Investigation of the Interactions between LOV Domains by Spectroscopic Techniques

Abstract

Plants have adapted a variety of methods necessary for their survival such as stomatal opening and closing, plant growth, and photosynthesis. In order to regulate these processes, plants rely on sensing sunlight in their environment. The plant protein phototropin (PHOT) helps to sense sunlight and transmit the signal downstream to other regulatory proteins.1 To accomplish light sensing, PHOT contains a pair of blue light sensitive structures referred to as Light, Oxygen, or Voltage (LOV) domains, referred to as LOV1 and LOV2, which interact to regulate downstream cell signaling events. While the photomechanism of how LOV domains sense sunlight has been studied and characterized, the process of how the domains propagate the sensory signal is not well understood.2, 3 In this work, spectroscopic methods were employed to investigate the interaction of LOV domains in the dark and when exposed to blue light. Specifically, single molecule burst measurements and stopped-flow spectroscopy were used to detect Förster Resonance Energy Transfer (FRET) between the LOV domains in sample mixtures. First, single molecule burst measurements were used to probe monomer and dimer formation in samples of labeled LOV from the bacteria Rhodobacter sphaeroides (Rsp), followed by the attempt to determine the equilibrium dissociation constant (Kd) for Rsp LOV. Similar methods were then used to probe the interactions of LOV1 from PHOT of the alga Chlamydomonas reinhardtii (C.r.). The experimental methods by which we probed the LOV-LOV interactions are presented. A maximum entropy algorithm was used to generate FRET probability distributions from fluorescence measurements on LOV that was placed in the dark (dark-incubated), then exposed to blue light (light-exposed), and then incubated in the dark again (dark-reverted). To attempt to determine a dissociation constant (Kd), we measured the fluorescence for Rsp mixtures of varying concentration and analyzed the ratios of donor and acceptor channel photons detected to search for a change in the ratio when the concentration of the sample mixture was above or below equilibrium. LOV from Rsp was shown to increase FRET upon dark-reverted incubation of the proteins. The change in FRET may be affected by the concentration of the sample mixture after blue light exposure, the time the sample mixture spent waiting in the dark, and the amount of blue light exposure. In an attempt to determine Kd, we observed FRET between dark-reverted samples that was not accounted for by cross talk and direct excitation of the acceptor, but the data were not conclusive enough to determine a dissociation constant. For Chlamydomonas reinhardtii sample mixtures, we observed no noticeable change in the FRET efficiency between dark and light states of C.r. LOV1, possibly caused by having one of the two labeled LOV samples in the mixture be photoactive. Second, we investigated the interaction of C.r. LOV1 using stopped-flow spectroscopy to detect FRET between LOV dimers and gain understanding of the kinetics of the interactions. Plots of the data showed a change in the acceptor channel response of labeled C.r. LOV1 proteins after exposed to blue light. Based on the recovered time constants from two-component fits of the detector response curves, dimer formation between C.r. LOV1 has a fast (~101 s) and slow (~102-103 s) process we attribute to the formation of heterodimers within the mixing chamber and formation of light adapted homodimers prior to mixing.