Relaxation Dispersion Measurements Research Articles

Transverse relaxation optimized spectroscopy of NH2 groups in glutamine and asparagine side chains of proteins.

A transverse relaxation optimized spectroscopy (TROSY) approach is described for the optimal detection of NH2 groups in asparagine and glutamine side chains of proteins. Specifically, we have developed NMR experiments for isolating the slow-relaxing 15N and 1H components of NH2 multiplets. Although even modest sensitivity gains in 2D NH2-TROSY correlation maps compared to their decoupled NH2-HSQC counterparts can be achieved only occasionally, substantial improvements in resolution of the NMR spectra are demonstrated for asparagine and glutamine NH2 sites of a buried cavity mutant, L99A, of T4 lysozyme at 5ºC. The NH2-TROSY approach is applied to CPMG relaxation dispersion measurements at the side chain NH2 positions of the L99A T4 lysozyme mutant-a model system for studies of the role of protein dynamics in ligand binding.

Quantitative Characterization of Chain-Flipping of Acyl Carrier Protein of Escherichia coli Using Chemical Exchange NMR.

The acyl carrier protein of Escherichia coli, termed AcpP, is a prototypical example of type II fatty acid synthase systems found in many bacteria. It serves as a central hub by accepting diverse acyl moieties (4-18 carbons) and shuttling them between its multiple enzymatic partners to generate fatty acids. Prior structures of acyl-AcpPs established that thioester-linked acyl cargos are sequestered within AcpP's hydrophobic lumen. In contrast, structures of enzyme-bound acyl-AcpPs showed translocation of AcpP-tethered acyl chains into the active sites of enzymes. The mechanistic underpinnings of this conformational interplay, termed chain-flipping, are unclear. Here, using heteronuclear NMR spectroscopy, we reveal that AcpP-tethered acyl chains (6-10 carbons) spontaneously adopt lowly populated solvent-exposed conformations. To this end, we devised a new strategy to replace AcpP's thioester linkages with 15N-labeled amide bonds, which facilitated direct "visualization" of these excited states using NMR chemical exchange saturation transfer and relaxation dispersion measurements. Global fitting of the corresponding data yielded kinetic rate constants of the underlying equilibrium and populations and lifetimes of solvent-exposed states. The latter were influenced by acyl chain composition and ranged from milliseconds to submilliseconds for chains containing six, eight, and ten carbons, owing to their variable interactions with AcpP's hydrophobic core. Although transient, the exposure of AcpP-tethered acyl chains to the solvent may allow relevant enzymes to gain access to its active thioester, and the enzyme-induced selection of this conformation will culminate in the production of fatty acids.

Conformation selection by ATP-competitive inhibitors and allosteric communication in ERK2.

Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named 'L' and 'R,' where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

Conformation selection by ATP-competitive inhibitors and allosteric communication in ERK2

Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named ‘L’ and ‘R,’ where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P+1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

Insights into the Structure of Invisible Conformations of Large Methyl Group Labeled Molecular Machines from High Pressure NMR

Most proteins are highly flexible and can adopt conformations that deviate from the energetically most favorable ground state. Structural information on these lowly populated, alternative conformations is often lacking, despite the functional importance of these states. Here, we study the pathway by which the Dcp1:Dcp2 mRNA decapping complex exchanges between an autoinhibited closed and an open conformation. We make use of methyl Carr–Purcell–Meiboom–Gill (CPMG) NMR relaxation dispersion (RD) experiments that report on the population of the sparsely populated open conformation as well as on the exchange rate between the two conformations. To obtain volumetric information on the open conformation as well as on the transition state structure we made use of RD measurements at elevated pressures. We found that the open Dcp1:Dcp2 conformation has a lower molecular volume than the closed conformation and that the transition state is close in volume to the closed state. In the presence of ATP the volume change upon opening of the complex increases and the volume of the transition state lies in-between the volumes of the closed and open state. These findings show that ATP has an effect on the volume changes that are associated with the opening-closing pathway of the complex. Our results highlight the strength of pressure dependent NMR methods to obtain insights into structural features of protein conformations that are not directly observable. As our work makes use of methyl groups as NMR probes we conclude that the applied methodology is also applicable to high molecular weight complexes.

Modeling Molecular Interactions with Wetting and Non-Wetting Rock Surfaces by Combining Electron Paramagnetic Resonance and NMR Relaxometry.

Three types of natural rocks─Bentheimer and Berea sandstones, as well as Liège Chalk─have been aged by immersion in a bitumen solution for extended periods of time in two steps, changing the surface conditions from water-wet to oil-wet. NMR relaxation dispersion measurements were carried out on water and oil constituents, with saturated and aromatic molecules considered individually. In order to separate the different relaxation mechanisms discussed in the literature, 1H and 19F relaxation times were compared to 2H for fully deuterated liquids: while 2H relaxes predominantly by quadrupolar coupling, which is an intramolecular process, the remaining nuclei relax by dipolar coupling, which potentially consists of intra- and intermolecular contributions. The wettability change becomes evident in an increase of relaxation rates for oil and a corresponding decrease for water. However, this expected behavior dominates only for the spin-lattice relaxation rate R1 at very low field strengths and for the spin-spin relaxation rate R2, while high-field longitudinal relaxation shows a much weaker or even reverse trend. This is attributed in part to a change of radical concentration on the pore surface upon coverage of the native rock surface by bitumen as well as by the change of surface chemistry and roughness. EPR and DNP measurements quantify the change of volume vs surface radical concentration in the rocks, and an improved understanding of the role of relaxation via paramagnetic centers is obtained. By means of comparing different fluids and nuclei in combination with a defined wettability change of natural rocks, a refined model for molecular dynamics in conjunction with NMR relaxation dispersion is proposed.

On the potentially spurious contributions of cosolutes in protein 15N relaxation dispersion measurements

A rigorous quantification of the thermodynamics, kinetic and structural changes can be approached by relaxation dispersion methods, when such motions are in the microsecond to millisecond timescale. Albeit some past efforts, it is still unclear how cosolutes modulate the fitted parameters extracted from relaxation dispersion analyses. Here, we have studied how the systematic measurement of 15N relaxation dispersion on a well-studied enzyme, undergoing two-state chemical exchange, are affected in terms of the populations of the minor state (thermodynamics) and the exchange rates (kinetics) in the presence of different cosolutes, from ions to denaturants. The cosolute-induced changes are modest but can significantly affect protein function. Specifically, exchange rates can be associated to a subtle leverage in the exchange rates. For most canonical residues, i.e. without spurious effects caused by the cosolute, the chemical shift difference (Δω) between both states is essentially unaffected with respect to the expected shift of water resonance. Yet, solvent exposed residues do not always follow this canonical trend. Instead, residue-cosolute interactions are large enough to induce a residue specific shift that ultimately modulates the fitted parameter Δω. Unfortunately, solvent accessibility analysis is not accurate enough to a priori discriminate between canonical and non-canonical residues and a full experimental analysis at varying concentrations of the cosolute is required. This observation aims to act as a word of caution for measurements of relaxation dispersion at a single-solvent condition that contains large amounts of cosolutes.

A litmus test for classifying recognition mechanisms of transiently binding proteins

Partner recognition in protein binding is critical for all biological functions, and yet, delineating its mechanism is challenging, especially when recognition happens within microseconds. We present a theoretical and experimental framework based on straight-forward nuclear magnetic resonance relaxation dispersion measurements to investigate protein binding mechanisms on sub-millisecond timescales, which are beyond the reach of standard rapid-mixing experiments. This framework predicts that conformational selection prevails on ubiquitin’s paradigmatic interaction with an SH3 (Src-homology 3) domain. By contrast, the SH3 domain recognizes ubiquitin in a two-state binding process. Subsequent molecular dynamics simulations and Markov state modeling reveal that the ubiquitin conformation selected for binding exhibits a characteristically extended C-terminus. Our framework is robust and expandable for implementation in other binding scenarios with the potential to show that conformational selection might be the design principle of the hubs in protein interaction networks.

Structural basis for the recognition of the bacterial tyrosine kinase Wzc by its cognate tyrosine phosphatase Wzb

Bacterial tyrosine kinases (BY-kinases) comprise a family of protein tyrosine kinases that are structurally distinct from their functional counterparts in eukaryotes and are highly conserved across the bacterial kingdom. BY-kinases act in concert with their counteracting phosphatases to regulate a variety of cellular processes, most notably the synthesis and export of polysaccharides involved in biofilm and capsule biogenesis. Biochemical data suggest that BY-kinase function involves the cyclic assembly and disassembly of oligomeric states coupled to the overall phosphorylation levels of a C-terminal tyrosine cluster. This process is driven by the opposing effects of intermolecular autophosphorylation, and dephosphorylation catalyzed by tyrosine phosphatases. In the absence of structural insight into the interactions between a BY-kinase and its phosphatase partner in atomic detail, the precise mechanism of this regulatory process has remained poorly defined. To address this gap in knowledge, we have determined the structure of the transiently assembled complex between the catalytic core of the Escherichia coli (K-12) BY-kinase Wzc and its counteracting low-molecular weight protein tyrosine phosphatase (LMW-PTP) Wzb using solution NMR techniques. Unambiguous distance restraints from paramagnetic relaxation effects were supplemented with ambiguous interaction restraints from static spectral perturbations and transient chemical shift changes inferred from relaxation dispersion measurements and used in a computational docking protocol for structure determination. This structurepresents an atomic picture of the mode of interaction between an LMW-PTP and its BY-kinase substrate, and provides mechanistic insight into the phosphorylation-coupled assembly/disassembly process proposed to drive BY-kinase function.

Analysis of conformational exchange processes using methyl-TROSY-based Hahn echo measurements of quadruple-quantum relaxation.

Transverse nuclear spin relaxation is a sensitive probe of chemical exchange on timescales on the order of microseconds to milliseconds. Here we present an experiment for the simultaneous measurement of the relaxation rates of two quadruple-quantum transitions in CH-labelled methyl groups. These coherences are protected against relaxation by intra-methyl dipolar interactions and so have unexpectedly long lifetimes within perdeuterated biomacromolecules. However, these coherences also have an order of magnitude higher sensitivity to chemical exchange broadening than lower order coherences and therefore provide ideal probes of dynamic processes. We show that analysis of the static magnetic field dependence of zero-, double- and quadruple-quantum Hahn echo relaxation rates provides a robust indication of chemical exchange and can determine the signed relative magnitudes of proton and carbon chemical shift differences between ground and excited states. We also demonstrate that this analysis can be combined with established Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion measurements, providing improved precision in parameter estimates, particularly in the determination of H chemical shift differences.

Hydrogen/Deuterium Exchange and Nuclear Magnetic Resonance Spectroscopy Reveal Dynamic Allostery on Multiple Time Scales in the Serine Protease Thrombin.

A deeper understanding of how hydrogen/deuterium exchange mass spectrometry (HDX-MS) reveals allostery is important because HDX-MS can reveal allostery in systems that are not amenable to nuclear magnetic resonance (NMR) spectroscopy. We were able to study thrombin and its complex with thrombomodulin, an allosteric regulator, by both HDX-MS and NMR. In this Perspective, we compare and contrast the results from both experiments and from molecular dynamics simulations. NMR detects changes in the chemical environment around the protein backbone N-H bond vectors, providing residue-level information about the conformational exchange between distinct states. HDX-MS detects changes in amide proton solvent accessibility and H-bonding. Taking advantage of NMR relaxation dispersion measurements of the time scale of motions, we draw conclusions about the motions reflected in HDX-MS experiments. Both experiments detect allostery, but they reveal different components of the allosteric transition. The insights gained from integrating NMR and HDX-MS into thrombin dynamics enable a clearer interpretation of the evidence for allostery revealed by HDX-MS in larger protein complexes and assemblies that are not amenable to NMR.

NMR T1 dispersion of crude oils from 10 kHz to 20 MHz

In this article T1 dispersion measurements on a set of crude oils that span a viscosity range of 0.7 cP up to 2·104 cP are reported. Larmor frequencies were varied from 10 kHz up to 20 MHz. The relaxation dispersion measurements are interpreted in terms of the threshold viscosity model, which allows model applicability tests presented in literature to be extended to Larmor frequencies below 1 MHz. It is shown that the measurements can be equally well described by assuming a proton-proton interaction process as by assuming proton-electronic spin interactions. A power-law frequency dependence of the threshold viscosity parameter is observed for both types of spin interactions. Modifications to the description of rotational diffusion in the threshold viscosity model are suggested that may account for the observed frequency dependence in the threshold viscosity parameter. Without modifications, the threshold viscosity model is shown to have a limited application range of η ≥ 40 cP and ω0/2π ≥ 700 kHz, in which model parameters have a physically justifiable order of magnitude for the set of crude oils studied. Outside this range, the threshold viscosity model can still be applied as a parametrization of T1 dispersion effects for the complete set of experiments presented in this study. This explicit viscosity and Larmor frequency dependent parametrization may be used as first-order approximation to T1 dispersion in crude oils for which only oil viscosity is known and may be applied to polarization level and pulse sequence simulations in low field NMR relaxometry studies up to 20 MHz for a wide range of crude oil viscosities, and in particular for better interpretation of NMR flow measurements on crude oils.

Modeling of Hidden Structures Using Sparse Chemical Shift Data from NMR Relaxation Dispersion

NMR relaxation dispersion measurements report on conformational changes occurring on the μs-ms timescale. Chemical shift information derived from relaxation dispersion can be used to generate structural models of weakly populated alternative conformational states. Current methods to obtain such models rely on determining the signs of chemical shift changes between the conformational states, which are difficult to obtain in many situations. Here, we use a “sample and select” method to generate relevant structural models of alternative conformations of the C-terminal-associated region of Escherichia coli dihydrofolate reductase (DHFR), using only unsigned chemical shift changes for backbone amides and carbonyls (1H, 15N, and 13C′). We find that CS-Rosetta sampling with unsigned chemical shift changes generates a diversity of structures that are sufficient to characterize a minor conformational state of the C-terminal region of DHFR. The excited state differs from the ground state by a change in secondary structure, consistent with previous predictions from chemical shift hypersurfaces and validated by the x-ray structure of a partially humanized mutant of E. coli DHFR (N23PP/G51PEKN). The results demonstrate that the combination of fragment modeling with sparse chemical shift data can determine the structure of an alternative conformation of DHFR sampled on the μs-ms timescale. Such methods will be useful for characterizing alternative states, which can potentially be used for in silico drug screening, as well as contributing to understanding the role of minor states in biology and molecular evolution.

Probing conformational transitions towards mutagenic Watson\u2013Crick-like G\xb7T mismatches using off-resonance sugar carbon R1\u03c1 relaxation dispersion

NMR off-resonance R1ρ relaxation dispersion measurements on base carbon and nitrogen nuclei have revealed that wobble G·T/U mismatches in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-abundance, and mutagenic Watson–Crick-like conformations. As Watson–Crick-like G·T mismatches have base pairing geometries similar to Watson–Crick base pairs, we hypothesized that they would mimic Watson–Crick base pairs with respect to the sugar-backbone conformation as well. Using off-resonance R1ρ measurements targeting the sugar C3′ and C4′ nuclei, a structure survey, and molecular dynamics simulations, we show that wobble G·T mismatches adopt sugar-backbone conformations that deviate from the canonical Watson–Crick conformation and that transitions toward tautomeric and anionic Watson–Crick-like G·T mismatches restore the canonical Watson–Crick sugar-backbone. These measurements also reveal kinetic isotope effects for tautomerization in D2O versus H2O, which provide experimental evidence in support of a transition state involving proton transfer. The results provide additional evidence in support of mutagenic Watson–Crick-like G·T mismatches, help rule out alternative inverted wobble conformations in the case of anionic G·T−, and also establish sugar carbons as new non-exchangeable probes of this exchange process.

NMR Relaxation Dispersion Reveals Macrocycle Breathing Dynamics in a Cyclodextrin-based Rotaxane.

A distinctive feature of mechanically interlocked molecules (MIMs) is the relative motion between the mechanically bonded components, and often it is the functional basis for artificial molecular machines and new functional materials. Optimization of machine or materials performance requires knowledge of the underlying atomic-level mechanisms that control the motion. The field of biomolecular NMR spectroscopy has developed a diverse set of pulse schemes that can characterize molecular dynamics over a broad time scale, but these techniques have not yet been used to characterize the motion within MIMs. This study reports the first observation of NMR relaxation dispersion related to MIM motion. The rotary (pirouette) motion of α-cyclodextrin (αCD) wheels was characterized in a complementary pair of rotaxanes with pirouetting switched ON or OFF. 13C and 1H NMR relaxation dispersion measurements reveal previously unknown exchange dynamics for the αCD wheels in the pirouette-ON rotaxane with a rate constant of 2200 s-1 at 298 K and an activation barrier of ΔF‡ = 43 ± 3 kJ/mol. The exchange dynamics disappear in the pirouette-OFF rotaxane, demonstrating their switchable nature. The 13C and 1H sites exhibiting relaxation dispersion suggest that the exchange involves "macrocycle breathing", in which the αCD wheel fluctuates between a contracted or expanded state, the latter enabling diffusive rotary motion about the axle. The substantial insight from these NMR relaxation dispersion methods suggests similar dynamic NMR methods can illuminate the fast time scale (microsecond to millisecond) mechanisms of intercomponent motion in a wide range of MIMs.

Design of a fast field-cycling magnetic resonance imaging system, characterization and methods for relaxation dispersion measurements around 1.5 T.

The dependence of the nuclear magnetic resonance relaxation rate on the magnetic field has been widely studied, in particular, in biomedical areas with the objectives to better understand the underlying microscopic mechanisms in tissues and provide biomarkers of diseases. By combining fast-field cycling (FFC) and magnetic resonance imaging (MRI), it is possible to provide localized relaxation dispersion measurements in heterogeneous systems with recent demonstrations in solutions, biological samples, human beings, and small animals. We report here the developments and performances of a device designed for small animal FFC-MRI comprising a resistive insert technology operating inside a 1.5 T MRI system. Specific measurement methods were developed to characterize the system efficiency, response time, homogeneity, stability, and compensation. By adding a non-linear element in the system and using a dual amplifier strategy, it is shown that large field offsets can be produced during relaxation periods while maintaining precise field control during detection periods. The measurement of longitudinal nuclear magnetic relaxation dispersion (NMRD) profiles in the range of 1.08 T-1.92 T is reported, essentially displaying a linear variation in this range for common MRI contrast agents. The slopes of both the longitudinal and transverse relaxation dispersion profiles at 1.5 T are measured and validated, extending the capabilities of previous approaches. The performances of a longitudinal relaxation dispersion mapping method are finally reported, opening the way to quantitative preclinical dispersion imaging studies at a high FFC-MRI field.

Role of Protein Dynamics in the Function of p38 Kinase and Protein Tyrosine Phosphatase 1B

In order to become highly specific, kinases and phosphatase require a carefully orchestrated mode of action. These modes are driven by both protein conformation and dynamics. Here, we use biomolecular NMR spectroscopy to determine how dynamics influence the catalytic activity, substrate selectivity and allostery in the kinase p38 and the phosphatase PTP1B. The MAPK p38 is a ser/thr protein kinase that is essential for cell differentiation and autophagy. The exact events that lead to p38 activation, however, are not well understood. Thus, we used a variety of NMR relaxation measurements applied to different states of the activation pathway of p38, which includes phosphorylation, substrate, and ATP binding, to understand the activation and regulation of p38. Unexpectedly, our data showed that phosphorylation of p38 is important, but substrate binding is indeed key for the synchronization of the dynamics across the molecule to activate p38. And, it is this synchronization that allows for enhanced ATP recruitment and thus the full activity of p38. The tyrosine phosphatase PTP1B is essential for a variety of cellular processes including glucose metabolism, body mass regulation, and proliferation. We have determined that PTP1B uses rigid conformation changes to achieve its enzymatic activity that can be dynamically modulated by allosteric action. Interestingly, this allostery is mainly driven by dynamic changes. To gain further insights into PTP1B dynamics and to understand how the backbone and side-chain dynamics are correlated, we performed relaxation dispersion measurements to measure the side-chain motions in PTP1B. Here, we present the results of PTP1B side-chain dynamics and show how it regulates its overall function.

Fast Microsecond Dynamics of the Protein-Water Network in the Active Site of Human Carbonic Anhydrase II Studied by Solid-State NMR Spectroscopy.

Protein-water interactions have widespread effects on protein structure and dynamics. As such, the function of many biomacromolecules can be directly related to the presence and exchange of water molecules. While the presence of structural water sites can be easily detected by X-ray crystallography, the dynamics within functional water-protein network architectures is largely elusive. Here we use solid-state NMR relaxation dispersion measurements with a focus on those active-site residues in the enzyme human carbonic anhydrase II (hCAII) that constitute the evolutionarily conserved water pocket, key for CAs' enzymatic catalysis. Together with chemical shifts, peak broadening, and results of molecular dynamics (MD) and DFT shift calculations, the relaxation dispersion data suggest the presence of a widespread fast μs-time-scale dynamics in the pocket throughout the protein-water network. This process is abrogated in the presence of an inhibitor which partially disrupts the network. The time scale of the protein-water pocket motion coincides both with the estimated residence time of Zn-bound water/OH- in the pocket showing the longest lifetimes in earlier magnetic relaxation dispersion experiments as well as with the rate-limiting step of catalytic turnover. As such, the reorganization of the water pocket:enzyme architecture might constitute an element of importance for enzymatic activity of this and possibly other proteins.

Quantifying Microsecond Exchange in Large Protein Complexes with Accelerated Relaxation Dispersion Experiments in the Solid State

Solid state NMR is a powerful method to obtain information on the structure and dynamics of protein complexes that, due to solubility and size limitations, cannot be achieved by other methods. Here, we present an approach that allows the quantification of microsecond conformational exchange in large protein complexes by using a paramagnetic agent to accelerate 15N R1ρ relaxation dispersion measurements and overcome sensitivity limitations. The method is validated on crystalline GB1 and then applied to a >300 kDa precipitated complex of GB1 with full length human immunoglobulin G (IgG). The addition of a paramagnetic agent increased the signal to noise ratio per time unit by a factor of 5, which allowed full relaxation dispersion curves to be recorded on a sample containing less than 50 μg of labelled material in 5 and 10 days on 850 and 700 MHz spectrometers, respectively. We discover a similar exchange process across the β-sheet in GB1 in crystals and in complex with IgG. However, the slow motion observed for a number of residues in the α-helix of crystalline GB1 is not detected in the complex.

Gd3+:DOTA-Modified 2-Hydroxypropyl-β-Cyclodextrin/4-Sulfobutyl Ether-β-Cyclodextrin-Based Polyrotaxanes as Long Circulating High Relaxivity MRI Contrast Agents.

A family of five water-soluble Gd3+:1,4,7,10-tetraazacyclododecane-1,4,7-tetraacetic acid-modified polyrotaxane (PR) magnetic resonance contrast agents bearing mixtures of 2-hydroxypropyl-β-cyclodextrin and 4-sulfobutylether-β-cyclodextrin macrocycles threaded onto Pluronic cores were developed as long circulating magnetic resonance contrast agents. Short diethylene glycol diamine spacers were utilized for linking the macrocyclic chelator to the PR scaffold prior to Gd3+ chelation. The PR products were characterized by 1H NMR, gel permeation chromatography/multiangle light scattering, dynamic light scattering, and analytical ultracentrifugation. Nuclear magnetic relaxation dispersion and molar relaxivity measurements at 23 °C revealed that all the PR contrast agents displayed high spin-spin T1 relaxation and spin-lattice T2 relaxation rates relative to a DOTAREM control. When injected at 0.05 mmol Gd/kg body weight in BALB/c mice, the PR contrast agents increased the T1-weighted MR image intensities with longer circulation times in the blood pool than DOTAREM. Excretion of the agents occurred predominantly via the renal or biliary routes depending on the polyrotaxane structure, with the longest circulating L81 Pluronic-based agent showing the highest liver uptake. Proteomic analysis of PR bearing different β-cyclodextrin moieties indicated that lipoproteins were the predominant component associated with these materials after serum exposure, comprising as much as 40% of the total protein corona. We infer from these findings that Gd(III)-modified PR contrast agents are promising long-circulating candidates for blood pool analysis by MRI.