Domain interactions in antibody Fv and scFv fragments: effects on unfolding kinetics and equilibria.


Abstract

The equilibrium denaturation and unfolding kinetics of the domains V(L) and V(H) have been compared with those of the Fv and single-chain Fv (scFv) fragment of an engineered variant of the antibody McPC603 in the presence and absence of the antigen phosphorylcholine. The scFv fragment is significantly more stable than the isolated constituting domains. Antigen binding stabilizes the heterodimeric assembly even further. Domain dissociation and domain unfolding are coupled processes, giving rise to a highly cooperative unfolding transition. For the Fv fragment, cooperative unfolding is only observed in the presence of antigen. At low protein concentrations and in the absence of antigen, the Fv fragment is significantly destabilized, leading to quantitative domain dissociation before significant domain unfolding takes place. The kinetic unfolding of V(H), V(L) and the scFv fragment is monophasic. Unfolding of the scFv fragment is much slower, when extrapolated to zero denaturant, than either of the isolated domains, suggesting that the higher thermodynamic stability of the scFv fragment is at least partially due to a high-energy transition state for unfolding. These studies emphasize the enormous importance of mutual domain stabilization in engineering stable antibodies. Study holds ProTherm entries: 14785, 14786, 14787, 14788, 14789, 14790, 14791, 14792, 14793, 14794 Extra Details: single-chain Fv fragment; protein stability; two-domain protein;,kinetic stabilization; domain folding

Submission Details

ID: QdmVeYQw

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Jäger M;Plückthun A,FEBS Lett. (1999) Domain interactions in antibody Fv and scFv fragments: effects on unfolding kinetics and equilibria. PMID:10622716
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
7JG1 2020-07-18T00:00:00+0000 3.3 Dimeric Immunoglobin A (dIgA)
7JG2 2020-07-18T00:00:00+0000 3.3 Secretory Immunoglobin A (SIgA)
1MCP 1984-07-09T00:00:00+0000 2.7 PHOSPHOCHOLINE BINDING IMMUNOGLOBULIN FAB MC/PC603. AN X-RAY DIFFRACTION STUDY AT 2.7 ANGSTROMS
2CJU 2006-04-09T00:00:00+0000 2.5 Crystal structure of the TEPC15-Vk45.1 anti-2-phenyl-5-oxazolone NQ16- 113.8 scFv in complex with phOxGABA
2MCP 1984-10-15T00:00:00+0000 3.1 REFINED CRYSTAL STRUCTURE OF THE MC/PC603 FAB-PHOSPHOCHOLINE COMPLEX AT 3.1 ANGSTROMS RESOLUTION
15C8 1998-03-18T00:00:00+0000 2.5 CATALYTIC ANTIBODY 5C8, FREE FAB
1A0Q 1997-12-05T00:00:00+0000 2.3 29G11 COMPLEXED WITH PHENYL [1-(1-N-SUCCINYLAMINO)PENTYL] PHOSPHONATE
1A3L 1998-01-22T00:00:00+0000 1.95 CATALYSIS OF A DISFAVORED REACTION: AN ANTIBODY EXO DIELS-ALDERASE-TSA-INHIBITOR COMPLEX AT 1.95 A RESOLUTION
1AHW 1997-04-10T00:00:00+0000 3.0 A COMPLEX OF EXTRACELLULAR DOMAIN OF TISSUE FACTOR WITH AN INHIBITORY FAB (5G9)
1AI1 1996-11-06T00:00:00+0000 2.8 HIV-1 V3 LOOP MIMIC

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 L Ig heavy chain V region M603 P01837 IGKC_MOUSE
100.0 H Ig heavy chain V region M603 P01878 IGHA_MOUSE
91.9 H Ig heavy chain V region M603 P01794 HVM25_MOUSE
93.5 H Ig heavy chain V region M603 P01793 HVM24_MOUSE
94.3 H Ig heavy chain V region M603 P01790 HVM21_MOUSE
94.3 H Ig heavy chain V region M603 P01791 HVM22_MOUSE
95.1 H Ig heavy chain V region M603 P01792 HVM23_MOUSE
95.9 H Ig heavy chain V region M603 P01788 HVM19_MOUSE
95.9 H Ig heavy chain V region M603 P01787 HVM18_MOUSE
100.0 H Ig heavy chain V region M603 P01789 HVM20_MOUSE