Membrane-induced conformational changes in LHCII

The linear absorption spectra of LHCII (Fig. 1a) in detergent and in nanodiscs confirm its successful incorporation into nanodiscs in intact trimeric form, based on similar overall peak location and profiles (Supplementary Note 4, Supplementary Table 2 and Supplementary Fig. 11). A closer inspection of the spectra shows subtle changes in peak position and/or intensity in the Car S 2 states (470–510 nm) as well as the two Q y bands of Chls (640–690 nm), suggesting changes in the arrangement of both the Cars and Chls resulting from introduction of the membrane environment.

Fig. 1: Changes in pigment orientations upon incorporation into membrane discs. a Schematic illustration of the membrane disc containing a single trimeric LHCII complex (PDB 1RWT70). b Side view of the LHCII trimer. Chl a are displayed in green, Chl b in blue, luteins (Luts) in pink, neoxanthin (Neo) in purple, and violaxanthin (Vio) in orange. Roman numerals show the two pigment clusters perturbed upon disc formation. c Pigment-only side views of clusters I and II (top: I, bottom: II). d and e CD (top) and second-derivative CD spectra (bottom) of LHCII in detergent (gray) and in membrane discs (green), plotted for the Car S 2 d and Chl Q y absorption range e. Stick plots indicate the absorption peak wavelengths of the pigments shown in c. The peak positions for Chls are taken from ref. 50. Purple and pink shaded regions highlight membrane-induced changes in CD for the two domains I and II. Full size image

Circular dichroism (CD) spectra of LHCII in detergent and in discs provide a sensitive measure of the spatial configuration of pigments bound to the complex, because CD peak shape and intensity are directly related to the mutual orientation of the transition dipoles and the strength of their interactions39,40. Comparison of the CD spectra reveals two differences involving two peripheral Cars, neoxanthin (Neo), and lutein 1 (Lut1, Fig. 1b, c). First, the relative peak intensities between 474 and 492 nm (494 nm) change, which has been reported to originate from the interactions between the Soret band of the Chls b and Neo (Fig. 1d and Supplementary Note 5, Supplementary Fig. 12)30,39,41. A similar change in the peak ratio was previously observed in LHCII nanodiscs42. Second, the negative 492 nm peak redshifts by 2 nm, reported to originate from the interactions between the high-energy lutein (Lut1) and the Soret band of Chl a61239. Thus, the observed changes point to alterations in the spatial arrangement of Neo and Lut1 caused by the membrane. In contrast, we do not observe any difference in the CD signal at 500–510 nm, where the lower-energy lutein (Lut2) absorbs.

CD in the Chl Q y region (Fig. 1e) reveals a slight broadening of the 653 nm peak and a 2 nm redshift of the 682 nm peak, attributed to excitonic interactions between Chl a604-Chl b606 and Chl a610-Chl b608, and between Chls a611 and a612, respectively39. These are the Chls that are strongly coupled with Neo (Chl a604, Chl b606, Chl b608) and Lut1 (Chls a610, a611, a612)43, illustrated as domains I and II, respectively, in Fig. 1b, c. Given that Neo and Lut1 are the two Cars impacted upon incorporation into the membrane, we speculate that the observed changes in the rotational strengths of the Chls could arise from changes in their excitonic interactions with the neighboring Cars, rather than independent structural reorganization of the Chls in the membrane.

Perturbation of domain II, which contains the three lowest-energy Chl a pigments that form the emissive locus (Chls a610, a611, a612)44, is further supported by a reduction of fluorescence in the membrane. The steady-state fluorescence quantum yield and fluorescence lifetime are reduced in the membrane discs by 17% and 18%, respectively (Supplementary Note 6, Supplementary Figs. 13, 14 and Supplementary Table 3). The slight quenching of the fluorescence upon membrane insertion is consistent with previous results on LHCII nanodiscs28. The observed fluorescence lifetime (2.8 ns) is still significantly longer than that measured in vivo (<2 ns)45 or in crystals (1 ns)22, suggesting additional interactions are present in these systems due to the presence of multiple antenna complexes.

Domains I and II are located at the periphery of the trimeric LHCII complex (Fig. 1a, b). Compared to the counterparts located closer to the core that are shielded by the surrounding pigments and protein matrix, these domains are more exposed to the lipid bilayer. Thus, they are more susceptible to structural changes induced by the membrane, consistent with our results. In particular, a significant part of the conjugated chain of Neo protrudes outward from the protein matrix, which may allow severe twisting of the chain by environmental interactions. Such a distortion in the conjugated chain of Neo has, in fact, been predicted theoretically24.

Energetics and ultrafast dynamics of the peripheral Cars

Ultrabroadband 2DES was employed to determine the impact of the membrane on the photophysical pathways in LHCII. By using a laser spectrum with a significantly broader bandwidth than that in conventional 2DES46, we map out energy transfer and dissipation across the broad range of Car and Chl excited states. Supplementary Fig. 15, Supplementary Note 7 shows a representative ultrabroadband 2D spectrum of LHCII with the main spectral features labeled.

Figure 2a compares the 2D spectra of LHCII in the detergent and the membrane environment (T = 533 fs) in the frequency range of the Car S 2 states. Two major changes are observed. The first is increased transfer of the Car S 2 population into the dark S 1 state (S 2 → S 1 internal conversion), which results in decreased energy transfer to the lower-lying Chls, the competing pathway (Supplementary Note 7, Supplementary Figs. 16, 17). The relative population in S 1 is shown by the ratio of the magnitude of the S 1 excited-state absorption (ESA) to that of the initial ground-state bleach (GSB) of S 2 immediately after photoexcitation. The ratio increases by 40% in the membrane, showing the increase in transfer to S 1 (Fig. 2b, Supplementary Fig. 17, Supplementary Note 7). The increase is pronounced at the excitation frequencies of Neo and Lut1, showing 35−43% more efficient relaxation to the S 1 state. While the excitation frequency of Neo and that of violaxanthin (Vio) have a significant overlap8,47, and so the contribution from these two Cars cannot be distinguished (Supplementary Note 4, Supplementary Table 2), Neo is the likely origin of the increase based on the dramatic changes observed in the CD results. Unlike in the case of Neo and Lut1, the relaxation dynamics of Lut2 are independent of environment (Fig. 2b, c, Supplementary Fig. 18, Supplementary Note 7). Lut2 is located at the inner core of the trimeric LHCII (Fig. 1a, b), and thus relatively protected from direct exposure to the protein–lipid interface, as mentioned earlier. This may be the origin of its environment-independent dynamics. The Car–Chl cross peaks directly visualize energy transfer from the Car S 2 to the lower-lying Chl Q states, and so further report on Car S 2 dynamics. The cross-peak intensities decrease by 35% in the membrane (Fig. 2d, e), consistent with the increased S 1 to S 2 ratio shown in Fig. 2b.

Fig. 2: Impact of the membrane environment on energetics and relaxation dynamics of carotenoids. a Absorptive 2D spectrum of LHCII in detergent (left) and in the membrane (right) in the Car S 2 /S 1 region at T = 533 fs. Contour lines are drawn at 15% intervals. White dashed lines indicate the shift in Car S 1 → S N transition energy (\(\Delta {E}_{{{\rm{S}}}_{1}-{{\rm{S}}}_{{\rm{N}}}}\)). Colored sticks indicate the energy levels of the Car S 2 states. b Intensity of the Car S 1 ESA relative to the initial Car S 2 population at T = 533 fs in detergent (gray) and in membrane discs (green). The relative S 1 intensity was obtained by normalizing the S 1 ESA intensity to the initial S 2 GSB intensity immediately after photoexcitation (T = 30 fs). c Comparison of Car S 1 ESA decay constants in detergent (gray) and in membrane discs (green). Due to the limited temporal window of our 2DES measurement (T = 0−8 ps), we are unable to determine the accurate S 1 lifetimes and therefore confine our discussion to relative changes in these timescales. d Absorptive 2D spectrum of the Car–Chl cross peak region at T = 300 fs (in detergent). Colored sticks indicate the energy levels of the Car S 2 and Chl Q states. e Ratio of Car–Chl cross peak intensity obtained by dividing the sum of all cross peak intensities in the membrane by that in detergent. Error bars in b, c, and e are s.d. from three independent measurements. f Projection of the 2D spectra shown in a onto the ω t -axis for a 600 cm−1 ω τ interval centered at ω τ = 20,000 cm−1 (gray: detergent, green: membrane). g A closer view of the boxed region in f, where both traces are normalized to the same scale to emphasize the energy shift. Full size image

The second major change is a blueshift of the Car S 1 ESA by ~200 cm−1, indicating that the S 1 → S N energy gap increases in the membrane (Fig. 2a, f, g and Supplementary Figs. 19, 20, Supplementary Note 7). This blueshift can originate from either a redshift in S 1 energy or a blueshift in S N energies. The former is more likely, because S N is a broad manifold of multiple higher-lying states that are unlikely to all shift in a correlated manner, especially given the environment-independent transition energy of the S 2 state. This energy level shift is an environment-induced static effect present at all waiting times, separate from a dynamic shift due to vibrational cooling of the hot S 1 state48,49. We do additionally observe dynamic shifts in the S 2 −S 1 zero-crossing frequency in the initial 500 fs, where the contribution from vibrational cooling is significant (Supplementary Note 7, Supplementary Fig. 21). These dynamic effects are independent of environment. In contrast to the S 1 → S N transition, no energy shift is observed for the S 2 states.

Along with the changes in spectral features, we observe an acceleration of the decay of the S 1 population of Neo/Vio (54%) and Lut1 (53%) in the membrane (Fig. 2c and Supplementary Fig. 18, Supplementary Table 4, Supplementary Note 7). This can originate from two different processes: a decrease in the S 1 −S 0 energy gap, which speeds up non-radiative decay, or an increase in energy transfer to the energetically close-lying Chl Q y states, which accelerates the depletion of the S 1 population11. We attribute the acceleration of the decay to the former mechanism, faster non-radiative decay, based on two results. First, an increase in energy transfer from Car S 1 to Chl Q y would result in an increase in magnitude of the Car–Chl cross peaks on the timescale of the S 1 decay, and no such feature is observed. Second, the S 1 state likely redshifts in the membrane, as discussed above. Consistent with the trend observed in the S 1 to S 2 ratio, the kinetics of Lut2 is independent of environment (Supplementary Note 7, Supplementary Fig. 18).

Chl b to Chl a energy transfer

The relaxation dynamics of the Chls reveal two prominent changes in the membrane environment (Fig. 3, Supplementary Note 7). First, the energy transfer from Chl b to Chl a50,51,52 is slowed down in the membrane (Fig. 3a–c, Supplementary Figs. 22, 23, and Supplementary Table 5, Supplementary Note 7). The timescales of the energy transfer pathways, obtained by fitting the initial rise time of the cross peaks, become longer in the membrane, from 80(±20) to 132(±22) fs (Chl b → Chl a H ) and from 130(±20) to 225(±20) fs (Chl b → Chl a L ), indicating a 39–42% reduction in the energy transfer rates and resulting in diminished cross peak intensities in the membrane. The same trend is observed in the kinetics of the Chl b diagonal peak, which decays 40% slower in the membrane due to the decreased rate of energy transfer to Chl a (Fig. 3b). The energy transfer between the high-energy and low-energy Chl a pools (Chl a H and Chl a L ) is also slowed down, but to a much lesser extent (14%, Supplementary Fig. 23, Supplementary Note 7).

Fig. 3: Impact of the membrane environment on chlorophyll relaxation dynamics. a Absorptive 2D spectrum of LHCII in detergent (left) and in the membrane (right) in the Chl Q y region at T = 533 fs. Colored sticks indicate the energy levels of the Chl Q y states. Contour lines are drawn at 15% and 5% intervals for positive and negative signals, respectively. b–e Waiting time traces of the peaks labeled in a: Chl b diagonal peak (b, cyan box in a), Chl b → Chl a energy transfer cross peak (c, red box in a), Chl SE (d, pink box in a), and Car S 1 ESA upon excitation of the terminal Chls (e, blue box in a). Insets in b and c show longer-timescale dynamics. The traces were generated by integrating the 2D intensity over frequency intervals of 100 cm−1 (ω τ ) × 100 cm−1 (ω t ) for b, c, and 300 cm−1 (ω τ ) × 400 cm−1 (ω t ) for d, e around the following center frequencies: (ω τ , ω t ) = (15,540, 15,300) b, (15,540, 14,750) c, (14,925, 14,470) d, (14,925, 18,400) (e, in cm−1). Full size image

The specific pigment structural changes responsible for the observed deceleration of Chl b → Chl a energy transfer cannot definitively be identified. Although LHCII is thought to compact overall in the membrane environment as compared to in a detergent micelle, the slower Chl b → Chl a energy transfer observed here suggests that the specific pigments involved actually move further apart. As discussed above, several Chl bs form a strongly coupled pigment cluster with Neo (domain I in Fig. 1b, c), the Car that is positioned to most easily undergo large structural motions24, which may induce displacement of these Chl bs. Even minor perturbations to inter-pigment distances can significantly change the dynamics due to the nonlinear relationship between distance and energy transfer rate53,54.

Low-energy Chl a to Car S 1 energy transfer

The second prominent change appears on the red side of the lower-energy Chl a pool (a L ). This pool consists of the three Chl as in domain II that interact strongly with Lut1 and form the terminal locus of energy, collecting energy from higher-lying states and emitting fluorescence in isolated LHCIIs44,55. The waiting time traces of the red half of the Chl stimulated emission (SE) reveal pronounced rapid decay components with time constants and amplitudes of 350(±30) fs (39%) in detergent and 270(±20) fs (53%) in the membrane, followed by slower decays of several ps. In LHCII, there are picosecond-timescale vibrational relaxation processes56,57 as well as the nanosecond-timescale fluorescence. Because of the limited temporal range of our 2DES apparatus, we do not fully characterize these slower processes and thus the collectively fit them as a single long-timescale component (Supplementary Note 7 and Supplementary Fig. 26). A representative time trace from the center of this region is shown in Fig. 3d. The amplitude of the sub-ps decay component increases as the emission frequency decreases, and is non-negligible only when the red side of the Chl a L band is probed, which corresponds to the red half of the Chl a emission (Supplementary Note 7, Supplementary Fig. 24). The biexponential decay kinetics of Chl a L imply two subpopulations with different levels of quenching, likely reflecting a quenched conformation and an unquenched one58,59. Recent transient absorption studies on CP29, a minor antenna complex homologous to LHCII, found a similar biexponential decay of the terminal Chl a excited state, which was attributed to the coexistence of quenched and unquenched conformations60. The coexistence of multiple conformations with distinct photophysics is further supported by single-molecule fluorescence measurements that identified unquenched and quenched conformations of LHCII61,62 and other homologous complexes63,64.

The presence of a rapid, sub-ps decay component points towards an energy sink that accepts energy from the terminal locus. Notably, we find concurrent rise at the excitation frequency of Chl a L and emission frequency of Car S 1 ESA, which indicates that the Car S 1 states are the energy sink populated by energy transfer from the terminal Chl as (Fig. 3e). Although this region of the 2D spectrum contains a contribution from Chl ESA18, the absence of an increase in Chl a population on the corresponding timescale supports the assignment that the rise originates from the ESA of the Car S 1 instead of Chl states (Supplementary Note 7, Supplementary Fig. 25). Following energy transfer from the Chls, the Car S 1 state dissipates the excitation energy via a picosecond non-radiative decay process, as mentioned earlier. This is a clear and direct observation of the dissipative energy transfer pathway from the emissive Chl a locus into the dark S 1 state of the Cars, one of the mechanisms of photoprotection proposed but not well understood10,18,19,25,65. Correlated decay of Chl a and rise of Car S 1 , similar to those identified here but on a slower timescale (2.1 ps), have been observed in a high light-inducible protein (Hlip), a cyanobacterial ancestor of plant antenna complexes, and assigned to Chl-to-Car energy transfer65. In contrast, in previous experiments on LHCII, differences in the kinetics of unquenched and quenched samples were observed, yet no rise of the Car S 1 ESA was detected, which was attributed to excitonic mixing of the Chl and Car states19 or inverted kinetics10,25 following data processing and/or kinetic modeling.