Gong Rui, Vu Bang K, Feng Yang, Prieto DaRue A, Dyba Marzena A, Walsh Joseph D, Prabakaran Ponraj, Veenstra Timothy D, Tarasov Sergey G, Ishima Rieko, Dimitrov Dimiter S
Protein Interactions Group, CCRNP, CCR, National Institutes of Health, Frederick, Maryland 21702, USA.
J Biol Chem. 2009 May 22;284(21):14203-10. doi: 10.1074/jbc.M900769200. Epub 2009 Mar 23.
The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising as scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. However, previous work has shown that an isolated murine CH2 domain is relatively unstable to thermally induced unfolding. To explore unfolding mechanisms of isolated human CH2 and increase its stability gamma1 CH2 was cloned and a panel of cysteine mutants was constructed. Human gamma1 CH2 unfolded at a higher temperature (T(m) = 54.1 degrees C, as measured by circular dichroism) than that previously reported for a mouse CH2 (41 degrees C). One mutant (m01) was remarkably stable (T(m) = 73.8 degrees C). Similar results were obtained by differential scanning calorimetry. This mutant was also significantly more stable than the wild-type CH2 against urea induced unfolding (50% unfolding at urea concentration of 6.8 m versus 4.2 m). The m01 was highly soluble and monomeric. The existence of the second disulfide bond in m01 and its correct position were demonstrated by mass spectrometry and nuclear magnetic resonance spectroscopy, respectively. The loops were on average more flexible than the framework in both CH2 and m01, and the overall secondary structure was not affected by the additional disulfide bond. These data suggest that a human CH2 domain is relatively stable to unfolding at physiological temperature, and that both CH2 and the highly stable mutant m01 are promising new scaffolds for the development of therapeutics against human diseases.
免疫球蛋白（Ig）恒定区CH2结构域对于抗体效应功能至关重要。分离出的CH2结构域有望作为构建包含多种结合物文库的支架，这些结合物也可赋予一些效应功能。然而，先前的研究表明，分离出的小鼠CH2结构域对热诱导的解折叠相对不稳定。为了探究分离出的人CH2结构域的解折叠机制并提高其稳定性，克隆了γ1 CH2并构建了一组半胱氨酸突变体。人γ1 CH2在比先前报道的小鼠CH2（41℃）更高的温度下解折叠（通过圆二色性测量，熔解温度T(m)=54.1℃）。一个突变体（m01）非常稳定（T(m)=73.8℃）。差示扫描量热法也得到了类似结果。该突变体在抵抗尿素诱导的解折叠方面也比野生型CH2显著更稳定（在尿素浓度为6.8 m时50%解折叠，而野生型为4.2 m）。m01高度可溶且为单体形式。分别通过质谱和核磁共振光谱证实了m01中第二个二硫键的存在及其正确位置。在CH2和m01中，环平均比骨架更灵活，并且整体二级结构不受额外二硫键的影响。这些数据表明，人CH2结构域在生理温度下相对稳定，并且CH2和高度稳定的突变体m01都是开发针对人类疾病治疗药物的有前景的新支架。
