• Journal of the Korean Magnetic Resonance Society
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• v.18 no.1
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• pp.1-4
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• 2014

It is generally understood that protein-protein interactions proceed via transient encounter complexes that rapidly evolve into the functional stereospecific complex. Direct detection and characterization of the encounter complexes, however, been difficult due to their low population and short lifetimes. Recent application of NMR paramagnetic relaxation enhancement first visualized the structures of the encounter complex ensemble, and allowed the characterization of their physicochemical properties. Further, rational protein mutations that perturbed the encounter complex formation provided a clue to the target search pathway during protein-protein association. Understanding the structure and dynamics of encounter complexes will provide useful information on the mechanism of protein association.

• Journal of the Korean Magnetic Resonance Society
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• v.22 no.4
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• pp.76-81
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• 2018

Investigation of the protein-protein interaction mode at atomic resolution is essential for understanding on the underlying functional mechanisms of proteins as well as for discovering druggable compounds blocking deleteriou interprotein interactions. Solution NMR spectroscopy provides accurate and precise information on intermolecular interactions even for weak and transient interactions, and it is also markedly useful for examining the change in the conformation and dynamics of target proteins upon binding events. In this mini-review, we comprehensively describe three unique and powerful methods of solution NMR spectroscopy, paramagnetic relaxation enhancement (PRE), pseudo-contact shift (PCS), and residual dipolar coupling (RDC), for the study on protein-protein interactions.

• Journal of the Korean Magnetic Resonance Society
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• v.19 no.3
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• pp.137-142
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• 2015

NMR-based structural studies on membrane proteins are appreciated quite challenging due to various reasons, generally including the narrow dispersion of NMR spectra, the severe peak broadening, and the lack of long range NOEs. In spite of the poor biophysical properties, structural studies on membrane proteins have got to go on, considering their functional importance in biological systems. In this review, we provide a simple overview of the techniques generally used in structural studies of membrane proteins by solution NMR, with experimental examples of a helical membrane protein, caveolin 3. Detergent screening is usually employed as the first step and the selection of appropriate detergent is the most important for successful approach to membrane proteins. Various tools can then be applied as specialized NMR techniques in solution that include sample deteuration, amino-acid selective isotope labeling, residual dipolar coupling, and paramagnetic relaxation enhancement.

• Journal of the Korean Magnetic Resonance Society
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• v.24 no.2
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• pp.47-52
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• 2020

Liquid-liquid phase separation (LLPS) of biomolecules, a newly-found phase behavior of molecules in the liquid phase, has shown to its relationship to various biological function and misfolding diseases. Extensive studies have increasingly revealed a general mechanism of LLPS and characterized the liquid droplet; ho wever, intermolecular interactions of proteins and structural states of LLPS-inducing proteins inside of the droplet remain largely unknown. Solution NMR spectroscopy has emerged as a powerful approach as it provides invaluable information on protein intermolecular interactions and structures at the atomic and residue level. We herein comprehensively address useful techniques of solution NMR including the effect of paramagnetic relaxation enhancement for the study on the LLPS and droplet based on recent studies.

• Investigative Magnetic Resonance Imaging
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• v.5 no.1
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• pp.33-37
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• 2001

Purpose : The water exchange rate between bulk water and bound water is an important parameter in deciding the efficiency of paramagnetic contrast agents. In this study, we evaluated the water exchange rates of various Gd-chelates using oxygen-17 NMR technique. Material and Methods : The samples (Gd-DTPA, Gd-DTPA-BMA, Gd-DOTA, Gd-EOB-DTPA) were prepared by mixing 5% $^{17}O-enriched$ water (Isotech, USA). The pH of the samples was adjusted to physiological value [pH=7.0] by buffer solution. The variable temperature $^{17}O-NMR$ measurements were performed using Bruker-600 (14.1 T, 81.3 MHz) spectrometer. Bruker VT-1000 temperature control units were used to stabilize the temperature. The $^{17}O$ spin-spin relaxation times (T2) were measured using Carr-Purcell-Meiboom-Gill (CPMG)I pulse sequence with 24 echo trains. The variable temperature T2 relaxation data were then fitted into Solomon-Bloembergen equations using least square fit algorithm to estimate the water exchange times. Results : From the measured $^{17}O-NMR$ relaxation rates, the determined water exchange rates at 300K are $0.42{\;}{\mu}s$ for Gd-DTPA, $1.99{\;}{\mu}s$ for Gd-DTPA-BMA, $0.27{\;}{\mu}s$ for Gd-DOTA, and $0.11{\;}{\mu}s$ for Gd-EOB-DTPA. The Gd-DTPA-BMA showed slowest exchange whereas Gd-EOB-DTPA had fastest water exchange rate. In addition, it was found that the water exchange rates (${\tau}_m$) of all samples had exponential temperature dependence with different decay constant. Conclusion : $^{17}O-NMR$ relaxation rate measurements, when combined with variable temperature technique, provide a solid tool for studying water exchange rate, which is very important in investigating the detailed mechanism of relaxation enhancement effect of the paramagnetic contrast agents.

• Biomedical Science Letters
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• v.11 no.3
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• pp.301-305
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• 2005

MR spectroscopy of intracellularly located $^{133}Cs$ has been used to monitor the uptake of Gd-EOB-DTPA by the isolated rat liver. As shown by ${31}P$ spectroscopy, accumulation of $^{133}Cs$ ions in hepatocytes does not produce detectable effects on the metabolism. The hepatic internalization of Gd-EOB-DTPA was followed by the paramagnetic relaxation enhancement of the intracellular $^{133}Cs$ ions, and confirmed by parallel quantitations of Gd and Cs run by inductively coupled plasma analysis of liver samples and aliquots of perfusate. Two peaks are observed at -22.0 and -23.5 ppm, with respect to the line of the external reference arbitarily set to 0 ppm. Upon rinsing of the extracellular compartment with regular K-H free of CsCl, the high-field resonance disappears within 20min. The intracellular concentration was confirmed by ICP, which gives a $Cs^+$ content of $22.0\pm3.5mM$. The relaxation data significantly underestimate the Gd content, suggesting a potential compartmentation of $Cs^+$ and the contrast agent.

• Investigative Magnetic Resonance Imaging
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• v.3 no.2
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• pp.159-166
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• 1999

Purpose : To evaluate the effect of rotational correlation time (${\tau}_R$) and the possible related changes of other parameters, ${\tau}_M,{\;}{\tau}_S,{\;}and{\;}(\tau}_V$ of gadolinium (Gd) chelate on T1 relaxation enhancement in two pool model. Materials and Methods : The NMRD (Nuclear Magnetic Relaxation Dispersion) profiles were simulated from 0.02 MHz to 800 MHz proton Larmor frequency for different values of rotational correlation times based on Solomon-Bloembergen equation for inner-sphere relaxation enhancement. To include both unbound pool (pool A) and bound pool (pool B), the relaxivity was divided by contribution from unbound pool and bound pool. The rotational correlation time for pool A was fixed at the value of 0.1 ns, which is a typical value for low molecular weight complexes such as Gd-DTPA in solution and ${\tau}_R$ for pool B was changed from 0.1 ns to 20 ns to allow the slower rotation by binding to macromolecule. The fractional factor of was also adjusted from 0 to 1.0 to simulate different binding ratios to macromolecule. Since the binding of Gd-chelate to macromolecule cab alter the electronic environment of Gd ion and also the degree of bulk water access to hydration site of Gd-chelate, the effects of these parameters were also included. Results : The result shows that low field profiles, ranged from 0.02 to 40 MHz, and dominated by contribution from bound pool, which is bound to macromolecule regardless of binding ratios. In addition, as more Gd-chelate bound to macromolecule, sharp increase of relaxivity at higher field occurs. The NMRD profiles for different values of ${\tau}_S$ show the enormous increase of low field profile whereas relaxivity at high field is not affected by ${\tau}_S$. On the other hand, the change in ${\tau}$V does not affect low field profile but strongly in fluences on both inflection fie이 and the maximum relaxivity value. The results shows a fluences on both inflection field and the maximum relaxivity value. The results shows a parabolic dependence of relaxivity on ${\tau}_M$. Conclusion : Binding of Gd-chelate to a macromolecule causes slower rotational tumbling of Gd-chelate and would result in relaxation enhancement, especially in clinical imaging field. However, binding to macromolecule can change water enchange rate (${\tau}_M$) and electronic relaxation ($T_le$) vis structural deformation of electron environment and the access of bulk water to hydration site of metal-chelate. The clinical utilities of Gd-chelate bound to macromolecule are the less dose requirement, the tissue specificity, and the better perfusion and intravascular agents.

• Investigative Magnetic Resonance Imaging
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• v.10 no.1
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• pp.20-25
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• 2006

Purpose : To evaluate the NMR relaxation properties of newly developed high performance paramagnetic complexes. Materials and methods : 4-aminomethylcyclohexane carboxylic acid (0.63g, 4 mmol) was mixed with the suspension solution of DMF (15mL) and DTPA-bis-anhydride (0.71g, 2 mmol) to synthesize the ligand. The ligand was then mixed with Gd2O3 (0.18g, 0.5 mmol) to synthesize Gd-chelate. For the measurement of magnetic relaxivity of paramagnetic compounds, the compounds were diluted to 1mM and then the relaxation times were measured at 1.5T(64 MHz). Inversion-recovery pulse sequence was employed for T1 relaxation measurement and CPMG(Carr-Purcell-Meiboon-Gill) pulse sequence was employed for T2 relaxation measurement. Using MATLAB(Version 7.1) program, T1 magnetic relaxation map, R1 map, T2 magnetic relaxation map and R2 map were developed to represent magnetic relaxation time and magnetic relaxivity as image. Results : Compared to $R1=4.9mM^{-1}sec^{-1}$ and $R2=4.8mM^{-1}sec^{-1}$ of Omniscan (Gadodiamide), which is commercially available paramagnetic MR agent, R1 of SUK090(Gd-C32H74N5O24) was $12.46mM^{-1}sec^{-1}$ and R1 of SUK091(Gd-C34H78N5O24) was $12.77mM^{-1}sec^{-1}$. However, R1 of SUK092(Gd-C30H56N5O17) was decreased to $2.09mM^{-1}sec^{-1}$. In case of R2, SUK090(Gd-C32H74N5O24) was $8.76mM^{-1}sec^{-1}$ and SUK091(Gd-C34H78N5O24) was $7.60mM^{-}1sec^{-1}$ whereas SUK092(Gd-C30H56N5O17) was decreased to $1.82mM^{-1}sec^{-1}$. Conclusion : Among three new paramagnetic complexes, SUK090(Gd-C32H74N5O24) and SUK091(Gd-C34H78N5O24) showed higher T1, T2 magnetic relaxation rates than that of commercially available paramagnetic MR agent and thus expected to have more contrast enhancement effect.

• Investigative Magnetic Resonance Imaging
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• v.4 no.1
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• pp.27-33
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• 2000

Purpose: To determine the electronic spin relaxation times, $T_{le}$, of three commercially available Gd-chelated MR contrast agents, Gd-DTPA, Gd-DTPA-BMA and Gd-DOTA, using Electron Paramagnetic Resonance(EPR) technique. Material and Methods: The paramagnetic MR contrast agents, Gd-DTFA(Magnevist) , Gd-DTFA-BMA(OMNISCAN) and Gd-DOTA(Dotarem), were used for this study, The EPR spectra of these contrast agents, which were prepared 2:1 methanol/water solution, were obtained at low temperatures, from $-160^{\circ}C~20^{\circ}C$. The glassy-state EPR spectra for these contrast agents were then fitted by the simulation spectra generated with different zero-field splitting (ZFS) parameters by a computer simulation program 'GEN', which generates the EPR powder spectrum using a given ZFS in $3{\times}3$ tensor. Finally, the spin relaxation times of the contrast agents were then determined from the $T_{2e}$, D, and E values of the best simulation spectra using the McLachlan's theory of average relaxation rate. Results: The electronic transverse spin relaxation times, $T_{2e}'s$, of Gd-DTPA, Gd-DTPA-BMA and Gd-DOTA were 0.113ns, 0.147ns and 1.81ns respectively. The g-values were 1.9737, 1.9735 and 1.9830 and the electronic spin relaxation times, $T_{1e}'s$, were 18.70ns, 33.40ns and $1.66{\mu}s$, respectively. Conclusion: The results of these studies reconfirm that the paramagnetic MR contrast agents with larger ZFS parameters should have shorter $T_{1e}'s$. Among three contrast agents used for this study, Gd-DOTA chelated with cyclic ligand structure shows better electronic property then the others with linear structure. Thus, it is concluded that the exact determination of ZFS parameters is the important factor in evaluating relaxation enhancement effect of the agents and in developing new contrast agents.

• Investigative Magnetic Resonance Imaging
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• v.6 no.1
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• pp.35-40
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• 2002

Purpose : To evaluate the NMR relaxation properties and imaging characteristics of tissue-specificity for a newly developed macromolecular MR agent. Materials and methods : Phthalocyanine (PC) was chelated with paramagnetic ion, Mn.2.01g (5.2 mmol) of Phthalocyanine was mixed with 0.37g (1.4 mmol) of Mn chloride at $310^{\circ}C$ for 36 hours and then purified by chromatography (CHC13/CH3OH 98/2 v/v, Rf, 0.76) to obtain 1.04g (46%) of MnPC (molecular weight= 2000d). The $T1}T2$ relaxivity of MnPC was measured in 1.5T(64 MHz) MR using 0.1 mM MnPC. The MR image characteristics of MnPC was evaluated using spin-echo (TR/TE=500/14 msec) and gradient-echo (FLASH) (TR/TE=80/4 msec, flip angle=60) techniques in 1.57 MR scanner. The images of rabbit liver were obtained every 10 minutes up to 4 hours. To study the effect of concentration on image, 20 mM, 50 mM, 100 mM of MnPC were tested. Results : The relaxivities of MnPC at 1.5T(64MHz) were Rl=7.28 $mM^{-1}S^{-1},{\;}R2=55.56mM^{-1}S^{-1}$. Compared to the values of Gd-DTPA (Rl[=4.8 $mM^{-1}S^{-1})$], R2[=5.2 $mM^{-1}S^{-1}])$]), both T1/T2 relaxivities of MnPC were higher than those of Gd-DTPA. For both of SE and FLASH techniques, the contrast enhancement reached maximum at 10 minutes after bolus injection and the enhancement continued for more than 2 hours. When compared with small molecular weight liver agents such as Gd-EOB-DTPA, Gd-BOPTA and MnDPDP, MnPC was characterized by more prolonged enhancement time. The time course of MR images also revealed biliary excretion of MnPC. Conclusion : We developed a new macromolecular MR agent, MnPC. The relaxivities of MnPC were higher than those of small molecular weight Gd-chelate. Hepatic uptake and biliary excretion of MnPC suggests that this agent is a new liver-specific MR agent.