Relaxation measurements of side-chain 13CH2-groups of uniformly 13C labeled human ubiquitin were performed at 600 MHz and 800 MHz magnetic field strength at 30°C. Dipole-dipole cross-correlated relaxation effects in T1 experiments were suppressed by the combination of radio-frequency pulses and pulsed field gradients during the relaxation delay leading to monoexponential relaxation decays that allow a more accurate extraction of the 13C T1 relaxation times. Heteronuclear 1H-13C NOEs obtained by using different proton saturation schemes indicate that the influence of cross-correlation is small. The experimental T1 and NOE data were interpreted in a model-free way in terms of a generalized order parameter and an internal correlation time. 相似文献
During ageing of the short-lived pollen grains of Cucurbita pepo L., water loss was examined in relation to viability using biophysical (1H-nuclear magnetic resonance, NMR) and cytological methods (fluorochromatic reaction test, freezefracture and scanning electron microscopy). A semi-logarithmic representation of the pollen weight loss demonstrated the complexity of the dehydration process. A the study of proton loss using 1H-NMR indicated that two major releases water of had taken place, each with different flux rates. Pulse 1H-NMR experiments showed the occurrene of non-exponential signal decay as a function of time, indicating the existence of different fractions of water in a pollen grain sample. These fractions leave the pollen grain at different times during pollen dehydration, and one of them (that of the so-called vital water) can be related to pollen viability. The quantity of protons giving a signal during pulse 1H-NMR experiments was very low when the pollen grains were judged to be dead according to the fluorochromatic test. Freeze-fracture replicas of these dead pollen grains (less than 25% water content) showed that the plasma membrane had become detached from the intine surface; this ultrastructural feature might therefore be involved in the loss of pollen viability.Abbreviations A
initial amplitude of the NMR signal
- A2
quantity of water charcterized by T2-2
- A5
quantity of water characterized by T2–5
- FCR
fluorochromatic reaction
- NMR
nuclear magnetic resonance
- T2
transverse relaxation time
- T2-2
T2 measured with 2 ms between each pulse of radiofrequency
- T2–5
T2 measured with 5 ms between each pulse of radiofrequency 相似文献
Human erythrocytes were incubated in a Ringer's solution enriched with 10–18% H217O. The longitudinal relaxation time (T1) of the 17O was determined separately in samples of red cell suspesions, packed cells, and supernatant. The longitudinal relaxation of 17O in erythrocyte suspensions was non-exponential, reflecting water exchange across the cell membranes as well as relaxation processes inside and outside the cell.The T1 of intracellular 17O is 4–5 times shorter than in the supernatant, similar to the enhancement of proton relaxation by hemoglobin in erythrocytes and free solution at the frequency applied (8.13 MHz). This datum is consistent with the thesis that hemoglobin modifies the NMR relaxation behavior of water inside cells and in free solution in the same way.The rate constant for water exchange was calculated to be 60 and 107 s−1 at 25 and at 37° C, respectively. The apparent activation energy for over the temperature range 23–37° C was 8.7±1.0 kcal/mole. 相似文献
Summary Relaxation times of 13C carbons of uniformly 13C/15N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T1, T2 and the heteronuclear NOE of 13C in uniformly 13C/15N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T1 and the heteronuclear NOE were similar to those of the corresponding 15N experiments published previously. The determination of T2 for the C-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T2 evolution period owing to the scalar coupling of C–C must be suppressed. Various different pulse sequences for the T2 evolution period were simulated in order to optimize the bandwidth for which reliable T2 relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual 1H–13C cross peaks, in a series of (1H, 13C)-ct-HSQC spectra with a modified CPMG sequence as well as a T1p sequence for the transverse relaxation time, to a single exponential using a simplex algorithm. 相似文献
The NMR paramagnetic relaxation enhancement (NMR-PRE) produced in the solvent proton resonance by manganese in the S0 and S2 states of the oxygen evolving center (OEC) has been recorded for three Photosystem II (PS II)-enriched preparations: (1) PS II-enriched thylakoid membrane fragments (TMF-2 particles); (2) salt-washed (2M NaCl) TMF-2 particles; and (3) the octylglucopyranoside (OGP)-solubilized PS II complex. The second and third preparations, but not the first, are depleted of the peripheral 17 and 23 kD polypeptides associated with the OEC. It has been proposed that depletion of these polypeptides increases the exposure of OEC manganese to the aqueous phase. The NMR-PRE response measures the quantity (T1m+m)-1, where T1m is the spin relaxation time and m is the mean residence time with respect to chemical exchange reactions of solvent protons in the manganese coordination sphere, and, thus, the NMR-PRE provides a direct measure of the solvent proton chemical exchange rate constant m-1. This study tested whether the 17 and 23 kD polypeptides shield the OEC from the solvent phase and whether their depletion enhances the S2 and S0 NMR-PRE signals by removing a kinetic barrier to the solvent proton chemical exchange reaction. The amplitude of the S2 NMR-PRE signal, measured in its chemical exchange-limited regime (m>T1m), is slightly decreased, rather than increased, in preparations (2) and (3) relative to (1), indicating that removal of the 17 and 23 kD polypeptides slightly slows, rather than accelerates, the rate-limiting steps of the solvent proton chemical exchange reactions. In addition, the lifetime of the S2 state was shortened several-fold in the solubilized PS II complex and in salt-washed TMF-2 membranes relative to untreated TMF-2 control samples. The S0 NMR-PRE signal, which is present in TMF-2 suspensions, was not detected in suspensions of the solubilized PS II complex, even though these samples contained high concentrations of active manganese centers (approximately double those of the TMF-2 control) and exhibited an S2 NMR-PRE signal of comparable amplitude to that of the TMF-2 preparation. These results suggest that the 17 and 23 kD extrinsic polypeptides do not shield the NMR-visible water binding site in the OEC from the aqueous phase, although their removal substantially alters the proton relaxation efficiency by shortening T1m.Abbreviations ADRY
acceleration of the deactivation reactions of the water splitting enzyme Y
- BBY
Photosystem II-enriched membrane fragments prepared by the method of Berthold et al. (1981)
- CCCP
carbonyl cyanide m-chlorophenyl hydrazone
- Chl
chlorophyll
- DCBQ
2,5-dichlorobenzoquinone
- MES
morpholinoethanesulfonate
- NMR
nuclear magnetic resonance
- OEC
oxygen evolving complex
- OGP
octylglucopyranoside
- PRE
paramagnetic relaxation enhancement
- PS II
Photosystem II
- SDS-PAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
- TMF-2
Photosystem II-enriched thylakoid membrane fragments prepared by the method of Radmer et al. (1986)
- T1, T2
longitudinal and transverse nuclear spin relaxation times 相似文献
Summary A set of three 3D (1H, 13C, 15N) triple-resonance correlation experiments has been designed to provide H1-H8 intraresidue sugar-to-base correlations in purines in an unambiguous and efficient manner. Together, the HsCsNb, HsCs(N)bCb, and HbNbCb experiments correlate the H1 sugar proton to the H8 proton of the attached base by means of the {H1, C1, N9, C8, H8} heteronuclear scalar coupling network. The assignment strategy presented here allows for unambiguous H1-H8 intraresidue correlations, provided that no two purines have both the same H1 and C1 chemical shifts and the same C8 and N9 chemical shifts. These experiments have yielded H1-H8 intraresidue sugar-to-base correlations for all five guanosines in the [13C, 15N] isotopically labeled RNA duplex r(GGCGCUUGCGUC)2. 相似文献
The 3D structure of two unlabeled FK506 analogs, (R)- and (S)-[18-OH]ascomycin, when bound to [U-13C,15N]FKBP were determined by isotope-filtered 2D NMR experiments. The structures for the R and S isomers that bind tightly to FKBP but lack immunosuppressive activity are compared to each other and to the conformation of the potent immunosuppressant, ascomycin, when bound to FKBP. The results are interpreted in terms of calcineurin binding to the FKBP/ascomycin complex. 相似文献
The affinity of concanavalin A (Con A) for simple saccharides has been known for over 50 years. However, the specificity of binding of Con A with cell-surface related carbohydrates has only recently been examined in detail. Brewer and coworkers [J Biol Chem (1986) 261:7306–10; J Biol Chem (1987) 262:1288–93; J Biol Chem (1987) 262:1294–99] have recently studied the binding interactions of a series of oligomannose and bisected hybrid type glycopeptides and complex type glycopeptides and oligosaccharides with Con A. The relative affinities of the carbohydrates were determined using hemagglutination inhibition measurements, and their modes of binding to the lectin examined by nuclear magnetic relaxation dispersion (NMRD) spectroscopy and quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves of Con A and the carbohydrates indicate that certain oligomannose and bisected hybrid type glycopeptides are bivalent for lectin binding. From the NMRD and precipitation data, two protein binding sites on each glycopeptide have been identified and characterized. Certain bisected complex type oligosaccharides also bind and precipitate Con A, while the corresponding nonbisected analogs bind but do not precipitate the protein. The precipitation data indicate that the bisected complex type oligosaccharides are also bivalent for lectin binding, while the nonbisected analogs are univalent. The NMRD and precipitation data are consistent with different mechanisms of binding of nonbisected and bisected complex type carbohydrates to Con A, including different conformations of the bound saccharides.Abbreviations Con A
Concanavalin A with unspecified metal ion content
- CMPL
Con A with Mn2+ and Ca2+ at the S1 and S2 sites respectively, in the locked conformation [12]; trisaccharide1, 3,6-di-O-(-d-mannopyranosyl)-d-mannose
- -MDM
methyl -d-mannopyranoside
- NMRD
nuclear magnetic relaxation dispersion, the magnetic field dependence of nuclear magnetic relaxation rates, in the present case, the longitudinal relaxation rate, 1/T1, of solvent protons 相似文献
Relaxation compensated constant-time Carr–Purcell–Meiboom–Gill relaxation dispersion experiments for amide protons are presented that detect s-ms time-scale dynamics of protein backbone amide sites. Because of their ten-fold larger magnetogyric ratio, much shorter 180° pulses can be applied to 1H than to 15N spins; therefore, off-resonance effects are reduced and a wider range of effective rf fields can often be used in the case of 1H experiments. Applications to [1H-15N]-ubiquitin and [1H-15N]-perdeuterated HIV-1 protease are discussed. In the case of ubiquitin, we present a pulse sequence that reduces artifacts that arise from homonuclear 3J(HN-H) coupling. In the case of the protease, we show that relaxation dispersion of both 1H and 15N spins provides a more comprehensive picture of slow backbone dynamics than does the relaxation dispersion of either spin alone. We also compare the relative merits of 1H versus 15N transverse relaxation measurements and note the benefits of using a perdeuterated protein to measure the relaxation dispersion of both spin types. 相似文献
13CHD2 methyl isotopomers are particularly useful to study methyl dynamics in proteins because, as compared with other methyl isotopomers, the 13C relaxation mechanism for this isotopomer is straightforward. However, in the case of proteins, where ()2 1, the refocused INEPT pulse sequence does not completely suppress unwanted 13CH3 signals. The presence of weak 13CH3 peaks is usually not a serious problem for smaller proteins because there are relatively few methyl signals and they are sharp; however, signal overlap becomes more common as the size of the protein increases. We overcome this problem by preparing a protein using a 98% D2O cell culture medium containing 3-13C pyruvic acid, 50–60% deuterated at the 3-position, and 4-13C 2-ketobutyric acid, 98% and 62% deuterated at the 3- and 4-positions, respectively. This approach significantly reduces the population of the CH3 isotopomer while optimizing the production of 13CHD2, the isotopomer desired for 13C relaxation measurements. In larger proteins where the deuterium T2 may be too short to measure accurately, we also suggest the alternative measurement of the proton T2 of the 13CH2D methyl isotopomer, because these protons are well-isolated from other protons in these highly deuterated samples. 相似文献
The paramagnetic effect of a spin-labeled sulfonyl fluoride, 4-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-p-fluorosulfonylbenzamide (p-V), when bound to the active site serine residue of the proteases, bovine plasma-activated protein C (APC) and des(1–41)-light-chain-activated protein C (GDAPC), on the longitudinal relaxation rate (T1) of Tl+ bound to these same proteins has been examined by 205Tl+-NMR spectroscopy. The substantial shortening by bound p-V of the T1 for Tl+ has been employed to estimate the distances between Tl+ and the unpaired electron on each protein surface. Assuming that a single cation-binding site exists on each enzyme, electron-nuclear distances of 3.4–3.9 Å have been calculated for each protein. This suggests that the removal of 41 amino acid residues and, concomitantly, all -carboxyglutamic acid, from the amino-terminal of the light chain of APC, does not significantly affect the protein topography in the region of the molecule probed by this technique. 相似文献
The nuclear magnetic resonance (NMR) of water protons in live and glycerinated muscle, suspensions of glycerinated myofibrils, and solutions of several muscle proteins has been studied. T1 and T2, measured on partially hydrated proteins by pulsed spin-echo techniques, decreased as the ratio of water to protein decreased, showing that the water which is tightly bound by the protein has short relaxation times. In live muscle fibers the pulse techniques showed that, after either a 180 or a 90° pulse, the relaxation of the magnetization is described by a single exponential. This is direct evidence that a fast exchange of protons occurs among the phases of the intracellular water. The data can be fitted with a model in which the bulk of the muscle water is in a phase which has properties similar to those of a dilute salt solution, while less than 4-5% of the total water is bound to the protein surface and has short relaxation times. Measurements of T1 and T2 in protein solutions showed that no change in the proton relaxation times occurred when heavy meromyosin was bound to actin, when myofibrils were contracted with adenosine triphosphate (ATP), or when globular actin was polymerized. 相似文献
Image contrast in clinical MRI is often determined by differences in tissue water proton relaxation behavior. However, many aspects of water proton relaxation in complex biological media, such as protein solutions and tissue are not well understood, perhaps due to the limited empirical data.
Principal Findings
Water proton T1, T2, and T1ρ of protein solutions and tissue were measured systematically under multiple conditions. Crosslinking or aggregation of protein decreased T2 and T1ρ, but did not change high-field T1. T1ρ dispersion profiles were similar for crosslinked protein solutions, myocardial tissue, and cartilage, and exhibited power law behavior with T1ρ(0) values that closely approximated T2. The T1ρ dispersion of mobile protein solutions was flat above 5 kHz, but showed a steep curve below 5 kHz that was sensitive to changes in pH. The T1ρ dispersion of crosslinked BSA and cartilage in DMSO solvent closely resembled that of water solvent above 5 kHz but showed decreased dispersion below 5 kHz.
Conclusions
Proton exchange is a minor pathway for tissue T1 and T1ρ relaxation above 5 kHz. Potential models for relaxation are discussed, however the same molecular mechanism appears to be responsible across 5 decades of frequencies from T1ρ to T1. 相似文献
The conformation and dynamic structure of single-stranded poly(inosinic acid), poly(I), in aqueous solution at neutral pH have been investigated by nmr of four nuclei at different frequencies: 1H (90 and 250 MHz), 2H (13.8 MHz), 13C (75.4 MHz), and 31P (36.4 and 111.6 MHz). Measurements of the proton-proton coupling constants and of the 1H and 13C chemical shifts versus temperature show that the ribose is flexible and that base-base stacking is not very significant for concentrations varying from 0.04 to 0.10M in the monomer unit. On the other hand, the proton T1 ratios between the sugar protons, T1 (H1′)/T1 (H3′), indicate a predominance of the anti orientation of the base around the glycosidic bond. The local motions of the ribose and the base were studied at different temperatures by measurements of nuclear Overhauser enhancement (NOE) of protonated carbons, the ratio of the proton relaxation times measured at two frequencies (90 and 250 MHz), and the deuterium quadrupolar transverse relaxation time T2. For a given temperature between 22 and 62°C, the 13C-{1H} NOE value is practically the same for seven protonated carbons (C2, C8, C1′, C2′, C3′, C4′, C5′). This is also true for the T1 ratio of the corresponding protons. Thus, the motion of the ribose–base unit can be considered as isotropic and characterized by a single correlation time, τc, for all protons and carbons. The τc values determined from either the 13C-{1H} NOE or proton T1 ratios, T1(90 MHz)/T1(250 MHz), and/or deuterium transverse relaxation time T2 agree well. The molecular motion of the sugar-phosphate backbone (O-P-O) and the chemical-shift anisotropy (CSA) were deduced from T1 (31P) and 31P-{1H} NOE measurements at two frequencies. The CSA contribution to the phosphorus relaxation is about 12% at 36.4 MHz and 72% at 111.6 MHz, corresponding to a value of 118 ppm for the CSA (σ = σ∥ ? σ?). Activation energies of 2–6 kcal/mol for the motion of the ribose–base unit and the sugarphosphate backbone were evaluated from the proton and phosphorus relaxation data. 相似文献
Summary Five different types of experiment are described which make it possible to measure various relaxation rates of selected protons in crowded spectra of macromolecules such as proteins: longitudinal spin-lattice relaxation rates =1/T1, transverse relaxation rates =1/T2 measured under conditions of free precession, transverse relaxation rates 1LOCK=1/T1 measured under conditions of spin-locking, and transverse relaxation rates DQC=1/T2DQC and ZQC=1/T2ZQC of double- and zero-quantum coherences. The surprisingly large discrepancy between the transverse rates t and t is discussed in detail. To separate overlapping proton signals, the experimental schemes involve one or several magnetization transfer steps, using a doubly selective homonuclear Hartmann-Hahn method. Numerous variants of the basic ideas can be conceived, depending on the extent of signal overlap and on the topology of the networks of scalar couplings. Applications are shown to H and H of Tyr23, to H, H and H of Cys30, and to H and H of Ala24 in bovine pancreatic trypsin inhibitor (BPTI). 相似文献
Summary Conformational rate processes in aqueous solutions of uniformly 15N-labeled pancreatic trypsin inhibitor (BPTI) at 36°C were investigated by measuring the rotating frame relaxation times of the backbone 15N spins as a function of the spin-lock power. Two different intramolecular exchange processes were identified. A first local rate process involved the residues Cys38 and Arg39, had a correlation time of about 1.3 ms, and was related to isomerization of the chirality of the disulfide bond Cys14-Cys38. A second, faster motional mode was superimposed on the disulfide bond isomerization and was tentatively attributed to local segmental motions in the polypeptide sequence-Cys14-Ala15-Lys16-. The correlation time for the overall rotational tumbling of the protein was found to be 2 ns, using the assumption that relaxation is dominated by dipolar coupling and chemical shift anistropy modulated by isotropic molecular reorientation.Abbreviations BPTI
basic pancreatic trypsin inhibitor
- 2D
two-dimensional
- COSY
2D correlation spectroscopy
- TOCSY
2D total correlation spectroscopy
- RF
radio frequency
- CW
continuous wave
- TPPI
time-proportional phase incrementation
- CSA
chemical shift anisotropy
- T1
longitudinal relaxation time
- T2
transverse relaxation time
- T1
relaxation time in the rotating frame , correlation time for overall rotational reorientation of the protein
-
exs
,
exf
,
correlation times for two conformational exchange processes (slow and fast). 相似文献
Tocopheramines (TNH2) and tocotrienamines (T3NH2) are analogues of tocopherols (TOH) and tocotrienols in which phenolic OH is replaced by NH2. It was shown in previous studies that TNH2 and T3NH2 act as potent antioxidants. In this study we compared the one-electron oxidation of TNH2/T3NH2 by diphenyl picryl hydrazyl (DPPH) and galvinoxyl (GOX) radicals with the one of α-TOH as a reference compound using ESR spectroscopy, stopped flow spectrophotometry and density functional theory (DFT) calculations. ESR spectroscopy revealed the presence of tocopheramine radicals during electrochemical oxidation of α-TNH2. Kinetic measurements demonstrated that in apolar n-hexane TNH2/T3NH2 derivatives reacted two to three orders of magnitude slower than α-TOH with the model radicals. DFT calculations indicated that this correlates well with the higher bond dissociation energy (BDE) for N–H in TNH2 than for O–H in α-TOH in pure H-atom transfer (HAT). In the more polar medium ethanol TNH2/T3NH2 derivatives partially reacted faster than α-TOH depending on the reaction partner. DFT calculations suggest that this is due to reaction mechanisms alternative to HAT. According to thermochemistry data sequential proton loss and electron transfer (SPLET) is more favored for α-TOH in ethanol than for TNH2. Therefore, for TNH2 a contribution of the alternative mechanism of sequential electron transfer–proton transfer (SET–PT) could be a possible explanation. These data show that the antioxidant reactivity strongly depends on the structure, reaction partners and environment. According to these findings TNH2/T3NH2 should be superior as antioxidants over α-TOH in polar head group regions of membranes but not in the apolar core of lipid bilayers. 相似文献
The relaxation rates (1 / T1 and 1 / T2) in cysts have already been analyzed in terms of materials such as albumin, cholesterol, manganese, iron, and copper. However,
the relaxivities of these materials have not been determined yet. In this work, five sets containing the ions, albumin, and
cholesterol were prepared by addition of increasing concentration of one material to each set. The relaxation times in these
sets were measured by MRI, and the relaxation rates were fitted versus concentrations. The slopes of the fits were used as
relaxivities. The (r1, r2) values of manganese, iron, and copper in mM−1 s−1, and those of albumin and cholesterol in (g/dl)−1 s−1 were found to be (32.64, 89.77), (0.31, 1.19), (0.5, 1.479), (0.01, 0.066) and (0.03, 0.458), respectively. The r2/r1 ratio ranged from 2.75 to 15.27. Manganese is an efficient relaxer, but iron and copper are poor ones. Albumin and cholesterol
are efficient relaxers for only T2. The contribution of water associated with native manganese of the cystic fluid to T1 was 0.268 s−1, whereas those of water associated with native manganese, albumin, cholesterol, and iron to T2 were 0.736, 0.185, 0.092, and 0.076 s−1, respectively. The other contributions were much smaller than 0.076 s−1. Manganese is most likely the compound altering T1-weighted images between different jaw cysts, whereas manganese and albumin are most likely the compounds altering the T2-weighted images. Present data suggest that such alterations may be used to separate jaw cysts from other jaw masses. The
high r2/r1 suggests that T2 is a more convenient parameter than T1 for diagnostic use.
This work is a part of the PhD thesis of U. Nezih Yilmaz supervised by R. Guner. 相似文献