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

Abstract

The intracellular environment is overcrowded with a range of molecules (small and large), all of which influence protein conformation. As a result, understanding how proteins fold and stay functional in such crowded conditions is essential. Several in vitro experiments have looked into the effects of macromolecular crowding on different proteins. However, there are hardly any reports regarding small molecular crowders used alone and in mixtures to observe their effects on the structure and stability of the proteins, which mimics of the cellular conditions. Here we investigate the effect of different mixtures of crowders, ethylene glycol (EG) and its polymer polyethylene glycol (PEG 400 Da) on the structural and thermal stability of myoglobin (Mb). Our results show that monomer (EG) has no significant effect on the structure of Mb, while the polymer disrupts its structure and decreases its stability. Conversely, the additive effect of crowders showed structural refolding of the protein to some extent. Moreover, the calorimetric binding studies of the protein showed very weak interactions with the mixture of crowders. Usually, we can assume that soft interactions induce structural perturbations while exclusion volume effects stabilize the protein structure; therefore, we hypothesize that under in vivo crowded conditions, both phenomena occur and maintain the stability and function of proteins.

Size-Dependent Interplay of Volume Exclusion Versus Soft Interactions: Cytochrome c in Macromolecular Crowded Environment

“…Moreover, such reports suggest that in the medium ( in vitro ), two processes monitor structural changes of proteins simultaneously and can be mimicked with cellular conditions where the milieu is highly crowded and proteins are surrounded by various sizes and shapes of other macromolecules ( Minton, 1981 ; Ellis, 2001 ; Goodsell, 2012 ; Sarkar et al, 2013b ; Parray et al, 2020a ; Parray et al, 2021b ). In the case of PEGs, exclusion volume effect is not only the mechanism but soft interactions also play an important role in stabilizing intermediate states.…”

“…Our research group has exploited a spectrum of different sizes of PEG, other crowding agents (ficoll and dextran), and some mixture solutions of crowders to measure their effect on heme-proteins in order to infer the significance of macromolecular crowders of different shapes and sizes in the cellular environments ( Parray et al, 2017 ; Nasreen et al, 2018 ; Parray et al, 2019 ; Parray et al, 2020a ; Parray et al, 2020b ; Parray et al, 2020c ; Parray et al, 2020d ; Nasreen et al, 2020 ; Parray et al, 2021a ; Parray et al, 2021b ). The goal of the study was always to concentrate mostly on the advances through in vitro and in silico methods to measure the implications of varying sizes of macromolecular crowders on biophysical characteristics and conformational changes in proteins.…”

“…These observations suggest that PEG 10 denatures cyt c (increases the hydrodynamic size) and PEG 20 results in structure compaction at high concentrations (decreases the hydrodynamic size). Thus, it can be said that PEG 10 results in the perturbation of the tertiary structure of cyt c without change in the secondary structure; however, PEG 20 leads the protein to a more structured form ( Qu et al, 2012 ; Parray et al, 2020c ; Das and Sen, 2020 ; Parray et al, 2021b ).…”

“…Earlier, we have studied the effects of various sizes of PEGs (PEG 400, PEG 10, and PEG 4000), their monomer (ethylene glycol, EG), and mixtures of crowders by varying their concentrations on the structural stability of heme-proteins (cyt c and myoglobin or Mb) ( Parray et al, 2017 ; Parray et al, 2019 ; Parray et al, 2020a ; Parray et al, 2020b ; Parray et al, 2020c ; Parray et al, 2021a ; Parray et al, 2021b ). The aim of these studies was to reflect on the updating progress being made through in vitro and in silico approaches to analyze the consequences of various sizes of macromolecular crowders on proteins and processes that stabilize the cell’s ( in vivo ) environment and to understand mechanisms of stabilization and destabilization due to various types of interactions in such conditions ( Parray et al, 2019 ; Parray et al, 2020a ; Parray et al, 2021a ).…”

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 ].…”