共查询到8条相似文献,搜索用时 0 毫秒
1.
Ch. Geßner M. Stein S. P. J. Albracht W. Lubitz 《Journal of biological inorganic chemistry》1999,4(4):379-389
Electron nuclear double resonance (ENDOR) was applied to study the active site of the oxidized "ready" state, Nir, in the [NiFe] hydrogenase of Chromatium vinosum. The magnetic field dependence of the EPR was used to select specific subsets of molecules contributing to the ENDOR response
by stepping through the EPR envelope. Three hyperfine couplings could be clearly followed over the complete field range. Two
protons, H1 and H2, display a very similar large isotropic coupling of 12.5 and 12.6 MHz, respectively. Their dipolar coupling
is small (2.1 and 1.4 MHz, respectively). A third proton, H3, exhibits a small isotropic coupling of 0.5 MHz and a larger
anisotropic contribution of 3.5 MHz. Based on a comparison with structural data obtained from X-ray crystallography of single
crystals of hydrogenases from Desulfovibrio gigas and D. vulgaris and the known g-tensor orientation of Nir, an assignment of the 1H hyperfine couplings could be achieved. H1 and H2 were assigned to the β-CH2 protons of the bridging cysteine Cys533 and H3 could belong to a β-CH2 proton of Cys68 or to a protonated cysteine (-SH) of Cys68 or Cys530.
Received: 26 November 1998 / Accepted: 1 April 1999 相似文献
2.
Huan-Xiang Zhou 《Journal of biological inorganic chemistry》1997,2(1):109-113
Factors that contribute to the control of reduction potential by protein matrix are examined within a spherical protein model.
These include the nonpolar nature of protein matrices, solvent accessibility of the redox center, and net charges and dipoles
of surrounding amino acids. Simple rules on their effects are established. In particular, surface charges have little effect
on the reduction potential, and polar groups may either increase or decrease the reduction potential, depending on their orientations
relative to the redox center. The effects of complex formation, proton titration, and ionic strength are also discussed.
Received, accepted: 26 November 1996 相似文献
3.
The reduction potentials of blue copper sites vary between 180 and about 1000 mV. It has been suggested that the reason for
this variation is that the proteins constrain the distance between the copper ion and its axial ligands to different values.
We have tested this suggestion by performing density functional B3LYP calculations on realistic models of the blue copper
proteins, including solvent effects by the polarizable continuum method. Constraining the Cu-SMet bond length to values between 245 and 310 pm (the range encountered in crystal structures) change the reduction potential
by less than 70 mV. Similarly, we have studied five typical blue copper proteins spanning the whole range of reduction potentials:
stellacyanin, plastocyanin, azurin, rusticyanin, and ceruloplasmin. These studies included the methionine (or glutamine) ligand
as well as the back-bone carbonyl oxygen group that is a ligand in azurin and is found at larger distances in the other proteins.
The active-site models of these proteins show a variation in the reduction potential of about 140 mV, i.e., only a minor part
of the range observed experimentally (800 mV). Consequently, we can conclude that the axial ligands have a small influence
on the reduction potentials of the blue copper proteins. Instead, the large variation in the reduction potentials seems to
arise mainly from variations in the solvent accessibility of the copper site and in the orientation of protein dipoles around
the copper site.
Received: 7 April 1999 / Accepted: 26 July 1999 相似文献
4.
Elaborations to an earlier design of an electron paramagnetic resonance (EPR) spectroelectrochemical titrator are described. While maintaining the anaerobic capabilities of the original design, a number of modifications and revisions have been introduced. The most significant modification is the use of a detachable spectral cell, making the apparatus modular and adaptable for multiple forms of spectroscopy. Additional modifications include removable reference, auxiliary, and working electrodes; modifications to facilitate sample transfer; and adaptations for operation within an anaerobic chamber. This apparatus has been used successfully in the coulometric titration of a [4Fe-4S] enzyme, as measured by EPR spectroscopy. The midpoint reduction potential for the 2+/1+ couple in the [4Fe-4S] cluster of lysine 2,3-aminomutase is -479+/-5mV, a value that falls within the range typical of ferredoxin-like iron-sulfur clusters. 相似文献
5.
Jonathan P. Hannan Sharon L. Davy Geoffrey R. Moore Robert R. Eady C. R. Andrew 《Journal of biological inorganic chemistry》1998,3(3):282-291
Assignment of the resonance Raman (RR) spectrum of Ni(II)-substituted azurin II from Alcaligenes xylosoxidans (NCIMB 11015) using Ni isotope substitution reveals an anomalously low Ni-S(Cys) stretching frequency of 349?cm–1, suggesting the presence of significant axial-ligand bonding interactions. The X-ray crystal structure of Ni(II)-substituted azurin from Pseudomonas aeruginosa shows that there are two potential axial ligands to the Ni ion: a peptide carbonyl O at a distance of 2.46?Å, together with a long-range interaction from a methionine sulfur (S′) at a distance of 3.30?Å. Comparison of the RR properties of Ni(II)-substituted azurin II with stellacyanin (which contains an axial carbonyl ligand, but no methionine) suggests that the interaction from the carbonyl oxygen ligand alone is not sufficient to account for the weak Ni azurin metal-thiolate bond. Instead, it appears that a Ni-methionine bonding interaction is also required to explain the low Ni-S(Cys) stretching frequency in Ni(II)-substituted azurin II. This hypothesis is supported by NMR studies which show a large paramagnetic shift for the protons of the methionine side-chain. Thus, it appears that Ni-substituted azurin II is best described as five-coordinate, and that significant Ni(II)-methionine bonding interactions can occur at a distance of 3.3?Å. 相似文献
6.
During oogenesis in Drosophila, several mRNAs and proteins are localized to discrete regions of the developing oocyte, resulting in a mature oocyte with
a well-defined anterior–posterior axis. The product of the swallow (sww) gene is required for the localization of two different mRNAs during oogenesis, bicoid (bcd) and Adducin-like/hu-li tai shao (hts). We initiated a detailed characterization of the phenotypes associated with each of eight sww alleles as a means of investigating the role of sww in oogenic patterning. RNA localization defects in various sww mutants were examined by radioactive in situ hybridization to paraffin sections. Using this technique, several previously
unreported RNA localization defects have been observed. Although bcd RNA localization is often lost completely in sww oocytes, in a high proportion of cases, bcd RNA is localized inappropriately along the periphery of the mature oocyte. In several sww mutants, a portion of the bcd mRNA population becomes concentrated at the posterior pole of the oocyte during late oogenesis. Several sww mutations also result inoskar RNA localization defects, consistent with a global role for sww in cytoskeletal regulation or organization. A detailed temporal and spatial analysis of hts RNA localization in sww mutants and in drug-treated ovaries reveals many similarities to bcd RNA localization, and implies the two independent localization events are accomplished by the same mechanism.
Received: 10 January 2000 / Accepted: 9 March 2000 相似文献
7.
Christoph G.O. Baerwald C. C. Mok Mark S. Fife Mohammad Tikly Chak Sing Lau B. Paul Wordsworth Bill Ollier G.S. Panayi J.S. Lanchbury 《Immunogenetics》1999,49(10):894-899
The regulatory region of the corticotropin-releasing hormone (CRH) is highly conserved across species and plays a crucial
role in the response of the organism to stress. Release of CRH initiates a cascade of events leading to the release of cortisol
and the regulation of inflammatory and immune events. In this report we describe polymorphisms in the 5′ regulatory region
of the CRH gene in humans. We studied the distribution of CRH alleles in three different African populations, in white UK Caucasoids, and in a Chinese population. In the African and UK
populations we found three new polymorphisms which cosegregated, resulting in two alleles, A1 and A2. Gene frequencies for A1 and A2 were extremely divergent between the African and the UK populations. The African A1 frequency ranged from 0.27–0.3, while the UK Caucasoid frequency was 0.9. Compound alleles could be assigned by taking into
account the previously described biallelic polymorphism at position 225 in the CRH promoter. The A2B1 compound allele is the commonest in contemporary African human populations (allele frequency range 0.44–0.61) and was the
only allele observed in a population of chimpanzees from Sierra Leone. Wright's FST for the A2B1 allele over the four sampled populations was 0.612, a value exceeded in human populations only by loci which have apparently
been subject to natural selection. Taken together, these findings support A2B1 as the ancestral allele and suggest that the CRH genomic region may have been subject to strong disruptive selection throughout human evolution.
Received: 29 October 1998 / Revised: 24 March 1999 相似文献
8.
S. Van Campenhout R. M. D. Koebner G. Volckaert 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(2):328-336
Two wheat consensus primer sets, directed to ”early-methionine-labelled” (Em) gene sequences, were tested for their ability
to amplify beyond their original source. A range of widely diverse templates, including other Triticeae species and sample
monocot and dicot species, was assayed. Primer set EMC5/EMC3, amplifying the entire coding region with its intron and part
of the 3’ untranslated region, targets Triticeae and sorghum Em sequences. The other set, EMC5/EMCO31, directed to the coding
region and its intron, amplifies templates from all the grass species. Both primer sets fail to amplify Em sequences from
more distant monocots and the dicots. Using set EMC5/EMC3, we isolated and sequenced ten members of the rye Em gene family
from five different rye sources. Significant DNA sequence variation between wheat and rye sequences in the non-coding regions
was found, and this was used to develop seven sequence-specific primers. Twelve primer combinations were analysed, 7 of which
were Em-R1-specific, amplifying a product in at least one of the tested rye or rye-carrying genotypes but not in wheat. Four sets exhibited
clear amplification length polymorphisms which allowed discrimination between and within the rye sources. The primers also
discriminated between wheat-rye recombinants with proximal 1RL rye chromatin and those carrying distal 1RL rye chromatin.
These results show that wheat consensus primer sets can be used to isolate orthologous sequences, especially from species
that are used for alien gene transfer in wheat. Subsequently, species-specific assays can be designed that are useful tools
for this application.
Received: 7 December 1998 / Accepted: 21 July 1999 相似文献