Unfolding and refolding of a protein by cholesterol and cyclodextrin: a single molecule study. Issue 12 (27th February 2015)

• Record Type: Journal Article
• Authors: Ghosh, Shirsendu; Ghosh, Catherine; Nandi, Somen; Bhattacharyya, Kankan
• Is Part Of: Physical chemistry chemical physics. Volume 17:Issue 12(2015)
• Page Start: 8017
• Page End: 8027
• Publication Date: 2015-02-27
• Subjects: Chemistry, Physical and theoretical -- Periodicals; 541.3
• DOI: 10.1039/c5cp00385g
• Languages: English
• ISSNs: 1463-9076

Abstract

Abstract : Cholesterol induced unfolding of a globular protein, human serum albumin (HSA), and β-cyclodextrin induced refolding of the unfolded protein is demonstrated in this study. Abstract : Unfolding/refolding of a plasma protein, human serum albumin (HSA), is studied using fluorescence correlation spectroscopy (FCS) and single molecule fluorescence resonance energy transfer (sm-FRET). Addition of cholesterol causes unfolding of HSA resulting in an increase in the hydrodynamic diameter ( d H = 2 r H ) from 76 Å in the native state to 120 Å upon addition of 1 mM cholesterol. Addition of β-cyclodextrin to HSA (unfolded by cholesterol) restores the hydrodynamic diameter back to 78 Å. The cholesterol induced unfolding and β-cyclodextrin induced refolding are also monitored by measuring the distance between a FRET donor (CPM dye, D) and a FRET acceptor (Alexa 488, A) covalently attached to the protein (HSA). It is observed that the average D–A distance increases from 45 ± 1 Å at 0 mM cholesterol to 51 ± 1 Å at 1 mM cholesterol. Upon addition of β-cyclodextrin, the D–A distance is restored to 45 ± 1 Å. The binding study indicates that nearly 94% of HSA molecules remain bound to cholesterol in the absence of β-cyclodextrin and only 5% binds to cholesterol in the presence of β-cyclodextrin. As much as 57% of the HSA and 99% of the cholesterol molecules bind to β-cyclodextrin. Thus β-cyclodextrin removes cholesterol from HSA by hydrophobic binding to cholesterol ("strip off") and also, itself binds to HSA. The conformational dynamics results suggest that addition of β-cyclodextrin restores native like binding free energy and folding dynamics.