Grant: Discovery Grants Program - Individual

The cell is a highly crowded place, full of ions, sugars, osmolytes, nucleic acids and proteins. Interestingly, this crowdedness is an asset not a liability: crowding not only prevents detrimental cellular processes such as misfolding and aggregation, but also causes protein phase separation, from membrane-less organelles such as nucleoli, Cajal bodies, paraspeckles and stress granules are formed. Such diverse manifestations of crowding effects pose the following question: how different are protein reactions performed in a test tube from when they occur inside a living organism? Understanding how proteins function in such remarkably crowded conditions is not only a fascinating question from a biophysical chemistry perspective, but also becomes extremely relevant in situations where proteins are found outside their natural habitat (most notably in protein production applications or enzyme-mediated biomanufacturing conditions). In these cases, knowing how to use crowders so that they restrict and restrain proteins from accessing undesirable conformations would have great impact on the work of biotechnologists aiming to optimize protein-based applications. In this research program, I will clarify the mechanism of action of osmolytes. Osmolytes are a special class of crowder molecules that organisms produce to counteract the destabilizing effects of environmental stress. Because unfolded, inactive proteins take up much more space than folded ones, osmolytes were first thought to stabilize proteins just by reducing the amount of available space allocated to their co-solute proteins, doing nothing else (entropic effect). Further research has shown that this picture is incomplete: osmolytes have been shown to modulate how proteins interact with their environment (enthalpic effect). Despite this change in our thinking about osmolyte effects, it is still unclear if osmolytes stabilize proteins through direct protein-osmolyte interactions, or whether their effects are mediated through the solvent (water). Theoretical and computational studies hypothesize that solvent mediation is the dominant reason for osmolyte effects. Over the next 5 years, my group (consisting of 5 graduate trainee HQP) aims to test this hypothesis by first quantifying how osmolyte addition perturbs the thermodynamics and kinetics of a two paradigm protein reactions: protein folding and protein-protein interactions. I will then quantify how osmolyte addition affects the hydrogen bonding networks of the solvent system and the protein solvation shell. If the effects of different osmolytes on protein reactions correlate with their effects on hydrogen bonding, it is clear that hydrogen bond manipulation is a major mechanism of osmolyte action. Otherwise, osmolyte effects on protein reactions originate from how they specifically interact with protein species. The methods developed in this research program can then be expanded to study the effects of protein crowders upon protein reactions.