FCS Study of the Structural Stability of Lysozyme in the Presence of Morpholinium Salts

Abstract

Ability of the ionic liquids to alter the structural stability of proteins in aqueous solution is a topic of considerable interest in modern bioscientific research because of possible applications of these substances in protein purification and as refolding agents. A few early studies involving the imidazolium ionic liquids have demonstrated their role as both denaturants and refolding agents. As the influence of an ionic liquid on a given protein depends on the identity of both species, it is necessary to extend the studies to a wider number of ionic liquids and proteins to obtain insight into the mechanism of interaction between the two and to arrive at a comprehensive picture. It is in this context that we have studied the effect of two morpholinium salts, [Mor1,2][Br] and [Mor1,4][Br], differing in the alkyl chain length of cation, on chicken egg white lysozyme in its native and chemically denatured states employing primarily the fluorescence correlation spectroscopy (FCS) technique. Fluorescence signal of Alexa488-labeled lysozyme (A488-Lysz) has been used to determine the changes in hydrodynamic radius of protein in the presence of additives. The results reveal a conformational dynamics of lysozyme with a time constant of 56 ± 10 μs in its native state. It is observed, when in its native state, both the morpholinium salts induce structural changes of lysozyme. However, when in its unfolded state, [Mor1,4][Br] at low concentration compacts the protein, but at higher concentration, it stabilizes the unfolded state, unlike [Mor1,2][Br], which compacts lysozyme at both low and high concentrations. A comparison of the effect of these salts and arginine, a protein stabilizer, on lysozyme indicates that [Mor1,2][Br] is a superior compacting agent for the unfolded state of the protein compared to arginine.

Structural Stability and Conformational Dynamics of Cytochrome c in Hydrated Deep Eutectic Solvents

“…In contrast, fluorescence of free A488 remains constant in buffered solutions containing similar quantities of DES (Figure S6) and GdHCl . Hence, it is evident that the increase in fluorescence intensity of Cytc-A488 in the presence of the DESs is due to structural transformation of Cytc from its native to the unfolded state. , A comparison of the fluorescence data suggests a stronger influence of TMG-EG on the protein native structure compared to TMG-GL. , …”

“…The time constant (54 μs) corresponding to the exponential term (τ R ) in pure buffered solution is comparable to the time scales of conformational dynamics of many proteins in their native state. 16,17,21,28,33,47 The increase in τ R value with the increase in quantity of the DES (Figure 5 and Table 2) indicates a slowing down of the conformational dynamics and this effect is greater in the case of TMG-EG as compared to TMG-GL. A slower conformational fluctuation in the presence of TMG-EG is the consequence of complete unfolding of protein, due to which the quencher amino acids of the protein need to diffuse a longer distance for quenching the fluorescence of A488 dye.…”

“…16,21 A comparison of the fluorescence data suggests a stronger influence of TMG-EG on the protein native structure compared to TMG-GL. 17,33 The fluorescence decay profiles of Cytc-A488 in the presence of different quantities of DESs and GdHCl are shown in Figure 2 and Figure S7, respectively. The decay profiles, in all cases, are found to be biexponential in nature; I(t) = a 1 exp(−t/τ 1 ) + a 2 exp(−t/τ 2 ), where τ 1 and τ 2 are lifetime components and a 1 and a 2 are associated amplitudes.…”

“…This suggests that the additional exponential component arises from conformational fluctuations of the protein, which occur during the passage of Cytc-A488 through the observation volume. 33,35…”

“…However, addition of the DESs and GdHCl changes the viscosity and refractive index of the medium, which, if not corrected, can lead to inaccurate estimation of r H . 17,33,39 For this reason, we have estimated the r H values of Cytc-A488 (r H Cytc ) with the help of eq 6 employing the experimentally measured diffusion times of free R6G (τ D R6G ) and Cytc-A488 (τ D Cytc ) in the media and calculated (using eq 5) hydrodynamic radius of R6G (r H R6G = 5.92 Å). 34,38 We have used here R6G as a diffusion standard because of its rigid structure and independence of its hydrodynamic radius on the viscosity and refractive index of the medium.…”