Myoglobin Unfolding in Crowding and Confinement

Abstract

Crowding and confinement have often been used synonymously with regard to their effect on the structure and dynamics of proteins. In this work, we have investigated the unfolding of the protein myoglobin (Mb) entrapped in the confinement of the water pool of AOT reverse micelles and in the presence of some commonly used macromolecular crowding agents (Ficoll 70, Dextran 70, and Dextran 40). Our results reveal that confinement effects can be quite destabilizing in nature for Mb with the extent of distortion depending on a host of factors apart from the size of the confining cage. Effects of the crowding agents on myoglobin also show a deviation from the general notion that synthetic macromolecular crowding agents are always stabilizing in nature. Ficoll 70 was observed to be particularly destabilizing in its influence on Mb unfolding. Moreover, tryptophan lifetime studies point to the fact that the Trp-heme distance in Mb might not always be a reliable probe of the secondary structural dissolution of the protein.

Comprehensive studies on the nature of interaction between catalase and SiO 2 nanoparticle

“…In presence of SiO 2 NP, CAT undergoes a distortion in its secondary structure as observed from the obvious decrease in the intensity of its helical band at 208 nm and 220 nm. The above result was in agreement with the UV-vis studies [26].…”

Macromolecular crowding: Macromolecules friend or foe

“…Dextran 70 has been demonstrated to decrease the stability of rPrP C (properly folded prion protein) by 8°C and rPrP 80R by 12.5°C [40]. Similar to Dextran 70, Ficoll 70 was also shown to enhance unfolding of myoglobin by urea [29]. Furthermore, in a recent development by our laboratory [41], holo α-lactalbumin was observed to be destabilized by several degrees (in terms of T m , melting temperature) in the presence of polymer crowders due to reduction in its calcium-binding affinity.…”

“…All these opposing effects are described as "hostile or negative effect" in the entire manuscript. For instance, macromolecular crowding has been demonstrated to have a destabilizing influence on the stability of Myoglobin [29]. Recent investigations made by Fan et al suggested a decrease in the activity of recombinant human brain-type creatine kinase under crowded conditions [30].…”

Unusual domain movement in a multidomain protein in the presence of macromolecular crowders

“…Extensive research has been carried out to investigate the various underlying mechanisms and driving forces that are important for proteins, having different degrees of structural complexity, to fold in a physiologically relevant timescale. [6][7][8] Irrespective of whether one uses dilute buffer solutions or the crowded media, most of the folding studies have focused on either single domain proteins 9 or those that are a part of bigger proteins having multiple domains 10,11 or involving DNA. These macromolecules, popularly termed crowding agents, through their excluded volume effect, reduce the space available for the biological molecule of interest.…”