Downloaded by guest on September 30, 2021 hne nfloecneuigete hnepitfidn (CPF) finding point change either using fluorescence in changes to challenging local, remained have resolve. microsecond), various processes (i.e., in dynamical fast multiple function and However, on (4). impact systems their biological con- access and to dynamics approach powerful formational a as emerged Single-molecule has averaging. struc- spectroscopy ensemble conventional to individually in due measurements if obscured tural system are dynamics the protein These to individual controlled. multifunctionality an asyn- providing in and 3), sites simultaneously (2, discrete place several take thus, across and can chronously fast dynamics are dynamics Local ener- the and local. commonly, temporal, conditions Most spatial, (3). natural across scales under occur getic dynamics survival These to 2). critical (1, are perturbations thermal therefore, and and environmental to which response structures, in X- time-varying emerge protein to from However, lead those microscopy. dynamics on conformational as electron based and such studied crystallography models, often ray structural is static function and well-resolved, structure between ship B harvesting protein spectroscopy uncover fluorescence single-molecule to quench- method for this basis of ability mechanistic function. These the a sites. illustrating quenching provide ing, the pro- as therefore, we serve findings, that data, previ- pigments computational with specific and combination pose spectroscopic, light-harvesting in sun- structural, the results sites in ous of our These fluctuations regulation Considering protein. robust to machinery. provides the response which within in light, on controlled sites timescales, dynamics individually quenching milliseconds are parallel two of tens quantitate at and likely and microseconds identify of technique hundreds we Through dynamics, this LHCSR1, applying level. rapid By to hidden single-molecule processes. parallel previously the multiple resolve including at we analysis and advances, by function, and these this states correlation apply lifetime model life- the we fluorescence multicomponent of expand the a estimation we of developing the function Here, improving correlation by moss. the time the energy, in of is excess quenching analysis (LHCSR1) an quenching for 1 responsible Related by Stress protein light Complex high Green Light-Harvesting function. and under biological on survive impact identification and plants preventing origin over multiplicity, molecular average the their of often and Experiments dynamics proteins. survival. these individual and for simultaneously in essential occur which rapidly to changes, is processes dynamical conformational blocks multiple local allows often building are func- protein dynamics and Protein their structure environmental the of 2018) continuous in 13, tion December flexibility to review therefore, for (received subjected and 2019 11, fluctuations, are April approved and systems IL, Urbana, Biological Urbana–Champaign, at Illinois of University Murphy, J. Catherine by Edited and Italy; Verona, a www.pnas.org/cgi/doi/10.1073/pnas.1821207116 Kondo Toru spectroscopy correlation single-molecule by observed LHCSR1 protein the photosynthetic in dynamics millisecond and Microsecond eateto hmsr,MsahstsIsiueo ehooy abig,M 02139; MA Cambridge, Technology, of Institute Massachusetts Chemistry, of Department h urn tt-fteati hrceiaino yaisvia dynamics of characterization is state-of-the-art current The ue ocryotawd ag ffntos h relation- The functions. of range wide a struc- out protein carry sophisticated to evolved tures have systems iological | opoohmclquenching nonphotochemical a,1,2 c eateto ilg n itcnlg,Uiest fPva 70 ai,Italy Pavia, 27100 Pavia, of University Biotechnology, and Biology of Department es .Gordon B. Jesse , a let Pinnola Alberta , | | htsnhtclight photosynthetic rti dynamics protein b,c uaDall’Osto Luca , utpednmccmoet a akn.A eut the result, a As lacking. describe was to function components model A dynamic (iii) occur. individual multiple they within if dynamics even independent molecules, from multiple photons of prevents the separation interspersal This ensembles, the interspersed. diffusing were Because molecules to different characterized. applied be with was could robust associated analysis for emitter processes magnitude fluorescence dynamic of simple one order an Only by (ii) lifetime estimation. required in were separated States be (i) imple- to in limitations. previous major However, occurring three (13). had processes mentations timescales dynamical different on multiple parallel and microsec- the timescale to without down ond data dynamics stream accesses photon approach func- this the This correlation from In binning. directly the dynam- lifetime 12). on the (11, fast of based tion identified information resolve are lifetime both substates fluorescence to analysis, use method and a ics as introduced recently analyses. intensity-based of in ignored indicator is FRET it powerful lifetime-based yet for a measurements, and is chromoproteins for lifetime microseconds. conformation to fluorescence down the dynamics However, accessing rate, the arrival in fluctuations photon inten- characterizes contrast, analysis In function dynamics. correlation fast sity obscuring data, analysis photon CPF the (10). bins analysis function correlation intensity or (5–9) 1073/pnas.1821207116/-/DCSupplemental. y at online information supporting contains article This 2 1 the under Published Submission.y Direct PNAS a is article This interest.y of J.B.G. conflict no and declare T.K. paper. authors tools; the research; The wrote reagents/analytic performed G.S.S.-C. new and T.K. T.K. contributed and research; R.B. data; analyzed and designed L.D., A.P., G.S.S.-C. J.B.G., and T.K., T.K. contributions: Author rsn drs:Dprmn fCeity oouUiest,9087 edi Japan.y Sendai, 980-8578 University, Tohoku Chemistry, of Department address: Present owo orsodnemyb drse.Eal knomteuo [email protected]. or [email protected] Email: addressed. be may correspondence whom To h htpoetv ehnssi re plants. of green of ana- in understanding mechanisms quenching Our structure-based photoprotective effect. the a control photoprotective enables that a approach is lytical motions which protein protein sunlight, the excess local to two iden- method we (LHCSR1), this tify 1 Related applying Stress Complex By Light-Harvesting proteins. fluo- we single processes the dynamical in Here, of multiple function characterize challenging. to correlation lifetime the rescence is using method responsible a pho- develop dynamics conformational of growth identifying and complexity that robust states the means for but proteins critical sunlight, tosynthetic is variable highly plants stochas- under in and are fast, flexibility Photoprotection small, are this tic. biologi- they underlying because of observe, dynamics to robustness the difficult the yet for systems, essential cal is flexibility Protein Significance Dfloecnelftm orlto 2-L)aayi was analysis (2D-FLC) correlation lifetime fluorescence 2D b oet Bassi Roberto , b eateto itcnlg,Uiest fVrn,37134 Verona, of University Biotechnology, of Department NSlicense.y PNAS b n areaS Schlau-Cohen S. Gabriela and , www.pnas.org/lookup/suppl/doi:10. y NSLts Articles Latest PNAS | a,1 f6 of 1 y

CHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY 2D-FLC analysis has been unable to separate small changes in ring at two different sites in parallel within LHCSR1. Within the fluorescence lifetime (i.e., local conformational dynamics) and context of structural models (30), we identified the likely molec- characterize multiple conformational dynamics occurring in par- ular origin of the dynamics, uncovering a molecular basis for allel at discrete sites within the protein, limiting the applicability the mechanism of NPQ in photosynthetic LHCs. This advance of the method. enables the study of chromoproteins as described here as well as One class of chromoproteins that exhibits multiple and FRET-based studies of complex systems. rapid dynamics is photosynthetic Light-Harvesting Complexes (LHCs). LHCs absorb light and transfer the energy to a reac- Results tion center, where photoelectric conversion occurs. Under high Determination of the Correlation Function of the Fluorescence Life- light (sunny), LHCs also dissipate excess energy as heat through time. 2D-FLC analysis uncovers protein conformations that a process known as nonphotochemical quenching (NPQ), which exhibit different fluorescence lifetimes (i.e., amounts of quench- is activated by conformational changes (14, 15). In unicellu- ing) and the transitions between them. To study conforma- lar algae and mosses, Light-Harvesting Complex Stress Related tions with closely spaced lifetimes, the analysis required two 1 (LHCSR1) is the LHC responsible for dissipation (16–27). steps, which are shown in Fig. 1. First, the number of states Single-molecule spectroscopy of LHCSR1 revealed frequent and their lifetimes were estimated from the lifetime distri- transitions between bright (photoactive) and dim (quenched) bution, and second, these states were used for the 2D-FLC states (9). These transitions emerge from conformational dynam- analysis. The introduction of step 1, reported here, enhanced ics that vary the configuration between pigments, likely chloro- the reproducibility, stability, and applicability to complex sys- phylls a (Chls a), which are the fluorescence emitters, and tems compared with previous implementations of the 2D-FLC carotenoids, which can quench Chl a (28). A pH drop and con- analysis. version of carotenoids from violaxanthin (Vio) into zeaxanthin In step 1, the fluorescence decay profile (Fig. 1B) is a his- (Zea) are both induced in the organism under high light (20, togram of the emission times t, which are the photon arrival times 29). Single-molecule experiments showed that the conforma- relative to excitation (Fig. 1A). The decay profile is fitted with the tional dynamics changed with both pH and carotenoid conver- maximum entropy method to obtain a fluorescence lifetime dis- sion, resulting in enhanced quenching efficiency (9). In contrast, tribution. The fluorescence lifetime distribution exhibits peaks an LHC primarily responsible for light harvesting (LHCB1) that reflect the number and associated lifetimes of the states, lacked these dynamics (9). While these results demonstrated which are shown for a two-state model system in Fig. 1C. that the conformational dynamics of LHCSR1 control quench- In step 2 of the 2D-FLC analysis, each photon pair separated ing, the underlying dynamics were unresolved due to limited by an interval of ∆T , as shown in Fig. 1A, provides a set of time resolution that averaged over multiple dynamical pro- emission times (t 0, t 00) that are histogrammed for all pairs in cesses. Furthermore, the switching mechanism and quenching the photon stream to generate a 2D fluorescence decay (2D-FD) site remained unclear, as LHCSR1 contains eight Chls a and profile (e.g., Fig. 1D for ∆T = 10−1 s). The 2D-FDs are inverse four carotenoids (24), where each Chl a–carotenoid pair can be a Laplace transformed by fitting with the lifetime states identified quenching site. (two states in the case of LHCSR1 and LHCB1) (a discussion of In this study, we generalize the 2D-FLC analysis for complex the analysis with three lifetime states is in SI Appendix, Fig. S8) states and dynamics by introducing a preestimation of lifetime to generate the 2D-FLC map (e.g., Fig. 1E for ∆T = 10−1 s). state, which enables robust and reliable separation of states In the 2D-FLC map, the diagonal peaks are the autocorrela- with closely spaced lifetimes, and developing a model corre- tion of individual lifetime states, whereas the off-diagonal peaks lation function to understand multiple independent dynamics. are the cross-correlation from processes, such as interconversion Application of the generalized analysis to single-molecule data between states. from immobilized LHCSR1 and LHCB1 resolves previously hid- The global fitting of 2D-FDs at various ∆T values gives the den conformations of these proteins, including their fluorescence temporal dependence of the correlation intensity (Fig. 1F), i.e., brightness and lifetime, and it identifies and characterizes the the correlation function Gn (∆T ), which contains the fluores- associated multiple dynamical processes. Notably, we discovered cence intensity of each state and the transition rates between millisecond and microsecond photoprotective dynamics occur- states. In a simple two-state system, the peaks all exhibit a

A Experimental dataB Fluorescence decay C Lifetime distribution T Δ 12 pulse train Photons fluorescence  t ttt t inverse Probability correlation T photons histogram correlation Laplace Correlation fitting Quantitative 2D fluorescence decay 2D-FLC map estimation D transform EFGfunction analysis identification 1-1

’’ 1-2 t’’  2-2 t’  ’

ΔT (s ~ s) ΔT (s ~ s) Correlation Intensity Photons ΔT (s ~ s) ’’ t’’ 

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Fig. 1. 2D-FLC analysis of a two-state model system. (A) Schematic of the photon stream, where each photon (red circles) is detected at a measurement time T and an emission time t after excitation (black triangles). (B) The fluorescence decay profile is a histogram of t for all photons and is inverse Laplace transformed to generate the fluorescence lifetime distribution in C.(D) The 2D histogram of (t0, t00), which are the emission times for two photons separated by ∆T as in A, at ∆T from 10−4 to 101 s. (E) The 2D-FLC maps from inverse Laplace transform of the 2D histograms using the lifetime distribution as a global variable. (F) The correlation function Gn(∆T) is extracted from the 2D-FLC maps, where autocorrelations (1–1, 2–2) are diagonal peaks and cross-correlations (1–2) are cross-peaks. (G) Analysis of the correlation function within the context of structural models enables assignment to sites in the protein. Details are described in SI Appendix, sections S1–S3 and Fig. S2.

2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1821207116 Kondo et al. Downloaded by guest on September 30, 2021 Downloaded by guest on September 30, 2021 o uo n rs-orltosaesonin shown function are cross-correlations model and the auto- using for determined in of was element measurement shown (black) off-diagonal of are curve the sum to analysis the corresponds the (1–2) and in curve (P) used photons (T) and times LHCSR1- (M) molecules (B), of LHCSR1-V-5 numbers ( A), LHCSR1-V-7.5 in Z-7.5(C (Bottom) curve correlation ,2-L a at map 2D-FLC (Top), distribution Lifetime 2. Fig. with Associated A–F, Dynamics LHCs. Independent in NPQ Multiple of Identification 2 Fig. in shown as profile B temporal correlation the 2 changed (Fig. drop observed was 1 state of and peak diagonal the only also 2 drop, were (Fig. peaks 7.5 off-diagonal pH and at 2 observed state for peaks Diagonal ples. Zea to 2C, (Fig. 2 Vio in 2 Fig. state from role of Conversion lifetime a the observed. plays shortened intensities likely fluo- relative therefore, of and the rate (31–33) the emission decreases the quench- rescence annihilation fluences, through singlet–triplet low in at from identified described even ing However, states are S10). the (details section Appendix, distribution affect used lifetime not fluence fluorescence do excitation contribution low triplets a relatively the includes here, for states states, these triplet of from photophysics the While peaks two exhibited samples at all 2 of Fig. distribution in lifetime shown rescence As LHCB1s. and LHCSR1s as features a below. additional detail In at exhibit more states. in peaks increases discussed the the cross-peaks systems, of between the complex time interconversions more of on to the intensity corresponding to the time corresponding and time states, a the at decreases 1F peaks Fig. nal in illustrated as profile sigmoidal od tal. et Kondo DF BD CE AC Correlation Correlation and 8.5 9.5 4.8 4.9 DFCaayi ihtolftm ttswspromdfor performed was states lifetime two with analysis 2D-FLC  (ns) Probability  (ns) Probability respectively. 2, and 1 states to corresponding ns, 2–3 and <1 0 1 2 3 4 0 1 2 3 4 F, 10 10 01234 01234 Bottom ,LC1V75(E LHCB1-V-7.5 (D), LHCSR1-Z-5 ), -4 -4 A–F C, i H75Za+p . pH7.5 Zea +pH7.5 Vio +pH7.5 21 12 12 12 .Mroe,cneso rmVot e n pH and Zea to Vio from conversion Moreover, Middle). 21 12 12 12 DFCo igeLCR n HB ne ifrn conditions. different under LHCB1 and LHCSR1 single of 2D-FLC i H5Za+p pH5 Zea +pH5 Vio +pH5 T =26394s P =2.6x10 M =123 T =22146s P =3.3x10 M =117 niaigcagsi dynamics. in changes indicating Bottom,   010 10 010 10   (ns) (ns) , T T 20 -2 20 -2 (s) (s) Middle h ope rfieo h orlto uvs(i.2 (Fig. curves correlation the of profile complex The and 7 7 1-2 1-2 LHCSR1 0.6 0 2 0 sdfeetfo sig- a from different is S9) Fig. Appendix, SI

xii ignlpasfrsae1i l sam- all in 1 state for peaks diagonal exhibit Intensity Intensity Correlation 3.4 3.9  (ns) Correlation  (ns) Probability 7 8 Probability 0 1 2 3 4 0 1 2 3 4 10 10 01234 01234 A, -4 -4 T =24202s P =2.4x10 M =109 T =30370s P =3.9x10 M =121 and C,   010 10 010 10   (ns) (ns) T T 20 -2 20 -2 (s) (s) 7 7 ,adLC1V5(F LHCB1-V-5 and ), E, 1-2 ∆T 1-2 . S9 Fig. Appendix , SI 0.2 0 4 0 .Hwvr npH on However, Middle).

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SI Intensity Intensity eto S3 section com- , Appendix dynamic multiple (SI of composed ponents system a for developed was 2 Fig. function in model lines (all fitting correlation from LHCB1 the extracted were and to dynamics LHCSR1 and states for These in 2 are LHCSR1-V-7.5 and for in 1 summarized states are parameters of rates sition as properties dynamic corre- 1G. these The Fig. estimate component. in illustrated can dynamic analysis a fitting to lation rise giving each emitters, the Chl Given of LHCB1. number and 2 LHCSR1 Fig. for in nonzero emissive is shown multiple cross-correlation As of protein). one a for within dynamics sites site to wells corresponds emissive energy each free single multiple mul- (i.e., on for separately a nonzero occur is that (i.e., dynamics cross-correlation tiple landscape base The occur energy protein). that a free within dynamics simple multiple a for on zero is in cross-correlation shown base simulations the S4 in Fig. Appendix, illustrated SI as landscape energy free shortest LHCB1 the base and at the cross-correlation Furthermore, LHCSR1 components. in in multiple dynamics from are system generated the are that two-state (examples suggesting simple component S3), a Fig. dynamic for one found with curve correlation moidal exnhn(e) n i 3,4) h Chls The 41). (30, Vio and (Neo), neoxanthin in Chls those six to a, as clues provide which context (30), the structure LHCSR1. in crystal LHCB1 be ini- in its We sites alternatively of dynamics. quenching may observed potential the sites discuss for tially other responsible fully system, or consis- the given partially are However, of 35–40). proposed (24, complexity data sites previous the and quenching data The our with are 3. tent origin Fig. likely their in components. and illustrated dynamic dynamics, the states, of conformational origin The structural the to to possible as is speculate it data, computational spec- and previous combina- measurements, crystallography, in transition troscopic X-ray results and from our information states, considering with quenched By tion states. and the active between the rates in intensity lifetime esti- fluorescence and quantitatively components, dynamic we of LHCB1, number the and mated LHCSR1 of curves LHCB1. relation in Sites Dynamic of Assignment Structural the Discussion enhance changes these of sta- enrichment Both drop Zea efficiency. quenching state. pH below, quenched the detail the and more bilizes component, in active-biased discussed the and suppresses shown As 3 processes. multiple Fig. significantly of in confirm- separation are our components, of deviations the validity the between These ing differences S2). the Table energy than and smaller free section S13, the , Appendix Fig. (SI in population S8, 16.1% state (25.3% the of in 15.5–48.9% 8.0% deviations and were difference to calcu- rates leading transition were average), in the data on in SDs LHCSR1-V-7.5 SDs dynamics, The of extracted lated. components the dynamic To of the dynamics. uncov- reliability conformational processes the the these demonstrate of of dependence the Resolution in pigment analyses, 34). resolved ered be previous (9, not in studies could which previous out dynamics, averaged fast identified were and which dynam- for multiple processes, separated sufficient and therefore, ical were fit, two LHCSR1-V-7.5 This samples. and for other S10), the Fig. needed Appendix, components were (SI dynamic LHCB1-7.5 timescales three curve, different correlation on the of profile the i.3sosdarm fteitniis ieie,adtran- and lifetimes, intensities, the of diagrams shows 3 Fig. ssonin shown As orcrtnis w uen Lt;Lt n Lut2), and Lut1 (Luts; luteins two carotenoids, four b, A–F, HB otisegtChls eight contains LHCB1 S11, Fig. Appendix, SI a ntepoen easg hst efo multiple from be to this assign we protein, the in n icse in discussed and Bottom i.S3adTbeS2 ). Table and S13 Fig. Appendix, SI and n SDs and S1, Table Appendix, SI ∆ i.S9. Fig. Appendix, SI T G a eal) oreproduce To details). has The . S5 section Appendix, SI 10 = s (∆T NSLts Articles Latest PNAS −4 A–F ) a eoto the on report can s ssona solid as shown as , yfitn h cor- the fitting By h base the Bottom, a r organized are IAppendix, SI G G n s | (∆T (∆T f6 of 3 ) )

CHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY LHCSR1 LHCB1 Vio + pH 7.5 Zea + pH 7.5 pH 7.5 ACVio Zea EStroma Lut2 3 QA(Lut2 site) 3 QA(Lut2 site) 3 QA ) ) ) s s 602 ns 168 2 11 6 414 2 2 357160 610 603 e(n e( e(n 365 < 0.1 125 60 10 < 0.1 237 611

m 12 37 612 i im im 30 219 < 0.1 t 613 1 1 e 1 fet 604 i ifet 49 614 L 25 Lut L Lut Lif 122 Lut1 0 (Lut1 site) 0 (Lut1 site) 0 Lumen 0 1 0 1 0 1 Intensity (a.u.) Intensity (a.u.) Intensity (a.u.) Vio + pH 5 Zea + pH 5 pH 5 BDLut Lut FStroma 3 QA3 QA3 QA (Neo site) (Neo site) ) Neo s 602 n 2 840 2 ( 2 97 610 603 506 9 e(ns) 44 1 787 611 m me 612 < 0.1 i t 73 117 613 1 1 < 0.1 e 1 feti < 0.1 604 239 i 350 614 Lif L Lifetime (ns) Lut Lut 615 Lut1 0 (Lut1 site) 0 (Lut1 site) 0 Lumen 0 1 0 1 0 1 Intensity (a.u.) Intensity (a.u.) Intensity (a.u.)

Fig. 3. Dynamics of LHCSR1 and LHCB1. Schematic of protein dynamics (Left), free energy surface (Center), and structural model (Right) in LHCSR1-V- 7.5 (A), LHCSR1-V-5 (B), LHCSR1-Z-7.5(C), LHCSR1-Z-5 (D), LHCB1-V-7.5 (E), and LHCB1-V-5 (F). Transitions occur between short-lifetime [quenched (Q)] and long-lifetime [active (A)] states. In the schematics, circle size and arrow thickness reflect the relative population and transition rate between the states, respectively. The transition rate (in 1/s) is labeled on each arrow. The free energy surface (in wavenumbers) was calculated as described in SI Appendix, section S7. All dynamics and free energy parameters are listed in SI Appendix, Table S1. The components in blue, red, and green are likely conformational fluctuations of the pigments in blue, red, and green, respectively, as illustrated in the structural model. As shown in E and F, Chls a cluster into Chl a610– 611–612 (blue), Chl a602–603 (red), Chl a613–614 (gray), and Chl a604 (green). These clusters neighbor carotenoids, Lut1 (blue), Lut2 (red), Neo (green), and Vio (gray).

into three clusters, Chl a610–611–612, Chl a602–603, and Chl major candidate for quenching (39), might be included in or even a613–614, as well as monomeric Chl a604 (nomenclature from dominate the static quenched component if the Lut2 is shifted ref. 30), although our preparation may lack Chl a611, Chl b607, toward the Chl a602–603 cluster. At low pH, the change in the and Vio. The Chls a contain the lowest energy levels, which are polarizability of the detergent shell around the hydrophobic core the emissive sites that appear as dynamic components in the of the molecule likely causes a small decrease in the diame- 2D-FLC analysis (42–44). ter of LHCB1 (47, 48). The decrease in diameter decreases the The 2D-FLC analysis identified three dynamic components in distance between the pigments, which would increase quench- LHCB1 (Fig. 3E): active-biased (blue), quenched-biased (red), ing throughout the protein, making assignment of quenching and almost static (gray) dynamics. The active-biased component sites particularly challenging. The active-biased dynamic com- (Fig. 3E, blue) exhibits high fluorescence intensity in the active ponent (Fig. 3F, green) likely corresponds to the Chl a604 state and large energy dissipation in the quenched state. This quenched by neighboring Neo, because the free energy land- component likely corresponds to the Lut1/Chl a610–(611)–612 scape of this component exhibits a different pH dependence in site (37, 45). This cluster is the main energy sink (42–44) and LHCB1 and LHCSR1 (i.e., the quenched state is stabilized in would, if quenched, produce the significant change in fluores- LHCSR1). As discussed below, it is the region around Neo that cence intensity observed. Additionally, Lut1 and Chl a612 inter- is affected by protonation and therefore, would differ between act strongly (38, 39), which would further enhance the change the two proteins. The conformational change around Neo has in intensity. Furthermore, computational results found that the been proposed to red shift the neighboring Chl a604 (42). In Lut1/Chl a612 interaction has the largest and thus, slowest fluc- this case, the Chl a604 becomes an energy sink instead of tuations in accordance with the slower dynamics observed for this Chl a602–603 and Chl a613–614. Calculations showed that Neo component (35). The fast quenched-biased component (Fig. 3E, can quench Chl a604 (39), although it is likely to only par- red) likely corresponds to the Lut2/Chl a602–603 site (37, 45), tially quench as observed, because of the higher energy level which shows higher flexibility and thus, faster dynamics in molec- and longer lifetime of its S1 state compared with the other ular dynamics (MD) simulations (36). Furthermore, the weaker carotenoids (49). interaction of Lut2 and Chl a603 compared with that of Lut1 and Chl a612 (35) would be expected to produce a smaller change in Multiple Independent Regulatory Dynamics in LHCSR1. The 2D-FLC fluorescence intensity between the active and quenched states as analysis identified three dynamic components in LHCSR1-V- observed in the fast quenched-biased component. In this way, 7.5 (Fig. 3A): active-biased fast dynamics (red), quenched-biased dynamic rates can be used in parallel to predicted quenching slow dynamics (blue), and an almost static component (gray). yields to assign the components to likely sites. However, as addi- LHCSR1 contains eight Chl binding sites, six of which are homol- tional structural and spectroscopic information emerges, these ogous to those in LHCB1 (17), that each embed Chl a and four assignments can be either further validated or refined. The static carotenoids but with two Lut in the Lut1 and Neo sites and two component (Fig. 3E, gray) likely includes unquenched emitters Vio in the Lut2 and Vio sites (25). Similar to LHCB1-7.5, in as the active state, i.e., Chl a613–614 cluster and Chl a604 (37), LHCSR1-V-7.5, the two dynamic components (Fig. 3A, red and and photobleached/degraded complexes as the quenched state. blue) are likely to be associated with conformational fluctuations In LHCB1 at low pH, the 2D-FLC analysis identified two of the carotenoids in the Lut1 and Lut2 sites, while the static dynamic components, both of which have an enhanced quench- component (Fig. 3A, gray) would be due to unquenched emitters ing efficiency (Fig. 3F). The static component is stabilized into as well as photobleached/degraded complexes. In Zea-enriched the quenched state (Fig. 3F, blue) and likely corresponds to LHCSR1 (Fig. 3C), one of two components is almost static the Lut1/Chl a610–(611)–612 site because of the ability of Lut1 (Fig. 3C, red), and the other is more quenched biased (Fig. 3C, to quench the Chl a as discussed above. The equilibrium free blue), suggesting that Zea binding suppresses the active-biased energy difference shifted toward the quenched state by more dynamics and shifts the equilibrium slightly toward the quenched than 800 cm−1, possibly through displacement of Lut1 toward side. The active-biased component (Fig. 3A, red) likely cor- the Chl a (38, 46). A contribution from Lut2, which is also a responds to Vio in the Lut2 site interacting with neighboring

4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1821207116 Kondo et al. Downloaded by guest on September 30, 2021 Downloaded by guest on September 30, 2021 od tal. et Kondo Chl xed h aaiyo P,teLCcnpoolah The photobleach. can LHC the NPQ, of capacity the exceeds Photobleaching. on NPQ of Enhancement be may refined. sites or quenching proposed validated the the emerges, infor- mechanism spectroscopic by the and about controlled mation structural be additional As can proposed. they between sites interaction carotenoids, involve and mechanisms Chl (24). these mechanisms of two as first both the such Because for Fur- cooperatively, LHCSR1 in (59). occur observed Chls been may has between mechanisms excitonic transfer these charge the Chl–carotenoid thermore, (iv) mixed to and a Chl (56–58), (iii) and state from Chl 53–55), between transfer (24, transfer charge (ii) carotenoid energy 38), (24, (i) state S1 quenchers carotenoid proposals: energy primary main four the are in Lut Vio and (24). than pH Lut1 low rather the at that site confirming change Neo 3D), little the caused (Fig. Zea dynamics to the Vio in from Conversion to energy. closer tation binding tighter to lead Chl can the (50) to Neo Neo 3F contrast with the (Fig. compared in in LHCB1 Lut in side in Vio Lut quenched Neo of the of of instead toward dynamics those biased site The strongly Neo quencher. change, are the the to site as in system site the Lut Lut2 in of the sink use energy the the allowing cause likely to seems Lut 52) The other 51, the site. to Neo corresponds the quenched-biased likely in other green) orig- the 3B, quenching therefore, (Fig. to and component (24), contribution Lut main from the inates that showed quenching results transfer LHCB1, 5 in electron Chl seen and of component as Lut observation between blue) the the 3B, in (Fig. with site state efficiency, consistent Lut1 quenching quenched the Lut1 with overall at the associated quenching the stabilizing on increases by turning drop primarily 46), pH (40, The Neo protonation (38). twist particular, to In (51). thought of shell is effect detergent the the with in along change present the is residues specific the con- protonatable pH, of causes low effect which at Thus, LHCSR1, quenching. of and loop changes formational luminal the on compact residues ing and site Lut2 quenched the the stabilize in hydrophobicity (50). could state binding which higher helixes, the The transmembrane tighten the Lut1. can to Zea of corresponds 3A, likely quench- (Fig. component the blue) quenched-biased toward the system Accordingly, side. overall ing the shifting suppressed, was .Sha-oe S ta.(05 igemlcl dnicto fqece and quenched of identification Single-molecule (2015) al. et GS, pho- Schlau-Cohen and 8. Single-molecule conformational- (2013) WE Moerner RJ, Cogdell Watching J, Southall Q, Wang (2010) GS, Schlau-Cohen 7. WE time-resolved Moerner in points change RH, intensity of Goldsmith Detection 6. (2005) H Yang LP, Samor Watkins 5. B, Schuler M, Brucale 4. spec- fluorescence K H, Single-molecule Michel TJ, (2017) Aartsma C, GS Hofmann Schlau-Cohen 3. WJ, Chen T, Kondo 2. Kr 1. h htpyia ehns fNQrmisdbtd with debated, remains NPQ of mechanism photophysical The protonat- (29), acidifies lumen thylakoid the light, high Under nunhdsae fLHCII. of states unquenched states. USA emissive Sci between solution. Acad switch Natl complexes Proc LH2 in photosynthetic reveals proteins spectroscopy fluorescent single 186. of todynamics measurements. single-molecule proteins. disordered study. single-molecule A chromoprotein: USA a of landscape energy 157– pp systems. photosynthetic 40, of Vol troscopy Netherlands), The Dordrecht, G, (Springer, Garab B, Govindjee Cyanobac- 185. Demmig-Adams III, and eds Respiration, W Algae and Plants, Adams Photosynthesis in in quenching. Dissipation Advances fluorescence Energy teria, and non-photochemical Quenching controls Photochemical disorder protein How grTJ loi ,Hro ,AeadeMA a rnel (2014) R Grondelle van MTA, Alexandre P, Horton C, Ilioaia TPJ, uger eas ncneso rmVot e,ti component this Zea, to Vio from conversion on because a, ¨ 100:15534–15538. a nrysn n hs oeefiin unhn fexci- of quenching efficient more thus, and sink energy hmRev Chem 110:10899–10903. ∆ a nLCR 2) nebeLHCSR1-V- Ensemble (24). LHCSR1 in hsCe Lett Chem Phys J Hidcdcnomtoa hne(25, change conformational pH-induced 114:3281–3317. 21)Snl-oeuesuiso intrinsically of studies Single-molecule (2014) B ` ı hsCe B Chem Phys J hmRev Chem he 20)Drc bevto ftesi the in tiers of observation Direct (2003) J ohler ¨ re) h yrpoiiyof hydrophobicity The green). , 6:860–867. 109:617–628. 117:860–898. fteqecigrequired quenching the If a Chem Nat rcNt cdSci Acad Natl Proc 2:179– Non- 7 oet ,e l 21)Aayi fLcR,apoenesnilfrfeedback for essential (2010) T protein Morosinotto R, a the Bassi LhcSR3, GM, for Giacometti of C, critical Gerotto Analysis is A, Alboresi (2010) protein 18. al. light-harvesting et ancient G, An Bonente (2009) 17. al. et P, Graham 16. green Mechanism quenching: and fluorescence plants chlorophyll in Nonphotochemical control (2016) AV capture Ruban light Multi-level 15. (2016) O Kruse R, Bassi W, single- the Lutz at observed 14. dynamics protein Microsecond (2015) T Tahara K, Ishii T, Otosu 13. spectroscopy. correlation fluctuation in Lessons (2011) E Gratton MA, Digman 10. 2 si ,Thr 21)Todmninlfloecnelftm orlto spec- correlation lifetime fluorescence Two-dimensional spec- (2013) correlation T lifetime Tahara fluorescence K, Two-dimensional Ishii (2013) 12. T Tahara K, Ishii 11. ewrsSlrEeg oBoas(EB Gat650-EBt . and “HARVEST” R.B (PRIN) to Relevance L.D.). National 675006-SE2B to of Training (Grant 201795SBA3-004 Projects Initial (Grant Research (SE2B) Actions for the Biomass Curie to by Center Marie and to application A.P.) the I Energy for by Solar Phase NSF supported Networks also the Design was from Molecular Work CHE-1740645 LHCs. Quantum Grant devel- for 2D-FLC Innovation for Chemical Center NIH9P41EB015871 Grant and NIH by opment supported was work The ACKNOWLEDGMENTS. in described are analysis 2D-FLC S1–S3. sections the for procedures Detailed efre sdsrbdpeiul 9 n in and (9) previously described were LHCB1 and as LHCSR on performed spectroscopy single-molecule and of Preparation Methods and Materials accessing systems. biological seconds, of to aspects dynamics hidden microseconds previously from of proteins. ranging visualization in timescales simultaneous multifunctionality data on enables investigate single-molecule Apply- to approach to organism. tool This analysis powerful intact a 2D-FLC the is generalized from in the information interpreted ing be reported of cap- and results context or interpret, the mimic the help behaviors, fully complement, and not should complexity does here vivo system in pigments vitro the individual power- in ture of a the contributions Because been the such (34). disentangle have work, to comparative which tool and future composition, ful simulations, carotenoid for particular, MD of benchmark In analyses, studies useful mutation refined. point a or as validated is be knowledge may this available, sites become proposed data as or site additional the one quenching as from assigned However, the results above. favor discussed case, strongly tools each these In of biochemi- more data. spectroscopic, computational the existing and are on study cal, based this ones in probable proposed in most models photoprotection structural of The origin LHCSR1. molecular a as suggesting speculation quenching, enables Chls This the energetics component. the to each characterizing for LHCs, dynamics and of quenching that the dynamics the applied underlie resolved analysis and separated 2D-FLC data single-molecule developed to the here, demonstrated As Zea that Conclusions indicating delayed, LHCSR1- photobleaching. is to in resistance photobleaching contributes whereas initial the photobleaches, in Z-7.5, Vio preferentially the site with another associated LHCSR1-V- Lut2 component provide in active-biased may the Additionally, 7.5, which photoprotection. state, for quenched photobleach- mechanism initial the that stabilizes revealed S14–S19) ing Appendix, Figs. (SI and time illumination S9 of section function a as analysis 2D-FLC .KnoT ta.(07 igemlcl pcrsoyo HS1poendynam- protein LHCSR1 of spectroscopy Single-molecule (2017) al. et T, Kondo 9. ehnssuo adcolonization. land upon mechanisms patens alga green the in de-excitation photosynthesis. algal of regulation photodamage. from plants 1916. protecting in effectiveness and algae. level. molecule photosynthetic multi-timescale for responsible states distinct photoprotection. two identifies ics rsoy .Application. 2. troscopy. Principle. 1. troscopy. Chem Phys Rev rnsPatSci Plant Trends uat fetdo etdsiaincaiyteeouino photoprotection of evolution the clarify dissipation heat on affected mutants a 62:645–668. a Commun Nat n aoeod ihnteLCrsosbefor responsible LHC the within carotenoids and a Chem Nat hsCe B Chem Phys J 21:55–68. etakD.Jsi aa o epu comments. helpful for Caram Justin Dr. thank We hsCe B Chem Phys J 9:772–778. 6:7685. hayooa reinhardtii Chlamydomonas Nature 117:11414–11422. rcNt cdSiUSA Sci Acad Natl Proc 117:11423–11432. 462:518–521. NSLts Articles Latest PNAS eto S10 section Appendix, SI . 107:11128–11133. LSBiol PLoS ln Physiol Plant IAppendix, SI Physcomitrella 9:e1000577. 170:1903– | f6 of 5 Annu .

CHEMISTRY BIOPHYSICS AND COMPUTATIONAL BIOLOGY 19. Tokutsu R, Minagawa J (2013) Energy-dissipative supercomplex of photosystem II 39. Fox KF, et al. (2017) The carotenoid pathway: What is important for excitation associated with LHCSR3 in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA quenching in plant antenna complexes?. Phys Chem Chem Phys 19:22957–22968. 110:10016–10021. 40. Belgio E, Duffy CDP, Ruban AV (2013) Switching light harvesting complex II into pho- 20. Pinnola A, et al. (2013) Zeaxanthin binds to light-harvesting complex stress-related toprotective state involves the lumen-facing apoprotein loop. Phys Chem Chem Phys protein to enhance nonphotochemical quenching in Physcomitrella patens. Plant Cell 15:12253–12261. 25:3519–3534. 41. Barros T, Kuhlbrandt¨ W (2009) Crystallisation, structure and function of plant light- 21. Pinnola A, et al. (2015) Heterologous expression of moss light-harvesting com- harvesting complex II. Biochim Biophys Acta 1787:753–772. plex stress-related 1 (LHCSR1), the chlorophyll a-xanthophyll pigment-protein 42. Muh¨ F, El-Amine Madjet M, Renger T (2010) Structure-based identification of energy complex catalyzing non-photochemical quenching, in Nicotiana sp. J Biol Chem sinks in plant light-harvesting complex II. J Phys Chem B 114:13517–13535. 290:24340–24354. 43. Schlau-Cohen GS, et al. (2009) Pathways of energy flow in LHCII from two- 22. Pinnola A, et al. (2015) Light-harvesting complex stress-related proteins catalyze dimensional electronic spectroscopy. J Phys Chem B 113:15352–15363. excess energy dissipation in both photosystems of Physcomitrella patens. Plant Cell 44. Novoderezhkin V, Marin A, van Grondelle R (2011) Intra- and inter-monomeric trans- 27:3213–3227. fers in the light harvesting LHCII complex: The Redfield–Forster¨ picture. Phys Chem 23. Maruyama S, Tokutsu R, Minagawa J (2014) Transcriptional regulation of the stress- Chem Phys 13:17093–17103. responsive light harvesting complex genes in Chlamydomonas reinhardtii. Plant Cell 45. Lampoura SS, Barzda V, Owen GM, Hoff AJ, van Amerongen H (2002) Aggregation of Physiol 55:1304–1310. LHCII leads to a redistribution of the triplets over the central xanthophylls in LHCII. 24. Pinnola A, et al. (2016) Electron transfer between carotenoid and chlorophyll con- Biochemistry 41:9139–9144. tributes to quenching in the LHCSR1 protein from Physcomitrella patens. Biochim 46. Pandit A, et al. (2013) An NMR comparison of the light-harvesting complex II (LHCII) in Biophys Acta 1857:1870–1878. active and photoprotective states reveals subtle changes in the chlorophyll a ground- 25. Pinnola A, et al. (2017) Functional modulation of LHCSR1 protein from Physcomitrella state electronic structures. Biochim Biophys Acta 1827:738–744. patens by zeaxanthin binding and low pH. Sci Rep 7:11158. 47. Bassi R, Silvestri M, Dainese P, Moya I, Giacometti GM (1991) Effects of a non- 26. Kosuge K, et al. (2018) LHCSR1-dependent fluorescence quenching is mediated by ionic detergent on the spectral properties and aggregation state of the light- excitation energy transfer from LHCII to photosystem I in Chlamydomonas reinhardtii. harvesting chlorophyll a/b protein complex (LHCII). J Photochem Photobiol B, Biol 9: Proc Natl Acad Sci USA 115:3722–3727. 335–353. 27. Pinnola A, Bassi R (2018) Molecular mechanisms involved in plant photoprotection. 48. Moya I, Silvestri M, Vallon O, Cinque G, Bassi R (2001) Time-resolved fluorescence Biochem Soc Trans 46:467–482. analysis of the photosystem II antenna proteins in detergent micelles and liposomes. 28. Holleboom C-P, Walla PJ (2014) The back and forth of energy transfer between Biochemistry 40:12552–12561. carotenoids and chlorophylls and its role in the regulation of light harvesting. 49. Pol´ıvka T, Sundstrom¨ V (2004) Ultrafast dynamics of carotenoid excited states- from Photosynth Res 119:215–221. solution to natural and artificial systems. Chem Rev 104:2021–2072. 29. Johnson MP, Zia A, Ruban AV (2012) Elevated δ pH restores rapidly reversible pho- 50. Ruban AV, Johnson MP (2010) Xanthophylls as modulators of membrane protein toprotective energy dissipation in Arabidopsis chloroplasts deficient in lutein and function. Arch Biochem Biophys 504:78–85. xanthophyll cycle activity. Planta 235:193–204. 51. Ballottari M, et al. (2016) Identification of pH-sensing sites in the Light Harvest- 30. Liu Z, et al. (2004) Crystal structure of spinach major light-harvesting complex at ing Complex Stress-Related 3 protein essential for triggering non-photochemical 2.72 A˚ resolution. Nature 428:287–292. quenching in Chlamydomonas reinhardtii. J Biol Chem 291:7334–7346. 31. Rutkauskas D, Novoderezkhin V, Cogdell RJ, van Grondelle R (2004) Fluorescence 52. Liguori N, Roy LM, Opacic M, Durand G, Croce R (2013) Regulation of light harvesting spectral fluctuations of single LH2 complexes from Rhodopseudomonas acidophila in the green alga Chlamydomonas reinhardtii: The C-terminus of LHCSR is the knob strain 10050. Biochemistry 43:4431–4438. of a dimmer switch. J Am Chem Soc 135:18339–18342. 32. Gruber JM, Chmeliov J, Kruger¨ TPJ, Valkunas L, Van Grondelle R (2015) Singlet–triplet 53. Holt NE, et al. (2005) Carotenoid cation formation and the regulation of annihilation in single LHCII complexes. Phys Chem Chem Phys 17:19844–19853. photosynthetic light harvesting. Science 307:433–436. 33. Gruber JM, et al. (2016) Photophysics in single light-harvesting complexes II: From 54. Ahn TK, et al. (2008) Architecture of a charge-transfer state regulating light micelle to native nanodisks. Proc SPIE 9714:97140A. harvesting in a plant antenna protein. Science 320:794–797. 34. Tutkus M, Chmeliov J, Rutkauskas D, Ruban AV, Valkunas L (2017) Influence of 55. Dall’Osto L, et al. (2017) Two mechanisms for dissipation of excess light in monomeric the carotenoid composition on the conformational dynamics of photosynthetic and trimeric light-harvesting complexes. Nat Plants 3:17033. light-harvesting complexes. J Phys Chem Lett 8:5898–5906. 56. van Amerongen H, van Grondelle R (2001) Understanding the energy transfer func- 35. Liguori N, Periole X, Marrink SJ, Croce R (2015) From light-harvesting to photoprotec- tion of LHCII, the major light-harvesting complex of green plants. J Phys Chem B tion: Structural basis of the dynamic switch of the major antenna complex of plants 105:604–617. (LHCII). Sci Rep 5:15661. 57. Bode S, et al. (2009) On the regulation of photosynthesis by excitonic interactions 36. Liguori N, et al. (2017) Different carotenoid conformations have distinct functions in between carotenoids and chlorophylls. Proc Natl Acad Sci USA 106:12311–12316. light-harvesting regulation in plants. Nat Commun 8:1994. 58. Liao P-N, et al. (2011) Two-photon study on the electronic interactions between the 37. Di Valentin M, Biasibetti F, Ceola S, Carbonera D (2009) Identification of the sites of first excited singlet states in carotenoid- tetrapyrrole dyads. J Phys Chem A 115:4082– chlorophyll triplet quenching in relation to the structure of LHC-II from higher plants. 4091. Evidence from EPR spectroscopy. J Phys Chem B 113:13071–13078. 59. Muller¨ MG, et al. (2010) Singlet energy dissipation in the photosystem II light- 38. Ruban AV, et al. (2007) Identification of a mechanism of photoprotective energy harvesting complex does not involve energy transfer to carotenoids. Chemphyschem dissipation in higher plants. Nature 450:575–578. 11:1289–1296.

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