Use of FCS to Study Protein Denaturation and Aggregation

Abstract

Fluorescence Correlation Spectroscopy (FCS) is a powerful technique for studying the diffusive dynamics of fluorophore-tagged biomolecules and thereby drawing inferences about their conformational states and transitions. We have employed FCS to investigate the denaturation and aggregation pathways of bovine immunoglobulin (IgG), a multi-subunit all beta-sheet Y-shaped protein prone to aggregation. Each upper arm of the Y consists of a light and a heavy chain connected by cysteine-shy;cysteine disulphide bonds, and the two heavy chains are joined at the bend by more disulphides. While majority of proteins show continuous increase of hydrodynamic radius (Rg) when presented with increasing denaturant concentration, IgG exhibits strikingly different behavior under similar conditions. Its Rg initially decreases with increasing concentration of GdnHCl (from 0 to 3 M) and then goes through two pronounced maxima at 4 M and 7 M [GdnHCl]. We propose that the initial contraction is related to sheet-to-helix transition at the flexible bends of the Y-arms of the protein. The first maximum of Rg is attributed to electrostatic repulsions and their screening by Gdn+ and Cl- ions; the second is shown to arise from disulphide-mediated effects.Previous studies have proposed that IgG aggregation is initiated by intermolecular disulphide bond formation. We studied the aggregation kinetics of IgG by monitoring the time evolution of characteristic diffusion time (τD) under two different conditions: with the cysteine residues free to interact or blocked. The progress curves (τD vs.elapsed time) were sigmoidal in both cases, but the delay time was almost an order of magnitude longer, and the apparent polymerization rate half as fast, for the case where the cysteines were blocked than when they were free. This result indicates that intermolecular disulphide bond formation is a key factor driving the aggregation process of IgG.