Thermal unfolding of a llama antibody fragment : a two-state reversible process

J.M.J. Pérez, J.G. Renisio, J.J. Prompers, C.J. Platerink, van, C. Cambillau, H. Darbon, L.G.J. Frenken

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    135 Citaten (Scopus)

    Samenvatting

    Camelids produce functional "heavy chain" antibodies which are devoid of light chains and CH1 domains [Hamers-Casterman, C., et al. (1993) Nature 363, 446-448]. It has been shown that the variable domains of these heavy chain antibodies (the VHH fragments) are functional at or after exposure to high temperatures, in contrast to conventional antibodies [Linden van der, R. H. J., et al. (1999) Biochim. Biophys. Acta 1431, 37-44]. For a detailed understanding of the higher thermostability of these VHH fragments, knowledge of their structure and conformational dynamics is required. As a first step toward this goal, we report here the essentially complete 1H and 15N NMR backbone resonance assignments of a llama VHH antibody fragment, and an extensive analysis of the structure at higher temperatures. The H-D exchange NMR data at 300 K indicate that the framework of the llama VHH fragment is highly protected with a ¿Gex of >5.4 kcal/mol, while more flexibility is observed for surface residues, particularly in the loops and the two outer strands (residues 4-7, 10-13, and 58-60) of the ß-sheet. The CD data indicate a reversible, two-state unfolding mechanism with a melting transition at 333 K and a ¿Hm of 56 kcal/mol. H-D exchange studies using NMR and ESI-MS show that below 313 K exchange occurs through local unfolding events whereas above 333 K exchange mainly occurs through global unfolding. The lack of a stable core at high temperatures, observed for VHH fragments, has also been observed for conventional antibody fragments. The main distinction between the llama VHH fragment and conventional antibody fragments is the reversibility of the thermal unfolding process, explaining its retained functionality after exposure to high temperatures.
    Originele taal-2Engels
    Pagina's (van-tot)74-83
    TijdschriftBiochemistry
    Volume40
    Nummer van het tijdschrift1
    DOI's
    StatusGepubliceerd - 2001

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