Optical perturbation-response measurements of molecular biophysics

Abstract

This work pertains to, and relies on tools enabled by, the interactions between light and life. In Chapter 2, we explore the photophysics of a red fluorescent protein called mScarlet3. We discover that its brightness can respond enormously (>20%) to weak (milli-Tesla) magnetic fields in live E. coli. We probe the mScarlet3 response to magnetic fields while varying the illumination conditions and chemical environment. Through these experiments we elucidate the mechanism of its magnetic field sensitivity: an excited-state electron transfer from a reduced flavin to the mScarlet3 triplet state creates a magnetically sensitive radical pair, consistent with the mechanism responsible for many chemical magnetic field effects. In Chapter 3, we explore the transport of an intracellular small molecule second messenger, cyclic adenosine monophosphate (cAMP), by using light to both perturb and to image it. We use a fluorescent actuator of cAMP and a spectrally distinct reporter to locally perturb and map cAMP transport in mammalian cells. We find that cAMP freely diffuses in primary rat hippocampal neurons (D ≈ 130 μm2/s), and that micron-scale domains (5–40 μm) are sustained at steady-state. We introduce relations to describe how cell geometry and enzyme activity and localization strongly inform the concentration profiles of diffusible small molecules.