排序方式: 共有9条查询结果,搜索用时 15 毫秒
1
1.
Wienk HL Martínez MM Yalloway GN Schmidt JM Pérez C Rüterjans H Löhr F 《Journal of biomolecular NMR》2003,25(2):133-145
A quantitative J-correlation pulse sequence is described that allows simultaneous determination of one-bond and two-bond nitrogen-carbon coupling constants for protonated or deuterated proteins. Coupling constants are calculated from volume ratios between cross peaks and reference axial peaks observed in a single 3D spectrum. Accurate backbone 1
J
NC, 1
J
NC, and 2
J
NC coupling constants are obtained for the two [15N;13C]-labeled, medium-sized proteins flavodoxin and xylanase and for the [2H;15N;13C]-labeled, large protein DFPase. A dependence of one-bond and two-bond J
NC values on protein backbone torsion angles is readily apparent, in agreement with previously found correlations. In addition, the experiment is performed on isotropic as well as aligned protein to measure associated 15N-13C residual dipolar couplings. 相似文献
2.
3.
Perdeuteration in neutron crystallography is an effective method for determining the positions of hydrogen atoms in proteins. However, there is shortage of evidence that the high-resolution details of perdeuterated proteins are consistent with those of the nondeuterated proteins. In this study, we determined the X-ray structure of perdeuterated high-potential iron-sulfur protein (HiPIP) at a high resolution of 0.85 å resolution. The comparison of the nondeuterated and perdeuterated structures of HiPIP revealed slight differences between the two structures. The spectroscopic and spectroelectrochemical studies also showed that perdeuterated HiPIP has approximately the same characteristics as nondeuterated HiPIP. These results further emphasize the suitability of using perdeuterated proteins in the high-resolution neutron crystallography. 相似文献
4.
Neutron structure of the T26H mutant of T4 phage lysozyme provides insight into the catalytic activity of the mutant enzyme and how it differs from that of wild type
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Takeshi Hiromoto Flora Meilleur Rumi Shimizu Chie Shibazaki Motoyasu Adachi Taro Tamada Ryota Kuroki 《Protein science : a publication of the Protein Society》2017,26(10):1953-1963
T4 phage lysozyme is an inverting glycoside hydrolase that degrades the murein of bacterial cell walls by cleaving the β‐1,4‐glycosidic bond. The substitution of the catalytic Thr26 residue to a histidine converts the wild type from an inverting to a retaining enzyme, which implies that the original general acid Glu11 can also act as an acid/base catalyst in the hydrolysis. Here, we have determined the neutron structure of the perdeuterated T26H mutant to clarify the protonation states of Glu11 and the substituted His26, which are key in the retaining reaction. The 2.09‐Å resolution structure shows that the imidazole group of His26 is in its singly protonated form in the active site, suggesting that the deprotonated N?2 atom of His26 can attack the anomeric carbon of bound substrate as a nucleophile. The carboxyl group of Glu11 is partially protonated and interacts with the unusual neutral state of the guanidine moiety of Arg145, as well as two heavy water molecules. Considering that one of the water‐binding sites has the potential to be occupied by a hydronium ion, the bulk solvent could be the source for the protonation of Glu11. The respective protonation states of Glu11 and His26 are consistent with the bond lengths determined by an unrestrained refinement of the high‐resolution X‐ray structure of T26H at 1.04‐Å resolution. The detail structural information, including the coordinates of the deuterium atoms in the active site, provides insight into the distinctively different catalytic activities of the mutant and wild type enzymes. 相似文献
5.
《Structure (London, England : 1993)》2021,29(9):1003-1013.e4
- Download : Download high-res image (403KB)
- Download : Download full-size image
6.
Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins 总被引:1,自引:0,他引:1
Methods are described to correlate aromatic 1H 2/13C 2 or 1H 1/15N 1 with aliphatic 13C chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [15N, 1H]- and/or [13C, 1H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with -carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [15N, 1H]-TROSY-H(N)Car experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine -CH and tryptophan -NH groups. 相似文献
7.
Rajesh S Nietlispach D Nakayama H Takio K Laue ED Shibata T Ito Y 《Journal of biomolecular NMR》2003,27(1):81-86
A novel biosynthetic strategy is described for the preparation of deuterated proteins containing protons at the ring carbons of Phe, Tyr and Trp, using the aromatic amino acid precursor shikimic acid. Specific protonation at aromatic side chains, with complete deuteration at C/positions was achieved in proteins overexpressed in bacteria grown in shikimate-supplemented D2O medium. Co-expression of a shikimate transporter in prototrophic bacteria resulted in protonation levels of 62–79%, whereas complete labeling was accomplished using shikimate auxotrophic bacteria. Our labeling protocol permits the measurement of important aromatic side chain derived distance restraints in perdeuterated proteins that could be utilized to enhance the accuracy of NMR structures calculated using low densities of NOEs from methyl selectively protonated samples. 相似文献
8.
9.
1