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
1QGC 2000-01-26 STRUCTURE OF THE COMPLEX OF A FAB FRAGMENT OF A NEUTRALIZING ANTIBODY WITH FOOT AND MOUTH DISEASE VIRUS
2AJV 2006-02-14 1.5 Crystal Structure of Cocaine catalytic Antibody 7A1 Fab' in Complex with Cocaine
1RUR 2004-03-02 1.5 Crystal Structure (I) of native Diels-Alder antibody 13G5 Fab at pH 8.0 with a data set collected at SSRL beamline 9-1.
2AJU 2006-02-14 1.5 Cyrstal structure of cocaine catalytic antibody 7A1 Fab'
2AJS 2006-02-14 1.7 Crystal structure of cocaine catalytic antibody 7A1 Fab' in complex with heptaethylene glycol
1SEQ 2005-03-01 1.78 Fab MNAC13
1Q9O 2004-01-27 1.79 S45-18 Fab Unliganded
1DQQ 2000-01-19 1.8 CRYSTAL STRUCTURE OF ANTI-LYSOZYME ANTIBODY HYHEL-63
1NBY 2003-04-01 1.8 Crystal Structure of HyHEL-63 complexed with HEL mutant K96A
1KCV 2002-05-11 1.8 Crystal structure of antibody pc282
2AK1 2006-02-14 1.85 Crystal Structure of Cocaine catalytic Antibody 7A1 Fab' in Complex with benzoic acid
1NBZ 2003-04-01 1.85 Crystal Structure of HyHEL-63 complexed with HEL mutant K97A
2AJX 2006-02-14 1.85 Crystal Structure of Cocaine catalytic Antibody 7A1 Fab' in Complex with Transition State Analog
1RUQ 2004-03-02 1.86 Crystal Structure (H) of u.v.-irradiated Diels-Alder antibody 13G5 Fab at pH 8.0 with a data set collected in house.
1E4X 2001-07-12 1.9 crossreactive binding of a circularized peptide to an anti-TGFalpha antibody Fab-fragment
6FAB 1993-01-15 1.9 THREE-DIMENSIONAL STRUCTURE OF MURINE ANTI-P-AZOPHENYLARSONATE FAB 36-71. 1. X-RAY CRYSTALLOGRAPHY, SITE-DIRECTED MUTAGENESIS, AND MODELING OF THE COMPLEX WITH HAPTEN
1R3J 2003-11-25 1.9 potassium channel KcsA-Fab complex in high concentration of Tl+
1NDG 2003-06-03 1.9 Crystal structure of Fab fragment of antibody HyHEL-8 complexed with its antigen lysozyme
1ORS 2003-05-06 1.9 X-ray structure of the KvAP potassium channel voltage sensor in complex with an Fab
1E4W 2001-07-12 1.95 crossreactive binding of a circularized peptide to an anti-TGFalpha antibody Fab-fragment
1GPO 1998-04-01 1.95 CRYSTAL STRUCTURE OF THE RATIONALLY DESIGNED ANTIBODY M41 AS A FAB FRAGMENT
1OSP 1997-04-21 1.95 CRYSTAL STRUCTURE OF OUTER SURFACE PROTEIN A OF BORRELIA BURGDORFERI COMPLEXED WITH A MURINE MONOCLONAL ANTIBODY FAB
1A3L 1999-02-16 1.95 CATALYSIS OF A DISFAVORED REACTION: AN ANTIBODY EXO DIELS-ALDERASE-TSA-INHIBITOR COMPLEX AT 1.95 A RESOLUTION
25C8 1999-03-23 2.0 CATALYTIC ANTIBODY 5C8, FAB-HAPTEN COMPLEX
35C8 1999-03-23 2.0 CATALYTIC ANTIBODY 5C8, FAB-INHIBITOR COMPLEX
1UB5 2004-04-20 2.0 Crystal structure of Antibody 19G2 with hapten at 100K
2Q76 2008-03-18 2.0 Mouse anti-hen egg white lysozyme antibody F10.6.6 Fab fragment
1DQJ 2000-01-19 2.0 CRYSTAL STRUCTURE OF THE ANTI-LYSOZYME ANTIBODY HYHEL-63 COMPLEXED WITH HEN EGG WHITE LYSOZYME
1FNS 2000-10-18 2.0 CRYSTAL STRUCTURE OF THE VON WILLEBRAND FACTOR (VWF) A1 DOMAIN I546V MUTANT IN COMPLEX WITH THE FUNCTION BLOCKING FAB NMC4
1P2C 2004-02-17 2.0 crystal structure analysis of an anti-lysozyme antibody
1MH5 2003-09-23 2.1 The Structure Of The Complex Of The Fab Fragment Of The Esterolytic Antibody MS6-164 and A Transition-State Analog
2AJY 2006-02-14 2.1 Crystal Structure of Cocaine catalytic Antibody 7A1 Fab' in Complex with ecgonine methyl ester and benzoic acid
1MLB 1995-06-03 2.1 MONOCLONAL ANTIBODY FAB D44.1 RAISED AGAINST CHICKEN EGG-WHITE LYSOZYME
1NDM 2003-06-03 2.1 Crystal structure of Fab fragment of antibody HyHEL-26 complexed with lysozyme
1XGU 2005-09-06 2.1 Structure for antibody HyHEL-63 Y33F mutant complexed with hen egg lysozyme
1DQM 2000-01-19 2.1 CRYSTAL STRUCTURE OF ANTI-LYSOZYME ANTIBODY
1UB6 2004-04-20 2.12 Crystal structure of Antibody 19G2 with sera ligand
1CT8 1999-11-10 2.2 CATALYTIC ANTIBODY 7C8 COMPLEX
1KCU 2002-05-11 2.2 CRYSTAL STRUCTURE OF ANTIBODY PC287
1UWX 2005-06-15 2.2 P1.2 serosubtype antigen derived from N. meningitidis PorA in complex with Fab fragment
2CK0 2003-08-05 2.2 ANTI-ANTI-IDIOTYPIC ANTIBODY AGAINST HUMAN ANGIOTENSIN II, COMPLEX WITH A SYNTHETIC CYCLIC PEPTIDE
2H8P 2006-09-12 2.25 Structure of a K channel with an amide to ester substitution in the selectivity filter
1EMT 2000-11-01 2.25 FAB ANTIBODY FRAGMENT OF AN C60 ANTIFULLERENE ANTIBODY
2AJZ 2006-02-14 2.3 Crystal Structure of Cocaine catalytic Antibody 7A1 Fab' in Complex with ecgonine methyl ester
1A0Q 1999-03-02 2.3 29G11 COMPLEXED WITH PHENYL [1-(1-N-SUCCINYLAMINO)PENTYL] PHOSPHONATE
1K4D 2001-11-14 2.3 Potassium Channel KcsA-Fab complex in low concentration of K+
4KVC 2013-07-24 2.31 2H2 Fab fragment of immature Dengue virus
1R3I 2003-11-25 2.4 potassium channel KcsA-Fab complex in Rb+
1EHL 2001-02-21 2.4 64M-2 ANTIBODY FAB COMPLEXED WITH D(5HT)(6-4)T
1R3L 2003-11-25 2.41 potassium channel KcsA-Fab complex in Cs+
1FL3 2000-11-01 2.45 CRYSTAL STRUCTURE OF THE BLUE FLUORESCENT ANTIBODY (19G2) IN COMPLEX WITH STILBENE HAPTEN AT 277K
1FDL 1991-10-15 2.5 CRYSTALLOGRAPHIC REFINEMENT OF THE THREE-DIMENSIONAL STRUCTURE OF THE FAB D1.3-LYSOZYME COMPLEX AT 2.5-ANGSTROMS RESOLUTION
1KCS 2002-05-11 2.5 CRYSTAL STRUCTURE OF ANTIBODY PC282 IN COMPLEX WITH PS1 PEPTIDE
15C8 1999-03-23 2.5 CATALYTIC ANTIBODY 5C8, FREE FAB
1KB5 1998-04-08 2.5 MURINE T-CELL RECEPTOR VARIABLE DOMAIN/FAB COMPLEX
1KC5 2002-07-24 2.5 CRYSTAL STRUCTURE OF ANTIBODY PC287 IN COMPLEX WITH PS1 PEPTIDE
1RIH 2004-01-13 2.5 Crystal Structure of Fab 14F7, a unique anti-tumor antibody specific for N-glycolyl GM3
1CIC 1999-04-19 2.5 IDIOTOPE-ANTI-IDIOTOPE FAB-FAB COMPLEX; D1.3-E225
1CK0 2003-08-05 2.5 ANTI-ANTI-IDIOTYPIC ANTIBODY AGAINST HUMAN ANGIOTENSIN II, UNLIGANDED FORM
2CJU 2006-06-20 2.5 Crystal structure of the TEPC15-Vk45.1 anti-2-phenyl-5-oxazolone NQ16- 113.8 scFv in complex with phOxGABA
1FGN 1998-02-25 2.5 MONOCLONAL MURINE ANTIBODY 5G9-ANTI-HUMAN TISSUE FACTOR
1MLC 1995-06-03 2.5 MONOCLONAL ANTIBODY FAB D44.1 RAISED AGAINST CHICKEN EGG-WHITE LYSOZYME COMPLEXED WITH LYSOZYME
1OTS 2003-04-15 2.51 Structure of the Escherichia coli ClC Chloride channel and Fab Complex
1HI6 2001-02-08 2.55 ANTI-P24 (HIV-1) FAB FRAGMENT CB41 COMPLEXED WITH A PEPTIDE
1MF2 1997-12-31 2.6 ANTI HIV1 PROTEASE FAB COMPLEX
1HH6 2001-01-26 2.6 ANTI-P24 (HIV-1) FAB FRAGMENT CB41 COMPLEXED WITH A PEPTIDE
1C12 1999-08-14 2.6 INSIGHT IN ODORANT PERCEPTION: THE CRYSTAL STRUCTURE AND BINDING CHARACTERISTICS OF ANTIBODY FRAGMENTS DIRECTED AGAINST THE MUSK ODORANT TRASEOLIDE
1CFN 1999-03-30 2.65 ANTI-P24 (HIV-1) FAB FRAGMENT CB41 COMPLEXED WITH AN EPITOPE-RELATED PEPTIDE
1FJ1 2000-10-11 2.68 LYME DISEASE ANTIGEN OSPA IN COMPLEX WITH NEUTRALIZING ANTIBODY FAB LA-2
1FAI 1992-10-15 2.7 THREE-DIMENSIONAL STRUCTURE OF TWO CRYSTAL FORMS OF FAB R19.9, FROM A MONOCLONAL ANTI-ARSONATE ANTIBODY
1HH9 2001-01-12 2.7 ANTI-P24 (HIV-1) FAB FRAGMENT CB41 COMPLEXED WITH A PEPTIDE
1MCP 1985-01-02 2.7 PHOSPHOCHOLINE BINDING IMMUNOGLOBULIN FAB MC/PC603. AN X-RAY DIFFRACTION STUDY AT 2.7 ANGSTROMS
1CF8 1999-07-28 2.7 Convergence of catalytic antibody and terpene cyclase mechanisms: polyene cyclization directed by carbocation-pi interactions
1CFS 1999-03-31 2.75 ANTI-P24 (HIV-1) FAB FRAGMENT CB41 COMPLEXED WITH AN EPITOPE-UNRELATED PEPTIDE
2HG5 2006-09-12 2.75 Cs+ complex of a K channel with an amide to ester substitution in the selectivity filter
2F19 1992-10-15 2.8 THREE-DIMENSIONAL STRUCTURE OF TWO CRYSTAL FORMS OF FAB R19.9, FROM A MONOCLONAL ANTI-ARSONATE ANTIBODY
1PSK 1997-09-04 2.8 THE CRYSTAL STRUCTURE OF AN FAB FRAGMENT THAT BINDS TO THE MELANOMA-ASSOCIATED GD2 GANGLIOSIDE
1CFT 1999-03-31 2.8 ANTI-P24 (HIV-1) FAB FRAGMENT CB41 COMPLEXED WITH AN EPITOPE-UNRELATED D-PEPTIDE
1R3K 2003-11-25 2.8 potassium channel KcsA-Fab complex in low concentration of Tl+
1FRG 1994-05-31 2.8 CRYSTAL STRUCTURE, SEQUENCE, AND EPITOPE MAPPING OF A PEPTIDE COMPLEX OF AN ANTI-INFLUENZA HA PEPTIDE ANTIBODY FAB 26(SLASH)9: FINE-TUNING ANTIBODY SPECIFICITY
1AI1 1997-05-15 2.8 HIV-1 V3 LOOP MIMIC
1JRH 1998-03-25 2.8 COMPLEX (ANTIBODY/ANTIGEN)
4QNP 2015-02-11 2.8 Crystal structure of the 2009 pandemic H1N1 influenza virus neuraminidase with a neutralizing antibody
1CFQ 1999-03-30 2.8 ANTI-P24 (HIV-1) FAB FRAGMENT CB41
4KK8 2013-08-21 2.86 Structure of the E148Q mutant of CLC-ec1 deltaNC construct in 100mM fluoride
1FSK 2000-10-02 2.9 COMPLEX FORMATION BETWEEN A FAB FRAGMENT OF A MONOCLONAL IGG ANTIBODY AND THE MAJOR ALLERGEN FROM BIRCH POLLEN BET V 1
1IAI 1996-03-08 2.9 IDIOTYPE-ANTI-IDIOTYPE FAB COMPLEX
1AIF 1997-02-01 2.9 ANTI-IDIOTYPIC FAB 409.5.3 (IGG2A) FAB FROM MOUSE
3EJZ 2009-02-17 2.9 Structure of E203V mutant E.coli Cl-/H+ exchanger, CLC-ec1
1KCR 2002-05-11 2.9 CRYSTAL STRUCTURE OF ANTIBODY PC283 IN COMPLEX WITH PS1 PEPTIDE
3CK0 2003-08-05 3.0 ANTI-ANTI-IDIOTYPIC ANTIBODY AGAINST HUMAN ANGIOTENSIN II, COMPLEX WITH HUMAN ANGIOTENSIN II
1AHW 1998-02-25 3.0 A COMPLEX OF EXTRACELLULAR DOMAIN OF TISSUE FACTOR WITH AN INHIBITORY FAB (5G9)
3FB6 2010-05-19 3.0 KcsA Potassium channel in the partially open state with 16 A opening at T112
1FBI 1995-02-27 3.0 CRYSTAL STRUCTURE OF A CROSS-REACTION COMPLEX BETWEEN FAB F9.13.7 AND GUINEA-FOWL LYSOZYME
1OTT 2003-04-15 3.0 Structure of the Escherichia coli ClC Chloride channel E148A mutant and Fab Complex
1N5Y 2003-01-28 3.1 HIV-1 Reverse Transcriptase Crosslinked to Post-Translocation AZTMP-Terminated DNA (Complex P)
2MCP 1985-01-02 3.1 REFINED CRYSTAL STRUCTURE OF THE MC/PC603 FAB-PHOSPHOCHOLINE COMPLEX AT 3.1 ANGSTROMS RESOLUTION
4KK5 2013-08-21 3.17 Structure of the CLC-ec1 deltaNC construct in 20mM fluoride and 20mM bromide
3F7V 2010-05-19 3.2 KcsA Potassium channel in the open-inactivated state with 23 A opening at T112
1KNO 1996-01-29 3.2 CRYSTAL STRUCTURE OF THE COMPLEX OF A CATALYTIC ANTIBODY FAB WITH A TRANSITION STATE ANALOG: STRUCTURAL SIMILARITIES IN ESTERASE-LIKE ABZYMES
1ORQ 2003-05-06 3.2 X-ray structure of a voltage-dependent potassium channel in complex with an Fab
1IGY 1998-04-15 3.2 STRUCTURE OF IMMUNOGLOBULIN
1OTU 2003-04-15 3.3 Structure of the Escherichia coli ClC Chloride channel E148Q mutant and Fab Complex
3F7Y 2010-05-19 3.4 KcsA Potassium channel in the partially open state with 17 A opening at T112
1KEN 2002-04-24 3.5 INFLUENZA VIRUS HEMAGGLUTININ COMPLEXED WITH AN ANTIBODY THAT PREVENTS THE HEMAGGLUTININ LOW PH FUSOGENIC TRANSITION

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