A Novel Method to Measure Rotational Diffusion of Proteins in Whole Cells

Authors: Patra Holmes, Michelle Chong, Maureen Montes, Lika Chhit, Ericka Gonzales

Faculty Supervisor: Raymond M. Esquerra

Department: Chemistry & Biochemistry

The cell's interior is densely packed with macromolecules, a condition known as macromolecular crowding, which impairs protein functionality. Microviscosity measures the resistance proteins encounter while moving in such crowded spaces and is higher where crowding is more intense. However, measuring microviscosity accurately is difficult, yet crucial for understanding diseases like Alzheimer’s and Parkinson’s, where protein aggregation is common. To address this issue, we have developed a new instrument that implements time-resolved linear dichroism (TRLD) to measure rotational microviscosity using protein probes. Here, we present four bioengineered probes designed to measure cellular microviscosity, paving the way for a deeper understanding of its role in cellular function and disease. After measuring these probes with this novel method, we will compare their diffusion as a function of macromolecular crowders and cellular location: 1) Probes of different sizes and shapes (Mb-MBP, Mb-HaloTag, Mb-PIF). 2) Probe with different dynamics (rigid and flexible). 3) Probes that target different cellular locations (Mb-MBP, Mb-MBP-PelB). 4) Probes with light-controlled dimerization via optogenetic control. (Mb-PIF, PhyB). This method will allow for a better understanding of the interplay between protein structure and the heterogeneous intracellular environment and, ultimately, the role this relationship plays in disease.