Estimation of the available free energy in a LOV2-J alpha photoswitch

Abstract

Protein photosensors are versatile tools for studying ligand-regulated allostery and signaling. Fundamental to these processes is the amount of energy that can be provided by a photosensor to control downstream signaling events. Such regulation is exemplified by the phototropins--plant serine/threonine kinases that are activated by blue light via conserved LOV (light, oxygen and voltage) domains. The core photosensor of oat phototropin 1 is a LOV domain that interacts in a light-dependent fashion with an adjacent alpha-helix (J alpha) to control kinase activity. We used solution NMR measurements to quantify the free energy of the LOV domain-J alpha-helix binding equilibrium in the dark and lit states. These data indicate that light shifts this equilibrium by approximately 3.8 kcal mol(-1), thus quantifying the energy available through LOV-J alpha for light-driven allosteric regulation. This study provides insight into the energetics of light sensing by phototropins and benchmark values for engineering photoswitchable systems based on the LOV-J alpha interaction.

Figure 1. Schematic representations of phototropin domain organization and photoswitching mechanism

(a). Domain organization of oat (A. sativa) phototropin 1, with the locations of domain boundaries indicated by residue numbers. (b). Phototropin switching mechanism is regulated at the stage of LOV2-Jα interactions, which are chiefly intact in the dark. Illumination leads to the formation of a protein-flavin adduct (involving Cys 490 in A. sativa phot1 LOV2 and the C4(a) position of the FMN chromophore), distorting the LOV2 structure sufficiently to significantly weaken the LOV2-Jα interactions thus freeing the Jα helix and allowing it to unfold.

Figure 2. Conformational exchange dynamics at the LOV-Jα interface as detected by relaxation dispersion measurements

Representative relaxation dispersion curves of backbone amide groups (a: K534 backbone ¹⁵N amide) and sidechain methyl groups (b: L514 ¹³Cα 1 methyl) at 600 (black circles) and 800 MHz (red squares) are shown. Residues with R_ex > 4 s⁻¹ for ¹⁵N (c) and > 2 s⁻¹ for ¹³C (d) are colored yellow in the LOV2-Jα ribbon diagrams, while the FMN chromophore is shown in green. Figures 2c and d were prepared with PyMOL software.

Figure 3. ¹⁵N relaxation dispersion analyses of LOV2-Jα constructs containing V529A, V529E and V529 point mutations

These mutants give similar Δω values as the wild type protein but have different high energy state populations. (a) ¹⁵N |Δω_Rex| of various residues from all constructs. (b) Relaxation dispersion curves of the A524 backbone amide ¹⁵N in WT (black), V529A (green), V529E (blue) and V529N (red) LOV2-Jα proteins at 800 MHz.

Figure 4. The Jα helix is unfolded in the high energy conformation of dark state LOV2-Jα

(a) Cα chemical shift index of Jα residues (D522 to A542) in the LOV2-Jα (open bars) and in the corresponding residues in the peptide (filled bars). (b) ¹⁵N |Δω_Rex| obtained from relaxation dispersion analyses plotted against |Δω_unfolding| = |ω_peptide – ω_LOV-Jω|. Data are labeled with the number of the corresponding residue. Residues which deviate from the m=1 line are indicated in red on LOV2-Jα structure.

Figure 5. The Jα helix is largely unfolded in the lit state

(a,b). Overlay of methyl region ¹³C-¹H (a) and ¹⁵N-¹H (b) HSQC spectra of lit state LOV2-Jα (red) protein and the peptide (blue). (c,d). Expansions of ¹⁵N-¹H HSQC spectra of WT LOV2-Jα in the dark (black), lit (red) states and the peptide (blue) of the backbone amides for residues R526 (c) and I539 (d).