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

Literature Information

Publication Date 2015-08-28
DOI 10.1039/C5CP04908C
Impact Factor 3.676
Authors

Debmalya Bhunia, Rajdeep Chowdhury, Kankan Bhattacharyya


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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 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.

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Source Journal

Physical Chemistry Chemical Physics

Physical Chemistry Chemical Physics
CiteScore: 5.5
Self-citation Rate: 10.3%
Articles per Year: 3036

Physical Chemistry Chemical Physics (PCCP) is an international journal co-owned by 19 physical chemistry and physics societies from around the world. This journal publishes original, cutting-edge research in physical chemistry, chemical physics and biophysical chemistry. To be suitable for publication in PCCP, articles must include significant innovation and/or insight into physical chemistry; this is the most important criterion that reviewers and Editors will judge against when evaluating submissions. The journal has a broad scope and welcomes contributions spanning experiment, theory, computation and data science. Topical coverage includes spectroscopy, dynamics, kinetics, statistical mechanics, thermodynamics, electrochemistry, catalysis, surface science, quantum mechanics, quantum computing and machine learning. Interdisciplinary research areas such as polymers and soft matter, materials, nanoscience, energy, surfaces/interfaces, and biophysical chemistry are welcomed if they demonstrate significant innovation and/or insight into physical chemistry. Joined experimental/theoretical studies are particularly appreciated when complementary and based on up-to-date approaches.

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