Interaction of polyethylene glycol with cytochrome c investigated via in vitro and in silico approaches

Abstract

One of the significant proteins that have attracted research groups due to virtue of being a potent selective anticancer drug target and property of triggering apoptosis upon release in cytoplasm is cytochrome c (cyt c). The mechanical transformations due to the macromolecular crowding in membrane in the mammalian cell are proposed to be useful inductors of changes in volume. It is very interesting to know that mitochondrial function were observed to be improved by polyethylene glycol (PEG) interaction, which in turn inhibits the cyt c (a pro-apoptotic cell death factor). In this work, the effect of polyethylene glycol of molecular weight 4 kilo Dalton (PEG 4 kDa) was investigated to highlight the structural transformations (tertiary and secondary structure) in cyt c using a choice of spectroscopic techniques (including UV–Vis absorption, near-UV, far-UV and Soret circular dichroism and fluorescence spectroscopy), which shows noteworthy shifts in the secondary and tertiary structures at higher concentrations of PEG 4 kDa with small changes in the heme-globular interactions. The size distribution changes of native protein treated with various concentrations of the crowder were observed and analyzed by dynamic light scattering (DLS). The interaction studies of the crowder with the protein was observed and analyzed by FTIR, isothermal titration calorimetry, time resolved fluorescence and molecular docking. The investigations suggested that the structural changes in the protein occurred due to soft interactions of PEG 4 kDa, which usually destabilizes proteins. The experimental evidence in this study proposed that crowding could be another approach to mechanical super-competition and free of certain markers that could aid in the identification and control of various diseases. This study suggests that crowders at specific concentrations, which softly interact with proteins, can be exploited as remedy for various diseases.

Structural Refolding and Thermal Stability of Myoglobin in the Presence of Mixture of Crowders: Importance of Various Interactions for Protein Stabilization in Crowded Conditions

“…Macromolecular crowding progressively is achieving acceptance in research, i.e., protein folding, therefore showing its nature of action as double-edged sword. The macromolecular crowding shows both destabilizing and stabilizing effects on protein structure, stability and function, depending upon size, shape and concentrations of crowder molecules as well as nature of the protein [ 15 , 19 , 31 , 38 , 57 , 58 , 59 , 60 , 61 , 62 ]. A few examples of proteins are examined in the crowded system (mixtures of crowders), where one molecule counteracts another, resulting in structural or/and thermal stabilization [ 14 , 20 , 48 , 57 , 58 ].…”

“…The negatively charged macromolecules, shape and size of molecules, charges on the surfaces of pathways which acts as switches to regulate cytoplasmic transport of ions and pools having unequal “bulk” concentrations of ionic metabolites are just a few of such theories [ 37 ]. As a result, in crowded cellular conditions, the importance of various types of interactions, which play a significant role in protein folding to maintain structure and function, cannot be ignored [ 15 , 25 , 45 , 63 , 64 ]. In vitro crowding assays are now being designed with proteins, which better reflect bio-macromolecular environments under in vivo, allowing for hydrophobic bonding and screened electrostatic interactions [ 25 ].…”

“…The various in vitro studies of proteins in the presence of natural crowders (DNA, proteins, carbohydrate etc.) and synthetic crowders (Ficoll, dextran and PEGs) put together the information on how proteins interact in such crowded solutions, resulting in their stabilization and/or destabilization [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 15 ]. Such studies impersonate the state of crowding with that of in vivo which is highly crowded [ 16 , 17 ].…”

“…These intermediate structures in the cell influence these mechanisms to address emerging challenges in developing therapeutics and precision medicine [ 29 , 30 ]. To maintain the stability and function of different existing intermediate states (such as molten globule, pre-molten globule) various types of interactions (attractive and/or repulsive forces) play a significant role [ 15 , 22 , 24 , 31 , 32 , 33 ]. It has been reported that PEG-protein interaction induced contraction of NalD chains [ 34 ].…”

Atomistic Simulation of Lysozyme in Solutions Crowded by Tetraethylene Glycol: Force Field Dependence

“…Wang and coworkers indicated that the increase in the residual activity of lysozyme was ascribed to the fact that the addition of PEG suppressed enzyme aggregation via a strong PEG–protein interaction and stabilized the protein secondary structure somewhat [ 6 ]. On the contrary, soft interactions with PEG were also observed to induce structural changes and destabilize proteins [ 28 , 31 , 32 , 33 , 34 ]. For the nucleic acids, low molecular weight PEGs were observed to decrease their thermodynamic stability, while high molecular weight PEGs showed the opposite [ 35 ].…”