scholarly journals Three-Dimensional Structure of Different Functional Forms of the Vibrio cholerae Hemolysin Oligomer: a Cryo-Electron Microscopic Study

2009 ◽  
Vol 192 (1) ◽  
pp. 169-178 ◽  
Author(s):  
Somnath Dutta ◽  
Budhaditya Mazumdar ◽  
Kalyan K. Banerjee ◽  
Amar N. Ghosh
Keyword(s):  
Vibrio Cholerae ◽  
Three Dimensional ◽  
Water Soluble ◽  
Particle Methods ◽  
Dimensional Structure ◽  
Striking Difference ◽  
Electron Microscopic ◽  
Membrane Bilayer ◽  
Lectin Domain ◽  
Structural Differences

ABSTRACT Vibrio cholerae hemolysin (HlyA) is a 65-kDa water-soluble pore-forming toxin that causes lysis of eukaryotic cells by destroying selective permeability of the plasma membrane bilayer. The HlyA monomer self-assembles on the target cell surface to the more stable β-barrel amphipathic heptamer, which inserts into the membrane bilayer to form a diffusion channel. Deletion of the 15-kDa β-prism lectin domain at the C terminus generates a 50-kDa hemolysin variant (HlyA50) with an ∼1,000-fold decrease in hemolytic activity. Because functional differences are eventually dictated by structural differences, we determined three-dimensional structures of 65- and 50-kDa HlyA oligomers, using cryo-electron microscopy and single-particle methods. Our study clearly shows that the HlyA oligomer has sevenfold symmetry but that the HlyA50 oligomer is an asymmetric molecule. The HlyA oligomer has bowl-like, arm-like, and ring-like domains. The bowl-like domain is coupled with the ring-like domain, and seven side openings are present just beneath the ring-like domain. Although a central channel is present in both HlyA and HlyA50 oligomers, they differ in pore size as well as in shape of the molecules and channel. These structural differences may be relevant to the striking difference in efficiencies of functional channel formation by the two toxin forms.

Structural Role of rRNAs on the Ribosomal Subunits from E. Coli by Scanning Transmission Electron Microscopy

1981 ◽  
Vol 39 ◽  
pp. 424-425
Author(s):  
M. Boublik ◽  
N. Robakis ◽  
J.S. Wall
Keyword(s):  
Three Dimensional ◽  
Uranyl Acetate ◽  
Dimensional Structure ◽  
Electron Microscopic ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
Scanning Transmission ◽  
And Function

The three-dimensional structure and function of biological supramolecular complexes are, in general, determined and stabilized by conformation and interactions of their macromolecular components. In the case of ribosomes, it has been suggested that one of the functions of ribosomal RNAs is to act as a scaffold maintaining the shape of the ribosomal subunits. In order to investigate this question, we have conducted a comparative TEM and STEM study of the structure of the small 30S subunit of E. coli and its 16S RNA.The conventional electron microscopic imaging of nucleic acids is performed by spreading them in the presence of protein or detergent; the particles are contrasted by electron dense solution (uranyl acetate) or by shadowing with metal (tungsten). By using the STEM on freeze-dried specimens we have avoided the shearing forces of the spreading, and minimized both the collapse of rRNA due to air drying and the loss of resolution due to staining or shadowing. Figure 1, is a conventional (TEM) electron micrograph of 30S E. coli subunits contrasted with uranyl acetate.

scholarly journals The centromere-kinetochore complex: a repeat subunit model.

1991 ◽  
Vol 113 (5) ◽  
pp. 1091-1110 ◽  
Author(s):  
R P Zinkowski ◽  
J Meyne ◽  
B R Brinkley
Keyword(s):  
Three Dimensional ◽  
Protein Composition ◽  
Cytoplasmic Dynein ◽  
Dimensional Structure ◽  
Electron Microscopic ◽  
Microtubule Motor ◽  
A Subunit ◽  
Kinetochore Region ◽  
Subunit Organization

The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species.

scholarly journals Effects of ionizing radiations on proteins. Evidence of non-random fragmentations and a caution in the use of the method for determination of molecular mass

1990 ◽  
Vol 267 (2) ◽  
pp. 431-439 ◽  
Author(s):  
M Le Maire ◽  
L Thauvette ◽  
B de Foresta ◽  
A Viel ◽  
G Beauregard ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Molecular Mass ◽  
Three Dimensional ◽  
Water Soluble ◽  
Dimensional Structure ◽  
Intact Protein ◽  
Aspartate Transcarbamylase ◽  
Ionizing Radiations ◽  
Polypeptide Chains ◽  
Best Fit

We have reinvestigated the use of ionizing radiations to measure the molecular mass of water-soluble or membrane proteins. The test was performed by using the most straightforward aspect of the technique, which consists of SDS/PAGE analysis of the protein-fragmentation process. We found that exposure of purified standard proteins to increasing doses of ionizing radiation causes progressive fragmentation of the native protein into defined peptide patterns. The coloured band corresponding to the intact protein was measured on the SDS gel as a function of dose to determine the dose (D37.t) corresponding to 37% of the initial amount of unfragmented protein deposited on the gel. This led to a calibration curve between 1/D37.t and the known molecular mass of the standard proteins whose best fit gave Mr = 1.77 x 10(6)/D37.t at -78 degrees C, i.e. 35% higher than the generally accepted value at that temperature obtained from inactivation studies. However, we have to conclude that this method is useless to determine the state of aggregation of a protein, since, for all the oligomers tested, the best fit was obtained by using the protomeric molecular mass, suggesting that there is no energy transfer between promoters. Furthermore, SDS greatly increases the fragmentation rate of proteins, which suggests additional calibration problems for membrane proteins in detergent or in the lipid bilayer. But the main drawback of the technique arises from our observation that some proteins behaved anomalously, leading to very large errors in the apparent target size as compared with true molecular mass (up to 100%). It is thus unreliable to apply the radiation method for absolute molecular-mass determination. We then focused on the novel finding that discrete fragmentation of proteins occurs at preferential sites, and this was studied in more detail with aspartate transcarbamylase. N-Terminal sequencing of several radiolysis fragments of the catalytic chain of the enzyme revealed that breaks along the polypeptide chains are localized close to the C-terminal end. Examination of the three-dimensional structure of aspartate transcarbamylase suggests that radiolysis sites (fragile bonds) might be localized in connecting loops.

Topology models of anion exchanger 1 that incorporate the anti-parallel V-shaped motifs found in the EM structureThis paper is one of a selection of papers published in a Special Issue entitled CSBMCB 53rd Annual Meeting — Membrane Proteins in Health and Disease, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (2) ◽  
pp. 148-156 ◽  
Author(s):  
Teruhisa Hirai ◽  
Naotaka Hamasaki ◽  
Tomohiro Yamaguchi ◽  
Yohei Ikeda
Keyword(s):  
Anion Exchanger ◽  
Peer Review Process ◽  
Three Dimensional ◽  
Structural Similarity ◽  
Dimensional Structure ◽  
Membrane Bilayer ◽  
Anion Exchanger 1 ◽  
Advantages And Disadvantages ◽  
Health And Disease ◽  
Selection Of

We recently published the three-dimensional structure of the membrane domain of human erythrocyte anion exchanger 1 (AE1) at 7.5 Å resolution, solved by electron crystallography. The structure exhibited distinctive anti-parallel V-shaped motifs, which protrude from the membrane bilayer on both sides. Similar motifs exist in the previously reported structure of a bacterial chloride channel (ClC)-type protein. Here, we propose two topology models of AE1 that reflect the anti-parallel V-shaped structural motifs. One is assumed to have structural similarity with the ClC protein and the other is only assumed to have internal repeats, as is often the case with transporters. Both models are consistent with most topological results reported thus far for AE1, each having advantages and disadvantages.

Structural Differences in the Tracheal Tapetum of Diurnal Butterflies

1979 ◽  
Vol 34 (3-4) ◽  
pp. 284-287 ◽  
Author(s):  
Willi A. Ribi
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Receptor Cells ◽  
Screening Pigment ◽  
The Family ◽  
Tapetum Lucidum ◽  
Structural Differences ◽  
First Results ◽  
Physiological Differences

Abstract The three-dimensional structure of the tracheal tapetum lucidum, its reflection properties and the resulting eye glow hue were studied in members of the diurnal butterfly families Pieridae, Nymphalidae, Satyridae and Lycaenidae. Two main groups of tapeta can be distinguished by structural and physiological differences. Whereas in pierids the main tracheal trunk at the bottom of the rhabdom bifurcates into two side branches before bifurcating again more distally, the tracheal trunk in the members of the family Nymphalidae, Satyridae and Lycaenidae investigated first divides into four side brandies. A second bifurcation shortly after the first results in eight subbranches which are regularly arranged between adjoining receptor cells. The broad banded reflection colour from incidently illuminated tapetal structures (at the level of the first bifurcation) varies between and within families but does not change significantly within the same eye. Whereas in nymphalids, satyrids and lycaenids the eye glow hue corresponds with the colour of the tapetal reflection, in pierids it is dominated by the coloured receptor screening pigment.

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