The Importance of Conformation and of Equilibria in the Interaction of Globular Proteins and their Fragments with Antibodies

Crumpton, Michaël J.

doi:10.1002/9780470513286.ch6

Cited by 3 publications

(1 citation statement)

References 21 publications

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“…Antibodies can distinguish native proteins from denatured ones, and in recent years the conformational specificity of antibodies has been unambiguously established in a number of systems~for a review see Crumpton, 1986!. The pioneering work of Crumptoñ 1966!…”

mentioning

confidence: 99%

Antibody‐detected folding: Kinetics of surface epitope formation are distinct from other folding phases

2000

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The rate of macromolecular surface formation in yeast iso-2 cytochrome c and its site-specific mutant, N52I iso-2, has been studied using a monoclonal antibody that recognizes a tertiary epitope including K58 and H39. The results indicate that epitope refolding occurs after fast folding but prior to slow folding, in contrast to horse cytochrome c where surface formation occurs early. The antibody-detected~ad! kinetic phase accompanying epitope formation has k ad ϭ 0.2 s Ϫ1 and is ;40-fold slower than the fastest detectable event in the folding of yeast iso-2 cytochrome c~k 2f ; 8 s Ϫ1 !, but occurs prior to the absorbance-and fluorescence-detected slow folding steps~k 1a ; 0.06 s Ϫ1 ; k 1b ; 0.09 s Ϫ1 !. N52I iso-2 cytochrome c exhibits similar kinetic behavior with respect to epitope formation. A detailed dissection of the mechanistic differences between the folding pathways of horse and yeast cytochromes c identifies possible reasons for the slow surface formation in the latter. Our results suggest that non-native ligation involving H33 or H39 during refolding may slow down the formation of the tertiary epitope in iso-2 cytochrome c. This study illustrates that surface formation can be coupled to early events in protein folding. Thus, the rate of macromolecular surface formation is fine tuned by the residues that make up the surface and the interactions they entertain during refolding.

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confidence: 99%

Antibody‐detected folding: Kinetics of surface epitope formation are distinct from other folding phases

2000

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The use of Synthetic Peptides in the Delineation of Immunoglobulin Antigenic Epitopes and Fc Effector Functions

Stanworth

Burt

Hastings

2007

Novartis Foundation Symposia

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Localization of antigenic domains on the major subunits of Bordetella pertussis serotype 2 and 3 fimbriae

et al. 1994

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Antibody-binding domains on the major subunits of Bordetella pertussis serotype 2 (Fim2) and 3 fimbriae (Fim3) have been identified using synthetic peptides which were screened for recognition by anti-protein monoclonal antibodies (mAbs). The presence of non-contiguous f imbrial epitopes was demonstrated by both anti-Fim2 and anti-Fim3 mAbs, several of which recognized at least two peptides that were discontinuous in the amino acid sequence of the corresponding subunits. The specificity of one mAb, 51/24, directed against Fim2, was investigated by replacement-set analysis of a 10-residue peptide, and revealed that antibody binding to the peptide was dependent on the sequence NWPQs6 which is non-conserved in Fim3. Furthermore, proline at residue 95 was found to be essential for mAb 51/24 binding. The specific anti-Fim3 mAb, AG3A, was found to recognize the 10-residue carboxy-terminal peptide from both Fim3 and, unexpectedly, from Fim2. This result suggests that mAb AG3A serospecificity a t the protein level is determined by a conf ormational constraint which prevents mAb AG3A binding to the Fim2 C-terminal domain. Several free peptides containing amino acid residues which comprise part of the Fim2 and Fim3 epitopic domains were prepared as immunogens. One of these peptides was immunogenic in the mouse, indicating the location of a T-helper cell epitope within the peptide sequence, and induced a strong anti-peptide antibody response. The other peptides each required immunization as a conjugate with a carrier protein for anti-peptide antibody stimulation. All four anti-peptide antibody preparations only weakly recognized f imbriae-coated ELISA plates. The results of this investigation demonstrate that although short linear peptides can mimic sub-domains of non-contiguous f imbrial epitopes, they are, however, poor candidate antigens for stimulating an anti-f imbrial antibody response.

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The Importance of Conformation and of Equilibria in the Interaction of Globular Proteins and their Fragments with Antibodies

Cited by 3 publications

References 21 publications

Antibody‐detected folding: Kinetics of surface epitope formation are distinct from other folding phases

Antibody‐detected folding: Kinetics of surface epitope formation are distinct from other folding phases

The use of Synthetic Peptides in the Delineation of Immunoglobulin Antigenic Epitopes and Fc Effector Functions

Localization of antigenic domains on the major subunits of Bordetella pertussis serotype 2 and 3 fimbriae

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