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Sun Y Breydo L Makarava N Yang Q Bocharova OV Baskakov IV 《The Journal of biological chemistry》2007,282(12):9090-9097
Despite the ability of most proteins to form amyloid, very little is know about amyloid fibril structures and the factors that govern their stability. Using amyloid fibrils produced from full-length prion protein (PrP), we describe a reliable approach for determining both site-specific and global conformational stability of the fibrillar form. To measure site-specific stability, we produced six variants of PrP by replacing the residues at positions 88, 98, 127, 144, 196, and 230 with cysteine, labeled the new cysteines with the fluorescent dye acrylodan, and investigated their conformational status within the amyloid form in guanidine hydrochloride-induced denaturation experiments. We found that the fibrils labeled at positions 127, 144, 196, and 230 displayed cooperative unfolding and showed a very high C1/2 value similar to that observed for the global unfolding of the amyloid structure. The unfolding at residue 98 was also cooperative; however, it showed a C1/2 value substantially lower than that of global unfolding, whereas the unfolding of fibrils labeled at residue 88 was non-cooperative. These data illustrate that there are at least two independent cooperative folding domains within the amyloid structure of the full-length PrP. In addition, kinetic experiments revealed only a partial overlap between the region that constituted the fibrillar cross-beta core and the regions that were involved in nucleation. This result illustrates that separate PrP regions accounted for the nucleation and for the formation of the conformationally most stable fibrillar core. 相似文献
23.
Ranguelova K Girotto S Gerfen GJ Yu S Suarez J Metlitsky L Magliozzo RS 《The Journal of biological chemistry》2007,282(9):6255-6264
Catalase-peroxidase (KatG) from Mycobacterium tuberculosis, a Class I peroxidase, exhibits high catalase activity and peroxidase activity with various substrates and is responsible for activation of the commonly used antitubercular drug, isoniazid (INH). KatG readily forms amino acid-based radicals during turnover with alkyl peroxides, and this work focuses on extending the identification and characterization of radicals forming on the millisecond to second time scale. Rapid freeze-quench electron paramagnetic resonance spectroscopy (RFQ-EPR) reveals a change in the structure of the initially formed radical in the presence of INH. Heme pocket binding of the drug and knowledge that KatG[Y229F] lacks this signal provides evidence for radical formation on residue Tyr(229). High field RFQ-EPR spectroscopy confirmed a tryptophanyl radical signal, and new analyses of X-band RFQ-EPR spectra also established its presence. High field EPR spectroscopy also confirmed that the majority radical species is a tyrosyl radical. Site-directed mutagenesis, along with simulations of EPR spectra based on x-ray structural data for particular tyrosine and tryptophan residues, enabled assignments based on predicted hyperfine coupling parameters. KatG mutants W107F, Y229F, and the double mutant W107F/Y229F showed alteration in type and yield of radical species. Results are consistent with formation of a tyrosyl radical reasonably assigned to residue Tyr(229) within the first few milliseconds of turnover. This is followed by a mixture of tyrosyl and tryptophanyl radical species and finally to only a tyrosyl radical on residue Tyr(353), which lies more distant from the heme. The radical processing of enzyme lacking the Trp(107)-Tyr(229)-Met(255) adduct (found as a unique structural feature of catalase-peroxidases) is suggested to be a reasonable assignment of the phenomena. 相似文献
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Genome-wide analysis of nucleotide-level variation in commonly used Saccharomyces cerevisiae strains
Ten years have passed since the genome of Saccharomyces cerevisiae-more precisely, the S288c strain-was completely sequenced. However, experimental work in yeast is commonly performed using strains that are of unknown genetic relationship to S288c. Here, we characterized the nucleotide-level similarity between S288c and seven commonly used lab strains (A364A, W303, FL100, CEN.PK, summation 1278b, SK1 and BY4716) using 25mer oligonucleotide microarrays that provide complete and redundant coverage of the approximately 12 Mb Saccharomyces cerevisiae genome. Using these data, we assessed the frequency and distribution of nucleotide variation in comparison to the sequenced reference genome. These data allow us to infer the relationships between experimentally important strains of yeast and provide insight for experimental designs that are sensitive to sequence variation. We propose a rational approach for near complete sequencing of strains related to the reference using these data and directed re-sequencing. These data and new visualization tools are accessible online in a new resource: the Yeast SNPs Browser (YSB; http://gbrowse.princeton.edu/cgi-bin/gbrowse/yeast_strains_snps) that is available to all researchers. 相似文献
26.
Li J Marionneau C Koval O Zingman L Mohler PJ Nerbonne JM Anderson ME 《Channels (Austin, Tex.)》2007,1(5):387-394
Mice with genetic inhibition (AC3-I) of the multifunctional Ca(2+)/calmodulin dependent protein kinase II (CaMKII) have improved cardiomyocyte survival after ischemia. Some K(+) currents are up-regulated in AC3-I hearts, but it is unknown if CaMKII inhibition increases the ATP sensitive K(+) current (I(KATP)) that underlies ischemic preconditioning (IP) and confers resistance to ischemia. We hypothesized increased I(KATP) was part of the mechanism for improved ventricular myocyte survival during ischemia in AC3-I mice. AC3-I hearts were protected against global ischemia due to enhanced IP compared to wild type (WT) and transgenic control (AC3-C) hearts. IKATP was significantly increased, while the negative regulatory dose-dependence of ATP was unchanged in AC3-I compared to WT and AC3-C ventricular myocytes, suggesting that CaMKII inhibition increased the number of functional I(KATP) channels available for IP. We measured increased sarcolemmal Kir6.2, a pore-forming I(KATP) subunit, but not a change in total Kir6.2 in cell lysates or single channel I(KATP) opening probability from AC3-I compared to WT and AC3-C ventricles, showing CaMKII inhibition increased sarcolemmal I(KATP) channel expression. There were no differences in mRNA for genes encoding I(KATP) channel subunits in AC3-I, WT and AC3-C ventricles. The I(KATP) opener pinacidil (100 microM) reduced MI area in WT to match AC3-I hearts, while the I(KATP) antagonist HMR1098 (30 microM) increased MI area to an equivalent level in all groups, indicating that increased I(KATP) and augmented IP are important for reduced ischemic cell death in AC3-I hearts. Our study results show CaMKII inhibition enhances beneficial effects of IP by increasing I(KATP). 相似文献
27.
Duroux M Skovsen E Neves-Petersen MT Duroux L Gurevich L Petersen SB 《Proteomics》2007,7(19):3491-3499
The present work shows how UV 'light-induced molecular immobilisation' (LIMI) of biomolecules onto thiol reactive surfaces can be used to make biosensors, without the need for traditional microdispensing technologies. Using 'LIMI,' arrays of biomolecules can be created with a high degree of reproducibility. This technology can be used to circumvent the need for often expensive nano/microdispensing technologies. The ultimate size of the immobilised spots is defined by the focal area of the UV beam, which for a diffraction-limited beam can be less than 1 microm in diameter. LIMI has the added benefit that the immobilised molecules will be spatially oriented and covalently bound to the surface. The activity of the sensor molecules is retained. Antibody sensor arrays made using LIMI demonstrated successful antigen binding. In addition, the pattern of immobilised molecules on the surface is not restricted to conventional array formats. The ultimate consequence of the LIMI is that it is possible to write complex protein patterns using bitmaps at high resolution onto substrates. Thus, LIMI of biomolecules provides a new technological platform for biomolecular immobilisation and the potential for replacing present microdispensing arraying technologies. 相似文献
28.
Fina A. S Kurreeman Leonid Padyukov Rute B Marques Steven J Schrodi Maria Seddighzadeh Gerrie Stoeken-Rijsbergen Annette H. M van der Helm-van Mil Cornelia F Allaart Willem Verduyn Jeanine Houwing-Duistermaat Lars Alfredsson Ann B Begovich Lars Klareskog Tom W. J Huizinga Rene E. M Toes 《PLoS medicine》2007,4(12)
29.
Krásová Jarmila Mikula Ondřej Lavrenchenko Leonid A. Šumbera Radim Meheretu Yonas Bryja Josef 《Organisms Diversity & Evolution》2022,22(2):491-509
Organisms Diversity & Evolution - The Ethiopian highlands represent the largest part of the Eastern Afromontane Biodiversity Hotspot (EAMBH). Their fauna and flora are largely unique.... 相似文献
30.