Fluorescence fluctuation of an antigen–antibody complex: circular dichroism, FCS and smFRET of enhanced GFP and its antibody

Abstract

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 V_HH-His₆. From FCS measurements, the hydrodynamic radius (r_H) of EGFP and its antibody (V_HH-His₆) is found to be 24 ± 2 Å and 18 ± 2 Å, respectively. For the antigen–antibody complex (EGFP:anti-EGFP V_HH-His₆), r_H 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 V_HH-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.