共查询到20条相似文献,搜索用时 31 毫秒
1.
Keith L. Constantine Mark S. Friedrichs Valentina Goldfarb Philip D. Jeffrey Steven Sheriff Luciano Mueller 《Proteins》1993,15(3):290-311
The dynamic behavior of the polypeptide backbone of a recombinant anti-digoxin antibody VL domain has been characterized by measurements of 15N T1 and T2 relaxation times, 1H–15N NOE values, and 1H–2H exchange rates. These data were acquired with 2D inverse detected heteronuclear 1H–15N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R2 (= 1/T2). All characterized residues display low-amplitude picosecond timescale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R2 exchange contributions, which reflect motions on the microsecond to millisecond timescale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R2 exchange contributions. The measured 1H–2H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of β-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the VL domain, including the three hypervariable loops, undergo dynamic changes upon VL:VH association and/ or complexation with digoxin. Overall, the 26–10 VL domain exhibits relatively low flexibility on the ps–ns timescale. The possible functional consequences of this result are considered. © 1993 Wiley-Liss, Inc. 相似文献
2.
Yasushi Oda Haruki Nakamura Toshio Yamazaki Kuniaki Nagayama Mayumi Yoshida Shigenori Kanaya Morio Ikehara 《Journal of biomolecular NMR》1992,2(2):137-147
Summary Two-dimensional (2D)1H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the1H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D1H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged. 相似文献
3.
Ronald A. Venters Chih-Chin Huang Bennett T. Farmer II Ronald Trolard Leonard D. Spicer Carol A. Fierke 《Journal of biomolecular NMR》1995,5(4):339-344
Summary The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with 2H, 13C and 15N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments, 2H has been incorporated into HCA II in order to decrease the rates of 13C and 1HN T2 relaxation. NMR quantities of protein with essentially complete aliphatic 2H incorporation have been obtained by growth of E. coli in defined media containing D2O, [1,2-13C2, 99%] sodium acetate, and [15N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for 13C and 15N backbone NMR assignment studies, since the 13C and 1HN T2 relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential 2H isotopic shift observed for partially deuterated CHnDm moieties. 相似文献
4.
Pervushin K 《Journal of biomolecular NMR》2001,20(3):275-285
The interference between conformational exchange-induced time-dependent variations of chemical shifts in a pair of scalar coupled 1H and 15N spins is used to construct novel TROSY-type NMR experiments to suppress NMR signal loss in [15N,1H]-correlation spectra of a 14-mer DNA duplex free in solution and complexed with the Antp homeodomain. An analysis of double- and zero-quantum relaxation rates of base 1H–15N moieties showed that for certain residues the contribution of conformational exchange-induced transverse relaxation might represent a dominant relaxation mechanism, which, in turn, can be effectively suppressed by TROSY. The use of the new TROSY method for exchange-induced transverse relaxation optimization is illustrated with two new experiments, 2D h1
J
HN,h2
J
NN-quantitative [15N,1H]-TROSY to measure h1
J
HN and h2
J
NN scalar coupling constants across hydrogen bonds in nucleic acids, and 2D (h2
J
NN+h1
J
NH)-correlation-[15N,1H]-TROSY to correlate 1HN chemical shifts of bases with the chemical shifts of the tertiary 15N spins across hydrogen bonds using the sum of the trans-hydrogen bond coupling constants in nucleic acids. 相似文献
5.
Alexey G. Krushelnitsky Vladimir D. Fedotov Jiri Spevacek Jaroslav Straka 《Journal of biomolecular structure & dynamics》2013,31(2):211-224
Abstract Temperature dependencies of 1H non-selective NMR T1 and T2 relaxation times measured at two resonance frequencies and natural abundance l3C NMR relaxation times Tl and Tlr measured at room temperature have been studied in a set of dry and wet solid proteins—;Bacterial RNase, lysozyme and Bovine serum albumin (BSA). The proton and carbon data were interpreted in terms of a model supposing three kinds of internal motions in a protein. These are rotation of the methyl protons around the axis of symmetry of the methyl group, and fast and slow oscillations of all atoms. The correlation times of these motions in solid state are found around 10?11, 10?9 and 10?6 s, respectively. All kinds of motion are characterized by the inhomogeneous distribution of the correlation times. The protein dehydration affects only the slow internal motion. The amplitude of the slow motion obtained from the carbon data is substantially less than that obtained from the proton data. This difference can be explained by taking into account different relative inter- and intra- chemical group contributions to the proton and carbon second moments. The comparison of the solid state and solution proton relaxation data showed that the internal protein dynamics in these states is different: the slow motion seems to be few orders of magnitude faster in solution. 相似文献
6.
The H2A–H2B histone heterodimer folds via monomeric and dimeric kinetic intermediates. Within ~ 5 ms, the H2A and H2B polypeptides associate in a nearly diffusion limited reaction to form a dimeric ensemble, denoted I2 and I2?, the latter being a subpopulation characterized by a higher content of nonnative structure (NNS). The I2 ensemble folds to the native heterodimer, N2, through an observable, first-order kinetic phase. To determine the regions of structure in the I2 ensemble, we characterized 26 Ala mutants of buried hydrophobic residues, spanning the three helices of the canonical histone folds of H2A and H2B and the H2B C-terminal helix. All but one targeted residue contributed significantly to the stability of I2, the transition state and N2; however, only residues in the hydrophobic core of the dimer interface perturbed the I2? population. Destabilization of I2? correlated with slower folding rates, implying that NNS is not a kinetic trap but rather accelerates folding. The pattern of Φ values indicated that residues forming intramolecular interactions in the peripheral helices contributed similar stability to I2 and N2, but residues involved in intermolecular interactions in the hydrophobic core are only partially folded in I2. These findings suggest a dimerize-then-rearrange model. Residues throughout the histone fold contribute to the stability of I2, but after the rapid dimerization reaction, the hydrophobic core of the dimer interface has few fully native interactions. In the transition state leading to N2, more native-like interactions are developed and nonnative interactions are rearranged. 相似文献
7.
Probing water compartments and membrane permeability in plant cells by 1H NMR relaxation measurements
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1H NMR relaxation times (T1 and T2) in parenchyma tissue of apple can identify three populations of water with different relaxation characteristics. By following the uptake of Mn2+ ions in the tissue it is shown that the observed relaxation times originate from particular water compartments: the vacuole, the cytoplasm, and the cell wall/extracellular space.
Proton exchange between these compartments is controlled by the plasmalemma and tonoplast membranes. During the Mn2+ penetration experiment, conditions occur that cause the relaxation times of protons of cytoplasmic water to be much shorter than their residence time in the cytoplasm. Then the tonoplast permeability coefficient Pd for water can be calculated from the vacuolar T1 and T2 values to be 2.44 10-5 m·s-1.
相似文献8.
The titration of the (S)‐enantiomer of omeprazole with the (R)‐enantiomer in chloroform‐d1 is monitored by continuous‐flow capillary microcoil 1H NMR spectroscopy employing a microcoil with a detection volume of 1.5 µl. The observed changes of the 1H NMR chemical shifts indicate the formation of a heterochiral (R,S) dimer of omeprazole via its sulfinyl group and the NH group of the benzimidazole ring. Chirality 24:1074–1076, 2012. © 2012 Wiley Periodicals, Inc. 相似文献
9.
Rathi Saravanan 《生物化学与生物物理学报:生物膜》2010,1798(2):128-18428
Melittin, the major component of the honey bee venom, is a 26-residue hemolytic and membrane active peptide. Structures of melittin determined either in lipid environments by NMR or by use of X-ray demonstrated two helical regions at the N- and C-termini connected by a hinge or a bend at the middle. Here, we show that deletion of the hinge residues along with two C-terminal terminal Gln residues (Q25 and Q26), yielding a peptide analog of 19-residue or Mel-H, did not affect antibacterial activity but resulted in a somewhat reduced hemolytic activity. A diastereomer of Mel-H or Mel-dH containing d-amino acids [dV5, dV8, dL11 and dK16] showed further reduction in hemolytic activity without lowering antibacterial activity. We have carried out NMR structures, dynamics (H-D exchange and proton relaxation), membrane localization by spin labeled lipids, pulse-field-gradient (PFG) NMR and isothermal titration calorimetry (ITC) in dodecylphosphocholine (DPC) micelles, as a mimic to eukaryotic membrane, to gain insights into cell selectivity of these melittin analogs. PFG-NMR showed Mel-H and Mel-dH both were similarly partitioned into DPC micelles. ITC demonstrated that Mel-H and Mel-dH interact with DPC with similar affinity. The micelle-bound structure of Mel-H delineated a straight helical conformation, whereas Mel-dH showed multiple β-turns at the N-terminus and a short helix at the C-terminus. The backbone amide-proton exchange with solvent D2O demonstrated a large difference in dynamics between Mel-H and Mel-dH, whereby almost all backbone protons of Mel-dH showed a much faster rate of exchange as compared to Mel-H. Proton T1 relaxation had suggested a mobile backbone of Mel-dH peptide in DPC micelles. Resonance perturbation by paramagnetic lipids indicated that Mel-H inserted deeper into DPC micelles, whereas Mel-dH is largely located at the surface of the micelle. Taken together, results presented in this study demonstrated that the poor hemolytic activity of the d-amino acid containing analogs of antimicrobial peptides may be correlated with their flexible dynamics at the membrane surface. 相似文献
10.
Stefan Highsmith Mark Kubinec Devendra K. Jaiswal Hiromi Morimoto Philip G. Williams David E. Wemmer 《Journal of biomolecular NMR》1993,3(3):325-334
Summary The synthesis of [2-3H]ATP with specific activity high enough to use for 3H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single 3H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes.The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg2+, S1 converts [2-3H]ATP to [2-3H]ADP and the complex S1.Mg[2-3H]ADP has ADP bound in the active site. At 0°C, 1D 3H NMR spectra of S1.Mg[2-3H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-3H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-3H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 °C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (Vi) to produce S1.Mg[2-3H]ADP.Vi shifts both peaks slightly more upfield without chaning their widths or relative areas. 相似文献
11.
The molecular basis of anesthetic interaction with membrane proteins has been explored via determination of anesthetic effects on the structure and dynamics of the extended second transmembrane domain (TM2e) of the human neuronal nicotinic acetylcholine receptor (nAChR) β2 subunit in dodecylphosphocholine (DPC) micelles by 1H and 15N solution-state NMR. Both 1-chloro-1,2,2-trifluorocyclobutane (F3) and isoflurane, two volatile general anesthetics, induced nonuniform changes in chemical shifts among residues in TM2e. Saturation transfer difference NMR experiments further confirmed the direct anesthetic interaction with TM2e. A significant and more specific anesthetic interaction was observed on three leucine residues at the helix C-terminus. Although the TM2e helical structure remained after addition of anesthetics, plausible shortening and lengthening of helix hydrogen bonds were evidenced by periodic changes in backbone amide chemical shifts. The TM2e backbone dynamics were determined on the basis of the 15N relaxation rate constants, R1 and R2, and the 15N-[1H] NOE using the model-free approach. The global tumbling time (11.7 ns) of TM2e in micelles slightly increased (∼12.3-12.5 ns) in the presence of anesthetics. The order parameter, S2, exceeded 0.9 for all 15N-labeled residues, showing a restricted internal motion. Anesthetics appear to have minor effect on the TM2e's internal motion. This study provided the basis for subsequent more comprehensive studies of anesthetic effects on the transmembrane domain complex of neuronal nAChR. 相似文献
12.
Error estimation and global fitting in transverse-relaxation dispersion experiments to determine chemical-exchange parameters 总被引:2,自引:2,他引:0
Off-resonance effects can introduce significant systematic errors in R2 measurements in constant-time Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation dispersion experiments. For an off-resonance chemical shift of 500 Hz, 15N relaxation dispersion profiles obtained from experiment and computer simulation indicated a systematic error of ca. 3%. This error is three- to five-fold larger than the random error in R2 caused by noise. Good estimates of total R2 uncertainty are critical in order to obtain accurate estimates in optimized chemical exchange parameters and their uncertainties derived from χ2 minimization of a target function. Here, we present a simple empirical approach that provides a good estimate of the total error (systematic + random) in 15N R2 values measured for the HIV protease. The advantage of this empirical error estimate is that it is applicable even when some of the factors that contribute to the off-resonance error are not known. These errors are incorporated into a χ2 minimization protocol, in which the Carver–Richards equation is used fit the observed R2 dispersion profiles, that yields optimized chemical exchange parameters and their confidence limits. Optimized parameters are also derived, using the same protein sample and data-fitting protocol, from 1H R2 measurements in which systematic errors are negligible. Although 1H and 15N relaxation profiles of individual residues were well fit, the optimized exchange parameters had large uncertainties (confidence limits). In contrast, when a single pair of exchange parameters (the exchange lifetime, τex, and the fractional population, pa), were constrained to globally fit all R2 profiles for residues in the dimer interface of the protein, confidence limits were less than 8% for all optimized exchange parameters. In addition, F-tests showed that quality of the fits obtained using τex, pa as global parameters were not improved when these parameters were free to fit the R2 profiles of individual residues. Finally, nearly the same optimized global τex, pa values were obtained, when the 1H and 15N data sets for residues in the dimer interface, were fit independently; the difference in optimized global parameters, ca. 10%, was of marginal significance according to the F-test. 相似文献
13.
《Inorganica chimica acta》1986,121(2):113-117
1H and 2H NMR spectra of the title copper(II) complexes and its derivatives have been measured. In contrast with their 1H NMR spectra, 2H NMR spectra gave well resolved sharp signals, and demonstrated that two diastereomers attributable to two asymmetric ligand nitrogens are readily resolved. The remarkable linewidth-narrowing was found in the peripheral methyl groups, which make 2H NMR spectra very useful even for copper(II) complexes with a long electron spin relaxation time. By using 2H NMR spectra, meso-racemate equilibrium was pursued and examined in aqueous and acetonitrile solutions. 相似文献
14.
Ivano Bertini Mario Piccioli Maria Silvia Viezzoli Choi Ying Chiu Guy T. Mullenbach 《European biophysics journal : EBJ》1994,23(3):167-176
A mutated protein of human Cu(II)2Zn(II)2 SOD in which residues Phe50 and Gly51 at the dimer interface were substituted by Glu's, thus producing a monomeric species, has been characterized by electronic absorption spectroscopy, EPR, relaxivity and1H NMR techniques. Such substitutions and/or accompanying remodeling and exposure of the dimer interface to solvent, alter the geometry of the active site: increases in the axiality of the copper chromophore and the Cu-OH2 distance have been observed. The affinity of both metal binding sites for Co(II) is also altered. The observed NMR parameters of the Co(II) substituted derivative have been interpreted as a function of the decrease of rotational correlation time as a consequence of the lower molecular weight of the mutated protein. Sharper NMR signals are also obtained for the reduced diamagnetic enzyme. Results are consistent with an active site structure similar to that observed for the dimeric analog Thr137Ile characterized elsewhere. An observed proportional decrease in enzymatic activity and affinity for the N3-anion suggests the importance of electrostatic forces during substrate docking and catalysis. 相似文献
15.
Sergio Tamburini Pietro A Vigato Silvio Aime Mauro Andrea Cremonini 《Inorganica chimica acta》2004,357(5):1374-1380
Quantitative 2H NMR analysis at the natural abundance represents a well-recognized and efficient method for the identification of the origin of ethanol from different sources. An intrinsic limitation of the protocol used is the long time required, about 8 h, because of the long T1 values of the 2H resonances. In this work we propose the use a paramagnetic relaxation agent that significantly catalyzes the relaxation times and reduces the total time of the analysis. This agent is the macrocyclic Schiff base complex [Gd(H2L)(H2O)3(EtOH)](Cl)3 · 2 EtOH (H2L is the [1 + 1] macrocycle derived from the condensation of 3,3′-(3-oxapentane-1,5-diyldioxy)bis(2-hydroxybenzaldehyde) with 1,5-diamino-3-azamethylpentane), which is highly stable and soluble in the alcoholic solution used. Elemental analysis, IR and mass spectrometry have characterized this complex. The homogeneity of the complex and the correct Gd:Cl=1:3 ratio was established by SEM-EDS measurements. Further characterization of the paramagnetic complex has been achieved by measuring the magnetic field dependence of the 1H longitudinal nuclear magnetic relaxation time of a 1 mM solution in CH3OD with a field-cycling relaxometer. The GdIII ion accommodates up to four methanol molecules in its inner coordination sphere, whose rapid exchange with the bulk provides an efficient relaxation mechanism. The addition of about 37 mg of the complex to a solution of ethanol (3.0 g) and tetramethylurea (TMU) (1.5 g) results in the reduction of the experimental time of more than 50% with a S/N ratio compatible with that required for this application. 相似文献
16.
Summary Two-dimensional sequence-specific1H NMR resonance assignment methodology (Wüthrich, 1986) has been applied for the first time to a 18-kDa paramagnetic hemoprotein (cyano-metAplysia Mb) to identify all the hyperfine-shifted residues. The assignment was greatly facilitated by the fact that hyperfine shifts of residues impart a strong temperature dependence to the cross peaks, which aids location and identification, and provides improved spectral dispersion, particularly in the fingerprint region. 2D COSY and TOCSY were found to be surprisingly effective in locating the complete spin connectivities of all of the hyperfine-shifted residues, with the exception of the axially coordinated His95 imidazole ring, whose proton resonances were found to exhibit severe line broadening (> 400 Hz). Conventional 1D NOE and NOESY with short mixing times, combined with paramagnetic-induced relaxation effects, led to the successful assignment of even extremely broad proton signals. Three helical stretches and two loop regions were identified as the source of all hyperfine-shifted residues: the F helical residues 3–9, the E-helix residues 6–14, the G-helix residues 5–9, the FG-loop residues 1–4 and the CD-loop residues 1–4. These segments comprise all the residues that make contact with the heme and modulate the reactivity of the prosthetic group. The sequence-specific identifications of the active-site residues revealed that the solution structure ofAplysia metMbCN is fully consistent with that observed by X-ray diffraction in single crystals for a variety of other derivatives, except for the distal Arg66 (E10), which is turned into the heme pocket, as found only in the metMbF crystal structure (Bolognesi et al., 1990). The ready identification, by their temperature sensitivity, and the complete assignments of all hyperfine-shifted residues ofAplysia metMbCN demonstrate that sequence-specific assignment can be profitably applied to paramagnetic proteins, and that it should be possible to determine the solution structures of paramagnetic proteins, at least for low-spin complexes, by using NMR techniques used for diamagnetic proteins. 相似文献
17.
P. Viglino E. F. Orsega E. Argese R. Stevanato A. Rigo 《European biophysics journal : EBJ》1987,15(4):225-230
The dependence of the magnetic relaxation rates of1H and19F on temperature, frequency, pH and N
3
-
concentration, were measured in solutions of Manganese-containing superoxide dismutase ofBacillus stearothermophilus, and were compared to activity measurements, in order to obtain some information on the structure and dynamics at Mn(III) present in the active site of the enzyme. The experimental data lead us to hypothesize the presence of two binding sites in the coordination sphere of the enzyme bound Mn(III), which are accessible to water and anions and have different chemical and spectroscopic properties. NMR measurements carried out in the presence of competitive inhibitors and the pH dependence of both NMR relaxation rates suggest that F-, N
3
-
and OH- ions bind to one site, while a water molecule binds to the other one. The stability constant values of the complexes between these anions and the enzyme are reported. The influence of the anions on activity and the pH dependence of NMR parameters are discussed.Abbreviations MnSOD
Manganese containing superoxide dismutase 相似文献
18.
The two histidines of the insulin monomer play a vital role in the organization of insulin into insulin hexamers. The B10 histidines bind to zinc to form two-zinc insulin hexamer, and both the B5 and B10 histidines are implicated in the formation of four-zinc insulin hexamer. These two histidines are both accessible to solvent in the dimeric form of insulin, the predominant species present at pH 2–3. In the present work we report the first 500-MHz1H NMR studies of insulin. At this frequency all four proton resonances from the two histidines of each equivalent monomer are resolved. The resonances are assigned to the C(2)- and C(4)-imidazole protons of B5 His and B10 His employing Carr-Purcell pulse sequences to detect singlets and to observe approximateT 2 relaxation times. Zinc-free bovine insulin at pH 2.9 was examined at temperatures up to 60°C in acetate buffer and in urea of varying concentrations. The environments of B5 His in molecule I and molecule II of the dimer must be the same, with the same being true for B10 His, since a total of only four sharp resonances are seen. Our assignments for the two C(2) protons are consistent with those determined from recent studies of human (B5 Ala) insulin. 相似文献
19.
1H NMR relaxometry is applied for the investigation of pore size distributions in geological substrates. The transfer to humous
soil samples requires the knowledge of the interplay between soil organic matter, microorganisms and proton relaxation. The
goal of this contribution is to give first insights in microbial effects in the 1H NMR relaxation time distribution in the course of hydration of humous soil samples. We observed the development of the transverse
relaxation time distribution of the water protons after addition of water to air dried soil samples. Selected samples were
treated with cellobiose to enhance microbial activity. Besides the relaxation time distribution, the respiratory activity
and the total cell counts were determined as function of hydration time.
Microbial respiratory activities were 2–15 times higher in the treated samples and total cell counts increased in all samples
from 1×109 to 5×109 cells g−1 during hydration. The results of 1H NMR relaxometry showed tri-, bi- and mono-modal relaxation time distributions and shifts of peak relaxation times towards
lower relaxation times of all investigated soil samples during hydration. Furthermore, we found lower relaxation times and
merging of peaks in soil samples with higher microbial activity. Dissolution and hydration of cellobiose had no detectable
effect on the relaxation time distributions during hydration. We attribute the observed shifts in relaxation time distributions
to changes in pore size distribution and changes in spin relaxation mechanisms due to dissolution of organic and inorganic
substances (e.g. Fe3+, Mn2+), swelling of soil organic matter (SOM), production and release of extracellular polymeric substances (EPS) and bacterial
association within biofilms. 相似文献
20.
NMR spin relaxation in the rotating frame (R1ρ) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond time scale. Here, we use amide 1H off-resonance R1ρ relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using 15N or 13C relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The 1H off-resonance R1ρ data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 μs. This is in contrast to the case for 15N, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly 15N-enriched samples that were either highly enriched in 2H or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the 1H transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present 1H off-resonance R1ρ experiments are in excellent agreement with those determined previously using a combination of 15N laboratory-frame and off-resonance R1ρ relaxation data, with average values of
and 21 ± 3 μs, respectively. 相似文献