Thermal unfolding of a llama antibody fragment:: A two-state reversible process
Thermal unfolding of a llama antibody fragment:: A two-state reversible process
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DOI：
10.1021/bi0009082
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发表时间：
2001-01-09
期刊：
影响因子：
2.9
通讯作者：
Frenken, LGJ
中科院分区：
文献类型：
--
作者：
Pérez, JMJ;Renisio, JG;Frenken, LGJ
骆驼科动物产生缺乏轻链和CH 1结构域的功能性“重链”抗体[Hamers-Casterman，C.，等人（1993）Nature 363，446-448]。已经表明，这些重链抗体的可变结构域（V-HH片段）在暴露于高温时或之后是有功能的，与常规抗体相反[林登货车der，R. H. J等人（1999）Biochim. Biophys. Acta 1431，37-44]。为了详细了解这些V-HH片段的较高热稳定性，需要了解它们的结构和构象动力学。作为实现这一目标的第一步，我们在这里报告了伊拉马VHH抗体片段的基本上完整的H-1和N-15 NMR骨架共振归属，以及在较高温度下对结构的广泛分析。300 K下的H-D交换NMR数据表明，伊拉马V-HH片段的骨架被高度保护，Δ G（ex）> 5.4 kcal/mol，而对于表面残基，特别是β-折叠的环和两个外链（残基4-7、10-13和58-60），观察到更大的柔性。CD数据表明，在333 K和56 kcal/mol的DeltaH（m）的熔融转变的可逆，两个状态的去折叠机制，使用NMR和ESI-MS的H-D交换研究表明，低于313 K的交换发生通过局部去折叠事件，而高于333 K的交换主要发生通过整体去折叠。对于V-HH片段观察到的在高温下缺乏稳定的核心，也已经对于常规抗体片段观察到。伊拉马V-HH片段和常规抗体片段之间的主要区别是热解折叠过程的可逆性，这解释了其在暴露于高温后保留的功能性。
Camelids produce functional "heavy chain" antibodies which are devoid of light 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 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 V-HH fragments, knowledge of their structure and conformational dynamics is required. As a first step toward this goal, we report here the essentially complete H-1 and N-15 NMR backbone resonance assignments of a Ilama 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 Ilama V-HH fragment is highly protected with a DeltaG(ex) 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 beta -sheet. The CD data indicate a reversible, two-state unfolding mechanism with a melting transition at 333 K and a DeltaH(m) 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 V-HH fragments, has also been observed for conventional antibody fragments. The main distinction between the Ilama V-HH fragment and conventional antibody fragments is the reversibility of the thermal unfolding process, explaining its retained functionality after exposure to high temperatures.