title: An FCS study of unfolding and refolding of CPM-labeled human serum albumin: role of ionic liquid.
abstract: The effect of a room temperature ionic liquid (RTIL) on the conformational dynamics of a protein, human serum albumin (HSA), is studied by fluorescence correlation spectroscopy (FCS). For this, the protein was covalently labeled by a fluorophore, 7-dimethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). On addition of a RTIL ([pmim][Br]) to the native protein, the diffusion coefficient (D(t)) decreases and the hydrodynamic radius (R(h)) increases. This suggests that the RTIL ([pmim][Br]) acts as a denaturant when the protein is in the native state. However, addition of [pmim][Br] to a protein denatured by GdnHCl causes an increases in D(t) and decrease in R(h). This suggests that in the presence of GdnHCl addition of RTIL helps the protein to refold. In the native state, the conformational dynamics of protein is described by three distinct time constants: ~3.6 ± 0.7, ~29 ± 4.5, and 133 ± 23 μs. The faster components (~3.6 ± 0.7 and ~29 ± 4.5 μs) are ascribed to chain dynamics of the protein, while the slowest component (133 μs) is responsible for interchain interaction or concerted motion. On addition of [pmim][Br], the conformational dynamics of HSA becomes slower (~5.1 ± 1, ~43.5 ± 2.8, and ~311 ± 2.3 μs in the presence of 1.5 M [pmim][Br]). The time constants for the protein denatured by 6 M GdnHCl are 3.2 ± 0.4, 34 ± 6, and 207 ± 38 μs. When 1.5 M [pmim][Br] is added to the denatured protein (in 6 M GdnHCl), the time constants become ~5 ± 1, ~41 ± 10, and ~230 ± 45 μs. The lifetime histogram shows that, on addition of GdnHCl to HSA, the contribution of the shorter lifetime component decreases and vanishes at 6 M GdnHCl. The shorter lifetime component immediately reappears after addition of RTIL to unfolded HSA. This suggests recoiling of the unfolded protein by RTIL.
authors.affiliation: Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata, India.
authors.forename: Dibyendu Kumar
authors.initials: DK
authors.lastname: Sasmal
authors.forename: Tridib
authors.initials: T
authors.lastname: Mondal
authors.forename: Supratik
authors.initials: S
authors.lastname: Sen Mojumdar
authors.forename: Aparajita
authors.initials: A
authors.lastname: Choudhury
authors.forename: Rajat
authors.initials: R
authors.lastname: Banerjee
authors.forename: Kankan
authors.initials: K
authors.lastname: Bhattacharyya
mesh_terms: D003374:Coumarins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D052578:Ionic Liquids / Q000737:chemistry; D008958:Models, Molecular; D058849:Protein Refolding; D058767:Protein Unfolding; D012709:Serum Albumin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
chemical_list: D003374:Coumarins; D005456:Fluorescent Dyes; D052578:Ionic Liquids; D012709:Serum Albumin; C092582:7-dimethylamino-4-methylcoumarin