Identification of the binding site of two monoclonal antibodies to human protamine

Denzin, Lisa K.; Gulliver, Gene A.; Voss, Edward W.

doi:10.1016/0161-5890(93)90094-r

Cited by 24 publications

(13 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Antibody͞hapten recognition in the 4-4-20͞fluorescein model system used in this study has been characterized extensively by structural (11,12), thermodynamic (13), kinetic (14), computational (15), spectroscopic (16), and mutagenic (17) means. The 4-4-20 affinity for the fluorescein-biotin (FL-bio) hapten (K d ϭ 0.7 Ϯ 0.3 nM in PBS) is near the affinity ceiling of the tertiary immune response.…”

mentioning

confidence: 99%

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

Boder

Midelfort

Wittrup

2000

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

620

437

View full text Add to dashboard Cite

Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant Kd ‫؍‬ 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10 5 -10 7 yeast surfacedisplayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wildtype complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

show abstract

mentioning

confidence: 99%

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

Boder

Midelfort

Wittrup

2000

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

620

437

View full text Add to dashboard Cite

show abstract

“…To satisfy these criteria, the Fv derivative of mAb 4-4-20, a high affinity murine anti-fluorescein antibody, has been synthesized. mAb 4-4-20 was a suitable antibody for this study due to its high degree of structural characterization (7,33,34) and the previous construction and characterization of SCA 4-4-20 (7,35). SCA 4-4-20 has been studied extensively in terms of active site environment (36,37), antigen binding structure (13), and thermodynamics (8,38).…”

mentioning

confidence: 99%

Comparative Properties of the Single Chain Antibody and Fv Derivatives of mAb 4-4-20

Mallender¹,

Carrero

Voss

1996

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

and light chain (V L ) variable domains. These two domains associate noncovalently to form the smallest functional antibody protein capable of antigen binding that most closely approximates the Ig molecule (1, 2). These proteins have been previously found to be less stable in terms of domain-domain association than Fab fragments due to the lack of covalent bonds between the two variable domains (3, 4). Single chain antibody (SCA) molecules have been produced to diminish this instability by the introduction of an interdomain linker peptide (5, 6). SCA proteins often mimic the parent antibody active site in terms of antigen binding and structural properties with usually some reduction in affinity for antigen (7-9). Recently, Fv proteins have been engineered to possess an interdomain disulfide linkage, effectively disallowing dissociation of the two domains (10). Due to their small size and amenability to genetic engineering, recombinant Fv proteins have been widely applied in the study of antibody active site structure-function (7, 11-13), idiotypy and metatypy (14 -17), antibody bivalency and bispecificity (18 -21), and in vivo immunodiagnostics and therapy (10,22,23).Fv molecules have been efficacious proteins in the study of antibody active site structure-function and protein stability. Studies involving comparative analysis of Fv protein with other immunoglobulin constructs afford unique opportunities for determining domain-domain interactions and the effects these interactions exert upon the intrinsic conformational and antigen binding properties of the variable domains. Being covalently coupled by an interdomain linker, SCA proteins have been suggested to possess greater interdomain stability than their Fv counterparts due to the favorable entropic effect of domain coupling (6,8). This would, in turn, suggest that in the appropriate Fv molecule (one with high affinity for antigen), interdomain associative properties would dictate the overall affinity displayed for antigen because only associated V L /V H proteins would bind antigen. In previous studies, dissociation constants for the V L /V H association in Fv molecules varied from 10 Ϫ7 to Ͼ10 Ϫ9 M (3, 24 -26). These Fv molecules also displayed similar dissociation constants for their respective antigens (10 Ϫ7 to Ͼ10 Ϫ9 M), further supporting some correlation between interdomain and active site/antigen interactions. Further analysis of V L /V H association constants in relation to antigen affinity would allow identification of components necessary for the production of stable Fv molecules and novel variable domain proteins.Fv molecules have been especially useful in the study of idiotypy and metatypy. Antibody idiotype and metatype are immunologically resolved markers of active site structural and conformational determinants in the unliganded and liganded state, respectively (review in Refs. 27 and 28). Indeed, the transition between the idiotypic and metatypic states upon ligand binding emphasizes the dynamic properties of antibody

show abstract

“…The formation of the high stability antibody‐fluorescein complex indicates that the dominant mechanism for the observed fluorescence suppression is static quenching. A charge‐transfer process that involves an aromatic amino acid side chain located nearby bound fluorescein was suggested in the early studies of anti‐fluorescein antibodies and propagated into further anti‐fluorescein antibody publications . Later, it was defined as an ultrafast photo‐induced electron transfer that occurs in the binding site of the anti‐fluorescein mAb 4‐4‐20 and involves Tyr37 L as the donor and fluorescein as the acceptor .…”

Section: Discussionmentioning

confidence: 99%

“…In the 1980s, several anti‐fluorescein monoclonal antibodies were produced and characterized . Two of these, mAbs 4‐4‐20 and 9‐40, were later crystallized and methodically studied with biophysical methods and site‐directed mutagenesis …”

Section: Introductionmentioning

confidence: 99%

Three‐dimensional structure, binding, and spectroscopic characteristics of the monoclonal antibody 43.1 directed to the carboxyphenyl moiety of fluorescein

et al. 2016

View full text Add to dashboard Cite

Unlike other known anti-fluorescein antibodies, the monoclonal antibody 43.1 is directed toward the fluorescein's carboxyl phenyl moiety. It demonstrates a very high affinity (KD ∼ 70 pM) and a fast association rate (kon ∼ 2 × 10(7) M(-1 ) s(-1) ). The three-dimensional structure of the Fab 43.1-fluorescein complex was resolved at 2.4 Å resolution. The antibody binding site is exclusively assembled by the CDR loops. It is comprised of a 14 Å groove-shaped entrance leading to a 9 Å by 7 Å binding pocket. The highly polar binding pocket complementary encloses the fluorescein's carboxyphenyl moiety and tightly fixes it by multiple hydrogen bonds. The fluorescein's xanthene ring is embedded in the more hydrophobic groove and stacked between the side chains of Tyr37L and of Arg99H providing conditions for an excited state electron transfer process. In comparison to fluorescein, the absorption spectrum of the complex in the visible region is shifted to the "red" by 23 nm. The complex demonstrates a very weak fluorescence (Φc = 0.0018) with two short lifetime components: 0.03 ns (47%) and 0.8 ns (24%), which reflects a 99.8% fluorescein emission quenching effect upon complex formation. The antibody 43.1 binds fluorescein with remarkable affinity, fast association rate, and strongly quenches its emission. Therefore, it may present a practical interest in applications such as molecular sensors and switches.

show abstract

Identification of the binding site of two monoclonal antibodies to human protamine

Cited by 24 publications

References 50 publications

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

Comparative Properties of the Single Chain Antibody and Fv Derivatives of mAb 4-4-20

Three‐dimensional structure, binding, and spectroscopic characteristics of the monoclonal antibody 43.1 directed to the carboxyphenyl moiety of fluorescein

Contact Info

Product

Resources

About