An FCS Study of Unfolding and Refolding of CPM-Labeled Human Serum Albumin: Role of Ionic Liquid

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• 作者：Dibyendu Kumar Sasmal ; Tridib Mondal ; Supratik Sen Mojumdar ; Aparajita Choudhury ; Rajat Banerjee ; Kankan Bhattacharyya
• 刊名：Journal of Physical Chemistry B
• 出版年：2011
• 出版时间：November 10, 2011
• 年：2011
• 卷：115
• 期：44
• 页码：13075-13083
• 全文大小：1113K
• 年卷期：v.115,no.44(November 10, 2011)
• ISSN：1520-5207

文摘

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 卤 0.7, 29 卤 4.5, and 133 卤 23 渭s. The faster components (3.6 卤 0.7 and 29 卤 4.5 渭s) are ascribed to chain dynamics of the protein, while the slowest component (133 渭s) is responsible for interchain interaction or concerted motion. On addition of [pmim][Br], the conformational dynamics of HSA becomes slower (5.1 卤 1, 43.5 卤 2.8, and 311 卤 2.3 渭s in the presence of 1.5 M [pmim][Br]). The time constants for the protein denatured by 6 M GdnHCl are 3.2 卤 0.4, 34 卤 6, and 207 卤 38 渭s. When 1.5 M [pmim][Br] is added to the denatured protein (in 6 M GdnHCl), the time constants become 5 卤 1, 41 卤 10, and 230 卤 45 渭s. 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.