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Three Markov models (Dayhoff, Proportional and Poisson models; Hasegawa et al., 1992a) for amino acid substitution during evolution were used for maximum likelihood analyses of proteins coded for in mitochondrial DNA in estimating a phylogenetic tree among human, bovine and murids (mouse and rat) with chicken as an outgroup. It turned out that Dayhoff model is the most appropriate model among the alternatives in approximating the amino acid substitutions of proteins coded for in mitochondrial DNA. In spite of the presence of the complete sequence data of mitochondrial genomes, we could not resolve the trichotomy among human, bovine and murids, probably because the time length separating two branching events among these three lines was short and because chicken is too distant from mammals to be used as an outgroup. It was suggested that the average substitution rate of amino acids coded for in mitochondrial DNA is lower along the bovine line than those along the human or murid lines. Advantages of amino acid sequence analysis over nucleotide sequence analysis in phylogenetic study were discussed. 相似文献
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The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19-26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD) and an equivalent phage optimized peptide (12/1) were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design. 相似文献
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The amino acid sequence of the C-terminal domain of the elongation factor G (EF-G) has been studied. The polypeptide chain of the domain consists of 228 amino acid residues, and contains no tryptophan or cysteine residues. To determine its structure, the peptides obtained as a result of the fragment digestion by staphylococcal glutamic protease, cyanogen bromide cleavage, and tryptic hydrolysis of the fragment modified by maleic anhydride have been analyzed, as well as peptides obtained after hydrolyses of cyanogen bromide fragments with chymotrypsin, thermolysin and trypsin. 相似文献
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All the desmosine-containing elastolytic peptides of bovine ligamentum-nuchae elastin have now been examined for amino acid sequences C-terminal to the cross-links. In addition, amino acid residues C-terminal to lysine residues in bovine tropoelastin were also examined. No tyrosine C-terminal to cross-links in bovine elastin or C-terminal to lysine in tropoelastin was detected. Apparently all the tyrosine residues C-terminal to lysine residues in pig tropoelastin are replaced with phenylalanine in bovine tropoelastin. All the data presented are consistent with the scheme proposed for the formation of desmosine and isodesmosine cross-links of elastin by Gerber & Anwar [(1975) Biochem. J. 149, 685--695]. 相似文献
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A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results. 相似文献
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Component PP3 is a phosphoglycoprotein isolated from bovine milk with unknown biological function, which displays in its C-terminal region a basic amphipathic alpha-helix, a feature often involved in membrane association. According to that, the behaviour of PP3 and of a synthetic peptide from the C-terminal domain (residues 113-135) was investigated in lipid environment. Conductance measurements indicated that the peptide was able to associate and form channels in planar lipid bilayers composed of neutral or charged phospholipids. Electrostatic interactions seemed to promote voltage-dependent channel formation but this was not absolutely required since the pore-forming ability of the 113-135 C-terminal peptide was also detected with the zwitterionic lipid bilayer. Additionally, a spectroscopic study using circular dichroism argues that the peptide adopts an alpha-helical conformation in interaction with neutral or charged micelles. Thus, the conducting aggregates in bilayers might be composed of a bundle of peptides in helical conformation. Besides, similar conductance measurements performed with the whole PP3 protein did not induce any channel fluctuations. However, with the latter, an early breakdown of the bilayers occurred, a finding that can be tentatively explained by a massive incorporation of PP3. In the light of the present results, it could be inferred that PP3 membrane attachment may be achieved by oligomerization of the C-terminal amphipathic helical region. 相似文献
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Monica Y. Burgos Zepeda Kevin Alessandri Dorothée Murat Chahrazade El Amri Elie Dassa 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(4):755-761
The Uup protein belongs to a subfamily of soluble ATP-binding cassette (ABC) ATPases that have been implicated in several processes different from transmembrane transport of molecules, such as transposon precise excision. We have demonstrated previously that Escherichia coli Uup is able to bind DNA. DNA binding capacity is lowered in a truncated Uup protein lacking its C-terminal domain (CTD), suggesting a contribution of CTD to DNA binding. In the present study, we characterize the role of CTD in the function of Uup, on its overall stability and in DNA binding. To this end, we expressed and purified isolated CTD and we investigated the structural and functional role of this domain. The results underline that CTD is essential for the function of Uup, is stable and able to fold up autonomously. We compared the DNA binding activities of three versions of the protein (Uup, UupΔCTD and CTD) by an electrophoretic mobility shift assay. CTD is able to bind DNA although less efficiently than intact Uup and UupΔCTD. These observations suggest that CTD is an essential domain that contributes directly to the DNA binding ability of Uup. 相似文献
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p53 binds single-stranded DNA ends through the C-terminal domain and internal DNA segments via the middle domain. 总被引:14,自引:4,他引:10
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G Bakalkin G Selivanova T Yakovleva E Kiseleva E Kashuba K P Magnusson L Szekely G Klein L Terenius K G Wiman 《Nucleic acids research》1995,23(3):362-369
We have previously reported that wild-type p53 can bind single-stranded (ss) DNA ends and catalyze renaturation of ss complementary DNA molecules. Here we demonstrate that p53 can also bind to internal segments of ss DNA molecules via a binding site (internal DNA site) distinct from the binding site for DNA ends (DNA end site). Using p53 deletion mutants, the internal DNA site was mapped to the central region (residues 99-307), while the DNA end site was mapped to the C-terminal domain (residues 320-393) of the p53 protein. The internal DNA site can be activated by the binding of ss DNA ends to the DNA end site. The C-terminal domain alone was sufficient to catalyze DNA renaturation, although the central domain was also involved in promotion of renaturation by the full-length protein. Our results suggest that the interaction of the C-terminal tail of p53 with ss DNA ends generated by DNA damage in vivo may lead to activation of non-specific ss DNA binding by the central domain of p53. 相似文献
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To obtain site-specific information about individual EF-hand motifs, the EF-hand Ca(2+)-binding loops from site III and site IV of calmodulin (CaM) were inserted separately into a non-Ca(2+)-binding cell adhesion protein, domain 1 of CD2 (denoted as CaM-CD2-III-5G-52 and CaM-CD2-IV-5G-52). Structural analyses using various spectroscopic methods have shown that the host protein CD2 retains its native structure after the insertion of the 12-residue loops. The Tb(3+) fluorescence enhancement upon formation of a Tb(3+)-protein complex and the direct competition by La(3+) and Ca(2+) suggest that native Ca(2+)-binding pockets are formed in both engineered proteins. Moreover, as revealed by NMR, both Ca(2+) and La(3+) specifically interact with the residues at the grafted EF-loop. The CaM-CD2-III-5G-52 has stronger affinities to Ca(2+), Tb(3+) and La(3+) than CaM-CD2-IV-5G-52, indicating differential intrinsic metal-binding affinities of the EF-loops. 相似文献
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Sato K Nakamura T Mizuguchi M Miura K Tada M Aizawa T Gomi T Miyamoto K Kawano K 《FEBS letters》2003,553(3):232-238
Epiregulin (EPR), a novel member of epidermal growth factor (EGF) family, is a ligand for ErbB-1 and ErbB-4 receptors. The binding affinity of EPR for the receptors is lower than those of other EGF-family ligands. The solution structure of EPR was determined using two-dimensional nuclear magnetic resonance spectroscopy. The secondary structure in the C-terminal domain of EPR is different from other EGF-family ligands because of the lack of hydrogen bonds. The structural difference in the C-terminal domain may provide an explanation for the reduced binding affinity of EPR to the ErbB receptors. 相似文献
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Pasta SY Raman B Ramakrishna T Rao ChM 《The Journal of biological chemistry》2002,277(48):45821-45828
Several small heat shock proteins contain a well conserved alpha-crystallin domain, flanked by an N-terminal domain and a C-terminal extension, both of which vary in length and sequence. The structural and functional role of the C-terminal extension of small heat shock proteins, particularly of alphaA- and alphaB-crystallins, is not well understood. We have swapped the C-terminal extensions between alphaA- and alphaB-crystallins and generated two novel chimeric proteins, alphaABc and alphaBAc. We have investigated the domain-swapped chimeras for structural and functional alterations. We have used thermal and non-thermal models of protein aggregation and found that the chimeric alphaB with the C-terminal extension of alphaA-crystallin, alphaBAc, exhibits dramatically enhanced chaperone-like activity. Interestingly, however, the chimeric alphaA with the C-terminal extension of alphaB-crystallin, alphaABc, has almost lost its activity. Pyrene solubilization and bis-1-anilino-8-naphthalenesulfonate binding studies show that alphaBAc exhibits more solvent-exposed hydrophobic pockets than alphaA, alphaB, or alphaABc. Significant tertiary structural changes are revealed by tryptophan fluorescence and near-UV CD studies upon swapping the C-terminal extensions. The far-UV CD spectrum of alphaBAc differs from that of alphaB-crystallin whereas that of alphaABc overlaps with that of alphaA-crystallin. Gel filtration chromatography shows alteration in the size of the proteins upon swapping the C-terminal extensions. Our study demonstrates that the unstructured C-terminal extensions play a crucial role in the structure and chaperone activity, in addition to generally believed electrostatic "solubilizer" function. 相似文献
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An amino acid substitution in biobreeding rat corticosteroid binding globulin results in reduced steroid binding affinity 总被引:1,自引:0,他引:1
BioBreeding (BB) rats are derived from an outbred colony of Wistar rats and are used as a model of autoimmune diabetes mellitus. A corticosteroid binding globulin (CBG) variant with reduced affinity for glucocorticoids has now been found in the blood of these animals. The dissociation rate constants of BB CBG for cortisol (4.42 nM) and corticosterone (1.43 nM) are both about 50% higher than those associated with Wistar CBG, but no obvious difference in the steroid binding specificity of BB and Wistar CBGs was detected. Purified BB and Wistar CBGs exhibit the same size heterogeneity when examined by polyacrylamide gel electrophoresis under denaturing conditions, and the sizes of their respective hepatic mRNAs are identical. The genetic basis for this abnormality was therefore determined by comparing the cDNA sequences for BB and Wistar CBG, and this revealed a point mutation that results in a single amino acid substitution at residue 276 (Ile in BB CBG and Met in Wistar CBG). To confirm that this mutation is responsible for the reduced steroid binding affinity associated with BB CBG, the cDNAs for rat CBG-Ile276 and CBG-Met276 were expressed in Chinese hamster ovary cells. The steroid binding affinities of the CBGs secreted by these cells were essentially identical with those observed in the corresponding serum samples from these two rat strains. The amino acid substitution identified in BB rat CBG therefore clearly accounts for the reduction in its steroid binding affinity, and further analysis of this and other natural CBG variants may reveal important information about the CBG steroid binding site. It is also possible that this mutation may contribute to the etiology of pathological abnormalities that are characteristic of the BB rat. 相似文献
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The complete amino acid sequence of a non-hemorrhagic fibrino(geno)lytic enzyme (VlF) isolated from Vipera lebetina venom has been determined. VlF was subjected to separate enzymatic and chemical digestions. Resulting fragments were purified by RP-HPLC and subjected for sequencing by automated Edman degradation. The amino terminus of VlF was determined by mass spectrometry. VlF was shown to be composed of 202 residues having a relative molecular mass of 22,826 Da and containing a zinc-binding site and a catalytically active residue. It displayed significant sequence similarities with many other mature metalloproteinases reported from snake venoms. Sequence comparison of hemorrhagic and non-hemorrhagic mature metalloproteinases revealed the presence at the C-terminal part of the enzymes of two residues common to only hemorrhagic metalloproteinases and two others shared by only non-hemorrhagic ones. 相似文献
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Rosamaria Avarello Antonio Pedicini Angela Caiulo Orsetta Zuffardi Marco Fraccaro 《Human genetics》1992,89(2):247-249
Summary In situ hybridization, under low stringency conditions with two alphoid DNA probes (pY1 and p82H) labeled with digoxigenin-dUTP, decorated all the centromeres of the human karyotype. However, signals were also detected on the long arm of chromosome 2 at approximately q21.3–q22.1. Since it is supposed that human chromosome 2 originated by the telomeric fusion of two ancestral primate chromosomes, these findings indicate that not only the telomeric sequences, but also the ancestral centromere (or at least its alphoid sequences), have been conserved. 相似文献