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This study presents an approach to identifying surface residues on membrane proteins that are exposed toward the membrane-aqueous interface. The method employs a lipid Ni(II) chelate that localizes the metal ion to a region near the membrane-aqueous interface. Lateral diffusion of the lipid chelate results in Heisenberg exchange (HE) with nitroxide side chains in the protein only if direct contact occurs between the paramagnetic species during a collision. Thus, HE serves as a signature for residues facing the bilayer in the neighborhood of the membrane-aqueous interface. To evaluate the method, 13 surface residues on the extracellular half of KcsA, a prokaryotic potassium channel of known structure, were examined for HE with the Ni(II) chelate. The HE rate between the two species is found to depend strongly on the vertical position of the nitroxide with respect to the membrane-aqueous interface. Nitroxides introduced near the interface experience relatively high HE rates, whereas nitroxides that are immersed in the bilayer interior or sterically sheltered from collision experience low or undetectable rates. The results indicate that residues near the interface can be identified on the basis of their high rates of collision with the headgroup region of the bilayer. 相似文献
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Alicia P. Higueruelo Adrian Schreyer G. Richard J Bickerton Tom L. Blundell Will R. Pitt 《PloS one》2012,7(12)
Efforts to increase affinity in the design of new therapeutic molecules have tended to lead to greater lipophilicity, a factor that is generally agreed to be contributing to the low success rate of new drug candidates. Our aim is to provide a structural perspective to the study of lipophilic efficiency and to compare molecular interactions created over evolutionary time with those designed by humans. We show that natural complexes typically engage in more polar contacts than synthetic molecules bound to proteins. The synthetic molecules also have a higher proportion of unmatched heteroatoms at the interface than the natural sets. These observations suggest that there are lessons to be learnt from Nature, which could help us to improve the characteristics of man-made molecules. In particular, it is possible to increase the density of polar contacts without increasing lipophilicity and this is best achieved early in discovery while molecules remain relatively small. 相似文献
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Adrian C. Kanaar 《BMJ (Clinical research ed.)》1941,1(4188):549-552
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We have previously reported that in vitro HCV infection of cells of hepatocyte origin attenuates complement system at multiple steps, and attenuation also occurs in chronically HCV infected liver, irrespective of the disease stage. However, none of these regulations alone completely impaired complement pathways. Modulation of the upstream proteins involved in proteolytic processing of the complement cascade prior to convertase formation is critical in promoting the function of the complement system in response to infection. Here, we examined the regulation of C2 complement expression in hepatoma cells infected in vitro with cell culture grown virus, and validated our observations using randomly selected chronically HCV infected patient liver biopsy specimens. C2 mRNA expression was significantly inhibited, and classical C3 convertase (C4b2a) decreased. In separate experiments for C3 convertase function, C3b deposition onto bacterial membrane was reduced using HCV infected patient sera as compared to uninfected control, suggesting impaired C3 convertase. Further, iC3b level, a proteolytically inactive form of C3b, was lower in HCV infected patient sera, reflecting impairment of both C3 convertase and Factor I activity. The expression level of Factor I was significantly reduced in HCV infected liver biopsy specimens, while Factor H level remained unchanged or enhanced. Together, these results suggested that inhibition of C3 convertase activity is an additional cumulative effect for attenuation of complement system adopted by HCV for weakening innate immune response. 相似文献
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A method for the separation and partial purification of the three forms of nitrate reductase present in wild-type soybean leaves 总被引:4,自引:1,他引:3
A rapid and simple purification method was used to separate and purify nitrate reductases (NR) from Williams soybean leaves. Blue Sepharose columns were sequentially eluted with 50 millimolar NADPH and 50 millimolar NADH, thus separating NAD(P)H:NR from NADH:NRs. Subsequent purification of the collected peaks on a fast protein liquid chromatography-Mono Q column enabled separation of two NADH:NRs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the subunit relative molecular mass for all three NR forms (constitutive NAD(P)H:NR [pH 6.5], EC 1.6.6.2; constitutive NADH:NR [pH 6.5], EC not assigned; and inducible NADH:NR [pH 7.5], EC 1.6.6.1) was approximately 107 to 109 kilodaltons. All three NRs showed similar spectra with absorption maxima at 413 and 273 nanometers in the oxidized state, and with the characteristics of a cytochrome b type heme upon reduction with NADH (absorption maxima at 556, 527, and 424 nanometers). The technique developed provides an improved separation of the three NR forms from soybean leaves. The similarity of the NRs with regard to their cytochrome b556 type heme content and in relative molecular mass indicated that other differences must exist to account for the different kinetic and physical properties previously reported. 相似文献
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DNA replication in maize leaf protoplasts 总被引:1,自引:0,他引:1
H. Wang Adrian J. Cutler M. Saleem Larry C. Fowke 《Plant Cell, Tissue and Organ Culture》1989,18(1):33-46
Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of 3H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca2+ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU
5-bromo-2-deoxyuridine
- DMSO
dimethyl sulfoxide
- n-PG
n-propyl gallate
- PBS
phosphate-buffered saline
Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday 相似文献