Mechanisms of Antibody Binding to a Protein

Getzoff, E. D.; Geysen, H. Mario; Rodda, Stuart J.; Alexander, Hannah; Tainer, John A.; Lerner, R.A.

doi:10.1126/science.3823879

Cited by 199 publications

(81 citation statements)

References 43 publications

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“…Another possibility is main chain elements involved in some unusual turn or chain reversal within the amyloid motif. Detailed structures of the epitopes of anti-protein Abs are normally characterized either by protein crystallography (42) or by mutational analysis of antigen fragments (43) or intact protein (44). Because amyloid has yet to be crystallized, and because WO1 and WO2 bind to many amyloids regardless of amino acid sequence, it is clear that the further structural analysis of the WO1͞WO2 epitope(s) will be challenging.…”

Section: Resultsmentioning

confidence: 99%

Conformational Abs recognizing a generic amyloid fibril epitope

O’Nuallain

Wetzel

2002

Proc. Natl. Acad. Sci. U.S.A.

307

304

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Disease-related amyloid fibrils appear to share a common, but poorly understood, structure. We describe here the generation and preliminary characterization of two conformation-specific mAbs, WO1 and WO2, that bind to the amyloid fibril state of the Alzheimer's peptide A␤(1-40) but not to its soluble, monomeric state. Surprisingly, these Abs also bind to other disease-related amyloid fibrils and amyloid-like aggregates derived from other proteins of unrelated sequence, such as transthyretin, islet amyloid polypeptide, ␤2-microglobulin, and polyglutamine. At the same time, WO1 and WO2 do not bind to the native protein precursors of these amyloids, nor do they bind to other kinds of protein aggregates. This new class of Abs associated with a fundamental amyloid-folding motif appear to recognize a common conformational epitope with little apparent dependence on amino acid side chain information. These Abs should contribute to the understanding of amyloid structure, assembly, and toxicity and also may benefit the development of diagnostic and therapeutic agents for amyloid diseases.A myloid fibrils are highly insoluble, ordered protein aggregates involved in a number of human diseases (1, 2), including Alzheimer's disease (3) and type II diabetes (4). Although the protein components of amyloid fibrils from various disease states differ considerably from each other in primary sequence, all amyloid fibrils share common features, including a high degree of ␤-sheet in a classical ''cross-␤'' pattern, a fibrillar morphology in electron microscopy, and the ability to bind and alter the spectroscopic properties of heteroaromatic dyes Congo red and thioflavin T (ThT) (5, 6). Although these common properties suggest that amyloid fibrils must share deeper similarities at the molecular level, the extent of similarity between the polypeptide-folding patterns of different amyloids is unknown. Details of the nature of the amyloid fold remain obscure because of technical limitations to obtaining high resolution structural information on large, insoluble, heterodisperse aggregates.Although mAbs have previously proved useful in the structural analysis of globular proteins, their use in the characterization of amyloid fibril structure has been limited. Most of the anti-fibril Abs generated in an immune response to fibrils tend to be directed at unstructured portions of the amyloidogenic peptide not involved in fibril structure. In a recent characterization of the Ab response in mice injected with amyloid ␤ protein (A␤) fibrils, it was found that the majority of the Abs are directed at the N-terminal 12 residues of the peptide and are capable of crossreacting strongly with the monomeric peptide (7). This agrees well with the results of limited proteolysis studies of A␤ fibrils indicating an exposed, unstructured N-terminal region in the aggregate (8). Thus, such Abs tell us about those parts of the amyloidogenic peptide that are not involved in fibril structure but little about the nature of fibril structure itself.Identification of c...

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Section: Resultsmentioning

confidence: 99%

Conformational Abs recognizing a generic amyloid fibril epitope

O’Nuallain

Wetzel

2002

Proc. Natl. Acad. Sci. U.S.A.

307

304

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“…It is of interest to know whether the conformational change adopted by a loop (a side chain, ligand, etc.) upon binding has been induced by the other protein (induced fit 5,6 ) or alternatively the free loop already interconverts among different microstates where one of them is selected upon binding (selected fit 7 ). This analysis requires calculating p m values, which are also needed for a correct analysis of NMR and X-ray data of flexible macromolecules.…”

Section: I1 the Role Of Free Energy In Structural Biologymentioning

confidence: 99%

Stability of the Free and Bound Microstates of a Mobile Loop of α-Amylase Obtained from the Absolute Entropy and Free Energy

Cheluvaraja

Meirovitch

2007

J. Chem. Theory Comput.

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Abstract:The hypothetical scanning molecular dynamics (HSMD) method is a relatively new technique for calculating the absolute entropy, S, and free energy, F, from a given sample generated by any simulation procedure. Thus, each sample conformation, i, is reconstructed by calculating transition probabilities that their product leads to the probability of i, hence to the entropy. HSMD is an exact method where all interactions are considered, and the only approximation is due to insufficient sampling. In previous studies HSMD (and HS Monte Carlo -HSMC) has been applied very successfully to liquid argon, TIP3P water, self-avoiding walks, and peptides in a R-helix, extended, and hairpin microstates. In this paper HSMD is developed further as applied to the flexible 7-residue surface loop, 304-310 (Gly-His-Gly-Ala-Gly-GlySer) of the enzyme porcine pancreatic R-amylase. We are mainly interested in entropy and free energy differences ∆S ) S free -S bound (and ∆F)F free -F bound ) between the free and bound microstates of the loop, which are obtained from two separate MD samples of these microstates without the need to carry out thermodynamic integration. As for peptides, we find that relatively large systematic errors in S free and S bound (and F free and F bound ) are cancelled in ∆S (∆F) which is thus obtained efficiently with high accuracy, i.e., with a statistical error of 0.1-0.2 kcal/mol (T)300 K) using the AMBER force field and AMBER with the implicit solvation GB/SA. We provide theoretical arguments in support of this cancellation, discuss in detail the problems involved in the computational definition of a microstate in conformational space, suggest potential ways for enhancing efficiency further, and describe the next development where explicit water will replace implicit solvation.

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“…This indicated that deformation of the antigen induced by binding to the trapping antibody influenced recognition by monoclonal antibody. Structural differences induced by antibody binding have been demonstrated in other protein systems (Getzoff et al, 1987). SB2 provided a negative control because no differentiation was evident between top, middle or bottom components in the affinity assay or the liquid phase competition assay.…”

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confidence: 99%

Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

Kalmar

Eastwell

1989

Journal of General Virology

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SUMMARYRabbit antisera produced against two comoviruses (cowpea mosaic virus and cowpea severe mosaic virus) were used in plate-trapped ELISA and liquid phase competition ELISA. In the latter, the top component competed against bound unfractionated virus more effectively than did nucleoprotein components. One murine monoclonal antibody elicited to cowpea mosaic virus and three monoclonal antibodies to cowpea severe mosaic virus exhibited differential binding to top component, relative to either middle or bottom components in plate-trapped or antibody-trapped ELISAs. Differential binding to centrifugal components of virus by two of these monoclonal antibodies was maintained in liquid phase competition assays, These data suggest that the encapsidation of RNA alters the configuration of the virion surface in the vicinity of the antibody epitopes. Ribonuclease digestion of intact or denatured virus did not affect the binding of monoclonal antibodies.Like other comoviruses, cowpea mosaic virus (CPMV) and cowpea severe mosaic virus (CPSMV) are resolved into three major fractions by density gradient centrifugation (Bruening, 1969). The slowest sedimenting top component consists of empty capsids whereas the middle and bottom components are nucleocapsids containing one molecule of either RNA 2 (Mr 1"37 x 106) or RNA 1 (Mr 2.02 x 106), respectively. PAGE does not show differences between the protein composition of the three components (Geelen et al., 1972;Wu & Bruening, 1971) and rabbit antisera prepared against unfractionated virus reacts with all components in Ouchterlony double-diffusion assays (Bruening & Agrawal, 1967). This paper reports that liquid phase competition assays utilizing rabbit antisera reveal a difference between the binding of antibodies to empty capsids and to nucleoprotein virions. Antigenic differences between top component and middle or bottom components were confirmed by assays with monoclonal antibodies.CPSMV-DG and CPMV-SB isolates were originally obtained from G. Bruening (Department of Plant Pathology, University of California, Davis, Ca., U.S.A.), propagated in cowpeas (Vigna unguiculata Walp. cv. California Black Eye) and isolated as previously described (Bruening, 1969). Approximately 5 mg virus was suspended in 0-5 ml gradient buffer (50 mM-KH2PO, pH 7.0, 2 mM-disodium EDTA) and layered onto 11 ml of 39 ~ (w/w) caesium chloride in gradient buffer. Gradients were centrifuged for 36 h at 36000 r.p.m, and 4 °C in an SW 40Ti rotor (Beckman) and fractionated by side-puncture. Fractions were diluted at least 10-fold in gradient buffer and pelleted by centrifugation at 45000r.p.m. for 2h. The pellets were resuspended in gradient buffer. Concentrations of virus components were calculated using A260 at 1 mg/ml of 6.2, 10-0 and 8.1 for middle component, bottom component and unfractionated virus and A2so at 1 mg/ml of 1.28 for top component (Geelen et al., 1972).The preparation of rabbit antisera and of the panel of monoclonal antibodies produced by hybridomas have been described (Kalmar & Eastwell,...

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Mechanisms of Antibody Binding to a Protein

Cited by 199 publications

References 43 publications

Conformational Abs recognizing a generic amyloid fibril epitope

Conformational Abs recognizing a generic amyloid fibril epitope

Stability of the Free and Bound Microstates of a Mobile Loop of α-Amylase Obtained from the Absolute Entropy and Free Energy

Serological Differentiation between Top Component and Nucleoprotein Components of Comoviruses

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