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61.
Two groups control light-induced schiff base deprotonation and the proton affinity of asp(85) in the Arg(82)His mutant of bacteriorhodopsin 总被引:1,自引:0,他引:1
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ES Imasheva SP Balashov TG Ebrey N Chen RK Crouch DR Menick 《Biophysical journal》1999,77(5):2750-2763
Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 &mgr;s to 75 &mgr;s with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release. 相似文献
62.
Structural analysis of human neutrophil migration: Centriole, microtubule, and microfilament orientation and function during chemotaxis 总被引:41,自引:21,他引:20
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Orientation of nucleus, centriole, microtubules, and microfilaments within human neutrophils in a gradient of chemoattractant (5 percent Escherichia coli endotoxin-activated serum) was evaluated by electron microscopy. Purified neutropils (hypaque-Ficoll) were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 μm) micropore filters permitted pseudopod penetration, but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus (activated serum above cells) resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Coordinated orientation of the entire neutrophil population did not occur in buffer (random migration) or in a uniform concentration of activated serum (activated random migration). Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e. cellular asymmetry with linear alignment of nucleus, centriole, microtubule array, and pseudopods. Thus, activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil populayion. In related studies, cytochalasin B and colchicines were used to explore the role of microfilaments and microtubules in the neutrophil orientation and migration response to activated serum. Cytochalasin B (3.0 μg/ml) prevented migration and decreased the microfilaments seen, but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicines, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10(-8) to 10(-7) M. These colchicines effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, a formation of narrow pseudopods within a substrate, and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicines, but not lumicolchicine, minimally decreased activated random migration and markedly inhibited directed migration, but had not effect on random migration. These studies show that, although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are essential for normal pseudopod formation and orientation, and maximal unidirectional migration during chemotaxis. 相似文献
63.
Worldwide patterns of mitochondrial DNA differentiation in the harbor seal (Phoca vitulina) 总被引:3,自引:0,他引:3
Stanley HF; Casey S; Carnahan JM; Goodman S; Harwood J; Wayne RK 《Molecular biology and evolution》1996,13(2):368-382
The harbor seal (Phoca vitulina) has one of the broadest geographic
distributions of any pinniped, stretching from the east Baltic, west across
the Atlantic and Pacific Oceans to southern Japan. Although individuals may
travel several hundred kilometers on annual feeding migrations, harbor
seals are generally believed to be philopatric, returning to the same areas
each year to breed. Consequently, seals from different areas are likely to
be genetically differentiated, with levels of genetic divergence increasing
with distance. Differentiation may also be caused by long-standing
topographic barriers such as the polar sea ice. We analyzed samples of 227
harbor seals from 24 localities and defined 34 genotypes based on 435 bp of
control region sequence. Phylogenetic analysis and analysis of molecular
variance showed that populations in the Atlantic and Pacific Oceans and
east and west coast populations of these oceans are significantly
differentiated. Within these four regions, populations that are
geographically farthest apart generally are the most differentiated and
often do not share genotypes or differ in genotype frequency. The average
corrected sequence divergence between populations in the Atlantic and
Pacific Oceans is 3.28% +/- 0.38% and those among populations within each
of these oceans are 0.75% +/- 0.69% and 1.19% +/- 0.65%, respectively. Our
results suggest that harbor seals are regionally philopatric, on the scale
of several hundred kilometers. However, genetic discontinuities may exist,
even between neighboring populations such as those on the Scottish and east
English coasts or the east and west Baltic. The mitochondrial data are
consistent with an ancient isolation of populations in both oceans, due to
the development of polar sea ice. In the Atlantic and Pacific, populations
appear to have been colonized from west to east with the European
populations showing the most recent common ancestry. We suggest the recent
ancestry of European seal populations may reflect recolonization from Ice
Age refugia after the last glaciation.
相似文献
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65.
Non-nucleoside inhibitors of the measles virus RNA-dependent RNA polymerase complex activity: Synthesis and in vitro evaluation 总被引:1,自引:0,他引:1
Sun A Chandrakumar N Yoon JJ Plemper RK Snyder JP 《Bioorganic & medicinal chemistry letters》2007,17(18):5199-5203
High-throughput screening has identified 1-methyl-3-(trifluoromethyl)-N-[4-(pyrrolidinylsulfonyl)phenyl]-1H-pyrazole-5-carboxamide 16677 as a novel and potent (IC(50)=35-145 nM) inhibitor against multiple primary isolates of diverse measles virus (MV) genotypes currently circulating worldwide. The synthesis of 16677 and several analogs together with effects on MV replication is described. The most potent analog displays nanomolar inhibition against the MV and a selectivity ratio (CC(50)/IC(50)) of ca. 16,500. 相似文献
66.
Mislay Avila Mojtaba Khosravi Lisa Alves Nadine Ader-Ebert Fanny Bringolf Andreas Zurbriggen Richard K. Plemper Philippe Plattet 《Journal of virology》2015,89(2):1445-1451
Membrane fusion for morbillivirus cell entry relies on critical interactions between the viral fusion (F) and attachment (H) envelope glycoproteins. Through extensive mutagenesis of an F cavity recently proposed to contribute to F''s interaction with the H protein, we identified two neighboring hydrophobic residues responsible for severe F-to-H binding and fusion-triggering deficiencies when they were mutated in combination. Since both residues reside on one side of the F cavity, the data suggest that H binds the F globular head domain sideways. 相似文献
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69.
Two domains that control prefusion stability and transport competence of the measles virus fusion protein
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Doyle J Prussia A White LK Sun A Liotta DC Snyder JP Compans RW Plemper RK 《Journal of virology》2006,80(3):1524-1536
Most viral glycoproteins mediating membrane fusion adopt a metastable native conformation and undergo major conformational changes during fusion. We previously described a panel of compounds that specifically prevent fusion induced by measles virus (MV), most likely by interfering with conformational rearrangements of the MV fusion (F) protein. To further elucidate the basis of inhibition and better understand the mechanism of MV glycoprotein-mediated fusion, we generated and characterized resistant MV variants. Spontaneous mutations conferring drug resistance were confirmed in transient assays and in the context of recombinant virions and were in all cases located in the fusion protein. Several mutations emerged independently at F position 462, which is located in the C-terminal heptad repeat (HR-B) domain. In peptide competition assays, all HR-B mutants at residue 462 revealed reduced affinity for binding to the HR-A core complex compared to unmodified HR-B. Combining mutations at residue 462 with mutations in the distal F head region, which we had previously identified as mediating drug resistance, causes intracellular retention of the mutant proteins. The transport competence and activity of the mutants can be restored, however, by incubation at reduced temperature or in the presence of the inhibitory compounds, indicating that the F escape mutants have a reduced conformational stability and that the inhibitors stabilize a transport-competent conformation of the F trimer. The data support the conclusion that residues located in the head domain of the F trimer and the HR-B region contribute jointly to controlling F conformational stability. 相似文献
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