Here are some of the projects that we’re working on

The Sheets/Heikal Group been developing new ways to sense cellular environments using fluorescent protein-based biosensors. The protein-based sensor, which was developed by our collaborator, Arnold Boersma, is based on coupling two fluorescent proteins by a flexible linker that responds to the environment. When the two fluorescent proteins are close, Förster resonance energy transfer (FRET) occurs.

We have developed new ways to measure FRET using fluorescence lifetime, time-resolved fluorescence anisotropy, and fluorescence correlation spectroscopy. Fluorescence lifetime and anisotropy are ensemble methods that measure changes on the nanosecond timescale, whereas fluorescence correlation spectroscopy is a single-molecule technique that measures changes on the microsecond timescale. We’ve characterized two families of sensors. One family detects changes in macromolecular crowding, and the other reports changes in ionic strength.

The interior of the cell is crowded with organelles and biomolecules, which contrasts with the buffered solutions that are typically used in conventional biochemical studies. Crowded conditions can impact the functions, interactions, and dynamics of biomolecules. These biological functions are also affected by localized changes in ionic strength, or salt concentrations. We are investigating the physical and chemical effects of macromolecular crowding (synthetic polymers and proteins) and ionic strength on protein interactions and dynamics.

This complexity requires the acquisition of single-molecule information, together with bulk studies, to understand the length- and time-scale dependence associated with crowding effects or ionic strength on protein association kinetics and activities. To investigate these effects, we use ultrafast laser-based spectroscopy to measure the rotational and translational diffusion and molecular volume of a given molecule as a function of crowding agent or salt concentration. We investigate these dynamics in bulk solution and live cells.