• Bulletin of the Korean Chemical Society
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• v.34 no.5
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• pp.1401-1406
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• 2013

This study tested the feasibility of observing H/D exchange of intact protein by top-down electron capture dissociation (ECD) mass spectrometry for the investigation of protein structure. Ubiquitin is selected as a model system. Local structural information was obtained from the deuteration levels of c and $z^{\cdot}$ ions generated from ECD. Our results showed that ${\alpha}$-helix region has the lowest deuteration level and the C-terminal fraction containing a highly mobile tail has the highest deuteration level, which correlates well with previous X-Ray and HDX/NMR analyses. We studied site-specific H/D exchange kinetics by monitoring H/D exchange rate of several structural motives of ubiquitin. Two hydrogen bonded ${\beta}$-strands showed similar HDX rates. However, the outer ${\beta}$-strand always has higher deuteration level than the inner ${\beta}$-strand. The HDX rate of the turn structure (residues 8-11) is lower than that of ${\beta}$-strands (residues 1-7 and residues 12-17) it connects. Although isotopic distribution gets broader after H/D exchange which results in a limited number of backbone cleavage sites detected, our results demonstrate that this method can provide valuable detailed structural information of proteins. This approach should also be suitable for the structural investigation of other unknown proteins, protein conformational changes, as well as protein-protein interactions and dynamics.

• Journal of the Korean Magnetic Resonance Society
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• v.19 no.3
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• pp.112-118
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• 2015

Hsp31 protein is one of the members of DJ-1 superfamily proteins and has a dimeric structure of which molecular weight (MW) is 62 kDa. The mutation of DJ-1 is closely related to early onset of Parkinson's disease. Hsp31 displays $Zn^{+2}$-binding activity and was first reported to be a holding chaperone in E. coli. Its additional glyoxalase III active has recently been characterized. Moreover, an incubation at $60^{\circ}C$ induces Hsp31 protein to form a high MW oligomer (HMW) in vitro, which accomplishes an elevated holding chaperone activity. The NMR technique is elegant method to probe any local or global structural change of a protein in responses to environmental stresses (heat, pH, and metal). Although the presence of the backbone chemical shifts (bbCSs) is a prerequisite for detailed NMR analyses of the structural changes, general HSQC-based triple resonance experiments could not be used for 62 kDa Hsp31 protein. Here, we prepared the per-deuterated Hsp31 and performed the TROSY-based triple resonance experiments for the bbCSs assignment. Here, detailed processes of per-deuteration and the NMR experiments are described for other similar NMR approaches.

• Journal of Photoscience
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• v.11 no.3
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• pp.95-99
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• 2004

Optical absorption and emission studies have been carried out to understand the effect of deuterium on the solvent dependent photophysical properties of curcumin in deuterated solvents such as $CDCl_3,\;(CD_3)_2SO,\;(CD_3)_2CO,\;CD_3OD\;and\;CD_3CN$. Optical absorption spectral studies showed that there is no significant shift in absorption maxima compared to the non-deuterated solvent. The fluorescence maxima shows significant shift with polarity of solvent but not much affected by the deuteration. The fluorescence quantum yield of curcumin increased marginally in almost all the deuterated solvents, indicating reduction in the non-radiative pathways. The fluorescence decay was biexponential in all the solvents and the average fluorescence lifetime was not much affected with deuteration, but showed decrease with increasing solvent polarity. Based on these studies, it is concluded that intermolecular hydrogen transfer is only partially responsible for the excited state deactivation of curcumin.

• Bulletin of the Korean Chemical Society
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• v.23 no.5
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• pp.727-735
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• 2002

Proton coupled carbon-13 relaxation experiment was performed to investigate the effect of vicinal protons on spin-lattice relaxation of methylene carbon-13 in n-undecane. A BIRD type pulse sequence was employed as a way to check the validity of describing the 13CH2 moiety as an isolated AX2 spin system. The results show that the presence of vicinal protons exerts substantial influence on the relaxation of methylene carbon-13, indicating that it is not a very good approximation to treat a methylene moiety as an isolated AX2 spin system.

• Analytical Science and Technology
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• v.7 no.2
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• pp.213-224
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• 1994

Several isotopomers of cyclooctanone were prepared by selective deuterium substitution. Intrinsic isotope effects on $^{13}C$ NMR chemical shifts of these isotopomers were investigated systematically at low temperature. These istope effects were discussed in relation to the preferred boat-chair conformation of cyclooctanone. Deuterium isotope effects on NMR chemical shifts have been known for a long time. Especially in a conformationally mobile molecule, isotope perturbation could affect NMR signals through a combination of isotope effects on equilibria and intrinsic effects. The distinction between intrinsic and nonintrinsic effects is quite difficult at ambient temperature due to involvement of both equilibrium and intrinsic isotope effects. However if equilibria between possible conformers of cyclooctanone are slowed down enough on the NMR time scale by lowering temperature, it should be possible to measure intrinsic isotope shifts from the separated signals at low temperature. $^{13}C$ NMR has been successfully utilized in the study on molecular conformation in solution when one deals with stable conformers or molecules were rapid interconversion occurs at ambient temperature. The study of dynamic processes in general requires analysis of spectra at several temperature. Anet et al. did $^1H$ NMR study of cyclooctanone at low temperature to freeze out a stable conformation, but were not able initially to deduce which conformation was stable because of the complexity of alkyl region in the $^1H$ NMR spectrum. They also reported the $^1H$ and $^{13}C$ NMR spectra of the $C_9-C_{16}$ cycloalkanones with changing temperature from $-80^{\circ}C$ to $-170^{\circ}C$, but they did not report a variable temperature $^{13}C$ NMR study of cyclooctanone. For the analysis of the intrinsic isotope effect with relation to cylooctanone conformation, $^{13}C$ NMR spectra are obtained in the present work at low temperatures (up to $-150^{\circ}C$) in order to find the chemical shifts at the temperature at which the dynamic process can be "frozen-out" on the NMR time scale and cyclooctanone can be observed as a stable conformation. Both the ring inversion and pseudorotational processes must be "frozen-out" in order to see separate resonances for all eight carbons in cyclooctanone. In contrast to $^1H$ spectra, slowing down just the ring inversion process has no apparent effects on the $^{13}C$ spectra because exchange of environments within the pairs of methylene carbons can still occur by the pseudorotational process. Several isotopomers of cyclooctanone were prepared by selective deuterium substitution (fig. 1) : complete deuterium labeling at C-2 and C-8 positions gave cyclooctanone-2, 2, 8, $8-D_4$ : complete labeling at C-2 and C-7 positions afforded the 2, 2, 7, $7-D_4$ isotopomer : di-deuteration at C-3 gave the 3, $3-D_2$ isotopomer : mono-deuteration provided cyclooctanone-2-D, 4-D and 5-D isotopomers : and partial deuteration on the C-2 and C-8 position, with a chiral and difunctional case catalyst, gave the trans-2, $8-D_2$ isotopomer. These isotopomer were investigated systematically in relation with cyclooctanone conformation and intrinsic isotope effects on $^{13}C$ NMR chemical shifts at low temperature. The determination of the intrinsic effects could help in the analysis of the more complex effects at higher temperature. For quantitative analysis of intrinsic isotope effects, the $^{13}C$ NMR spectrum has been obtained for a mixture of the labeled and unlabeled compounds because the signal separations are very small.

• Journal of the Korean Chemical Society
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• v.28 no.1
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• pp.14-19
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• 1984

The stability difference of metmyoglobin in $H_2O$ and $D_2O$ at pH 5.7 and pH 7.0 toward pressure denaturation is studied. Metmyoglobin is denatured in $D_2O$ at smaller pressure than in $H_2O$. The stability difference in $H_2O$ and $D_2O$ is more pronounced at pH 5.7 than at pH 7. The main reasons for the stability difference in $H_2O$ and $D_2O$are the difference in positive charge due to $H^+$and $D^+$ binding to the protein in $H_2O$ and $D_2O$, and the structural change that accompany deuteration.

• Journal of the Korean Chemical Society
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• v.33 no.2
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• pp.238-246
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• 1989

1,3-Oxathiolane sulfoxide 4 in which the sulfoxide oxygen and the 2-methyl group are on the same face of the oxathiolane ring undergoes a sigmatropic rearrangement to produce a ring expansion product. The structure of this product would be dihydro-1,4-oxathiin 6 or isomeric exo compound 7. This paper describes physical and chemical methods to determine the correct structure of the two alternatives. Thus, $^1HNMR$, UV spectroscopies, and mass spectrometry showed that the product actually obtained had the structure 6. It was also found that from deuteration reactions of the product the compound 7 was initaly formed and then tautomerized to endo compound 6.

• Journal of Photoscience
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• v.1 no.1
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• pp.9-14
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• 1994

Exposure of isolated intact mitochondria to near UV to visible light resulted in not only loss of respiration, the most well-documented phenomenon regarding phototoxic effects in the respiring organelles, but also lipid peroxidation of membranes and mitochondrial swelling; these turned out to be O$_2$-dependent and thus prevented by anaerobiosis, enhanced by a partial deuteration of the suspension medium, and suppressed by the presence of a singlet oxygen ($^1O_2$) scavenger. Measurements of the spectral dependence of such detrimental effects of light on mitochondrial structure and function revealed that all the resulting spectra bear a significant resemblance to the action spectrum for photogeneration of $^1O_2$ from mitochondrial membranes, which in turn carries the spectral characteristics of light absorption by mitochondrial Fe-S centers. Futhermore, destructing the Fe-S centers by a mercurial treatment of mitochondria brought about a striking reduction of the light-induced membrane peroxidation and swelling of mitochondria. These results are consistent with the suggestion that the impairment of functional, structural integrity of mitochondria caused by strong irradiation is directly related to the production of $^1O_2$ in mitochondria, photosensitized by the Fe-S centers. This paper also presents kinetic data which indicate that, among various membrane-bound protein systems associated with mitochondrial energy metabolism, the respiratory chain is the primary target for photodamage.

• 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.

• Publications of The Korean Astronomical Society
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• v.29 no.2
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• pp.29-34
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• 2014

We have observed the deuterated methanol, $CH_3OD$, toward the hot core MM1 in the massive star-forming region DR21 (OH) using the Submillimeter Array with a high angular resolution of about 1 arcsecond. The position of the hot core associated with the sub-core MM1a was confirmed to coincide with the continuum peak where an embedded young stellar object is located. The column density of $CH_3OD$ was found to be about $(2{\pm}1){\times}10^{16}cm^{-2}$ toward the MM1a center. The abundance ratio $CH_3OD/CH_3OH$ was measured to be ~ 0.45, which is about the median value for low mass star-forming cores but much larger than those of the massive star-forming cores. The ratio is believed to change depending on, for example, the chemical condition, the temperature and the density of the source. This ratio may further depend on the evolutionary phase especially in the massive-star-forming cores. The sub-core MM1a is thought to be in the very early phase of star formation. This large abundance ratio found in this source indicates that even the massive star-forming cores, during a relatively short period in the very early stage of star formation, may also show a chemical state resulted from the cold and dense pre-collapsing phase, the enhanced deuteration as found in low mass star-forming cores.