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排序方式: 共有114条查询结果,搜索用时 31 毫秒
41.
Matthew Biancalana Koki Makabe Shude Yan Shohei Koide 《Protein science : a publication of the Protein Society》2015,24(5):841-849
Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model β-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain “clusters” at highly solvent-exposed positions in the flat, single-layer β-sheet of Borrelia outer surface protein A (OspA). This unusual β-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the β-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of β-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA β-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating β-sheet conformation in protein design. 相似文献
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Manato Suda Miyuki Katoh Kazuya Toda Kiriko Matsumoto Koichiro Kawaguchi Sei-ichi Kawahara Yasunao Hattori Hiroshi Fujii Hidefumi Makabe 《Bioorganic & medicinal chemistry letters》2013,23(17):4935-4939
Synthesis of procyanidin B2 and B3 gallate derivatives, 3-O-gallate, 3″-O-gallate, and 3,3″-di-O-gallate, were synthesized using equimolar condensation mediated by Yb(OTf)3. Synthesized compounds showed significant antitumor effects against human prostate PC-3 cell lines. Their activities were weaker than well-known EGCG and prodelphinidin B3. 相似文献
45.
Makabe K Nakanishi T Tsumoto K Tanaka Y Kondo H Umetsu M Sone Y Asano R Kumagai I 《The Journal of biological chemistry》2008,283(2):1156-1166
To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized. 相似文献
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The catalytic domains of murine Golgi alpha1,2-mannosidases IA and IB that
are involved in N-glycan processing were expressed as secreted proteins in
P.pastoris . Recombinant mannosidases IA and IB both required divalent
cations for activity, were inhibited by deoxymannojirimycin and
kifunensine, and exhibited similar catalytic constants using
Manalpha1,2Manalpha-O-CH3as substrate. Mannosidase IA was purified as a 50
kDa catalytically active soluble fragment and shown to be an inverting
glycosidase. Recombinant mannosidases IA and IB were used to cleave
Man9GlcNAc and the isomers produced were identified by high performance
liquid chromatography and proton-nuclear magnetic resonance spectroscopy.
Man9GlcNAc was rapidly cleaved by both enzymes to Man6GlcNAc, followed by a
much slower conversion to Man5GlcNAc. The same isomers of Man7GlcNAc and
Man6GlcNAc were produced by both enzymes but different isomers of
Man8GlcNAc were formed. When Man8GlcNAc (Man8B isomer) was used as
substrate, rapid conversion to Man5GlcNAc was observed, and the same
oligosaccharide isomer intermediates were formed by both enzymes. These
results combined with proton-nuclear magnetic resonance spectroscopy data
demonstrate that it is the terminal alpha1, 2-mannose residue missing in
the Man8B isomer that is cleaved from Man9GlcNAc at a much slower rate.
When rat liver endoplasmic reticulum membrane extracts were incubated with
Man9GlcNAc2, Man8GlcNAc2was the major product and Man8B was the major
isomer. In contrast, rat liver Golgi membranes rapidly cleaved
Man9GlcNAc2to Man6GlcNAc2and more slowly to Man5GlcNAc2. In this case all
three isomers of Man8GlcNAc2were formed as intermediates, but a distinctive
isomer, Man8A, was predominant. Antiserum to recombinant mannosidase IA
immunoprecipitated an enzyme from Golgi extracts with the same specificity
as recombinant mannosidase IA. These immunodepleted membranes were enriched
in a Man9GlcNAc2to Man8GlcNAc2- cleaving activity forming predominantly the
Man8B isomer. These results suggest that mannosidases IA and IB in Golgi
membranes prefer the Man8B isomer generated by a complementary mannosidase
that removes a single mannose from Man9GlcNAc2.
相似文献
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N Shindo-Okada H Nagahara Z Yamaizumi O Makabe S Nishimura 《Nucleic acids symposium series》1988,(19):129-130
50.
Variations in nitrogen-15 natural abundance of plant and soil systems in four remote tropical rainforests, southern China 总被引:1,自引:0,他引:1
Ang Wang Yun-Ting Fang De-Xiang Chen Keisuke Koba Akiko Makabe Yi-De Li Tu-Shou Luo Muneoki Yoh 《Oecologia》2014,174(2):567-580
The foliar stable N isotope ratio (δ15N) can provide integrated information on ecosystem N cycling. Here we present the δ15N of plant and soil in four remote typical tropical rainforests (one primary and three secondary) of southern China. We aimed to examine if (1) foliar δ15N in the study forests is negative, as observed in other tropical and subtropical sites in eastern Asia; (2) variation in δ15N among different species is smaller compared to that in many N-limited temperate and boreal ecosystems; and (3) the primary forest is more N rich than the younger secondary forests and therefore is more 15N enriched. Our results show that foliar δ15N ranged from ?5.1 to 1.3 ‰ for 39 collected plant species with different growth strategies and mycorrhizal types, and that for 35 species it was negative. Soil NO3 ? had low δ15N (?11.4 to ?3.2 ‰) and plant NO3 ? uptake could not explain the negative foliar δ15N values (NH4 + was dominant in the soil inorganic-N fraction). We suggest that negative values might be caused by isotope fractionation during soil NH4 + uptake and mycorrhizal N transfer, and by direct uptake of atmospheric NH3/NH4 +. The variation in foliar δ15N among species (by about 6 ‰) was smaller than in many N-limited ecosystems, which is typically about or over 10 ‰. The primary forest had a larger N capital in plants than the secondary forests. Foliar δ15N and the enrichment factor (foliar δ15N minus soil δ15N) were higher in the primary forest than in the secondary forests, albeit differences were small, while there was no consistent pattern in soil δ15N between primary and secondary forests. 相似文献