scholarly journals Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding sites on the β-chain of human haemoglobin by synthetic overlapping peptides encompassing the entire chain

1986 ◽  
Vol 234 (2) ◽  
pp. 453-456 ◽  
Author(s):  
N Yoshioka ◽  
M Z Atassi
Keyword(s):  
Binding Sites ◽  
Solid Phase ◽  
Three Dimensional ◽  
Binding Activity ◽  
Dimensional Structure ◽  
Synthetic Approach ◽  
Protein Chain ◽  
Beta Chain ◽  
Human Haemoglobin ◽  
Β Chain

A synthetic approach was employed to identify the haptoglobin-binding sites on the beta-chain of human haemoglobin. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen synthetic peptides (beta 1-15, beta 11-25 etc.) were examined for their ability to bind human haptoglobin by quantitative solid-phase radiometric titrations of 125I-labelled haptoglobin. Of these 14 peptides only peptides beta 11-25 and beta 131-146 bound haptoglobin significantly; peptide beta 21-35 exhibited a small binding activity as a consequence of the overlap with peptide beta 11-25. On this basis and by examination of the three-dimensional structure of haemoglobin, it was concluded that the beta-chain of haemoglobin has two binding sites for haptoglobin that reside in, but do not necessarily encompass all of, the regions beta 11-25 and beta 131-146.

scholarly journals Antigenic structure of human haemoglobin. Localization of the antigenic sites of the β-chain in three host species by synthetic overlapping peptides representing the entire chain

1986 ◽  
Vol 234 (2) ◽  
pp. 441-447 ◽  
Author(s):  
N Yoshioka ◽  
M Z Atassi
Keyword(s):  
Host Species ◽  
Structural Characteristics ◽  
Three Dimensional ◽  
Antigenic Structure ◽  
Synthetic Approach ◽  
Immune Recognition ◽  
Beta Chain ◽  
Human Haemoglobin ◽  
Antigenic Sites ◽  
Full Profile

A comprehensive synthetic approach is applied here to localize the continuous antigenic sites of the beta-chain of haemoglobin. The approach was based on the synthesis and purification of the following consecutive 15-residue peptides (each overlapping by five residues at both ends with the peptides preceding it and following it in the sequence): 1-15, 11-25 etc. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labelled anti-haemoglobin antibodies from three different host species. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition studies and by the binding specificity of antibodies isolated from peptide adsorbents. These studies established the full profile of antigenic beta-chain regions, which was found to be independent of the host species. Five major antigenic sites were localized, and their three-dimensional and structural characteristics are discussed in relation to the immune recognition of haemoglobin and other proteins.

scholarly journals Subunit interacting surfaces of human haemoglobin. Localization of the α-subunit-β-subunit interacting surfaces on the β-chain by a comprehensive synthetic strategy

1986 ◽  
Vol 234 (2) ◽  
pp. 457-461 ◽  
Author(s):  
N Yoshioka ◽  
M Z Atassi
Keyword(s):  
Binding Capacity ◽  
Binding Activity ◽  
The Other ◽  
Synthetic Approach ◽  
Beta Chain ◽  
Human Haemoglobin ◽  
Α Subunit ◽  
Alpha Chain ◽  
Synthetic Strategy ◽  
Interacting Surfaces

A synthetic approach is introduced for localization of subunit interacting surfaces in oligomeric proteins. It consists of studying the binding activity of consecutive uniform overlapping peptides encompassing an entire subunit to the other, radiolabelled, subunit. This permits the establishment of the full profile of peptides that bind the other intact subunit. This approach has been demonstrated with haemoglobin, and its application here with the beta-chain peptides has enabled the localization on the beta-chain of the submolecular regions responsible for its binding to alpha-chain in solution. There was good agreement between the binding surfaces found here in solution and those expected from the crystal structure. There were also, however, some significant differences in the levels of binding found in solution and those expected from the crystal. Peptide 21-35 possessed much higher binding activity than would be expected from its contribution to subunit association in the crystal. Conversely, other regions expected to possess considerable binding capacity for alpha-chain either showed low (peptides 111-125 and 121-135) or almost no binding (peptides 91-105 and 101-115) capacity. On the other hand, two interacting surfaces (within peptides 11-25 and 71-85) that make a contribution in solution do not appear to play a role in the crystal. It is concluded that the regions of subunit association in solution are close to, but not identical with, those in the crystal. The approach should serve as an effective method for localization of subunit interacting surfaces of unknown proteins, even those that can be isolated only in traces.

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.

Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

1998 ◽  
Vol 79 (01) ◽  
pp. 104-109 ◽  
Author(s):  
Osamu Takamiya
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Human Factor ◽  
Solid Phase ◽  
Three Dimensional ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Dimensional Structure ◽  
Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

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.

The Construction of New Proteins. II. Design, Synthesis and Conformational Studies of Folding Units with βαβ-Topology

1986 ◽  
Vol 41 (10) ◽  
pp. 1315-1322 ◽  
Author(s):  
Manfred Mutter ◽  
Karl-Heinz Altmann ◽  
Thomas Vorherr
Keyword(s):  
Solid Phase ◽  
Three Dimensional ◽  
Chemical Properties ◽  
Spectroscopic Studies ◽  
General Concept ◽  
Dimensional Structure ◽  
Design Synthesis ◽  
Phase Synthesis ◽  
Conformational Studies ◽  
Conformational Effects

The design, synthesis and preliminary conformational studies of two polypeptides exhibiting βαβ-type folding topologies are presented. In the design of the model peptides the general concept for the construction of new proteins developed in the preceeding paper was applied. According to this strategy, amphiphilic helices and β-sheets are linked together via hydrophilic loops to attain three-dimensional structures of higher order (‘supersecondary structures’). Com­puter-assisted molecular modelling served as a valuable tool for minimizing conformational con­straints within the molecules. The 38-residue peptide MI was synthesized using polyethylene glycol (PEG) as solubilizing polymeric support (‘Liquid-Phase synthesis'). Conformationally in­duced changes in the physico-chemical properties of the growing peptide chain stressed the significance of conformational effects in peptide synthesis reported earlier. Similar observations were made during the solid-phase synthesis of the 35-peptide MII. CD and IR spectroscopic studies revealed a high degree of secondary structure for both folding units. The present data strongly support the adoption of a three-dimensional structure for both models.

scholarly journals Structurally inherent antigenic sites. Localization of the antigenic sites of the α-chain of human haemoglobin in three host species by a comprehensive synthetic approach

1982 ◽  
Vol 203 (1) ◽  
pp. 201-208 ◽  
Author(s):  
A L Kazim ◽  
M Z Atassi
Keyword(s):  
Primary Structure ◽  
Host Species ◽  
Three Dimensional ◽  
Antigenic Structure ◽  
Synthetic Approach ◽  
Human Haemoglobin ◽  
Alpha Chain ◽  
Antigenic Sites ◽  
Full Profile ◽  
Related Proteins

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major ‘continuous’ antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of ‘structurally inherent antigenic sites’, namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.

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.

A structural proteomics filter: prediction of the quaternary structural type of hetero-oligomeric proteins on the basis of their sequences

2007 ◽  
Vol 40 (6) ◽  
pp. 986-989 ◽  
Author(s):  
Oliviero Carugo
Keyword(s):  
Structural Biology ◽  
Three Dimensional ◽  
Structural Type ◽  
Computational Method ◽  
Structural Proteomics ◽  
Dimensional Structure ◽  
Protein Chain ◽  
Oligomeric Proteins ◽  
Single Protein ◽  
A Chain

A protein chain can correspond to a monomeric protein or it can form, together with other chains, oligomeric assemblies, which can be either homo-oligomers or hetero-oligomers. In the latter case, the three-dimensional structure of the single protein chain is unlikely to be determined, since it will probably be difficult to express and crystallize. A computational method is presented here that allows one to predict if a chain participates in hetero-oligomeric assemblies, on the basis of its amino acid composition, with accuracy close to 80%. Such a technique should improve the success rate of structural biology projects.

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