共查询到20条相似文献,搜索用时 15 毫秒
1.
D.R. Muhandiram Philip E. Johnson Daiwen Yang Ouwen Zhang Lawrence P. McIntosh Lewis E. Kay 《Journal of biomolecular NMR》1997,10(3):283-288
A triple-resonance pulse scheme is described which records15N, NH correlations of residues that immediately follow amethyl-containing amino acid. The experiment makes use of a15N, 13C and fractionally deuterated proteinsample and selects for CH2D methyl types. The experiment isthus useful in the early stages of the sequential assignment process as wellas for the confirmation of backbone 15N, NH chemical shiftassignments at later stages of data analysis. A simple modification of thesequence also allows the measurement of methyl side-chain dynamics. This isparticularly useful for studying side-chain dynamic properties in partiallyunfolded and unfolded proteins where the resolution of aliphatic carbon andproton chemical shifts is limited compared to that of amide nitrogens. 相似文献
2.
David S. Wishart Colin G. Bigam Arne Holm Robert S. Hodges Brian D. Sykes 《Journal of biomolecular NMR》1995,5(1):67-81
Summary In this study we report on the 1H, 13C and 15N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide
Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the
possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because
all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide
a complete and internally consistent set of 1H, 13C and 15N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor
effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical
shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences
arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring
for both alanine and proline (i.e., the residues in the Y position) are found to depend strougly on the type of amino acid
substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in
determining peptide and protein chemical shifts. 相似文献
3.
Franklin J. Moy Andrew P. Seddon Ernest B. Campbell Peter Böhlen Robert Powers 《Journal of biomolecular NMR》1995,6(3):245-254
Summary The assignments of the 1H, 15N, 13CO and 13C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation >95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C, C and H to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C, C and H to the backbone amide 1H and 15N in the CBCANH and HNHA experiments. In addition, C and C chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the carbonyl correlations observed in the HNCO and HCACO experiments and C correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic 1H and 13C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H and H protons as well as 3JH
n
H coupling constants, amide exchange and 13C and 13C secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel -sheets (residues 30–34, 39–44, 48–53, 62–67, 71–76, 81–85, 91–94, 103–108, 113–118, 123–125 and 148–152) and a helix-like structure (residues 131–136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131–136 were defined as -strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128–138) instead of the -strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9–28. This is consistent with the X-ray structures of FGF-2, where the first 17–19 residues were ill defined. 相似文献
4.
《Nucleosides, nucleotides & nucleic acids》2013,32(5-8):1669-1672
Abstract It was found by 1H, 13C and 15N NMR study that substitution of 4,9-dihydro-4, 6-dimethyl-9-oxo-3-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl) imidazo [1,2-a]purine (wyosine triacetate, 1) at C2 position with electronegative groups CH3O and C6H5CH2O results in a noticeable electron distribution disturbance in the “left-hand” imidazole ring and a significant increase in the North conformer population of the sugar moiety. 相似文献
5.
Two triple resonance experiments, HNN and HN(C)N, are presented which correlate HN and 15N resonances sequentially along the polypeptide chain of a doubly (13C, 15N) labeled protein. These incorporate several improvements over the previously published sequences for a similar purpose and have several novel features. The spectral characteristics enable direct identification of certain triplets of residues, which provide many starting points for the sequential assignment procedure. The experiments are sensitive and their utility has been demonstrated with a 22 kDa protein under unfolding conditions where most of the standard triple resonance experiments such as HNCA, CBCANH etc. have limited success because of poor amide, C and C chemical shift dispersions. 相似文献
6.
7.
Summary Heteronuclear 2D (13C, 1H) and (15N, 1H) correlation spectra of (13C, 15N) fully enriched proteins can be acquired simultaneously with virtually no sensitivity loss or increase in artefact levels. Three pulse sequences are described, for 2D time-shared or TS-HSQC, 2D TS-HMQC and 2D TS-HSMQC spectra, respectively. Independent spectral widths can be sampled for both heteronuclei. The sequences can be greatly improved by combining them with field-gradient methods. By applying the sequences to 3D and 4D NMR spectroscopy, considerable time savings can be obtained. The method is demonstrated for the 18 kDa HU protein.Abbreviations HMQC
heteronuclear multiple-quantum coherence spectroscopy
- HSQC
heteronuclear single-quantum coherence spectroscopy
- HSMQC
heteronuclear single- and multiple-quantum coherence spectroscopy
- NOESY
nuclear Overhauser enhancement spectroscopy 相似文献
8.
9.
Jens Chr. Madsen Ole Winneche Sørensen Poul Sørensen Flemming M. Poulsen 《Journal of biomolecular NMR》1993,3(2):239-244
Summary NMR pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins are presented. These pulse sequences represent improvements over earlier experiments with respect to resolution and number of radiofrequency pulses. The experiments are useful for measuring JNH
, JNCO, JNC
, JH
N
CO and JH
N
H
. Applications to chymotrypsin inhibitor 2 (CI-2) are shown. 相似文献
10.
11.
Translocation of carbon and nitrogen within a single source-sink unit, comprising a trifoliated leaf, the axillary pod and the subtending internode, and from this unit to the rest of the plant was examined in soybean (Glycine max L. cv. Akishirome) plant by feeding 13CO2 and 15NO3. The plants were grown at two levels of nitrogen in the basal medium, i.e. low-N (2 g N m–2) and high-N (35 g N m–2) and a treatment of depodding was imposed by removing all the pods from the plant, except the pod of the source sink unit, 13 days after flowering. The plants at high-N accumulated more biomass in its organs compared to low-N and pod removal increased the weight of the vegetative organs. When the terminal leaflet of the source-sink unit was fed with 13CO2, almost all of the radioactive materials were retained inside the source-sink unit and translocation to rest of the plants was insignificant under any of the treatments imposed. Out of the13C exported by the terminal leaflet, less than half went into the axillary pod, as the lateral leaflets claimed equal share and very little material was deposited in the petiole. Pod removal decreased 13C export at high-N , but not at low-N. Similar to 13C, the source-sink unit retained all the 15N fed to the terminal leaflet at high-N. At low-N, the major part of 15N partitioning occurred in favour of the rest of the plant outside the source-sink unit, but removal of the competitve sinks from the rest of the plants nullified any partitioning outside the unit. Unlike the situation in 13C, no partitioning of 15N occurred in favour of the lateral leaflets from the terminal leaflet inside the unit. It is concluded that sink demand influences partitioning of both C and N and the translocation of carbon is different from that of nitrogen within a source-sink unit. The translocation of the N is more adjustive to a demand from other sink units compared to the C. 相似文献
12.
The linear analysis of chemical shifts (LACS) has provided a robust method for identifying and correcting 13C chemical shift referencing problems in data from protein NMR spectroscopy. Unlike other approaches, LACS does not require
prior knowledge of the three-dimensional structure or inference of the secondary structure of the protein. It also does not
require extensive assignment of the NMR data. We report here a way of extending the LACS approach to 15N NMR data from proteins, so as to enable the detection and correction of inconsistencies in chemical shift referencing for
this nucleus. The approach is based on our finding that the secondary 15N chemical shift of the backbone nitrogen atom of residue i is strongly correlated with the secondary chemical shift difference (experimental minus random coil) between the alpha and
beta carbons of residue i − 1. Thus once alpha and beta 13C chemical shifts are available (their difference is referencing error-free), the 15N referencing can be validated, and an appropriate offset correction can be derived. This approach can be implemented prior
to a structure determination and can be used to analyze potential referencing problems in database data not associated with
three-dimensional structure. Application of the LACS algorithm to the current BMRB protein chemical shift database, revealed
that nearly 35% of the BMRB entries have δ
15N values mis-referenced by over 0.7 ppm and over 25% of them have δ
1HN values mis-referenced by over 0.12 ppm. One implication of the findings reported here is that a backbone 15N chemical shift provides a better indicator of the conformation of the preceding residue than of the residue itself.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
13.
14.
Högberg Peter Johnnisson Christian Högberg Mona Högbom Lars Näsholm Torgny Hällgren Jan-Erik 《Plant and Soil》1995,168(1):125-133
Preliminary attempts to make retrospective studies of N balances and water stress in forest fertilization experiments by analyzing changes in the abundances of 15N and 13C, respectively, are discussed. Most evidence is from the Swedish Forest Optimum Nutrition Experiments, which have been running for two decades. Annual additions of N have been given either alone or in combination with other elements, notably P and K, every third year. Processes leading to loss of N, e.g. volatilization of ammonia, nitrification followed by leaching or denitrification, and denitrification alone, discriminate against the heavy isotope 15N. A correlation was found between fractional losses of added N and the change in 15N () during 19 years in current needles in a Scots pine forest, irrespective of source of N. Isotope effects were larger on urea than on ammonium nitrate plots (2 as compared to 9 15N ()) because of ammonia volatilization and higher rates of nitrification. They developed gradually over time, which opens possibilities to analyse the development of N saturation. However, the analysis may be confounded by shifts in 15N abundance of fertilizer N. In another trial, N isotope effects could be seen in both plants and soils 10 years after the last fertilization; they were smaller in soils because of a large pretreatment memory effect, but we expect them to persist there for decades.The enzyme RuBisCo discriminates strongly against the heavy isotope 13C during photosynthesis, but this effect becomes less expressed as stomata close because of water stress. The supply of N may also affect the 13C () via effects on rates of photosynthesis, and the source of N may have an influence directly via non-RubisCo carboxylations, and indirectly via effects on water use efficiency. In a trial with Norway spruce, the effect of N fertilization on the 13C () of current needles was strongly correlated with production and weakly so with foliar biomass a dry year, but not a wet year. This suggested that these variations are primarily related to induced differences in the balance between supply and demand for water. Hence, studies of {au13}C abundance can disentangle the role of water as such from its effects on mineralization of N and flow of N. 相似文献
15.
16.
A triple resonance NMR experiment is presented for the simultaneous recording of HNCA and HNCO data sets on 15N, natural abundance 13C samples. The experiment exploits the fact that transfers of magnetization from 15N to 13CO and from 15N to 13C (and back) proceed independently for samples that are not enriched in 13C. A factor of 2 in measuring time is gained by recording the two data sets simultaneously with no compromise in spectral quality. An application to a 0.5 mM 15N labeled sample of protein-L is presented with all expected correlations observed in spectra recorded with a cryogenic probe at 500 MHz. 相似文献
17.
18.
Recently, several TROSY-based experiments have been designed for backbone chemical shift assignment and measurement of the NOEs of 2H, 13C and 15N labeled proteins. Here, we present TROSY-enhanced NOESY experiments, namely the 2D S3E-NOESY-S3E, 3D TROSY-NOESY-S3E and S3E-NOESY-TROSY experiments. These experiments use the spin-state selective excitation method (S3E), and have the TROSY effect in all the indirectly and directly detected dimensions, and so provide optimal resolution for amide protons. The first two experiments provide an additional useful feature in that the diagonal peaks of the amide proton region are cancelled or greatly reduced, allowing clear identification of NOE cross peaks that are close to diagonal peaks. 相似文献
19.
Rasmus H. Fogh Dick Schipper Rolf Boelens Robert Kaptein 《Journal of biomolecular NMR》1995,5(3):259-270
Summary The 1H, 13C and 15N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CHn carbons, all amide (NH and NH2) nitrogens and 99.2% of the amide and CHn protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D
two-/three-/four-dimensional
- HSQC
heteronuclear single-quantum coherence
- HMQC
heteronuclear multiple-quantum coherence
- COSY
correlation spectroscopy
- TOCSY
total correlation spectroscopy
- NOE
nuclear Overhauser enhancement (connectivity)
- NOESY
2D NOE spectroscopy
Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667]. 相似文献
20.
Tyler RC Sreenath HK Singh S Aceti DJ Bingman CA Markley JL Fox BG 《Protein expression and purification》2005,40(2):268-278
Protocols have been developed and applied for the high-throughput production of [U-15N]- or [U-13C-, U-15N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B12, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of approximately 5, an average wet cell mass yield of approximately 9.5 g L(-1), and an average yield of approximately 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both 15N and 13C at levels greater than 95%. 1H-15N heteronuclear single quantum correlation spectroscopy as well as 13C; 15N-edited spectroscopy showed that the purified [U-15N]- and [U-13C, U-15N]-labeled proteins were suitable for structure analysis. 相似文献