Crowded milieu tuning the stability and activity of stem bromelain

Proteins in vivo are under an extremely crowded environment because of the presence of bulky and large biological macromolecules (known as crowders). These crowders affect the proper functioning and structure of proteins in a cell. During in vitro studies, we often ignore the effect of macromolecular crowding on protein stability. However, if a large concentration of crowder is used to examine protein stability, its effects on the functioning of protein inside the cell in a confined environment, as stated, can be understood. Keeping this in context, we investigated the effects of macromolecular crowding on stem bromelain (BM) with the help of different crowding agents of varying molecular weights such as dextran (40 kDa and 6 kDa) and ficoll (70 kDa). Activity and stability of BM was examined using UV-vis, fluorescence and circular dichroism (CD) spectroscopy. Furthermore, docking methods are used to complement the crowding effects on the stability of BM. We found that stability and activity of BM are dependent on the surrounding crowder molecules. Thermal flouresence results showed that, thermal stability of BM decreses with incresing concentration of crowder except dextran40. It was observed that the decrese in stability and activity can be related to the presence of soft interactions between crowder and BM. Thus,crowding does not always stabilize the native structure, instead, it depends on degree of disorder of protein structure and on two competing effects: the excluded volume, which favors compact states, and soft interactions, which favor extended conformers.