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排序方式: 共有750条查询结果,搜索用时 31 毫秒
1.
Rpn1 (109 kDa) and Rpn2 (104 kDa) are components of the 19S regulatory complex of the proteasome. The central portions of both proteins are predicted to have toroidal α-solenoid folds composed of 9-11 proteasome/cyclosome repeats, each ∼ 40 residues long and containing two α-helices and turns [A. V. Kajava, J. Biol. Chem. 277, 49791-49798, 2002]. To evaluate this prediction, we examined the full-length yeast proteins and truncated versions thereof consisting only of the repeat-containing regions by gel filtration, CD spectroscopy, and negative-staining electron microscopy (EM). All four proteins are monomeric in solution and highly α-helical, particularly the truncated ones. The EM data were analyzed by image classification and averaging techniques. The preponderant projections, in each case, show near-annular molecules 6-7 nm in diameter. Comparison of the full-length with the truncated proteins showed molecules similar in size and shape, indicating that their terminal regions are flexible and thus smeared to invisibility in the averaged images. We tested the toroidal model further by calculating resolution-limited projections and comparing them with the EM images. The results support the α-solenoid model, except that they indicate that the repeats are organized not as symmetrical circular toroids but in less regular horseshoe-like structures. 相似文献
2.
Crystallization of transmembrane proteins in cubo: mechanisms of crystal growth and defect formation
Qutub Y Reviakine I Maxwell C Navarro J Landau EM Vekilov PG 《Journal of molecular biology》2004,343(5):1243-1254
Crystallization of membrane proteins is a major stumbling block en route to elucidating their structure and understanding their function. The novel concept of membrane protein crystallization from lipidic cubic phases, "in cubo", has yielded well-ordered crystals and high-resolution structures of several membrane proteins, yet progress has been slow due to the lack of understanding of the molecular mechanisms of protein transport, crystal nucleation, growth, and defect formation in cubo. Here, we examine at molecular and mesoscopic resolution with atomic force microscopy the morphology of in cubo grown bacteriorhodopsin crystals in inert buffers and during etching by detergent. The results reveal that crystal nucleation occurs following local rearrangement of the highly curved lipidic cubic phase into a lamellar structure, which is akin to that of the native membrane. Crystals grow within the bulk cubic phase surrounded by such lamellar structures, whereby transport towards a growing crystalline layer is constrained to within an individual lamella. This mechanism leads to lack of dislocations, generation of new crystalline layers at numerous locations, and to voids and block boundaries. The characteristic macroscopic lengthscale of these defects suggests that the crystals grow by attachment of single molecules to the nuclei. These insights into the mechanisms of nucleation, growth and transport in cubo provide guidance en route to a rational design of membrane protein crystallization, and promise to further advance the field. 相似文献
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4.
Huanhuan Liao Hui He Yuan Chen Fangfa Zeng Jie Huang Li Wu Yong Chen 《Cytotechnology》2014,66(2):229-238
The effects of serial cell passaging on cell spreading, migration, and cell-surface ultrastructures have been less investigated directly. This study evaluated the effects of long-term serial cell passaging (totally 35 passages) on cultured human umbilical vein endothelial cells which were pre-stored at −80 °C as usual. Percentage- and spread area-based spreading assays, measurements of fluorescently labeled actin filaments, migration assay, and measurements of cell-surface roughness were performed and quantitatively analyzed by confocal microscopy or atomic force microscopy. We found that the abilities of cell spreading and migration first increased at early passages and then decreased after passage 15, in agreement with the changes in average length of actin filaments. Recovery from cold storage and effects of cell passaging were potentially responsible for the increases and decreases of the values, respectively. In contrast, the average roughness of cell surfaces (particularly the nucleus-surrounding region) first dropped at early passages and then rose after passage 15, which might be caused by cold storage- and cell passaging-induced endothelial microparticles. Our data will provide important information for understanding serial cell passaging and implies that for pre-stored adherent cells at −80 °C cell passages 5–10 are optimal for in vitro studies. 相似文献
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p53 is a human tumour suppressor which regulates multiple cellular processes, including cell growth, genomic stability and cell death. Recent works have demonstrated the bacterial redox protein azurin to enter cancer cells and induce apoptosis through p53 stabilization, resulting in a tumour growth regression. Azurin has been shown to bind p53 although many details of the complex formed by these two proteins are still poorly characterized. Here, we get insight into the kinetics of this complex formation, by exploring the interaction between p53 and azurin in their environment by single molecule force spectroscopy. To this aim, azurin has been linked to the atomic force microscope tip, whereas p53 has been immobilized onto a gold substrate. Therefore, by performing force-distance cycles we have detected specific recognition events between p53 and azurin, displaying unbinding forces of around 70 pN for an applied loading rate of 3 nN s(-1). The specificity of these events has been assessed by the significant reduction of their frequency observed after blocking the p53 sample by an azurin solution. Moreover, by measuring the rupture force as a function of the loading rate we have determined the dissociation rate constant of this complex to be approximately 0.1 s(-1). Our findings are here discussed in connection with results obtained in bulk experiments, with the aim of clarifying some molecular details of the p53-azurin complex that may help designing new anticancer strategy. 相似文献
7.
Tom Bongiorno Jacob Kazlow Roman Mezencev Sarah Griffiths Rene Olivares-Navarrete John F. McDonald Zvi Schwartz Barbara D. Boyan Todd C. McDevitt Todd Sulchek 《Journal of biomechanics》2014
Although it has been established that cellular stiffness can change as a stem cell differentiates, the precise relationship between cell mechanics and other phenotypic properties remains unclear. Inherent cell heterogeneity and asynchronous differentiation complicate population analysis; therefore, single-cell analysis was employed to determine how changes in cell stiffness correlate with changes in molecular biomarkers during differentiation. Design of a custom gridded tissue culture dish facilitated single-cell comparisons between cell mechanics and other differentiation biomarkers by enabling sequential measurement of cell mechanics and protein biomarker expression at the single cell level. The Young’s modulus of mesenchymal stem cells was shown not only to decrease during chemically-induced osteoblast differentiation, but also to correlate more closely with the day of differentiation than did the relative expression of the traditional osteoblast differentiation markers, bone sialoprotein and osteocalcin. Therefore, cell stiffness, a measurable property of individual cells, may serve as an improved indicator of single-cell osteoblast differentiation compared to traditional biological markers. Revelation of additional osteoblast differentiation indicators, such as cell stiffness, can improve identification and collection of starting cell populations, with applications to mesenchymal stem cell therapies and stem cell-based tissue engineering. 相似文献
8.
Jia X Gharibyan AL Öhman A Liu Y Olofsson A Morozova-Roche LA 《Journal of molecular biology》2011,(5):699-712
Parkinson's disease is a common neurodegenerative disorder characterized by α-synuclein (α-Syn)-containing Lewy body formation and selective loss of dopaminergic neurons in the substantia nigra. We have demonstrated the modulating effect of noopept, a novel proline-containing dipeptide drug with nootropic and neuroprotective properties, on α-Syn oligomerization and fibrillation by using thioflavin T fluorescence, far-UV CD, and atomic force microscopy techniques. Noopept does not bind to a sterically specific site in the α-Syn molecule as revealed by heteronuclear two-dimensional NMR analysis, but due to hydrophobic interactions with toxic amyloid oligomers, it prompts their rapid sequestration into larger fibrillar amyloid aggregates. Consequently, this process rescues the cytotoxic effect of amyloid oligomers on neuroblastoma SH-SY5Y cells as demonstrated by using cell viability assays and fluorescent staining of apoptotic and necrotic cells and by assessing the level of intracellular oxidative stress. The mitigating effect of noopept against amyloid oligomeric cytotoxicity may offer additional benefits to the already well-established therapeutic functions of this new pharmaceutical. 相似文献
9.
不同固定条件下细胞与活细胞的原子力显微镜实时观察 总被引:3,自引:0,他引:3
用原子力显微镜(atom force microscope,AFM)观察固定细胞的最佳条件并在生理溶液中对活细胞实时观察.用不同固定剂和同一固定剂的不同浓度处理细胞;不加任何固定剂而直接在生理溶液中对细胞进行AFM成像.以戊二醛为固定剂并使用0.5%~1%的浓度固定细胞,后用缓冲溶液漂洗,再对细胞进行成像时可获得质量良好的图像.直接在生理溶液中进行观察,成像质量低于使用固定剂的细胞,但保持了细胞的生活原貌.在用原子力显微镜高分辨率观察生理条件下细胞的特点时,需要在制样与观测系统两方面进行改进. 相似文献
10.
Andreas Stylianou Vasiliki Gkretsi Triantafyllos Stylianopoulos 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(7):1537-1546