title: Unfolding and refolding of a protein by cholesterol and cyclodextrin: a single molecule 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 (dH = 2rH) 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.
authors.affiliation: Department of Physical Chemistry, Indian Association For The cultivation of Science, Jadavpur, Kolkata 700 032, India. pckb@iacs.res.in.
authors.forename: Shirsendu
authors.initials: S
authors.lastname: Ghosh
authors.forename: Catherine
authors.initials: C
authors.lastname: Ghosh
authors.forename: Somen
authors.initials: S
authors.lastname: Nandi
authors.forename: Kankan
authors.initials: K
authors.lastname: Bhattacharyya
mesh_terms: D002784:Cholesterol / Q000737:chemistry*; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D057446:Hydrodynamics; D057927:Hydrophobic and Hydrophilic Interactions; D056004:Molecular Dynamics Simulation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000737:chemistry*
chemical_list: D012709:Serum Albumin; D047392:beta-Cyclodextrins; D002784:Cholesterol; C031215:betadex