scholarly journals Morphology of Isolated Gli349, a Leg Protein Responsible for Mycoplasma mobile Gliding via Glass Binding, Revealed by Rotary Shadowing Electron Microscopy

2006 ◽  
Vol 188 (8) ◽  
pp. 2821-2828 ◽  
Author(s):  
Jun Adan-Kubo ◽  
Atsuko Uenoyama ◽  
Toshiaki Arata ◽  
Makoto Miyata
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Gel Filtration ◽  
Solid Surfaces ◽  
Membrane Protrusion ◽  
Molecular Image ◽  
Peptide Length ◽  
20 Nm ◽  
Molecular Images ◽  
Rotary Shadowing

ABSTRACT Several species of mycoplasmas rely on an unknown mechanism to glide across solid surfaces in the direction of a membrane protrusion at the cell pole. Our recent studies on the fastest species, Mycoplasma mobile, suggested that a 349-kDa protein, Gli349, localized at the base of the membrane protrusion called the neck, forms legs that stick out from the neck and propel the cell by repeatedly binding to and releasing from a solid surface, based on the energy of ATP hydrolysis. Here, the Gli349 protein was isolated from mycoplasma cells and its structure was analyzed. Gel filtration analysis showed that the isolated Gli349 protein is monomeric. Rotary shadowing electron microscopy revealed that the molecular structure resembles the symbol for an eighth note in music. It contains an oval foot 14 nm long in axis. From this foot extend three rods in tandem of 43, 20, and 20 nm, in that order. The hinge connecting the first and second rods is flexible, while the next hinge has a distinct preference in its angle, near 90 degrees. Molecular images revealed that a monoclonal antibody that can bind to the position at one-third of the total peptide length from the N terminus bound to a position two-thirds from the foot end, suggesting that the foot corresponds to the C-terminal region. The amino acid sequence was assigned to the molecular image, and the topology of the molecule in the gliding machinery is discussed.

scholarly journals Structural Evidence that the P/Q Domain of ZipA Is an Unstructured, Flexible Tether between the Membrane and the C-Terminal FtsZ-Binding Domain

2002 ◽  
Vol 184 (15) ◽  
pp. 4313-4315 ◽  
Author(s):  
Tomoo Ohashi ◽  
Cynthia A. Hale ◽  
Piet A. J. de Boer ◽  
Harold P. Erickson
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
Transmembrane Domain ◽  
Persistence Length ◽  
Binding Domain ◽  
Globular Domain ◽  
Cell Division Protein ◽  
20 Nm ◽  
Rotary Shadowing

ABSTRACT The cell division protein ZipA has an N-terminal transmembrane domain and a C-terminal globular domain that binds FtsZ. Between them are a charged domain and a P/Q domain rich in proline and glutamine that has been proposed to be an unfolded polypeptide. Here we provide evidence obtained by electron microscopy that the P/Q domain is a flexible tether ranging in length from 8 to 20 nm and invisible in rotary shadowing electron microscopy. We estimated a persistence length of 0.66 nm, which is similar to the persistence lengths of other unfolded and unstructured polypeptides.

scholarly journals Evidence of a defined spatial arrangement of hyaluronate in the central filament of cartilage proteoglycan aggregates

1995 ◽  
Vol 307 (2) ◽  
pp. 595-601 ◽  
Author(s):  
M Mörgelin ◽  
M Paulsson ◽  
D Heinegård ◽  
U Aebi ◽  
J Engel
Keyword(s):  
Electron Microscopy ◽  
Core Protein ◽  
Gel Filtration ◽  
Spatial Arrangement ◽  
Link Protein ◽  
Original Length ◽  
Central Filament ◽  
Proteoglycan Aggregates ◽  
Rotary Shadowing

Aggregates of proteoglycans from the Swarm rat chondrosarcoma reassembled in vitro have been studied by rotary-shadowing electron microscopy, and shown to be similar to native structures that have never been dissociated [Mörgelin, Engel, Heinegård and Paulsson (1992) J. Biol. Chem. 267, 14275-14284]. A hyaluronate with defined chain length (HAshort) has now been prepared by autoclaving high-Mr hyaluronate and fractionation to a narrow size distribution by gel filtration. Proteoglycan monomers, core protein, hyaluronate-binding region and link protein were combined with HAshort. Free chains of HAshort and reconstituted complexes with proteoglycan, link protein and aggrecan fragments were examined by electron microscopy after rotary shadowing. Length measurements showed that the hyaluronate was condensed to about half of its original length on binding intact aggrecan monomers, any aggrecan fragment or link protein alone. This strongly implies that hyaluronate adopts a defined spatial arrangement within the central filament of the aggregate, probably different from its secondary structure in solution. No differences in length were observed between link-free and link-stabilized aggregates.

scholarly journals Binding sites of calmodulin and actin on the brain spectrin, calspectin.

1983 ◽  
Vol 97 (2) ◽  
pp. 574-578 ◽  
Author(s):  
S Tsukita ◽  
S Tsukita ◽  
H Ishikawa ◽  
M Kurokawa ◽  
K Morimoto ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Complex Formation ◽  
Binding Sites ◽  
Actin Binding ◽  
Calmodulin Binding ◽  
20 Nm ◽  
Labeling Method ◽  
The Brain ◽  
Brain Spectrin ◽  
Rotary Shadowing

We used rotary-shadowing electron microscopy to map the calmodulin-and actin-binding sites on the brain spectrin, calspectin (or fodrin). Calspectin dimers appeared as rods 110 nm long and joined in a head-to-head manner to form tetramers 220 nm long. We determined calmodulin-binding sites by a ferritin-labeling method combined with biotin-avidin complex formation. Ferritin particles were found to attach to the head parts of calspectin dimers at a position 10-20 nm from the top of the head. The number of the calmodulin-binding sites seemed to be only one for each dimer and two for each tetramer. In contrast, the actin-binding sites were localized at the tail ends of the calspectin molecules. The tetramers attached to muscle F-actin with their tail ends and often cross-linked adjacent filaments. The results are discussed in view of the analogy to the erythrocyte spectrin.

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

1969 ◽  
Vol 27 ◽  
pp. 424-425
Author(s):  
Lee F. Ellis ◽  
Richard M. Van Frank ◽  
Walter J. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Interferon Inducer ◽  
Virus Particles ◽  
Staining Methods ◽  
Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

CRYO-Electron Microscopy of the Acto-Myosin-ATP Complex

1990 ◽  
Vol 48 (1) ◽  
pp. 248-249
Author(s):  
John Trinickt ◽  
Howard White
Keyword(s):  
Electron Microscopy ◽  
Atp Hydrolysis ◽  
Myosin Head ◽  
Bound State ◽  
Cross Linking ◽  
Weakly Bound ◽  
Cryo Electron Microscopy ◽  
Myosin Subfragment 1 ◽  
Attached State ◽  
High Fraction

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

scholarly journals Oligomerization and Activity of the Helicase Domain of the Tobacco Mosaic Virus 126- and 183-Kilodalton Replicase Proteins

2003 ◽  
Vol 77 (6) ◽  
pp. 3549-3556 ◽  
Author(s):  
Sameer P. Goregaoker ◽  
James N. Culver
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Virus Replication ◽  
Protein Interaction ◽  
Gel Filtration ◽  
Helicase Domain ◽  
Protein Protein Interaction ◽  
Replicase Proteins

ABSTRACT A protein-protein interaction within the helicase domain of the Tobacco mosaic virus (TMV) 126- and 183-kDa replicase proteins was previously implicated in virus replication (S. Goregaoker, D. Lewandowski, and J. Culver, Virology 282:320-328, 2001). To further characterize the interaction, polypeptides covering the interacting portions of the TMV helicase domain were expressed and purified. Biochemical characterizations demonstrated that the helicase domain polypeptides hydrolyzed ATP and bound both single-stranded and duplexed RNA in an ATP-controlled fashion. A TMV helicase polypeptide also was capable of unwinding duplexed RNA, confirming the predicted helicase function of the domain. Biochemically active helicase polypeptides were shown by gel filtration to form high-molecular-weight complexes. Electron microscopy studies revealed the presence of ring-like oligomers that displayed six-sided symmetry. Taken together, these data demonstrate that the TMV helicase domain interacts with itself to produce hexamer-like oligomers. Within the context of the full-length 126- and 183-kDa proteins, these findings suggest that the TMV replicase may form a similar oligomer.

A Facile Fynthesis of ZnS Nanostructures via Liquid-Solid Reactions

2014 ◽  
Vol 979 ◽  
pp. 184-187
Author(s):  
Weerachon Phoohinkong ◽  
Thitinat Sukonket ◽  
Udomsak Kitthawee
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Inorganic Salts ◽  
Chloride Salt ◽  
X Ray ◽  
Commercial Grade ◽  
Transmission Electron ◽  
Salt Addition ◽  
20 Nm ◽  
Scanning Electron

Zinc sulfide (ZnS) nanostructures are important materials for many technologies such as sensors, infrared windows, transistors, LED displays, and solar cells. However, many methods of synthesizing ZnS nanostructures are complex and require expensive equipment. In this study, a liquid-solid chemical reaction without surfactant was used to synthesize ZnS at room temperature. In addition, commercial grade zinc oxide (ZnO) particles were used as a precursor. The effect of the addition of acids and inorganic salts were investigated. The products were characterized by field emission scanning electron microscopy (FESEM) coupled with energy-dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM). The results show that the nanoparticles of ZnS were obtained in hydrochloric acid and acetic acid addition. The diameters were in the range of 10 to 20 nm and 50 to 100 nm, respectively. In the case of a sodium chloride salt addition, a ZnS structure was obtained with a particle size of approximately 5 nm and a flake-like morphology.

scholarly journals Movements of Mycoplasma mobile gliding machinery detected by high-speed atomic force microscopy

2021 ◽  
Author(s):  
Kohei Kobayashi ◽  
Noriyuki Kodera ◽  
Taishi Kasai ◽  
Yuhei O Tahara ◽  
Takuma Toyonaga ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Internal Structure ◽  
Sodium Azide ◽  
High Speed ◽  
Atp Hydrolysis ◽  
Solid Surfaces ◽  
Force Microscopy ◽  
Special Mechanism ◽  
Atomic Force ◽  
Internal Structures

ABSTRACTMycoplasma mobile, a parasitic bacterium, glides on solid surfaces, such as animal cells and glass by a special mechanism. This process is driven by the force generated through ATP hydrolysis on an internal structure. However, the spatial and temporal behaviors of the internal structures in living cells are unclear. In this study, we detected the movements of the internal structure by scanning cells immobilized on a glass substrate using high-speed atomic force microscopy (HS-AFM). By scanning the surface of a cell, we succeeded in visualizing particles, 2 nm in hight and aligned mostly along the cell axis with a pitch of 31.5 nm, consistent with previously reported features based on electron microscopy. Movements of individual particles were then analyzed by HS-AFM. In the presence of sodium azide, the average speed of particle movements was reduced, suggesting that movement is linked to ATP hydrolysis. Partial inhibition of the reaction by sodium azide enabled us to analyze particle behavior in detail, showing that the particles move 9 nm right, relative to the gliding direction, and 2 nm into the cell interior in 330 ms, then return to their original position, based on ATP hydrolysis.IMPORTANCEThe Mycoplasma genus contains bacteria generally parasitic to animals and plants. Some Mycoplasma species form a protrusion at a pole, bind to solid surfaces, and glide by a special mechanism linked to their infection and survival. The special machinery for gliding can be divided into surface and internal structures that have evolved from rotary motors represented by ATP synthases. This study succeeded in visualizing the real-time movements of the internal structure by scanning from the outside of the cell using an innovative high-speed atomic force microscope, and then analyzing their behaviors.

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