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1.
Carbonyl 13C′ relaxation is dominated by the contribution from the 13C′ chemical shift anisotropy (CSA). The relaxation rates provide useful and non-redundant structural information in addition to dynamic parameters. It is straightforward to acquire, and offers complimentary structural information to the 15N relaxation data. Furthermore, the non-axial nature of the 13C′ CSA tensor results in a T1/T2 value that depends on an additional angular variable even when the diffusion tensor of the protein molecule is axially symmetric. This dependence on an extra degree of freedom provides new geometrical information that is not available from the NH dipolar relaxation. A protocol that incorporates such structural restraints into NMR structure calculation was developed within the program Xplor-NIH. Its application was illustrated with the yeast Fis1 NMR structure. Refinement against the 13C′ T1/T2 improved the overall quality of the structure, as evaluated by cross-validation against the residual dipolar coupling as well as the 15N relaxation data. In addition, possible variations of the CSA tensor were addressed. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

2.
Rotational diffusion properties have been derived for the DNA dodecamer d(CGCGAATTCGCG)2 from 13C R1 and R1 measurements on the C1, C3, and C4 carbons in samples uniformly enriched in 13C. The narrow range of C-H bond vector orientations relative to the DNA axis make the analysis particularly sensitive to small structural deviations. As a result, the R1/R1 ratios are found to fit poorly to the crystal structures of this dodecamer, but well to a recent solution NMR structure, determined in liquid crystalline media, even though globally the structures are quite similar. A fit of the R1/R1 ratios to the solution structure is optimal for an axially symmetric rotational diffusion model, with a diffusion anisotropy, D||/D, of 2.1±0.4, and an overall rotational correlation time, (2D||+4D)–1, of 3.35 ns at 35 °C in D2O, in excellent agreement with values obtained from hydrodynamic modeling.  相似文献   

3.
Model-free analysis has been extensively used to extract information on motions in proteins over a wide range of timescales from NMR relaxation data. We present a detailed analysis of the effects of rotational anisotropy on the model-free analysis of a ternary complex for dihydrofolate reductase (DHFR). Our findings show that the small degree of anisotropy exhibited by DHFR (D||/D=1.18) introduces erroneous motional models, mostly exchange terms, to over 50% of the NH spins analyzed when isotropic tumbling is assumed. Moreover, there is a systematic change in S2, as large as 0.08 for some residues. The significant effects of anisotropic rotational diffusion on model-free motional parameters are in marked contrast to previous studies and are accentuated by lowering of the effective correlation time using isotropic tumbling methods. This is caused by the preponderance of NH vectors aligned perpendicular to the principal diffusion tensor axis and is readily detected because of the high quality of the relaxation data. A novel procedure, COPED (COmparison of Predicted and Experimental Diffusion tensors) is presented for distinguishing genuine motions from the effects of anisotropy by comparing experimental relaxation data and data predicted from hydrodynamic analyses. The procedure shows excellent agreement with the slow motions detected from the axially symmetric model-free analysis and represents an independent procedure for determining rotational diffusion and slow motions that can confirm or refute established procedures that rely on relaxation data. Our findings show that neglect of even small degrees of rotational diffusion anisotropy can introduce significant errors in model-free analysis when the data is of high quality. These errors can hinder our understanding of the role of internal motions in protein function.  相似文献   

4.
Because the overall tumbling provides a major contribution to protein spectral densities measured in solution, the choice of a proper model for this motion is critical for accurate analysis of protein dynamics. Here we study the overall and backbone dynamics of the B3 domain of protein G using 15N relaxation measurements and show that the picture of local motions is markedly dependent on the model of overall tumbling. The main difference is in the interpretation of the elevated R 2 values in the -helix: the isotropic model results in conformational exchange throughout the entire helix, whereas no exchange is predicted by anisotropic models that place the longitudinal axis of diffusion tensor almost parallel to the helix axis. Due to small size (fast tumbling) of the protein, the T 1 values have low sensitivity to NH bond orientation. The diffusion tensor derived from orientation dependence of R 2/R 1 is anisotropic (D par/D perp=1.4), with a small rhombic component. In order to distinguish the correct picture of motion, we apply model-independent methods that are sensitive to conformational exchange and do not require knowledge of protein structure or assumptions about its dynamics. A comparison of the CSA/dipolar cross-correlation rate constants with 15N relaxation rates and the estimation of R ex terms from relaxation data at 9.4 and 14.1 T indicate no conformational exchange in the helix, in support of the anisotropic models. The experimentally derived diffusion tensor is in excellent agreement with theoretical predictions from hydrodynamic calculations; a detailed comparison with various hydrodynamic models revealed optimal parameters for hydrodynamic calculations.  相似文献   

5.
The motional properties of the cyclic enterobacterial common antigen (cECA), consisting of four trisaccharide repeat units, have been investigated by carbon-13 spin relaxation. R1, R2 and NOE relaxation parameters have been determined at three magnetic field strengths. The data were interpreted within the model-free framework to include the possibility of motional anisotropy, and overall as well as local dynamical parameters were fitted separately for each ring carbon. The motional anisotropy was addressed by assuming an axially symmetric diffusion tensor, which was fitted from the overall correlation times for each site in the sugar residues using the previously determined crystal structure. The data were found to be in agreement with an oblate shape of the molecule, and the values for Diso and were in good agreement with translational diffusion data and an estimate based on calculation of the moment of inertia tensor, respectively. The local dynamics in cECA were found to be residue-dependent. Somewhat lower values for the order parameters, as well as longer local correlation times, were observed for the -linked ManNAcA residue compared to the two -linked residues in the trisaccharide repeat unit.  相似文献   

6.
Soluble ammonia monooxygenase (AMO) from Nitrosomonas europaea was purified to homogeneity and metals in the active sites of the enzyme (Cu, Fe) were analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were obtained for a type 2 Cu(II) site with g|| = 2.24, A|| = 18.4 mT and g = 2.057 as well as for heme and non heme iron present in purified soluble AMO from N. europaea. A second type 2 Cu(II) EPR signal with g|| = 2.29, A|| = 16.1 mT and g = 2.03 appeared in the spectrum of the ferricyanide oxidized enzyme and was attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu2+ with total copper determined by inductively coupled plasma-mass spectrometry (ICP-MS) suggests that there are six paramagnetic Cu2+ and three diamagnetic Cu1+ per heterotrimeric soluble AMO (two paramagnetic and one diamagnetic Cu per αβγ-protomer). A trigonal EPR signal at g = 6.01, caused by a high-spin iron, indicative for cytochrome bound iron, and a rhombic signal at g = 4.31, characteristic of specifically bound Fe3+ was detectable. The binding of nitric oxide in the presence of reductant resulted in a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex. Inactivation of soluble AMO with acetylene did neither diminish the ferrous signal nor the intensity of the Cu2+-EPR signal.  相似文献   

7.
Gametophores of mosses Mnium undulatum and Polytrichum commune were submerged in distilled water or in calcium chloride solution (0.9 mM Ca2+) to induce hypoxia. The net photosynthetic (PN) and dark respiration rate (RD) were measured in the air containing 300–400 μmol(CO2)·mol−1(air) and 0.21 mol(O2)·mol−1(air). PN of M. undulatum gametophores decreased to 58 % of the control after 1-h submersion in water, whereas to 80 % of the control in P. commune gametophores. A smaller decrease in PN was observed when the gametophores were immersed in CaCl2 solution. In hypoxia, RD in the tested mosses species was a little higher than in the control.  相似文献   

8.
Dinitrosyl iron complexes (DNICs) with various thiol ligands, the known donors of nitric oxide, markedly inhibited aidB gene expression in E. coli cells by destroying the [4Fe-4S]2+ center of its regulator protein Fnr. Therewith, the cells accumulated DNICs in the protein-bound form, identified by the EPR signal with g = 2.04 and g = 2.014. Subsequent addition of sulfur sources L-cysteine or N-acetylcysteine, DTT as well as Na2S to the DNIC-treated cells significantly restored the reporter gene expression. Simultaneously, the above-specified EPR signal was partly or completely replaced with a narrower signal (g = 2.032, g = 2.02) identical to that of DNICs with persulfide (R-S-S) ligands, which result from interaction of S2− with thiols; inorganic sulfide proved to be the most efficient agent. These data corroborate the central role of S2− in recovery of the protein [4Fe-4S] center disrupted by the NO donors.  相似文献   

9.
The stereodynamics for H++HD and its isotopic variant D++HD were studied with a quasi-classical trajectory (QCT) method at a collision energy of 0.7 eV on the ground 11A′ potential energy surface (PES). The polarization-dependent differential cross-sections (PDDCSs) in the center-of-mass frame are presented here. Furthermore, the distribution of the angle between k and j′, p(θ r ) and the distribution of the dihedral angle p(ϕ r ) were calculated and are discussed. The results indicate that isotopic substitution exerts substantial effects on the differential cross-section and the product’s rotational polarization.  相似文献   

10.
Heavy water (H218O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the 18O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% 18O for DNA extracted from soil incubated with the addition of H218O. Four experiments were conducted to test how the atom% 18O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% 18O of added H2O. In the time series experiment, the atom% 18O of DNA increased linearly (R 2 = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R 2 = 0.991, p = 0.006) between the log10 of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log10 of the atom% 18O of DNA. For the experiment where soil moisture was manipulated, the atom% 18O of DNA increased with higher soil moisture until soil moisture reached 30%, above which 18O enrichment of DNA declined as soils became more saturated. When the atom% 18O for H2O added was varied, there was a positive linear relationship between the atom% 18O of the added water and the atom% 18O of the DNA. Results indicate that quantification of 18O incorporated into DNA from H218O has potential to be used as a proxy for microbial growth in soil.  相似文献   

11.
A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by ≈ 17-fold and ≈ 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (Vt) of 38 to 45 mV and high resistance (Rt) of 800 to 1400 Ω • cm2 measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10−4 M, ouabain 10−4 M, bumetanide 10−4 M), K+-free solutions, or organic solutes such as D-glucose 10 mM or DL-alanine 0.5 mM failed to affect Vt or Rt. Bafilomycin-A1 (3 to 5 μM) decreased Vt by ≈ 40% and short-circuit current (Isc) by ≈ 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H+-ATPase located in the basal membrane. Gradients of H+ were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO3), driven by the basal V-H+-ATPase.  相似文献   

12.
Methyl 13CHD2 isotopomers of all methyl-containing amino-acids can be observed in residually protonated samples of large proteins obtained from [U-13C,1H]-glucose/D2O-based bacterial media, with sensitivity sufficient for a number of NMR applications. Selective detection of some subsets of methyl groups (Alaβ, Thrγ2) is possible using simple ‘out-and-back’ NMR methodology. Such selective methyl-detected ‘out-and-back’ NMR experiments allow complete assignments of threonine γ2 methyls in residually protonated, [U-13C,1H]-glucose/D2O-derived samples of an 82-kDa enzyme Malate Synthase G. [U-13C,1H]-glucose/D2O-derived protein samples are relatively inexpensive and are usually available at very early stages of any NMR study of high-molecular-weight systems.  相似文献   

13.
An approach for generating efficient RNnnS, nk {\rm{RN}}_{n}^{\nu_{\rm{S}}, {\nu_{\rm{k}}}} symmetry-based dual channel RF pulse schemes for γ-encoded broadband 15N–13C dipolar recoupling at high magic angle spinning frequencies is presented. The method involves the numerical optimisation of the RF phase-modulation profile of the basic “R” element so as to obtain heteronuclear double quantum dipolar recoupling sequences with satisfactory magnetisation transfer characteristics. The basic “R” element was implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by a RF phase and amplitude values. The performance characteristics of the sequences were evaluated via numerical simulations and 15N–13C chemical shift correlation experiments. Employing such 13C–15N double-quantum recoupling sequences and the multiple receiver capabilities available in the current generation of NMR spectrometers, the possibility to simultaneously acquire 3D NCC and CNH chemical shift correlation spectra is also demonstrated.  相似文献   

14.
To gain insight into the metabolic design of the amino acid carrier systems in fish, we injected a bolus of 15N amino acids into the dorsal aorta in mature rainbow trout (Oncorhynchus mykiss). The plasma kinetic parameters including concentration, pool size, rate of disappearance (R d), half-life and turnover rate were determined for 15 amino acids. When corrected for metabolic rate, the R d values obtained for trout for most amino acids were largely comparable to human values, with the exception of glutamine (which was lower) and threonine (which was higher). R d values ranged from 0.9 μmol 100 g−1 h−1 (lysine) to 22.1 μmol 100 g−1 h−1 (threonine) with most values falling between 2 and 6 μmol 100 g−1 h−1. There was a significant correlation between R d and the molar proportion of amino acids in rainbow trout whole body protein hydrolysate. Other kinetic parameters did not correlate significantly with whole body amino acid composition. This indicates that an important design feature of the plasma-free amino acids system involves proportional delivery of amino acids to tissues for protein synthesis.  相似文献   

15.
Oriented solid-state NMR is the most direct methodology to obtain the orientation of membrane proteins with respect to the lipid bilayer. The method consists of measuring 1H-15N dipolar couplings (DC) and 15N anisotropic chemical shifts (CSA) for membrane proteins that are uniformly aligned with respect to the membrane bilayer. A significant advantage of this approach is that tilt and azimuthal (rotational) angles of the protein domains can be directly derived from analytical expression of DC and CSA values, or, alternatively, obtained by refining protein structures using these values as harmonic restraints in simulated annealing calculations. The Achilles’ heel of this approach is the lack of suitable experiments for sequential assignment of the amide resonances. In this Article, we present a new pulse sequence that integrates proton driven spin diffusion (PDSD) with sensitivity-enhanced PISEMA in a 3D experiment ([1H,15N]-SE-PISEMA-PDSD). The incorporation of 2D 15N/15N spin diffusion experiments into this new 3D experiment leads to the complete and unambiguous assignment of the 15N resonances. The feasibility of this approach is demonstrated for the membrane protein sarcolipin reconstituted in magnetically aligned lipid bicelles. Taken with low electric field probe technology, this approach will propel the determination of sequential assignment as well as structure and topology of larger integral membrane proteins in aligned lipid bilayers.  相似文献   

16.
The relationships between dark respiration rate (R D) and net photosynthetic rate (P N) in Quercus ilex L. shrubs growing at the Botanical Garden in Rome were analysed. Correlation analysis of the data sets collected in the year 2006 confirmed the dependence among the considered leaf traits, in particular, R D was significantly (p<0.05) correlated with P N (r = 0.40). R D and P N increased from March to May [1.40±0.10 and 10.1±1.8 μmol(CO2) m−2 s−1 mean values of the period, respectively], when air temperature was in the range 14.8–25.2 °C, underlining the highest metabolic activity in the period of the maximum vegetative activity that favoured biomass accumulation. On the contrary, the highest R D [1.60±0.02 μmol(CO2) m−2 s−1], associated to the lowest P N rates (44 % of the maximum) and carbon use efficiency (CUE) in July underlined the mobilization of stored material during drought stress by a higher air temperature (32.7 °C).  相似文献   

17.
In hypertonic solutions made by adding nonelectrolytes, K+ channels of squid giant axons opened at usual asymmetrical K+ concentrations in two different time courses; an initial instantaneous activation (I IN) and a sigmoidal activation typical of a delayed rectifier K+ channel (I D). The current–voltage relation curve for I IN was fitted well with Goldman equation described with a periaxonal K+ concentration at the membrane potential above −10 mV. Using the activation–voltage curve obtained from tail currents, K+ channels for I IN are confirmed to activate at the membrane potential that is lower by 50 mV than those for I D. Both I IN and I D closed similarly at the holding potential below −100 mV. The logarithm of I IN/I D was linearly related with the osmolarity for various nonelectrolytes. Solute inaccessible volumes obtained from the slope increased with the nonelectrolyte size from 15 to 85 water molecules. K+ channels representing I D were blocked by open channel blocker tetra-butyl ammonium (TBA) more efficiently than in the absence of I IN, which was explained by the mechanism that K+ channels for I D were first converted to those for I IN by the osmotic pressure and then blocked. So K+ channels for I IN were suggested to be derived from the delayed rectifier K+ channels. Therefore, the osmotic pressure is suggested to exert delayed-rectifier K+ channels to open in shrinking rather hydrophilic flexible parts outside the pore than the pore itself, which is compatible with the recent structure of open K+ channel pore.  相似文献   

18.
The resonance scattering spectral probe for Pb2+ was obtained using aptamer-modified AuPd Nanoalloy. In the pH 7.0 Na2HPO4–NaH2PO4 buffer solution, the aptamer interacted with AuPd nanoalloy particles to form stable aptamer-AuPd nanoalloy probe for Pb2+ that is stable in high concentration of salt. The probe combined with Pb2+ ions to form a G-quadruplex and to release AuPd nanoalloy particles that aggregate to form big particles which led the resonance scattering (RS) intensity enhancing. The reaction solution was filtered by 0.15 μm membrane to obtain the filtration containing aptamer-AuPd nanoalloy probe that has strong catalytic effect on the electrodeless nickel particle plating reaction between Ni(II) and PO23− that exhibited a strong RS peak at 508 nm. The RS intensity at 508 nm decreased when the Pb2+ concentration increased. The decreased intensity (ΔI 508nm) is linear to the concentration of 0.08–42 nM Pb2+, with regress equation of DI508nm = 16.3 c + 1.5 \Delta {I_{{5}0{\rm{8nm}}}} = {16}.{3}\,c + {1}.{5} , correlation coefficient of 0.9965, and detection limit of 0.04 nM Pb2+. The RS assay was applied to the analysis of Pb2+ in wastewater, with satisfactory results.  相似文献   

19.
Two new bismacrocyclic Gd3+ chelates containing a specific Ca2+ binding site were synthesized as potential MRI contrast agents for the detection of Ca2+ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca2+-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L1) or by a propylene (L2) unit [H4BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H3DO3A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd3+ complexes towards Ca2+ and Mg2+ was studied by 1H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca2+ binding to Gd2L1 and Gd2L2, respectively, with a distinct selectivity of Gd2L1 towards Ca2+ compared with Mg2+. For Ca2+ binding, association constants of log K = 1.9 (Gd2L1) and log K = 2.7 (Gd2L2) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu3+ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca2+ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca2+ addition. A 1H nuclear magnetic relaxation dispersion and 17O NMR study on Gd2L1 in the absence and in the presence of Ca2+ was performed to assess the microscopic parameters influencing relaxivity. On Ca2+ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.  相似文献   

20.
Effects of intracellular Mg2+ on a native Ca2+-and voltage-sensitive large-conductance K+ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca2+ ([Ca2+]i) of 10−5–10−4 M, addition of 1–10 mM Mg2+ increased the open probability (Po) of the channel, which shifted the Po –membrane potential (Vm) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg2+-induced increase in Po was suppressed at a relatively low [Ca2+]i (10−5.5–10−6 M). Dwell-time histograms have revealed that addition of Mg2+ mainly increased Po by extending open times at 10−5 M Ca2+ and extending both open and closed times simultaneously at 10−5.5 M Ca2+. Since our data showed that raising the [Ca2+]i from 10−5 to 10−4 M increased Po mainly by shortening the closed time, extension of the closed time at 10−5.5 M Ca2+ would result from the Mg2+-inhibited Ca2+-dependent activation. At a constant Vm, adding Mg2+ enhanced the sigmoidicity of the Po–[Ca2+]i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg2+ on this channel is to elevate Po by lengthening the open time, while extension of the closed time at a relatively low [Ca2+]i results from a lowering of the sensitivity to Ca2+ of the channel by Mg2+, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.  相似文献   

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