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Single-domain antibody fragments with high conformational stability
Mireille Dumoulin1, Katja Conrath, Annemie Van Meirhaeghe
1Laboratoire d'Enzymologie, Centre d'Ingénerie des Protéines, Institut de Chimie B6, Université de Liége, B-4000 Liége, Belgium.
Protein Science : a Publication of the Protein Society
|February 16, 2002
Summary
Camelid heavy-chain antibody fragments exhibit remarkable stability and reversible unfolding, offering significant potential for biotechnological and medical applications due to their unique properties.
Area of Science:
- Biochemistry
- Structural Biology
- Immunology
Background:
- Camelid heavy-chain antibodies offer unique single-domain antigen-binding fragments (VHHs).
- Understanding the biophysical properties of VHHs is crucial for their application.
Purpose of the Study:
- To investigate the equilibrium folding properties and thermodynamic stability of VHH fragments.
- To characterize their unfolding mechanisms and reversibility under various denaturing conditions.
Main Methods:
- High-pressure unfolding monitored by Fourier transform infrared spectroscopy, fluorescence, circular dichroism, and surface plasmon resonance spectroscopy.
- Denaturation using guanidinium chloride, urea, temperature, and pressure.
- Affinity measurements and optical spectroscopic methods.
Main Results:
- VHH fragments undergo reversible, cooperative unfolding via a two-state mechanism with chemical denaturants.
- Thermally-induced denaturation shows partial irreversibility due to CDR loop refolding issues.
- VHH fragments display high thermal resistance (apparent Tm = 60-80°C) and conformational stability (ΔG(H2O) = 30-60 kJ/mol).
Conclusions:
- Camelid VHH fragments possess exceptional thermodynamic stability, surpassing conventional antibody fragments.
- Their reduced size, enhanced solubility, and high stability make them highly promising for biotechnological and medical applications.