Information transfer in biological signaling pathways often takes the form of stimulus-induced changes in protein structure that consequently alter a functional output. Many signaling proteins are composed of linked arrays of modular domains, which cooperatively function to control activity1,2
. In such proteins, sensor domains receive inputs related to changes in the local environment, e.g.
the binding of a metabolite or other protein, or the alteration of a bound cofactor by a change in redox state. These events change the interactions of the sensors with other domains, which subsequently transmit the signal downstream through changes in their activities. A critical parameter in the construction of such sensors is the magnitude of input-induced changes in the energetics of the sensor domain and its interactions with downstream effectors. It is this change in energy, and the corresponding change in interaction equilibria, that determines the dynamic range of the sensor along with several other fundamental signaling properties.
Several examples of this principle are provided by photosensory proteins, which have evolved to sense and respond to light across the UV/visible spectrum at a wide range of intensities3
. These sensory processes are typically achieved through protein domains that bind light-absorbing chromophores and convert photon energy into structural, dynamic and functional changes. One class of these proteins are phototropins, a group of blue light-activated serine/threonine kinases that control a range of biological responses in algae and plants, including phototropism, chloroplast migration and stomatal opening4
. Phototropins sense light through two LOV (Light-Oxygen-Voltage) domains, LOV1 and LOV2, which are located on the N-terminal side of the conserved kinase domain ()5
. LOV domains are a subgroup of the larger PAS (Per-ARNT-Sim) domain family, whose members function as sensors of a wide variety of environmental processes throughout biology6,7
. To sensitize phototropin to blue light, both LOV1 and LOV2 contain non-covalently bound FMN chromophores ()8
. Blue light absorption induces formation of a covalent adduct between a conserved cysteine residue in the LOV domain and the C4a carbon of the isoalloxazine ring of FMN9
. While the functional significance of light absorption by LOV1 is still unclear, adduct formation in LOV2 is crucial for light-dependent enhancement of phototropin kinase activity as assessed in vitro
by autophosphorylation and in planta
by phototropism and other responses10
. Crystal structures and high-resolution solution NMR data of several LOV domains11–16
show they all adopt typical PAS domain folds, consisting of a mixed α/β fold of approximately 110 amino acid residues surrounding the flavin chromophore. Solution NMR studies13
and a subsequent crystal structure15
of a fragment of the Avena sativa
phototropin 1 (AsPhot1) containing the LOV2 domain plus a 40 residue C terminal extension have revealed an additional 20 residue α-helix, Jα, that packs onto the PAS core in the dark state. As demonstrated by NMR and other biophysical data, light induces conformational changes in the LOV2 domain that lead to Jα unfolding and dissociation from the LOV domain after the Cys-FMN adduct is formed13,17
. Complementary biochemical studies have shown that destabilization of the LOV2-Jα interaction by mutagenesis activates full-length Arabidopsis thaliana
phototropin 1 independent of light18
. Together, these data argue that dissociation of Jα – or more precisely, a shift in the LOV2-Jα binding equilibrium to favor the helix-dissociated state – upon photon absorption is the principal mechanism by which phototropin is controlled by light ().
Schematic representations of phototropin domain organization and photoswitching mechanism
Despite substantial progress in understanding the structural mechanisms of phototropin signaling, the energetics of this core LOV2-Jα switch have not yet been explored. Here we use NMR spectroscopy to quantify the equilibria between the helix-bound and helix-dissociated conformations of LOV2-Jα in the dark and lit states, yielding an estimate of the energy that is potentially available in this photoswitch to achieve this change in functional states. Using NMR 15N and 13C relaxation dispersion analyses, we found that the Jα helix and its binding surface on LOV2 undergo substantial fluctuations on micro- to millisecond timescales in the dark. Fitting these dispersion data to a model for two-site exchange, we determined that the populations of the low- and high-energy conformations are 98.4% and 1.6%, respectively, with a corresponding free energy difference of 2.4 kcal mol−1 across this equilibrium. By comparing the fitted chemical shifts of the high energy state to those directly measured in an isolated Jα peptide, we suggest that Jα is unfolded and dissociated in the high energy conformation despite the lack of a Cys-FMN adduct. Systematic differences between the chemical shifts of the Jα peptide and those of a photoactivated sample suggest that the LOV2-Jα system samples an analogous bound-dissociated equilibrium for Jα after blue-light absorption. A quantitative analysis of these differences indicates that the population of the helix-dissociated conformation in the lit state is ~91%, corresponding to approximately –1.4 kcal mol−1 of free energy across the equilibrium. Thus, we determined that the primary effect of light absorption and the corresponding Cys-FMN adduct formation is to shift the LOV2-Jα binding equilibrium by ~3.8 kcal mol−1. This provides the first estimate of the energy potentially available through the LOV2-Jα photoswitch to activate the kinase domain in AsPhot1, and also yields a critical value for the engineering of artificial photoswitches where the LOV2-Jα interaction is coupled to regulation of the activity of other proteins.