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.

Effect of ionic liquid on the native and denatured state of a protein covalently attached to a probe: Solvation dynamics study

“…14 Similarly, the decrease in the long component from ∼15 ns (in 1.5 M RTIL or 6 M GdnHCl added to the native HSA) to ∼12.5 ns (in the presence of both 1.5 M RTIL and 6 M GdnHCl) may be due to decrease in r h of the protein. 14 These observations are consistent with our previous FCS results. 14 The relatively faster component (∼4 ns in native protein and ∼1.5 ns in presence of other additives) may be assigned to the segmental chain motion of the protein (HSA) or the local motion of the probe (CPM) molecule.…”

“…Addition of 1.5 M RTIL to the CPM labeled HSA denatured by 6 M GdnHCl causes a decrease in the hydrodynamic radius (r h ) of the protein. 14 The 5 nm red shift caused by RTIL (with respect to HSA unfolded by 6 M GdnHCl) in spite of decrease in the hydrodynamic radius (r h ) implies an increase in local polarity. This may be because of the presence of a large number of ions of the RTIL and GdnHCl around the probe replaces the water molecules.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14] There are many reports that some proteins retain their biological activity when dissolved in some RTILs. [2][3][4] This is an important observation since proteins are sometimes unstable when handled in vitro, and stabilizing agents are required to stabilize them.…”

“…In this work, we study the solvation dynamics of a probe (CPM) covalently attached to the lone cysteine group 14 The FCS study does not provide any information on the effect of RTIL and GdnHCl on the microenvironment and solvation dynamics of the fluorescent probe (CPM) covalently attached to the protein (HSA). 14 In this work, we demonstrate that in the presence of RTIL and GdnHCl, though the protein is structurally more compact and the r h is similar to the protein in the native state, the local environment of CPM is very different from that in the native state.…”

Compatibility of Ionic Liquids with Enzymes

“…Many efforts with different techniques such as spectroscopy, molecular dynamics simulation have been paid to explore the dynamic structure and conformation of protein in ionic liquid in recent years [109][110][111][112][113][114][115][116][117][118][119][120][121] …”