Dynamical and allosteric regulation of photoprotection in light harvesting complex II

Abstract

Major light-harvesting complex of photosystem II (LHCII) plays a dual role in light-harvesting and excited energy dissipation to protect photodamage from excess energy. The regulatory switch is induced by increased acidity, temperature or both. However, the molecular origin of the protein dynamics at the atomic level is still unknown. We carried out temperature-jump time-resolved infrared spectroscopy and molecular dynamics simulations to determine the energy quenching dynamics and conformational changes of LHCII trimers. We found that the spontaneous formation of a pair of local α-helices from the 3₁₀-helix E/loop and the C-terminal coil of the neighboring monomer, in response to the increased environmental temperature and/or acidity, induces a scissoring motion of transmembrane helices A and B, shifting the conformational equilibrium to a more open state, with an increased angle between the associated carotenoids. The dynamical allosteric conformation change leads to close contacts between the first excited state of carotenoid lutein 1 and chlorophyll pigments, facilitating the fluorescence quenching. Based on these results, we suggest a unified mechanism by which the LHCII trimer controls the dissipation of excess excited energy in response to increased temperature and acidity, as an intrinsic result of intense sun light in plant photosynthesis.

Keywords: FTIR; LHCII photoprotection; T-jump; conformational dynamics and allostery; excited energy transfer; fluorescence quenching; protein switch.

Figure 1

Crystal structure and the fluorescence decay kinetics of LHCII trimers. (a) Schematic structures of LHCII trimers (PDB: 1RWT) along with pigments (stick model) in highlighted in one subunits. (b) Fluorescence decay curves of LHCII trimers at room temperature (RT) under different conditions as indicated, excited at 400 nm, and acquired by a streak camera with an instrument response factor (IRF) of 0.13 ns. The fluorescence decay curves for the aggregated LHCII trimer are fitted with a bi-exponential process (weakly aggregated condition: pH 5.6, β-DM: 0.003% (w/v); heavily aggregated condition: pH 5.6, β-DM: 0.0003% (w/v)) and that for non-aggregated is fitted by a mono-exponential decay (non-aggregated condition: pH 5.6, β-DM: 0.03% (w/v)). (c) Globally fitted fluorescence lifetimes for the fast and slow components at varied temperatures for the aggregated LHCII trimer (β-DM: 0.006% (w/v)) at two different pH as indicated. (d) Photos of the sucrose density gradient ultracentrifugation separation of the thermal incubated LHCII trimer for 5 min at varied temperatures as indicated and under two different aggregated conditions (aggregated condition: pH 5.6, β-DM: 0.003% (w/v); non-aggregated condition: pH 5.6, β-DM: 0.03% (w/v)). LHCII trimers corresponding to 124 μg of Chl were loaded on the sucrose gradient, and the details in fractionation were described in “Sample preparation”. (e, f) Temperature-dependent population variation of a three-state model, i.e., harvesting (H), quenching (Q), and denaturing (D) states, of non-aggregated (β-DM: 0.03% (w/v)) and aggregated LHCII trimers (β-DM: 0.006% (w/v)) at pH of 7.7 (e) and 5.6 (f) respectively (color online).

Figure 2

Temperature-induced electronic couplings between Lut1 and Chl and configurational twists of lutein for LHCII trimers. Distribution of computed electronic couplings between Lut1 and Chl. The computed electronic couplings are given as dashed curves for Lut1-Chl610, and as solid curves for Lut1-Ch612 at low (270–290 K) in blue, medium (300–320 K) in orange, and high (330–360 K) in maroon temperature ranges. The lowest unoccupied molecular orbital (LUMO) of Lut1 is shown along with its relative proximity to Chl610 and Chl612. Multistate density functional theory was used along with the CAM-B3LYP functional and 6– 31G(d) basis set in combined quantum-mechanical/molecular mechanical (QM/MM) calculations, in which the pigments are treated by MSDFT embedded in the rest of the protein and solvent system represented by molecular mechanics (color online).

Figure 3

IR spectral assignments and correlation between 3₁₀-helix and α-helix in response to increased temperature and acidity for LHCII trimers. (a) Second-derivative FTIR spectra and IR assignments of LHCII trimers and monomers at RT. (b) Time-resolved spectral diffusion induced by a T-jump of ΔT=17°C. Thermal-induced transition of 3₁₀-helix/loop to α-helices is revealed by the complementary kinetics, (c) Acidity-induced spectral variation of LHCII in the amide I′ region for α-helices (1,651 cm⁻¹) and 3₁₀-helix/loop (1,659 cm⁻¹) at pH 5.4, 7.5 and 9.6. The isosbestic point indicates the acidity-induced interconversion between the two secondary structures, (d) Comparison of the secondary derivative spectra of wild-type (WT) LHCII trimers and S123G mutants at pH 7.4 with a β-DM concentration of 0.03% (w/v) at RT (color online).

Figure 4

Experimental T-jump time-resolved IR spectra and a-helices transition of LHCII trimers from computations, (a) Differential optical absorption (ΔOD) of IR spectra of LHCII trimers at RT from T-jump values (ΔT) of 17 and 15 °C delayed by 8 μs. Positive and negative values indicate population increase and decrease of the corresponding structural elements, (b) Time-resolved spectral diffusion at selected frequencies induced by a T-jump of ΔT=17°C. The overlap in bleaching kinetics at 1,608 and 1,660 cm⁻¹ indicates a cooperative conversion of 3₁₀-helix E/loop and antiparallel β-strands to α-helices. Details of kinetics analyses are given in Figure S7. (c, d) The distribution of helical contents of the residue 97–107 of subunit Ml vs. that of the residue 221–229 of the neighboring subunit M2 based on structures obtained from MD trajectories at 280 K (c) and 340 K (d). The helical content characterizes the number of six residue segments of the protein that are in an α-helical configuration, (e) Representative structures of the 3₁₀-helix E of Ml and C-terminal residues of M2 (blue) at 280 K. (f) A snapshot structure depicting α-helix transitions (red) at 340 K in the same regions as in (e). Note that the nascent α-helix E has been extended by two residues from Trp97-Phe105 of the initial 3₁₀-helix to Trp97-Glu107 (color online).

Figure 5

Representative structures and populations of LHCII trimers at different temperatures revealed by MD simulations. (a) Illustration of PCI modes at the lumenal interface, highlighting the intrusion motion of helix D and E towards each other. The coloring of the backbone from blue to red corresponds to average structures as temperature increases. (b) A snapshot structure from the MD trajectory at 280 K (low T), illustrating key interactions in the close proximity of TM helix A (cyan) and B (purple). One end of helix D (orange) forms a cluster of hydrophobic contacts with the helix B, involving V80, L84, and L206 in the yellow shade, denoted as Lever I. The extended loop of 3₁₀-helix E forms a second cluster of hydrophobic contacts, involving W97, FI 94 and F195 (in the yellow shade), specified as Lever II. K99 near the beginning of 3₁₀-helix E forms a salt bridge with E94, shown as the stick model, (c) A representative snapshot structure from the MD trajectory at 340 K (high T), depicting structural variations from that shown in (b). The same color scheme as that in (b) is used, superimposing to the structure of (b) in light grey. Noticeable changes are (1) the switch of the hydrogen bond of E94 from K99 in (b) to Q103 in (c), (2) the shifts of both α-helix D and E towards each other, indicated by arrows, and (3) overall tilts of TM helix A and B away from one another, (d, e) Distributions of average distances of Lever I and Lever II, respectively, from MD trajectories obtained at temperatures ranging from 270 to 360 K. Notice the relative similar distributions of each clusters of hydrophobic contacts at different temperatures. The density is given in the number of pairs per bin (0.05 Å) throughout, (f) Distribution of the distance of helix D and E at different temperatures, where the distance is represented by W97-Cα at helix E and P205-Cα at helix D. (g) Contact distance of E94-Q103 at different temperatures. The formation hydrogen bond with Q103 increases significantly as temperature increases, and the existence of hydrogen bonds is more likely attributed to the conformational change and intrusion motions of helix E. (h, i) Distributions of average hydrogen bond distances between Arg70 and Glul80 (h), Glu65 and Argl85 (i) at different temperatures, (j) Correlation of the angle between carotenoid Lut1 and Lut2 with that of TM helix A and B. (k) Distribution of the distance between Lut1 and Chl612, characterized by the minimal distance between Mg atoms of Chl and the C atoms in the conjugated π-system of lutein, at different temperatures (color online).

Figure 6

Dynamical allosteric regulation mechanism for light-harvesting and photoprotection of LHCII trimers. (a, b) LHCII trimers act as a molecular machine, in which TM helices constitute the wedges that pivot around the fulcrum anchored by the salt bridge between Arg70 and Glul80, Glu65 and Argl85 (Figure 5(h, i)), and the hydrophobic contacts connecting the helix D and E (Figure 5(b)) are the lever. The signal for photoprotection, the increase in acidity, environmental steady-state temperature together with transient temperature rising due to NPQ or both, induces a local structural transition to convert the 310-helix E and C-terminal loop into two α-helices, which triggers a switch of the hydrogen bond of Glu94 from Lys99 in the light-harvesting state (left) to Gin 103 in the photoprotection state (right) (see Figure 5(b, c)). In turn, α-helix D and E are pulled closer against TM helices A and B, shifting its conformational equilibrium to a wider intercrossing angle. The net effect of the overall conformation change of TM helices is to reduce the contact distance between Lut1 and Chl612, enhancing their electronic coupling in favor of excited energy transfer from excited Chls to the SI dark state of Lut1. ΔT: transient temperature rising; ΔpH: pH gradient (color online).

Scheme 1

Relations among three conformers for LHCII trimers.