Exploring the Size-Scaling of Microviscosity within the Crowded Environment of the Cell
By: Patra Holmes, Michelle Chong, Lika Chhit, Maureen Montes
Department: Chemistry & Biochemistry
Faculty Advisor: Dr. Raymond Esquerra
Macromolecular crowding within the cell creates a viscous environment that directly affects how proteins fold and function. The impact of crowding on protein motion can be quantified as microviscosity – the resistance to motion a molecule experiences. Rotational microviscosity is particularly important for proteins since folding and functional motions occur over shorter distances (nanometers) than the motions associated with translational microviscosity (micrometers). Previous studies have probed rotational microviscosity using small molecules. However, the rotational microviscosity specific to proteins remains poorly understood. Microviscosity determined using small molecules may not extend to how proteins experience the same environment since proteins have a larger surface area. To address this knowledge gap, we have developed time-resolved linear dichroism (TRLD), a novel instrument that can accurately determine the rotational microviscosity of a protein probe in whole cells.
Our hypothesis is that the effects of macromolecular crowding on microviscosity differ for proteins compared to small molecules. To test this hypothesis, we have constructed a dual probe for rotational microviscosity consisting of a heme protein linked to a haloalkane dehalogenase (HaloTag, Promega) that allows for a small molecular rotor to be covalently attached. This will enable microviscosity measurements of the larger protein complex and the small rotor within the same immediate environment. The microviscosity of the protein will be determined using TRLD and the Stokes-Einstein-Debye relations. For comparison, the microviscosity of the small rotor will be simultaneously determined from its fluorescence lifetime and the Förster-Hoffman equation. The dual probe will first be tested in crowding agents that mimic the cellular environment to calibrate microviscosity to TRLD and fluorescence signals. Finally, microviscosity in situ will be measured in E. coli. Investigating the size-scaling of microviscosity between proteins and small molecules will deepen our foundational understanding of the specific relationship between protein folding and function and the crowded intracellular environment.