Unusual effects of crowders on heme retention in myoglobin

Abstract

Edited by Stuart FergusonKeywords: Macromolecular crowder Glucose and sucrose Excluded volume effect Heme retention BSA Lysozyme Soft interaction a b s t r a c t Myoglobin (Mb) undergoes pronounced heme loss under denaturing conditions wherein the proximal histidine gets protonated. Our data show that macromolecular crowding agents (both synthetic and protein based) can appreciably influence the extent of heme retention in Mb. Interestingly, glucose and sucrose, the monomeric constituents of dextran and ficoll-based crowders were much more effective in preventing heme dissociation of Mb, albeit, at much higher concentrations. The protein crowders BSA and lysozyme show very interesting results with BSA bringing about the maximum heme retention amongst all the crowding agents used while lysozyme induced heme dissociation even in the native state of Mb. The stark difference that these protein crowders exhibit when interacting with the heme protein is a testament to the varied interaction potentials that a test protein might be exposed to in the physiological (crowded) milieu.

Effects of Ethylene Glycol on the Structure and Stability of Myoglobin Using Spectroscopic, Interaction, and In Silico Approaches: Monomer Is Different from Those of Its Polymers

“…Significant efforts have been devoted to explore the potential effects of macromolecular crowding on protein folding and crowder–protein interactions. Structural and thermodynamic studies of proteins in crowded and confined conditions in vitro − have been exploited to describe in vivo protein behavior, which is affected by dominance of various types of interactions occurring in cells. − In 2008, Zhou et al presented a thoughtful argument in their review in which polyethylene glycol (PEG) should be avoided as a crowding agent precisely because of its potential for the favorably interaction with the proteins that it is intended to be crowded. As a result, PEG is now fast-becoming something of an exile in the macromolecular crowding community.…”

Interactions Under Crowding Milieu: Chemical-Induced Denaturation of Myoglobin is Determined by the Extent of Heme Dissociation on Interaction with Crowders

“…Since in the case of soft interactions, chemical interactions are happening, it may be suggested that the natures of crowding agents can be a significant factor in the degree of destabilization of proteins. Crowding agents, i.e., low molecular size PEG 400 Da and intermediate sizes (PEG 10 kDa, PEG 8 kDa) [3,57,58], ficoll, dextran [33] and proteins (lysozyme and BSA) [3] also destabilize Mb, and in another report it shows that glucose, an osmolyte, also destabilizes Mb [67]. However, it was reported that glucose and sucrose, the monomeric constituents of dextran-and ficoll-based crowder prevents the heme dissociation at higher concentrations [3].…”

“…In myoglobin, porphyrin ring of the heme moiety acts as a chromophore and shows characteristic absorption peak at 409 nm, which originates from the interaction between heme and globin (folded protein) and is a signature of the native myoglobin [1]. Absorption of Mb in the Soret region is extremely sensitive to the milieu of heme and therefore generally has been exploited to monitor disruption in the heme pocket due to change in experimental condition [2,3]. GdmCl-induced denaturation of Mb in the presence of ficoll 70 ( Figure S1A) and dextran 70 ( Figure S1B) was measured in the wavelength range 350-450 nm.…”

“…Since the intracellular environment is highly crowded, it becomes very imperative and interesting to know how protein folds and functions in the cellular environment. Various natural and artificial crowder molecules are used with the purpose to investigate the structural, functional changes and type of interactions induced by crowding, in order to mimic cell like conditions [2][3][4]. As the cellular environment is composed of various biomolecules, which differ in shape, size and chemical nature [5], crowders having different shape, size and nature were used in order to study the effect of crowding [6,7].…”

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

“…Various studies on protein folding in crowded systems with a range of parameters (size, shape, and concentrations) were carried out to understand the phenomenon ( Van Den Berg et al, 1999 ; Van Den Berg et al, 2000 ; Galan et al, 2001 ; Zhou et al, 2004 ; Parray et al, 2019 ; Parray et al, 2020a ; Parray et al, 2020b ; Parray et al, 2020c ; Parray et al, 2020d ; Nasreen et al, 2020 ). Protein folding has been examined in the presence of macromolecular crowders like polyethylene glycols (PEGs), ficolls, dextran, and others under physiological environments ( Sasahara et al, 2003 ; Rawat et al, 2010 ; Qu et al, 2012 ; Zhang et al, 2012 ; Sarkar et al, 2013a ; Kundu et al, 2015 ; Shahid et al, 2015 ; Parray et al, 2017 ; Shahid 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 ). It is known that macromolecular crowding in cells has a massive effect on biological activity, but predictive studies are relatively unexplored when it comes to its implications.…”