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

Camelids produce functional "heavy chain" antibodies which are devoid of li ght chains and CHI 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 V-HH fragments) are functional at or after exposure to hig h temperatures, in contrast to conventional antibodies [Linden van der, R. H. J,, et al. (1999) Biochim. Biophys. Acta 1431, 37-44]. For a detailed un derstanding of the higher thermostability of these V-HH fragments, knowledg e of their structure and conformational dynamics is required. As a first st ep toward this goal, we report here the essentially complete H-1 and N-15 N MR backbone resonance assignments of a Ilama VHH antibody fragment, and an extensive analysis of the structure at higher temperatures, The H-D exchang e NMR data at 300 K indicate that the framework of the Ilama V-HH fragment is highly protected with a DeltaG(ex) of > 5.4 kcal/mol, while more flexibi lity is observed for surface residues, particularly in the loops and the tw o outer strands (residues 4-7, 10-13, and 58-60) of the beta -sheet. The CD data indicate a reversible, two-state unfolding mechanism with a melting t ransition at 333 K and a DeltaH(m) of 56 kcal/mol, H-D exchange studies usi ng NMR and ESI-MS show that below 313 K exchange occurs through local unfol ding events whereas above 333 K exchange mainly occurs through global unfol ding. The lack of a stable core at high temperatures, observed for V-HH fra gments, has also been observed for conventional antibody fragments. The mai n distinction between the Ilama V-HH fragment and conventional antibody fra gments is the reversibility of the thermal unfolding process, explaining it s retained functionality after exposure to high temperatures.