Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen.
In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90 degrees C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear. To examine in more detail the structural and thermodynamic aspects of the binding mechanism, an extensive CD, ITC, and NMR study was initiated. In this study the interaction between the llama VHH -R2 fragment and its antigen, the dye Reactive Red-6 (RR6) has been explored. The data show clearly that most of the VHH-R2 population at 80 degrees C is in an unfolded conformation. In contrast, CD spectra representing the complex between VHH-R2 and the dye remained the same up to 80 degrees C. Interestingly, addition of the dye to the denatured VHH-R2 at 80 degrees C yielded the spectrum of the native complex. These results suggest an induced refolding of denatured VHH-R2 by its antigen under these extreme conditions. This induced refolding showed some similarities with the well established "induced fit" mechanism of antibody-antigen interactions at ambient temperature. However, the main difference with the "induced fit" mechanism is that at the start of the addition of the antigen most of the VHH molecules are in an unfolded conformation. The refolding capability under these extreme conditions and the stable complex formation make VHHs useful in a wide variety of applications.
Dolk
Edward
E
Department of Molecular and Cellular Biology, University of Utrecht, Utrecht, The Netherlands.
van Vliet
Cees
C
Perez
Janice M J
JM
Vriend
Gert
G
Darbon
Hervé
H
Ferrat
Gilles
G
Cambillau
Christian
C
Frenken
Leon G J
LG
Verrips
Theo
T
D000818:Animals; D000936:Antigen-Antibody Complex; D002161:Camelids, New World / Q000276:immunology*; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000276:immunology; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D006358:Hot Temperature; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
D000936:Antigen-Antibody Complex; D006063:Chorionic Gonadotropin; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D010446:Peptide Fragments