Folding and interaction of subunits at the antibody combining site

Abstract

The Fv fragment derived from mouse myeloma protein 315 possessing anti-dinitrophenyl (DNP) activity, is composed of two subunits, the peptide chain VL and VH. In 8 M urea there is a complete dissociation of VL and VH and an approximately twofold increase in the fluorescence emission of Fv with a characteristic red shift of 11 nm. Upon dilution of Fv from 8 M urea into neutral buffer full regain of activity was observed, concomitant with regain of native fluorescence spectrum. The decrease in fluorescence upon dilution from 8 M urea was used to follow the renaturation process of Fv. At relatively high protein concentration (2.5 x 10(-6) M) two steps were observed during renaturation: a fast one, which is completed in less than 30 s, and a slower step, which proceeds for approximately 20 min. The fast process represents the refolding and association of VL and VH to form an active FV, whereas the slow step is attributed to the formation of "incorrect" associates between VL and VH which slowly reshuffle to the thermodynamically stable active FV. Indeed, at low protein concentration (1.5 x 10(-8 M) only the fast step is observed and renaturation is completed in less than 30s. The presence of hapten does not affect the rate of renaturation of FV. Reoxidation of FV completely reduced in 8 M urea was also found to yield a fully active Fv. Since either VL or VH have only one intrachain disulfide bond, reoxidation was performed at high protein concentration (3 mg/ml) in 8 M urea followed by dilution into neutral buffer. This demonstrates that variable domains not only exist in immunoglobulin structure but can also fold correctly independent of the rest of the peptide chains.

Protein folding

“…Finally, the experiments of Hochman et al (1976), in which a molecular fragment containing an antibody combining site composed of two non-covalently bound peptide subunits (F L and F H ) was regenerated by reoxidation of the reduced forms, demonstrated that the V h and F H domains (each of which contains only one disulphide bond) fold correctly and independently of each other and of the rest of the antibody molecule. Thus, in some cases, portions of a protein molecule have the capability of folding to the native structure, without requiring longrange interactions with other parts of the molecule.…”

Domain Interactions in the Fab Fragment: A Comparative Evaluation of the Single-chain Fv and Fab Format Engineered with Variable Domains of Different Stability

“…17,18 The smallest antibody entity retaining the antigen specificity of its parental antibody consists of a heterodimer of the V H and V L variable domains, the so-called Fv fragment. 19 In most Fv fragments, the dissociation constant between the V H and V L domains, which ranges from 10 K5 M to 10 K8 M, [20][21][22][23][24] is not sufficient to keep the domains associated at low concentrations of protein or under mildly destabilizing conditions. To avoid this problem in recombinantly produced constructs, the variable domains are usually connected by a genetically encoded flexible peptide linker, either in the orientation V H -linker-V L or V L -linker-V H , resulting in a single-chain Fv (scFv) fragment.…”

Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli.

“…It is known that antigen binding fragments of antibodies (1,2) can be refolded from denatured states with recovery of their specific binding activity (3)(4)(5)(6). The smallest such fragment that contains a complete binding site is termed Fv, consisting of an Mr 25,000 heterodimer of the VH and VL domains (2, 5-11).…”

“…After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between afirmity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 ± 0.9) x 107 M -1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [K. = (1.9 @ 0.2) x 108 M 'I under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.It is known that antigen binding fragments of antibodies (1,2) can be refolded from denatured states with recovery of their specific binding activity (3)(4)(5)(6). The smallest such fragment that contains a complete binding site is termed Fv, consisting of an Mr 25,000 heterodimer of the VH and VL domains (2, 5-11).…”