium-Labeled Asparaginase

Biologics are emerging as the most important class of drugs and are used to treat a large variety of pathologies. Most of biologics are proteins administered in large amounts, either by intramuscular injection or by intravenous infusion. Asparaginase is a large tetrameric protein assembly, currently used against acute lymphoblastic leukemia. Here, a gadolinium(III)-DOTA derivative has been conjugated to asparaginase, and its relaxation properties have been investigated to assess its efficiency as a possible theranostic agent. The field-dependent 1H longitudinal relaxation measurements of water solutions of gadolinium(III)-labeled asparaginase indicate a very large increase in the relaxivity of this paramagnetic protein complex with respect to small gadolinium chelates, opening up the possibility of its use as an MRI contrast agent

High Relaxivity Gd(III)–DNA Gold Nanostars: Investigation of Shape Effects on Proton Relaxation

Gadolinium(III) nanoconjugate contrast agents (CAs) have distinct advantages over their small-molecule counterparts in magnetic resonance imaging. In addition to increased Gd(III) payload, a significant improvement in proton relaxation efficiency, or relaxivity (<i>r</i>₁), is often observed. In this work, we describe the synthesis and characterization of a nanoconjugate CA created by covalent attachment of Gd(III) to thiolated DNA (Gd(III)–DNA), followed by surface conjugation onto gold nanostars (DNA–Gd@stars). These conjugates exhibit remarkable <i>r</i>₁ with values up to 98 mM^–1 s^–1. Additionally, DNA–Gd@stars show efficient Gd(III) delivery and biocompatibility <i>in vitro</i> and generate significant contrast enhancement when imaged at 7 T. Using nuclear magnetic relaxation dispersion analysis, we attribute the high performance of the DNA–Gd@stars to an increased contribution of second-sphere relaxivity compared to that of spherical CA equivalents (DNA–Gd@spheres). Importantly, the surface of the gold nanostar contains Gd(III)–DNA in regions of positive, negative, and neutral curvature. We hypothesize that the proton relaxation enhancement observed results from the presence of a unique hydrophilic environment produced by Gd(III)–DNA in these regions, which allows second-sphere water molecules to remain adjacent to Gd(III) ions for up to 10 times longer than diffusion. These results establish that particle shape and second-sphere relaxivity are important considerations in the design of Gd(III) nanoconjugate CAs

Long-Range Correlated Dynamics in Intrinsically Disordered Proteins

Intrinsically disordered proteins (IDPs) are involved in a wide variety of physiological and pathological processes and are best described by ensembles of rapidly interconverting conformers. Using fast field cycling relaxation measurements we here show that the IDP α-synuclein as well as a variety of other IDPs undergoes slow reorientations at time scales comparable to folded proteins. The slow motions are not perturbed by mutations in α-synuclein, which are related to genetic forms of Parkinson’s disease, and do not depend on secondary and tertiary structural propensities. Ensemble-based hydrodynamic calculations suggest that the time scale of the underlying correlated motion is largely determined by hydrodynamic coupling between locally rigid segments. Our study indicates that long-range correlated dynamics are an intrinsic property of IDPs and offers a general physical mechanism of correlated motions in highly flexible biomolecular systems

Nanodiamond–Gadolinium(III) Aggregates for Tracking Cancer Growth In Vivo at High Field

The ability to track labeled cancer cells in vivo would allow researchers to study their distribution, growth, and metastatic potential within the intact organism. Magnetic resonance (MR) imaging is invaluable for tracking cancer cells in vivo as it benefits from high spatial resolution and the absence of ionizing radiation. However, many MR contrast agents (CAs) required to label cells either do not significantly accumulate in cells or are not biologically compatible for translational studies. We have developed carbon-based nanodiamond–gadolinium­(III) aggregates (NDG) for MR imaging that demonstrated remarkable properties for cell tracking in vivo. First, NDG had high relaxivity independent of field strength, a finding unprecedented for gadolinium­(III) [Gd­(III)]–nanoparticle conjugates. Second, NDG demonstrated a 300-fold increase in the cellular delivery of Gd­(III) compared to that of clinical Gd­(III) chelates without sacrificing biocompatibility. Further, we were able to monitor the tumor growth of NDG-labeled flank tumors by <i>T</i>₁- and <i>T</i>₂-weighted MR imaging for 26 days in vivo, longer than was reported for other MR CAs or nuclear agents. Finally, by utilizing quantitative maps of relaxation times, we were able to describe tumor morphology and heterogeneity (corroborated by histological analysis), which would not be possible with competing molecular imaging modalities

Gd(III)-Labeled Peptide Nanofibers for Reporting on Biomaterial Localization <i>in Vivo</i>

Bioactive supramolecular nanostructures are of great importance in regenerative medicine and the development of novel targeted therapies. In order to use supra­molecular chemistry to design such nanostructures, it is extremely important to track their fate <i>in vivo</i> through the use of molecular imaging strategies. Peptide amphiphiles (PAs) are known to generate a wide array of supramolecular nanostructures, and there is extensive literature on their use in areas such as tissue regeneration and therapies for disease. We report here on a series of PA molecules based on the well-established β-sheet amino acid sequence V₃A₃ conjugated to macrocyclic Gd(III) labels for magnetic resonance imaging (MRI). These conjugates were shown to form cylindrical supra­molecular assemblies using cryogenic transmission electron microscopy and small-angle X-ray scattering. Using nuclear magnetic relaxation dispersion analysis, we observed that thermal annealing of the nanostructures led to a decrease in water exchange lifetime (τ_m) of hundreds of nanoseconds only for molecules that self-assemble into nanofibers of high aspect ratio. We interpret this decrease to indicate more solvent exposure to the paramagnetic moiety on annealing, resulting in faster water exchange within angstroms of the macrocycle. We hypothesize that faster water exchange in the nanofiber-forming PAs arises from the dehydration and increase in packing density on annealing. Two of the self-assembling conjugates were selected for imaging PAs after intra­muscular injections of the PA C₁₆V₃A₃E₃-NH₂ in the <i>tibialis anterior</i> muscle of a murine model. Needle tracts were clearly discernible with MRI at 4 days post­injection. This work establishes Gd(III) macrocycle-conjugated peptide amphiphiles as effective tracking agents for peptide amphiphile materials <i>in vivo</i> over the timescale of days

Mechanisms of Gadographene-Mediated Proton Spin Relaxation

Gd­(III) associated with carbon nanomaterials relaxes water proton spins at an effectiveness that approaches or exceeds the theoretical limit for a single bound water molecule. These Gd­(III)-labeled materials represent a potential breakthrough in sensitivity for Gd­(III)-based contrast agents used for magnetic resonance imaging (MRI). However, their mechanism of action remains unclear. A gadographene library encompassing GdCl₃, two different Gd­(III) complexes, graphene oxide (GO), and graphene suspended by two different surfactants and subjected to varying degrees of sonication was prepared and characterized for their relaxometric properties. Gadographene was found to perform comparably to other Gd­(III)–carbon nanomaterials; its longitudinal (<i>r</i>₁) and transverse (<i>r</i>₂) relaxivity are modulated between 12–85 mM^–1 s^–1 and 24–115 mM^–1 s^–1, respectively, depending on the Gd­(III)–carbon backbone combination. The unusually large relaxivity and its variance can be understood under the modified Florence model incorporating the Lipari–Szabo approach. Changes in hydration number (<i>q</i>), water residence time (τ_M), molecular tumbling rate (τ_R), and local motion (τ_fast) sufficiently explain most of the measured relaxivities. Furthermore, results implicated the coupling between graphene and Gd­(III) as a minor contributor to proton spin relaxation