scholarly journals Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis.

1994 ◽  
Vol 126 (2) ◽  
pp. 433-443 ◽  
Author(s):  
A McGough ◽  
M Way ◽  
D DeRosier
Keyword(s):  
Image Analysis ◽  
Smooth Muscle ◽  
Binding Sites ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Outer Face ◽  
Actin Binding Domain

The three-dimensional structure of actin filaments decorated with the actin-binding domain of chick smooth muscle alpha-actinin (alpha A1-2) has been determined to 21-A resolution. The shape and location of alpha A1-2 was determined by subtracting maps of F-actin from the reconstruction of decorated filaments. alpha A1-2 resembles a bell that measures approximately 38 A at its base and extends 42 A from its base to its tip. In decorated filaments, the base of alpha A1-2 is centered about the outer face of subdomain 2 of actin and contacts subdomain 1 of two neighboring monomers along the long-pitch (two-start) helical strands. Using the atomic model of F-actin (Lorenz, M., D. Popp, and K. C. Holmes. 1993. J. Mol. Biol. 234:826-836.), we have been able to test directly the likelihood that specific actin residues, which have been previously identified by others, interact with alpha A1-2. Our results indicate that residues 86-117 and 350-375 comprise distinct binding sites for alpha-actinin on adjacent actin monomers.

scholarly journals Evidence for a Conformational Change in Actin Induced by Fimbrin (N375) Binding

1997 ◽  
Vol 139 (2) ◽  
pp. 387-396 ◽  
Author(s):  
Dorit Hanein ◽  
Paul Matsudaira ◽  
David J. DeRosier
Keyword(s):  
Actin Filaments ◽  
Structural Changes ◽  
Three Dimensional ◽  
Binding Domain ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Binding Surface ◽  
Actin Binding Domain ◽  
Actin Structure ◽  
Signal Transduction Proteins

Fimbrin belongs to a superfamily of actin cross-linking proteins that share a conserved 27-kD actin-binding domain. This domain contains a tandem duplication of a sequence that is homologous to calponin. Calponin homology (CH) domains not only cross-link actin filaments into bundles and networks, but they also bind intermediate filaments and some signal transduction proteins to the actin cytoskeleton. This fundamental role of CH domains as a widely used actin-binding domain underlines the necessity to understand their structural interaction with actin. Using electron cryomicroscopy, we have determined the three-dimensional structure of F-actin and F-actin decorated with the NH2-terminal CH domains of fimbrin (N375). In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. In addition, a fit of the atomic model for the actin filament to the maps reveals the actin residues that line, the binding surface. The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375. This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself. Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.

scholarly journals Disruption of the utrophin–actin interaction by monoclonal antibodies and prediction of an actin-binding surface of utrophin

1998 ◽  
Vol 337 (1) ◽  
pp. 119-123 ◽  
Author(s):  
Glenn E. MORRIS ◽  
Nguyen thi MAN ◽  
Nguyen thi Ngoc HUYEN ◽  
Alexander PEREBOEV ◽  
John KENDRICK-JONES ◽  
...  
Keyword(s):  
Binding Sites ◽  
Three Dimensional ◽  
Binding Domain ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Functional Regions ◽  
Binding Surface ◽  
Actin Binding Domain ◽  
Actin Binding Site ◽  
Helical Region

Monoclonal antibody (mAb) binding sites in the N-terminal actin-binding domain of utrophin have been identified using phage-displayed peptide libraries, and the mAbs have been used to probe functional regions of utrophin involved in actin binding. mAbs were characterized for their ability to interact with the utrophin actin-binding domain and to affect actin binding to utrophin in sedimentation assays. One of these antibodies was able to inhibit utrophin–F-actin binding and was shown to recognize a predicted helical region at residues 13–22 of utrophin, close to a previously predicted actin-binding site. Two other mAbs which did not affect actin binding recognized predicted loops in the second calponin homology domain of the utrophin actin-binding domain. Using the known three-dimensional structure of the homologous actin-binding domain of fimbrin, these results have enabled us to determine the likely orientation of the utrophin actin-binding domain with respect to the actin filament.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

scholarly journals Three-dimensional structure of actin filaments and of an actin gel made with actin-binding protein.

1983 ◽  
Vol 96 (5) ◽  
pp. 1400-1413 ◽  
Author(s):  
R Niederman ◽  
P C Amrein ◽  
J Hartwig
Keyword(s):  
Actin Filaments ◽  
Binding Protein ◽  
Three Dimensional ◽  
Actin Binding ◽  
Dimensional Structure ◽  
Molar Ratio ◽  
Computer Assisted ◽  
Three Dimensional Structure ◽  
Actin Binding Protein ◽  
Critical Point Drying

Purified muscle actin and mixtures of actin and actin-binding protein were examined in the transmission electron microscope after fixation, critical point drying, and rotary shadowing. The three-dimensional structure of the protein assemblies was analyzed by a computer-assisted graphic analysis applicable to generalized filament networks. This analysis yielded information concerning the frequency of filament intersections, the filament length between these intersections, the angle at which filaments branch at these intersections, and the concentration of filaments within a defined volume. Purified actin at a concentration of 1 mg/ml assembled into a uniform mass of long filaments which overlap at random angles between 0 degrees and 90 degrees. Actin in the presence of macrophage actin-binding protein assembled into short, straight filaments, organized in a perpendicular branching network. The distance between branch points was inversely related to the molar ratio of actin-binding protein to actin. This distance was what would be predicted if actin filaments grew at right angles off of nucleation sites on the two ends of actin-binding protein dimers, and then annealed. The results suggest that actin in combination with actin-binding protein self-assembles to form a three-dimensional network resembling the peripheral cytoskeleton of motile cells.

scholarly journals Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments.

1988 ◽  
Vol 106 (3) ◽  
pp. 805-812 ◽  
Author(s):  
H L Yin ◽  
K Iida ◽  
P A Janmey
Keyword(s):  
Human Plasma ◽  
Binding Sites ◽  
Actin Filaments ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Assembly ◽  
Plasma Gelsolin ◽  
High Affinity ◽  
Binding Data ◽  
Actin Binding Domain

Gelsolin is a Ca2+- and polyphosphoinositide-modulated actin-binding protein which severs actin filaments, nucleates actin assembly, and caps the "barbed" end of actin filaments. Proteolytic cleavage analysis of human plasma gelsolin has shown that the NH2-terminal half of the molecule severs actin filaments almost as effectively as native gelsolin in a Ca2+-insensitive but polyphosphoinositide-inhibited manner. Further proteolysis of the NH2-terminal half generates two unique fragments (CT14N and CT28N), which have minimal severing activity. Under physiological salt conditions, CT14N binds monomeric actin coupled to Sepharose but CT28N does not. In this paper, we show that CT28N binds stoichiometrically and with high affinity to actin subunits in filaments, suggesting that it preferentially recognizes the conformation of polymerized actin. Analysis of the binding data shows that actin filaments have one class of CT28N binding sites with Kd = 2.0 X 10(-7) M, which saturates at a CT28N/actin subunit ratio of 0.8. Binding of CT28N to actin filaments is inhibited by phosphatidylinositol 4,5-bisphosphate micelles. In contrast, neither CT14N nor another actin-binding domain located in the COOH-terminal half of gelsolin form stable stoichiometric complexes with actin along the filaments, and their binding to actin monomers is not inhibited by PIP2. Based on these observations, we propose that CT28N is the polyphosphoinositide-regulated actin-binding domain which allows gelsolin to bind to actin subunits within a filament before serving.

Actin Filaments Decorated with Cytoskeletal Proteins

1998 ◽  
Vol 4 (S2) ◽  
pp. 458-459
Author(s):  
D. Hanein ◽  
S. Goldsmith ◽  
W. Lehman ◽  
R. Craig ◽  
I. Correia ◽  
...  
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Structural Systems ◽  
Calponin Homology Domain ◽  
Flexible Molecules ◽  
Actin Binding Domain

A superfamily of actin crosslinking proteins, the calponin-homology domain (CH) family is implicated in directing the assembly and controlling the organization of the cytoskeleton through a highly conserved actin binding domain (ABD). This domain, composed of a tandem pair of CH motifs, is found in actin crosslinking proteins such as fimbrin, alpha-actinin, spectrin, and dystrophin. In addition to its role in crosslinking actin filaments, a putative homologous ABD in intermediate filament-binding proteins, plectin, dystonin, and BPAGlnl, suggests a possible role in crosslinks between desmosomal and vimentin intermediate filaments and actin. Thus, the CH domain could integrate the functions of the actin and IF cytoskeletons by physically linking these structural systems. We present an atomic model for the complex of the CH domain and actin.In crosslinking proteins that organize actin into two and three dimensional networks or gels the ABDs lie at the ends of long, flexible molecules.

Electron Microscopy of Cytoplasmic Contractile Proteins

1978 ◽  
Vol 36 (3) ◽  
pp. 606-614 ◽  
Author(s):  
T.D. Pollard ◽  
P. Maupin
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Uranyl Acetate ◽  
Contractile Proteins ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Cytoplasmic Matrix ◽  
Computer Image Processing ◽  
Nonmuscle Cells

In this paper we review some of the contributions that electron microscopy has made to the analysis of actin and myosin from nonmuscle cells. We place particular emphasis upon the limitations of the ultrastructural techniques used to study these cytoplasmic contractile proteins, because it is not widely recognized how difficult it is to preserve these elements of the cytoplasmic matrix for electron microscopy. The structure of actin filaments is well preserved for electron microscope observation by negative staining with uranyl acetate (Figure 1). In fact, to a resolution of about 3nm the three-dimensional structure of actin filaments determined by computer image processing of electron micrographs of negatively stained specimens (Moore et al., 1970) is indistinguishable from the structure revealed by X-ray diffraction of living muscle.

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