CPMG Experiments Research Articles

Measuring Hydroxyl Exchange Rates in Glycans Using a Synergistic Combination of Saturation Transfer and Relaxation Dispersion NMR.

Molecular recognition events mediated by glycans play pivotal roles in controlling the fate of diverse biological processes such as cellular communication and the immune response. The affinity of glycans for their target receptors is governed primarily by the hydrogen bonds formed by hydroxyl groups decorating the glycan surface. Hydroxyl exchange rate constants are therefore vital parameters that report on glycan structure and dynamics. Here we present a strategy for characterizing hydroxyl hydrogen/deuterium (H/D) exchange in glycans that employs a synergistic combination of 13C chemical exchange saturation transfer (CEST) and Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG) NMR methods. We show that the combination of CEST and CPMG experiments facilitates the sensitive detection of the small (∼0.1 ppm) two-bond deuterium isotope shift on a 13C nucleus when the attached hydroxyl group fluctuates between protonated and deuterated states. This shift is leveraged for measuring site-specific kinetic H/D exchange rate constants as well as thermodynamic free energies of isotope fractionation. The CEST and CPMG modules are integrated with a selective J-cross-polarization scheme that provides the flexibility for rapid characterization of H/D exchange at a specific hydroxyl site. Moreover, our approach enables the precise isothermal measurement of hydroxyl exchange rate constants without the need for cumbersome isotope labeling. The H/D exchange rate constants of three different glycans assessed using this method highlight its potential for detecting transient intra- and intermolecular hydrogen bonds. In addition, the trends in H/D exchange rate constants establish site-specific steric accessibility as a key determinant of solvent exchange dynamics in glycans.

Beyond slow two-state protein conformational exchange using CEST: applications to three-state protein interconversion on the millisecond timescale.

Although NMR spectroscopy is routinely used to study the conformational dynamics of biomolecules, robust analyses of the data are challenged in cases where exchange is more complex than two-state, such as when a 'visible' major conformer exchanges with two 'invisible' minor states on the millisecond timescale. It is becoming increasingly clear that chemical exchange saturation transfer (CEST) NMR experiments that were initially developed to study systems undergoing slow interconversion are also sensitive to intermediate-fast timescale biomolecular conformational exchange. Here we investigate the utility of the amide 15N CEST experiment to characterise protein three-state exchange occurring on the millisecond timescale by studying the interconversion between the folded (F) state of the FF domain from human HYPA/FBP11 (WT FF) and two of its folding intermediates I1 and I2. Although 15N CPMG experiments are consistent with the F state interconverting with a single minor state on the millisecond timescale, 15N CEST data clearly establish an exchange process between F and a pair of minor states. A unique three-state exchange model cannot be obtained by analysis of 15N CEST data recorded at a single temperature. However, including the relative sign of the difference in the chemical shifts of the two minor states based on a simple two-state analysis of CEST data recorded at multiple temperatures, results in a robust three-state model in which the F, I1 and I2 states interconvert with each other on the millisecond timescale ( ~ 550s-1, ~ 1200s-1, ~ 5000s-1), with I1 and I2 sparsely populated at ~ 0.15% and ~ 0.35%, respectively, at 15°C. A computationally demanding grid-search of exchange parameter space is not required to extract the best-fit exchange parameters from the CEST data. The utility of the CEST experiment, thus, extends well beyond studies of conformers in slow exchange on the NMR chemical shift timescale, to include systems with interconversion rates on the order of thousands/second.

Dynamic allostery in substrate binding by human thymidylate synthase.

Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide substrate. We have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ~1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by 2H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity-a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme.

Investigation of the Low-Populated Excited States of the HIV-1 Nucleocapsid Domain.

The nucleocapsid domain (NCd), located at the C-terminus of the HIV-1 Gag protein, is involved in numerous stages of the replication cycle, such as the packaging of the viral genome and reverse transcription. It exists under different forms through the viral life cycle, depending on the processing of Gag by the HIV-1 protease. NCd is constituted of two adjacent zinc knuckles (ZK1 and ZK2), separated by a flexible linker and flanked by disordered regions. Here, conformational equilibria between a major and two minor states were highlighted exclusively in ZK2, by using CPMG and CEST NMR experiments. These minor states appear to be temperature dependent, and their populations are highest at physiological temperature. These minor states are present both in NCp7, the mature form of NCd, and in NCp9 and NCp15, the precursor forms of NCd, with increased populations. The role of these minor states in the targeting of NCd by drugs and its binding properties is discussed.

Oxygen saturation-dependent effects on blood transverse relaxation at low fields

ObjectiveBlood oxygenation can be measured using magnetic resonance using the paramagnetic effect of deoxy-haemoglobin, which decreases the textit{T}_{2} relaxation time of blood. This textit{T}_{2} contrast has been well characterised at the textit{B}_{{0}} fields used in MRI (1.5 T and above). However, few studies have characterised this effect at lower magnetic fields. Here, the feasibility of blood oximetry at low field based on textit{T}_{2} changes that are within a physiological relevant range is explored. This study could be used for specifying requirements for construction of a monitoring device based on low field permanent magnet systems.MethodsA continuous flow circuit was used to control parameters such as oxygen saturation and temperature in a sample of blood. It flowed through a variable field magnet, where CPMG experiments were performed to measure its textit{T}_{2}. In addition, the oxygen saturation was monitored by an optical sensor for comparison with the textit{T}_{2} changes.ResultsThese results show that at low textit{B}_{{0}} fields, the change in blood textit{T}_{2} due to oxygenation is small, but still detectable. The data measured at low fields are also in agreement with theoretical models for the oxy-deoxy textit{T}_{2} effect.Conclusiontextit{T}_{2} changes in blood due to oxygenation were observed at fields as low as 0.1 T. These results suggest that low field NMR relaxometry devices around 0.3 T could be designed to detect changes in blood oxygenation.

Studies of periodic seawater spray icing with unilateral NMR

Sea water ice has a complicated pore structure consisting of brine-filled pockets within a solid ice matrix. In this work, an unilateral Nuclear Magnetic Resonance instrument was used to characterize the evolution of sea-ice brine inclusions in two types of ice: stationary seawater ice and seawater spray ice formed by periodic spraying on horizontal and vertical surfaces. The portable unilateral NMR was capable of measuring very low amounts of brine (<10% of the water-filled volume). CPMG experiments were performed to extract the information on brine content and T2 distribution at temperatures between −6 °C and −16 °C. 1D imaging was used to spatially resolve the ice brine accumulation. The results show that the seawater spray ice growth, brine content (greater for the horizontal orientation than for the vertical one), and T2 distribution (unimodal for stationary ice and bimodal for spray ice) depend on temperature and surface orientation.

Quantification of Whole-Brain Oxygenation Extraction Fraction Among People with Sickle Cell Anemia without Stroke History

IntroductionAn increased oxygen extraction fraction (OEF) is an indicator of decreased cerebrovascular reserve and may help predict the risk of cerebral infarction in people with sickle cell anemia (SCA). MRI T2 oximetry can conveniently convert measured venous blood T2 values to venous oxygenation fraction (Yv) using predetermined hematocrit (Hct)-dependent calibration curves. Previously, calibration models established for healthy adults and neonates were applied to people with SCA. However, a recent study on SCA blood showed T2 dependence on Y, but not on Hct (Bush et al. ISMRM, 2017, #1898). This might be due to the variations in disease history and treatment status of the SCA cohort in that study. Here, we established a calibration curve specific to people with SCA without a history of stroke or recent transfusion. Whole-brain OEFs for individuals with SCA were derived using this calibration model.MethodsExperiments were performed on a 3T Philips Achieva scanner. Thirteen healthy volunteers (35 ± 8 yrs; 7M / 6F; Hct: 0.42+/-0.03) and ten people with SCA (25 ± 7 yrs; 4M / 6F; HbS: 82%+/-7%; HbF: 9%+/-4%; Hct: 0.24+/-0.05) with no history of stroke, recent transfusion, or renal impairment were enrolled after informed consent.Venous blood T2 values were measured in the internal jugular vein (IJV) using a 48 sec protocol (Qin et al. MRM, 2011, 65:471). In vitro T2 measurements were conducted on the same day on venous blood drawn from the healthy volunteers and patients with SCA under physiological conditions (T=37ºC) following established protocols. Nitrogen and air gas were used to prepare different oxygenation levels (ranging from ~50% to 100%) for each blood sample. CPMG experiments in vitro were performed within 1 min to prevent sedimentation of erythrocytes. Blood oxygenation was measured immediately after every measurement using a blood analyzer. Blood T2 values measured in vitro from both normal and SCA samples were separately fitted relative to Y and Hct using an empirical model proposed by Lu et al. (MRM, 2012, 67:42) (Fig. 1a). Then venous blood T2 values in healthy volunteers and people with SCA were converted to Yv using these calibration curves from normal and SCA blood and their individual Hct. The whole-brain OEF was calculated as 1-Yv.ResultsGood fitting was obtained for the results of the in vitro experiments. The relationship between blood T2, Y and Hct for both the normal and SCA blood samples are illustrated with surface plots in Figs. 1b,c. The red dots indicate the experimental data points and the mesh shows the model-fitted surface.In a Bland-Altman plots comparison of the predicted and measured Y for SCA blood samples, our calibration model for SCA blood corresponded well (Figs. 2a,b), while the SCA calibration model based on mixed SCA blood (Bush et al. ISMRM, 2017, #1898) (Figs. 2c,d) had a slight underestimation (~5%), and both the normal blood calibration models developed in this work and by Bush et al. (Bush et al. MRM, 2017, Epub ahead) underestimated Y by 10%-20% (Figs. 2e-h).Using our SCA in vitro blood model, the averaged whole-brain OEFs of participants with SCA were measured as 0.39+/-0.08 (Table 1), similar to the result (0.41+/-0.06) based on Bush-Wood SCA model, but 25% lower than the values of 0.52 estimated using a calibration models based on normal human blood. Participants with SCA showed similar whole-brain OEF (0.39+/-0.08 vs 0.40+/-0.04) with greater variation among participants (P<0.004, Table 1).Discussion and ConclusionWe developed an empirical blood T2-Y calibration model for SCA blood. For people with SCA, underestimations of Yv or overestimations of OEF were observed when using developed calibration models based on normal blood. This may be caused by the sickle erythrocytes' irregular shape and degree of hemoglobin S polymerization, which require specific calibrations. The average whole-brain OEF values estimated using our SCA blood calibration model did not differ significantly from healthy volunteers, which is consistent with an earlier PET study (Stroke 1986, 17:692), but contradictory to an earlier T2 oximetry study (Brain, 2016, 139:738) using a calibration model derived from normal blood. [Display omitted] DisclosuresStrouse:Sickle Cell Adult Provider Network: Membership on an entity's Board of Directors or advisory committees; HRSA-Sickle Cell Treatment Demonstration Program: Research Funding.

Low Volume in Vitro Diagnostic Proton NMR Spectroscopy of Human Blood Plasma for Lipoprotein and Metabolite Analysis: Application to SARS-CoV-2 Biomarkers.

The utility of low sample volume in vitro diagnostic (IVDr) proton nuclear magnetic resonance (1H NMR) spectroscopic experiments on blood plasma for information recovery from limited availability or high value samples was exemplified using plasma from patients with SARS-CoV-2 infection and normal controls. 1H NMR spectra were obtained using solvent-suppressed 1D, spin–echo (CPMG), and 2-dimensional J-resolved (JRES) spectroscopy using both 3 mm outer diameter SampleJet NMR tubes (100 μL plasma) and 5 mm SampleJet NMR tubes (300 μL plasma) under in vitro diagnostic conditions. We noted near identical diagnostic models in both standard and low volume IVDr lipoprotein analysis (measuring 112 lipoprotein parameters) with a comparison of the two tubes yielding R2 values ranging between 0.82 and 0.99 for the 40 paired lipoprotein parameters samples. Lipoprotein measurements for the 3 mm tubes were achieved without time penalty over the 5 mm tubes as defined by biomarker recovery for SARS-CoV-2. Overall, biomarker pattern recovery for the lipoproteins was extremely similar, but there were some small positive offsets in the linear equations for several variables due to small shimming artifacts, but there was minimal degradation of the biological information. For the standard untargeted 1D, CPMG, and JRES NMR experiments on the same samples, the reduced signal-to-noise was more constraining and required greater scanning times to achieve similar differential diagnostic performance (15 min per sample per experiment for 3 mm 1D and CPMG, compared to 4 min for the 5 mm tubes). We conclude that the 3 mm IVDr method is fit-for-purpose for quantitative lipoprotein measurements, allowing the preparation of smaller volumes for high value or limited volume samples that is common in clinical studies. If there are no analytical time constraints, the lower volume experiments are equally informative for untargeted profiling.

Real time monitoring by time-domain NMR of ring opening metathesis copolymerization of norbornene-based red palm olein monomer with norbornene

Changes in molecular mobility throughout the metathesis copolymerization of a norbornene functionalized with a palm olein monomer and a pure norbornene were monitored in situ by time-domain 1H NMR, using a ruthenium catalyst. Reaction progress to produce branched palm oil-based copolymers was studied as a function of the amounts of monomers and catalyst. Transverse relaxation rates were determined from CPMG experiments and the results were in accordance with the reactivity ratios, which indicated production of gradient copolymers. Properties of the resulting material observed through TD-NMR measurements of the isolated copolymers corroborated the type of the used monomers. TD-NMR demonstrated to be a useful tool for learning copolymerization reactions, particularly in ROMP, for the first time.

Opportunities and Challenges of Backbone, Sidechain, and RDC Experiments to Study Membrane Protein Dynamics in a Detergent-Free Lipid Environment Using Solution State NMR.

Whereas solution state NMR provided a wealth of information on the dynamics landscape of soluble proteins, only few studies have investigated membrane protein dynamics in a detergent-free lipid environment. Recent developments of smaller nanodiscs and other lipid-scaffolding polymers, such as styrene maleic acid (SMA), however, open new and promising avenues to explore the function-dynamics relationship of membrane proteins as well as between membrane proteins and their surrounding lipid environment. Favorably sized lipid-bilayer nanodiscs, established membrane protein reconstitution protocols and sophisticated solution NMR relaxation methods probing dynamics over a wide range of timescales will eventually reveal unprecedented lipid-membrane protein interdependencies that allow us to explain things we have not been able to explain so far. In particular, methyl group dynamics resulting from CEST, CPMG, ZZ exchange, and RDC experiments are expected to provide new and surprising insights due to their proximity to lipids, their applicability in large 100+ kDa assemblies and their simple labeling due to the availability of commercial precursors. This review summarizes the recent developments of membrane protein dynamics with a special focus on membrane protein dynamics in lipid-bilayer nanodiscs. Opportunities and challenges of backbone, side chain and RDC dynamics applied to membrane proteins are discussed. Solution-state NMR and lipid nanodiscs bear great potential to change our molecular understanding of lipid-membrane protein interactions.

A Second Backbone: The Contribution of a Buried Asparagine Ladder to the Global and Local Stability of a Leucine-Rich Repeat Protein.

Parallel β-sheet-containing repeat proteins often display a structural motif in which conserved asparagines form a continuous ladder buried within the hydrophobic core. In such "asparagine ladders", the asparagine side-chain amides form a repetitive pattern of hydrogen bonds with neighboring main-chain NH and CO groups. Although asparagine ladders have been thought to be important for stability, there is little experimental evidence to support such speculation. Here we test the contribution of a minimal asparagine ladder from the leucine-rich repeat protein pp32 to stability and investigate lattice rigidity and hydrogen bond character using solution nuclear magnetic resonance (NMR) spectroscopy. Point substitutions of the two ladder asparagines of pp32 are strongly destabilizing and decrease the cooperativity of unfolding. The chemical shifts of the ladder side-chain HZ protons are shifted significantly downfield in the NMR spectrum and have low temperature coefficients, indicative of strong hydrogen bonding. In contrast, the HE protons are shifted upfield and have temperature coefficients close to zero, suggesting an asymmetry in hydrogen bond strength along the ladder. Ladder NH2 groups have weak 1H-15N cross-peak intensities; 1H-15N nuclear Overhauser effect and 15N CPMG experiments show this to be the result of high rigidity. Hydrogen exchange measurements demonstrate that the ladder NH2 groups exchange very slowly, with rates approaching the global exchange limit. Overall, these results show that the asparagine side chains are held in a very rigid, nondynamic structure, making a significant contribution to the overall stability. In this regard, buried asparagine ladders can be considered "second backbones" within the cores of their elongated β-sheet host proteins.

Residue selective 15N CEST and CPMG experiments for studies of millisecond timescale protein dynamics

Proteins are intrinsically dynamic molecules and undergo exchanges among multiple conformations to perform biological functions. The CPMG relaxation dispersion and CEST experiments are two important solution NMR techniques for characterizing the conformational exchange processes on the millisecond timescale. Traditional pseudo 3D 15N CEST and CPMG experiments have certain limitations in their applications. For example, both experiments have low sensitivity for broadened resonances, and the process of optimizing sample conditions and experimental parameters are often time consuming. To overcome these limitations, we herein present a new set of residue selective 15N CEST and CPMG pulse sequences by employing the Hartmann-Hahn cross-polarization transfer of magnetization in both 1D and 2D schemes. Combined with frequency labeling in the indirect dimension using only a small number of increments, the pulse sequences in the 2D scheme can be applied on resonances in overlapped regions of the 1H-15N HSQC spectrum. The pulse sequences were further applied on several proteins, demonstrating their advantages over the traditional CEST and CPMG experiments under specific circumstances.

CPMG Experiments for Protein Minor Conformer Structure Determination.

CPMG relaxation dispersion NMR experiments have emerged as a powerful method to characterize protein minor states that are in exchange with a visible dominant conformation, and have lifetimes between ~0.5 and 5 milliseconds (ms) and populations greater than 0.5%. The structure of the minor state can, in favorable cases, be determined from the parameters provided by the CPMG relaxation dispersion experiments. Here, we go through the intricacies of setting up these powerful CPMG experiments.

Capturing Excited States in the Fast‐Intermediate Exchange Limit in Biological Systems Using 1H NMR Spectroscopy

AbstractChanges in molecular structure are essential for the function of biomolecules. Characterization of these structural fluctuations can illuminate alternative states and help in correlating structure to function. NMR relaxation dispersion (RD) is currently the only method for detecting these alternative, high‐energy states. In this study, we present a versatile 1H R1ρ RD experiment that not only extends the exchange timescales at least three times beyond the rate limits of 13C/15N R1ρ and ten times for CPMG experiments, but also makes use of easily accessible probes, thus allowing a general description of biologically important excited states. This technique can be used to extract chemical shifts for the structural characterization of excited states and to elucidate complex excited states.

Capturing Excited States in the Fast‐Intermediate Exchange Limit in Biological Systems Using 1H NMR Spectroscopy

Changes in molecular structure are essential for the function of biomolecules. Characterization of these structural fluctuations can illuminate alternative states and help in correlating structure to function. NMR relaxation dispersion (RD) is currently the only method for detecting these alternative, high-energy states. In this study, we present a versatile 1 H R1ρ RD experiment that not only extends the exchange timescales at least three times beyond the rate limits of 13 C/15 N R1ρ and ten times for CPMG experiments, but also makes use of easily accessible probes, thus allowing a general description of biologically important excited states. This technique can be used to extract chemical shifts for the structural characterization of excited states and to elucidate complex excited states.

Complexity of protein energy landscapes studied by solution NMR relaxation dispersion experiments.

The millisecond time scale motions in ribonuclease A (RNase A) were studied by solution NMR CPMG and off-resonance R1ρ relaxation dispersion experiments over a wide pH and temperature range. These experiments identify three separate protein regions termed Cluster 1, Cluster 2, and R33, whose motions are governed by distinct thermodynamic parameters. Moreover, each of these regions has motions with different pH dependencies. Cluster 1 shows an increase in activation enthalpy and activation entropy as the pH is lowered, whereas Cluster 2 exhibits the opposite behavior. In contrast, the activation enthalpy and entropy of R33 show no pH dependence. Compounding the differences, Δω values for Cluster 2 are characteristic of two-site conformational exchange, yet similar analysis for Cluster 1 indicates that this region of the enzyme exhibits conformational fluctuations between a major conformer and a pH-dependent average of protonated and deprotonated minor conformers.

D–T2 correlation using the inhomogeneity of single sided NMR devices

Single-sided NMR systems have become ubiquitous in industry and laboratory environments due to their low cost, low maintenance and capacity to evaluate quantity and quality of Hydrogen based materials. The performance of such devices has improved significantly over the last decade, providing increased field homogeneity, field strength and even controlled static field gradients. For a class of these devices, the configuration of the permanent magnets provides a linear variation of the magnetic field and can be used in diffusion measurements. However, magnet design depends directly on its application and, according to the purpose, the field homogeneity may significantly be compromised. This work introduces a new approach that extends the usability of diffusion-editing CPMG experiments to NMR devices with highly inhomogeneous magnetic fields, which do not vary linearly in space. Here we propose a method to determine a custom diffusion kernel based on the gradient distribution, which can be seen as a signature of each NMR device. This new diffusion kernel is then utilised in the 2D inverse Laplace transform (2D ILT) in order to determine diffusion-relaxation correlation maps of homogeneous multi-phasic fluids.

Measuring diffusion–relaxation correlation maps using non-uniform field gradients of single-sided NMR devices

Single-sided NMR systems are becoming a relevant tool in industry and laboratory environments due to their low cost, low maintenance and capacity to evaluate quantity and quality of hydrogen based materials. The performance of such devices has improved significantly over the last decade, providing increased field homogeneity, field strength and even controlled static field gradients. For a class of these devices, the configuration of the permanent magnets provides a linear variation of the magnetic field and can be used in diffusion measurements. However, magnet design depends directly on its application and, according to the purpose, the field homogeneity may significantly be compromised. This may prevent the determination of diffusion properties of fluids based on the natural inhomogeneity of the field using known techniques. This work introduces a new approach that extends the applicability of diffusion-editing CPMG experiments to NMR devices with highly inhomogeneous magnetic fields, which do not vary linearly in space. Herein, we propose a method to determine a custom diffusion kernel based on the gradient distribution, which can be seen as a signature of each NMR device. This new diffusion kernel is then utilised in the 2D inverse Laplace transform (2D ILT) in order to determine diffusion–relaxation correlation maps of homogeneous multi-phasic fluids. The experiments were performed using NMR MObile Lateral Explore (MOLE), which is a single-sided NMR device designed to maximise the volume at the sweet spot with enhanced depth penetration.

The Dynamic Functional Consequences of the Thrombin-Thrombomodulin Interaction

The coagulation cascade is critical to wound healing, but must be well regulated to prevent pathological clot formation. We previously investigated the backbone dynamics of a key component in this cascade, thrombin, and found that the loops surrounding the thrombin active site show dynamics on the ps-ns timescale when the active site is inhibitor-bound, but have dynamics on the μs-ms timescale when the active site is unoccupied. Thrombomodulin (TM) plays a key role in the regulation of the coagulation cascade by switching the function of thrombin from procoagulant to anticoagulant. A current molecular dynamics study suggests TM does this by influencing the slow timescale dynamics of thrombin, but the actual timescale of these motions is currently unknown. NMR measurements of chemical shift differences (CSDs) can be used to probe changes in the conformational ensemble upon TM binding. Other NMR experiments probe the timescales of motion including R1 and R2 relaxation experiments, which probe the ps-ns time regime, and TROSY Hahn Echo and CPMG experiments, which probe slow (μs-ms) timescale motions. To perform these NMR studies, our lab has developed a truncation of human TM (TM456m) with complete thrombin-altering activity. We have produced 15N-labeled human thrombin using an E. coli expression system and have collected HSQCs of unbound 15N-thrombin and 15N-thrombin in 1:1 complex with unlabeled TM456m. Binding of TM456m induces significant CSDs and differences in peak intensities throughout thrombin. Several residues in the TM binding site, including S36A, L65, I82, M84, and K110, become significantly perturbed upon binding. These perturbations propagate to several residues near the active site, including H57 and D102 of the catalytic triad. We hope that a complete understanding of how TM alters the dynamics and function of thrombin will aid in the design of safer anticoagulant drugs.

Relationship between Structural Flexibility and Function in the C-Terminal Region of the Heparin-Binding Domain of VEGF165

Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Previously, we reported that triamterene (Trm) inhibits VEGF-amyloid β (Aβ) interactions without affecting other biological activities of VEGF or Aβ [Jeong, K.-W., et al. (2011) Biochemistry 50, 4843-4854]. We further showed that molecular motions in the N-terminal disordered loop region of the heparin-binding domain (HBD) are important for interaction with Trm. To investigate the importance of motion at the C-terminal domain of HBD, we constructed a binding model of HBD with heparin octasaccharide (HOS) based on measurements of chemical shift changes and docking studies. Furthermore, the dynamic properties of the HBD-HOS and HBD-Trm-HOS complexes were assessed by measuring spin relaxation rates. The results showed that the HOS-binding site is composed of two basic clusters consisting of side chains of residues R13, R14, and K15 and residues K30, R35, and R49. When HOS binds, values for the heteronuclear nuclear Overhauser effect near HOS-binding sites increased dramatically. CPMG (Carr-Purcell-Meiboom-Gill sequence) experiments as well as an R2 relaxation experiment were undertaken to understand millisecond time-scale motions in HBD. There is large relaxation dispersion of residues at Trm- and HOS-binding sites in free HBD. C-Terminal residues such as S34, C48, and D51 near the HOS-binding sites continued to exhibit slow conformational motions in the HBD-Trm complex, while those slow motions disappeared in the bound conformation of HBD with HOS. Collectively, our results demonstrate that the inherent structural flexibilities of the C-terminal region of the HBD are important in the heparin binding process and that Trm does not inhibit VEGF-heparin interactions necessary for the biological activities of VEGF.