Bhunia Debmalya, Chowdhury Rajdeep, Bhattacharyya Kankan, Ghosh Surajit
Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata-700032, India.
Phys Chem Chem Phys. 2015 Oct 14;17(38):25250-9. doi: 10.1039/c5cp04908c.
The structure and dynamics of an antigen-antibody complex are monitored by circular dichroism (CD) spectroscopy, fluorescence correlation spectroscopy (FCS) and single molecule FRET (smFRET). In this work, the antigen is enhanced GFP (EGFP) and the antibody is anti-EGFP VHH-His6. From FCS measurements, the hydrodynamic radius (rH) of EGFP and its antibody (VHH-His6) is found to be 24 ± 2 Å and 18 ± 2 Å, respectively. For the antigen-antibody complex (EGFP:anti-EGFP VHH-His6), rH is 41 ± 3 Å. CD spectra indicate that the addition of guanidium hydrochloride (GdnHCl) causes unfolding of the antigen, its antibody and their complex, and a consequent increase in size is observed from FCS data. smFRET between EGFP (donor, D) and Alexa 594 (acceptor, A) bound to anti-EGFP VHH-His6 reveals a time dependent fluctuation in donor-acceptor distances. This suggests that the structure of the antigen-antibody complex is dynamic in nature and is not rigid.
通过圆二色性（CD）光谱、荧光相关光谱（FCS）和单分子荧光共振能量转移（smFRET）来监测抗原-抗体复合物的结构和动力学。在这项工作中，抗原是增强型绿色荧光蛋白（EGFP），抗体是抗EGFP VHH-His6。通过FCS测量发现，EGFP及其抗体（VHH-His6）的流体动力学半径（rH）分别为24±2 Å和18±2 Å。对于抗原-抗体复合物（EGFP:抗EGFP VHH-His6），rH为41±3 Å。CD光谱表明，添加盐酸胍（GdnHCl）会导致抗原、其抗体及其复合物发生解折叠，并且从FCS数据中观察到尺寸随之增加。与抗EGFP VHH-His6结合的EGFP（供体，D）和Alexa 594（受体，A）之间的smFRET揭示了供体-受体距离随时间的波动。这表明抗原-抗体复合物的结构本质上是动态的，而非刚性的。
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