Light-Harvesting Complex II Exhibits a Temperature-Dependent

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B: Liquids, Chemical and Dynamical Processes in Solution, Spectroscopy in Solution

Rigid versus Flexible Protein Matrix: Light-Harvesting Complex II Exhibits a Temperature-Dependent Phonon Spectral Density Maksym Golub, Leonid Rusevich, Klaus-Dieter Irrgang, and Joerg Pieper J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b02948 • Publication Date (Web): 16 Jun 2018 Downloaded from http://pubs.acs.org on June 19, 2018

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The Journal of Physical Chemistry

Rigid versus Flexible Protein Matrix: Light-Harvesting Complex II Exhibits a Temperature-Dependent Phonon Spectral Density Maksym Goluba, Leonid Rusevichb,c, Klaus-Dieter Irrgangd, and Jörg Piepera* a

University of Tartu, Institute of Physics, W. Ostwaldi 1, 50411 Tartu, Estonia

b

Institute of Physical Energetics, Krivu 11, LV-1006 Riga, Latvia

c

Institute of Solid State Physics, University of Latvia, Kengaraga 8, LV-1063 Riga, Latvia

d

Department of Life Science & Technology, Laboratory of Biochemistry, University for Applied Sciences, Berlin, Germany

*Author to whom correspondence should be addressed: Jörg Pieper University of Tartu, Institute of Physics, W. Ostwaldi 1, 50411 Tartu, Estonia. phone.:

+(372) 737 4627

fax:

+(372) 738 3033

email:

[email protected]

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Dynamics-function correlations are usually inferred when molecular mobility and protein function are simultaneously impaired at characteristic temperatures or hydration levels. In this sense, excitation energy transfer in the photosynthetic light-harvesting complex II (LHC II) is an untypical example, because it remains fully functional even at cryogenic temperatures relying mainly on interactions of electronic states with protein vibrations. Here, we study the vibrational and conformational protein dynamics of monomeric and trimeric light-harvesting complex II (LHC II) from spinach using inelastic neutron scattering (INS) in the temperature range between 20 and 305 K. INS spectra of trimeric LHC II reveal a distinct vibrational peak at ~2.4 meV. At temperatures above ~160 K, however, the inelastic peak shifts towards lower energies, which is attributed to vibrational anharmonicity. A more drastic shift is observed at about 240 K, which is interpreted in terms of a “softening” of the protein matrix along with the dynamical transition. Monomeric LHC II exhibits a larger degree of conformational mobility at physiological temperatures, which can be attributed to a higher number of solvent-exposed sidechains at the protein surface. The effects of the changes in protein dynamics on the spectroscopic properties of LHC II are considered in comparative model calculations. The absorption lineshapes of a pigment molecule embedded into LHC II are simulated for the cases of i) a rigid protein matrix, ii) a protein matrix with temperature dependent spectral density of protein vibrations, and iii) temperature-dependent electron-phonon coupling strength. Our findings indicate that vibrational and conformational protein dynamics affect the spectroscopic (absorption) properties of trimeric and monomeric LHC II at physiological temperatures.

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1. Introduction Photosynthetic light-harvesting complexes are large pigment-protein assemblies specialized to collect solar radiation and to efficiently transfer the resulting excitation energy to photochemically active reaction center complexes (for reviews see e.g. 1, 2, 3). The trimeric lightharvesting complex of photosystem II (LHC II) is the major antenna complex of green plants (see Fig. 1). Its structure is well characterized by X-ray crystallography

4, 5

revealing the presence of

14 chlorophyll (Chl) and four carotenoid (Car) molecules bound by each protein monomer. Center-to-center distances between Chls in the order of only 8-10 Å suggest sizeable excitonic interactions for efficient excitation energy transfer (EET), while a previous structure6 indicated rather weak excitonic delocalizations within Chl heterodimers

7, 8

. Trimeric LHC II may consist

of various combinations of three slightly different proteins denoted as Lhcb1-3, which are characterized by individual spectroscopic properties

9,10

. The native state of membrane-bound

LHC II may also be affected by solubilization using different detergent types and by membrane lipids 11. The specific structural arrangement of pigment molecules in LHC II provides the framework for efficient, ultrafast EET. A number of experimental time-resolved absorption and 2D spectroscopy studies have directly revealed time constants in the order of femto- to picoseconds for Chl-toChl11-14 and Car-to-Chl EET 15, 16. Several theoretical studies have modeled spectroscopic data in LHC II and thus provided deeper insight into the specific pathways of ultrafast EET 17-20. LHC II is also involved in non-photochemical quenching (NPQ) and thus in protection against excess energy (see 21 and references therein).

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Figure 1. Left panel: crystal structure of the LHC II monomer according to 4 (pdb 1RWT). Chl a molecules are shown in orange, Chl b in green, carotenoids in purple color. Right panel: schematic energy level diagram illustrating light absorption and excitation energy transfer in a model complex binding two pigment molecules. The electronic energy levels in the ground and excited states S0 and S1, respectively, are shown as thick black bars. Vibrational levels building on the electronic excited state are shown as thin black lines. The low-frequency protein vibrations (phonons) are illustrated by full lines, the higher-frequency pigment vibrations by dashed lines. Light absorption, i.e. the transition from the ground state of both pigments P1P2 into the excited state of the first pigment P1*P2 is illustrated by a blue arrow. Due to electron-vibrational coupling, light absorption cannot only occur to the excited electronic state S1, but into the whole spectral regions marked in orange (protein vibrations) and yellow (pigment vibrations). EET from pigment P1 to pigment P2 is depicted by red arrows showing the de-excitation of P1 and the concomitant excitation of P2 resulting in the excited state P1P2*. Due to electron-vibrational coupling, EET can occur between pigment molecules possessing energetically inequivalent electronic states resulting in spectrally and spatially directed EET.

However, most of the experimental and theoretical studies of EET in LHC II have been restricted to (individual) cryogenic temperatures. These temperatures are usually chosen to be below the onset of conformational dynamics in proteins so that calculations can be based on the static protein structure. It is remarkable that simulations of LHC II spectra appear to fit spectroscopic data well only up to about ~120 K 8, 22. Very few attempts were made to model spectroscopic data of LHC II over a wider temperature range up to physiological conditions

23, 24

. More recently, it

was shown that excited state positions and/or electron-phonon coupling are affected by temperature-dependent onset of protein dynamics

25

. The latter finding came as a surprise,

because LHC II does not undergo pronounced conformational changes during EET (but during NPQ, see above). In contrast, it is well accepted that protein dynamics is a prerequisite for conformational gating of electron transfer in reaction centers 26, 27.

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In addition to electronic pigment-pigment interactions determining the excited state positions in pigment-protein complexes, ultrafast EET processes in antenna complexes are mediated via the coupling of electronic transitions to low-frequency vibrations of the protein matrix, also referred to as electron-phonon coupling (for a review see 2). This is illustrated schematically in Fig. 1, see figure caption for details. Briefly, electron-phonon coupling broadens the spectral range of light absorption (see blue arrow in Fig. 1), because transitions are not only allowed between the purely electronic energy levels, but also to vibrational energy levels. In addition, electron-phonon coupling enables EET between energetically inequivalent pigment energy levels (see red arrows in Fig. 1) by dumping the energetic difference into vibrations. Any temperature-dependent change in excited state positions and coupling to vibrational frequencies may thus affect absorption lineshapes and EET between pigments. However, detailed information on the temperature dependence of protein vibrations is lacking. So far, electron-phonon coupling has been mainly investigated by high-resolution optical spectroscopies at cryogenic temperatures, which are referred to as spectral hole burning (SHB) and (difference-) fluorescence line-narrowing ((∆)FLN) (for a review see 2). The electron-phonon coupling strengths reported for antenna complexes are often weak or moderate with coupling constants S in the range of 0.5 – 1.5. The spectral shape of the protein vibrations referred to as one-phonon profile or spectral density (see below) is typically asymmetric with peak energies at about 2.5 meV equivalent to 20 cm-1, but can be as high as about 4.6 meV 28, 29. Highly structured one-phonon profiles were observed for the water soluble chlorophyll-binding protein WSCP30, 31. However, experimental techniques like SHB and FLN are technically restricted to temperatures below about 40 K 2, because these spectrally selective methods require a static (frozen) ensemble of protein conformations. As a result, electron-phonon coupling could not be investigated at temperatures higher than accessible by SHB and FLN. Therefore, an independent experimental approach is required to study the vibrational dynamics of photosynthetic pigment-protein complexes at elevated or even physiological temperatures. Inelastic and quasielastic neutron scattering (INS and QENS) are powerful experimental tools for direct investigations of vibrational and conformational protein dynamics (for reviews, 5



see

32-34

). Because of the high incoherent scattering cross section of hydrogen atoms, which are

almost homogeneously distributed in biomolecules, INS and QENS are widely used to study protein dynamics. QENS experiments have shown that proteins 35-39 and biological membranes 4043

display complex hydration-dependent conformational (diffusive) dynamics at physiological

temperatures above a dynamical transition. The latter transition occurs in the temperature range from 200 – 240 K depending on the particular protein and on its specific environment. It was also shown that protein dynamics is affected by intrinsic disorder, folding, or interaction with membranes lipids44, 45. Depending on the spectral resolution employed, QENS experiments probe different time scales of protein motions46,

47

, which in turn affects the observed dynamical

transition48, 49. Hydration water dynamics can also be studied by QENS using contrast variation50, 51

. INS spectra of proteins generally display a distinct inelastic peak centered at energies of 2-

7 meV representing an excess of protein vibrational modes compared to the Debye-like density of states (see e.g 52, 53). More recently, collective water dynamics was also studied in whole cells of green algae using inelastic coherent neutron scattering54. Furthermore, QENS data are complementary to molecular dynamics (MD) simulations of internal protein motions.34, 55-57 In photosynthesis research, INS has been employed to study protein vibrations of trimeric LHC II in the low temperature regime up to 120 K, where the dynamics was shown to be widely harmonic

53

. The data revealed an asymmetric inelastic peak at ~2.5 meV, which was in good

agreement with previous results from SHB and FLN experiments22, 33, 53. Complementary QENS experiments revealed the dynamical transition, i.e. the onset of conformational protein motions, at 240 K in trimeric LHC II

25

. More recent INS experiments on Photosystem II membranes

appeared to indicate a shift of the vibrational energies upon temperature increase.44 In the present study, we report novel INS data of monomeric LHC II measured over a wide temperature range between 20 and 305 K. The data are compared to INS spectra of trimeric LHC II which were previously analyzed in the restricted quasielastic region25 or were available for only three selected temperatures 58. The data characterize the functionally important protein vibrations of monomeric and trimeric LHC II pigment-protein complexes up to physiological temperatures.

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2. Materials and Methods Sample Preparation: Solubilized LHC II was isolated and prepared for neutron scattering experiments as reported before

53

. Briefly, trimeric LHC II was purified from spinach

Photosystem II membrane fragments by sucrose density gradient centrifugation in the presence of n-ß-dodecylmaltoside as described previously induced as described by Nussberger et al.

61

59, 60

. Monomerization of trimeric LHC II was

with some modifications outlined in ref.62. The

Chl a/b ratio (w/w) was determined to be 1,36 ± 0.6 (n=22). LHC II was lyophilized to extract H2O and then resuspended in D2O (99,9% D, Eurisotop, CEA group, Saclay, France) resulting in a final concentration of about 25 mg Chl/ml. This is equivalent to a protein mass of roughly 100 mg in a 1 ml sample cell. The buffer solution is composed of D2O, 25 mM Mes-NaOH, 10 mM CaCl2, 0.025 % w/v ß-dodecylmaltoside, and 30 % w/v sucrose at a pD-value of 6.7. This preparation was shown to be virtually free of aggregation of LHC II

63, 64

. The protein

composition and pigment content were analyzed before and after each neutron scattering experiment, the analyses yielded no indication for radiation damage. Experimental methods: INS experiments: INS spectra were recorded using the time-of-flight spectrometer V3 (Helmholtz Zentrum Berlin, Germany). The incident neutron wavelength was 5.1 Å (~3.2 meV) corresponding to an (elastic) Q range of 0.3 - 2.3 Å-1. The elastic resolution of ∆E = 0.117 meV was determined by vanadium standard runs. The samples were kept in cylindrical Aluminium cells having an effective volume of 1 ml. The sample temperature was maintained using an Orange Cryofurnace and stabilized by a Lakeshore temperature controller. The INS data were corrected for detector efficiency and sample-geometry dependent attenuation, normalized, as well as transferred to energy scale using the program package FITMO-4. Solubilized LHC II complexes and the D2O-containing buffer solution were studied separately in order to independently investigate the contribution of the solvent. Eventually, the buffer contribution was subtracted from the spectra according to the weight contribution of the buffer to the full sample weight to yield the INS spectra of the LHC II protein.

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Data Analysis: INS experiments: In the case of a protonated scatterer like a protein, the number of neutrons detected in a space angle element δΩ and an energy element δω in an inelastic scattering δ 2σ experiment is given by the double-differential cross section (for an overview see e.g. 65) δΩδω

k1 2 δ 2σ = binc S inc (Q, ω ) , δΩδω k 0

(1)

where k0 and k1 are the wave vectors of incident and scattered neutrons, respectively, Q is the momentum transfer vector, binc is the incoherent scattering length and Sinc(Q,ω) is the incoherent scattering function. Sinc(Q,ω) is not directly accessible in experiments, so that it has to be replaced by the experimental scattering function Sexp(Q,ω) given as  hω S exp (Q, ω ) = FN exp  −  2 kT

  R (Q, ω ) ⊗ S theo (Q, ω ) , 

(2)

 hω  which is the product of a normalization factor FN , the detailed balance factor exp  −  and  2 kT 

the convolution of an experimentally obtained resolution function R(Q,ω) with a theoretical model function Stheo(Q,ω) describing the dynamics of the sample system. The theoretical scattering function can be described by the following phenomenological model function:

S theo (Q, ω ) = e

− u2 Q2

   A0 (Q )δ (ω ) + ∑ An (Q )Ln (H n , ω ) + S in (Q, ω ) . n  

(3)

Here, the scattered intensity is the product of the Debye-Waller factor e

− u2 Q2

, characterized by

the “global” vibrational mean square displacement and the sum of three contributions: i) the elastic component A 0 (Q)δ (ω ) , ii) the quasielastic component ∑ An (Q )Ln (H n , ω ) and iii) the n

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inelastic contribution Sin(Q,ω) describing low-energy vibrational motions. Hn is the half-width at half maximum (HWHM). In the simplest case of an exponential protein relaxation, the lineshape function Ln (H n , ω ) is a Lorentzian, whose width is related to the characteristic decay time τ R of the relaxation process. A0(Q) and An(Q) are the elastic and quasielastic incoherent structure factors (EISF and QISF), respectively, which are related to each other according to

∑ A (Q) = 1 − A (Q) . n

0

(4)

n

For proper comparison to optical spectra (see below), we will model the inelastic scattering contribution Sin(Q,ω) by an asymmetric lineshape composed of a Gaussian at its low-energy side and a Lorentzian at its high-energy side. Similar asymmetric lineshapes can be achieved be employing the shape of a damped harmonic oscillator biological systems

42, 67

66

, which was also successfully used for

. INS spectra were simulated using the built-in fitting routines of Origin.

The fit quality achieved was evaluated by the χ2-values obtained. Calculation of optical spectra: Within the Franck–Condon and mean frequency approximations, the low-temperature single site absorption and fluorescence spectra of a chromophore embedded into an amorphous protein matrix are given by 68 ∞  e−S  L(ω ) = ∑  S R  dΩ 0 N (Ω 0 − ω C ) l R (ω − Ω 0 m Rω m ) R!  ∫ R=0 

(5)

where − and + correspond to absorption and fluorescence, respectively. The first term describes the zero-phonon line (ZPL), i.e. a transition without a net change in phonon quanta, having a Lorentzian lineshape l0 at frequency Ω. The phonon sideband (PSB) consists of all lR-terms with R=1,2,… corresponding to the one-phonon (R=1) peaking at ωm and further multi-phonon (R ≥ 2) transitions peaking at Rωm. Each profile lR (R>1) is obtained by folding the one-phonon profile l1 R-times with itself, so that the form of the one-phonon profile determines the shape of the whole PSB. The dimensionless Huang–Rhys factor S is a measure for the linear electron–phonon 9



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coupling strength and characterizes the average number of phonons accompanying a particular electronic transition. The one-phonon profile is composed of an asymmetric lineshape of a Gaussian at its low-energy side and a Lorentzian at its high-energy side. The one-phonon profile is related to the recently more common notation in terms of the phonon spectral density J(ω) by

l1 = J(ω)/S.

(6)

The spectrum for an ensemble of pigments embedded in an amorphous matrix is obtained by convolution with a Gaussian inhomogeneous distribution function N(Ω0 − ωc) peaking at ωc. It was shown before53, that the shape of l1 is widely similar to the shape of the inelastic peak in LHC II INS spectra.

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RESULTS

Figure 2. INS spectra of LHC II and of the buffer solution at 120 K: Frame A: INS spectra of trimeric LHC II (green circles) and of its buffer solution (blue circles) measured at 120 K at an elastic resolution of 0.117 meV and summed over all scattering angles resulting in Q = 1.46 Å-1.The inset shows the INS difference spectrum attributed to the protein contribution of trimeric LHC II (black circles). The red line is the fit function according to Eq. 2. Black arrows indicate inelastic peaks at about 2.4 and 6.5 meV. Frame B shows the equivalent data for the case of monomeric LHC II (green circles) and its buffer solution (blue circles). The protein contribution of monomeric LHC II is shown in the inset (black circles). Experimental conditions were the same as for the data in Frame A.

Separation of protein and buffer dynamics: INS spectra of trimeric and monomeric LHC II obtained with an elastic energy resolution of 117 µeV at 120 K are shown in Fig. 2 (green 11



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symbols in Frames A and B, respectively). As pointed out in refs.

53, 58

, these spectra contain

information about the dynamics of both LHC II complex and buffer solution. Therefore, INS spectra of the buffer solution were measured separately (blue symbols in Frames A and B of Fig. 2, respectively) and have to be properly subtracted

53, 69, 70

. The INS spectra of trimeric and

monomeric LHC II are rather similar and reveal two inelastic peaks at roughly 2.5 and 6.5 meV, respectively. The latter peak is also found in the buffer spectrum, so that it can be mainly attributed to the solvent. At the same time, the buffer contribution to the spectra of the solubilized complexes appears to be smaller in the low-energy range between about 2 and 4 meV. The buffer spectra were subtracted according to its weight contribution to the solubilized sample as described before in ref.

58

. The weight of LHC II was determined independently via absorbance

measurements. The resulting difference curves corresponding to the scattering functions S(Q,ω) of trimeric and monomeric LHC II complexes only are shown in the insets of Figure 2. As expected, the 120 K difference spectrum of trimeric LHC II reveals an inelastic peak at 2.4 meV and an asymmetric shape similar to the results for temperatures below 100 K reported in refs. 53, 58

. The buffer contribution was subtracted in the same way at all temperatures.

Temperature-dependent INS spectra: INS spectra of monomeric and trimeric LHC II were measured over a wide temperature range from 20 to 305 K. The resulting difference spectra representing monomeric and trimeric LHC II complexes only, i.e. without a contribution from the buffer solution, are shown for three representative temperatures in Fig. 3. The spectra of the low temperature regime below 240 K shown in Frame A of Fig. 3 exhibit a distinct asymmetric inelastic peak located at about 2.4 meV at 80 K and at lower temperatures. The data for T < 80 K (not shown) are very similar to those reported in

53

. The latter peak subsequently broadens and

shifts towards lower energies with increasing temperature. At the same time, the peak intensity increases with increasing temperature on the expense of the intensity of the elastic peak at 0 meV. Above 240 K (see Frame B of Fig. 2), however, the inelastic peak merges with the more pronounced quasielastic contribution visible around the elastic peak.

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80 K

0

10-1 10-2

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Figure 3. Temperature dependence of INS spectra (black circles) of trimeric (left column) and monomeric LHC II (right column) obtained after subtraction of the buffer contribution as shown in Figure 2. The spectra are obtained with an elastic resolution of 0.117 meV and summed over all scattering angles resulting in Q = 1.46 Å-1. Theoretical fits are 25 shown as red lines. Data of trimeric LHC II are taken from ref. . Copyright 2015 American Chemical Society.

The INS spectra of trimeric and monomeric LHC II complexes can be generally fitted according to Eqs. 2 and 3 using a model scattering function comprising a Gaussian resolution function

13



R(ω), a Lorentzian quasielastic component, and two asymmetric inelastic contributions composed of a Gaussian and a Lorentzian wing at its low- and high energy side, respectively. The fits are shown in Fig. 3 for some representative temperature values. The QISFs and the widths of the quasielastic component obtained for trimeric LHC II by Vrandecic et al.25 were taken as input parameters. Accordingly, the width (FWHM) of the quasielastic component is 507 µeV corresponding to a relaxation time of ~1.3 ps. In the case of trimeric LHC II, the two inelastic contributions are peaking at about 2.4 and 6.5 meV at 80 K. The latter peak positions remain virtually invariant for temperatures below ~160K, but shift to lower energies at higher temperatures. This effect is illustrated for two selected temperatures in Fig. 4. The inelastic peak positions determined by fitting are shown in Fig. 5 as blue squares and circles representing trimeric and monomeric LHC II complexes, respectively. The fit results for monomeric LHC II are rather similar, while the inelastic peaks appear to be slightly more distinct than in the case of trimeric LHC II. In addition, the low-energy inelastic peak of monomeric LHC II is shifted to slightly higher energies of about 2.7 meV at 80 K consistent with a smaller delocalization of the corresponding protein vibrations in the monomer. Again, the inelastic peak position shifts to lower energies with increasing temperature in the case of monomeric LHC II, although it reveals subtle differences compared with the temperature-dependent positions of the inelastic peaks of trimeric LHC II. The corresponding quasielastic incoherent structure factors (QISFs) obtained from the fits of Fig. 4 are also shown in Fig. 5 as black squares and circles representing trimeric and monomeric LHC II, respectively. The QISFs of both trimeric and monomeric LHC II exhibit a rather similar temperature dependence with a small increase above 80 K and a major transition at 240 K. The observation of the dynamical transition at about 240 K is consistent with refs.25, 57. As discussed in the latter references, the QISFs characterize the temperature dependence of the conformational protein flexibility, i.e. the large-amplitude motions of protein backbone and sidechains on the picosecond timescale. At first glance, the temperature dependence of the QISFs and inelastic peak positions appears to be only partly correlated, see Discussion for more details.

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Figure 4. Inelastic region of the INS spectra of trimeric (left) and monomeric LHC II (right) at 80 and 240 K: Left: INS spectra of trimeric LHC II at 80 (green squares) and 240 K (grey circles) The red line is the fit function according to Eq. 2, while the blue line shows lowest-energy inelastic contribution. Black arrows indicate inelastic peaks at about 2.4 and 2.1 meV, respectively. Right: the equivalent data are shown for the case of monomeric LHC II using the same color code. Black arrows indicate inelastic peaks at about 2.6 and 1.5 meV, respectively.

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Figure 5. The temperature dependence of the QISF (black) and of the inelastic peak position (blue). Full and open symbols correspond to trimeric and monomeric LHC II, respectively. Full black and dashed red lines interpolate the data for trimeric LHC II as a guide to the eye. The labels indicate: A) the appearance of internal protein dynamics at about 80 K, B) the onset of vibrational anharmonicity at about 160 K, and C) the dynamical transition at about 240 K. The 25 QISFs of trimeric LHC II (fixed as input parameters in the fits shown in Fig. 3) are taken from ref. . Copyright 2015 American Chemical Society.

Dependence of INS spectra on scattering angle: INS spectra of trimeric LHC II obtained at different scattering angles are shown in Fig. 6. The data were measured at a temperature of 80 K, where almost no quasielastic contribution is present, i.e. only harmonic vibrational motions are expected. Phenomenologically, it is visible that the intensity of the inelastic peak increases with increasing scattering angle on the expense of the elastic scattering as theoretically expected. Considering only the elastic contribution in Eq. 3, the natural logarithm of its intensity should follow a linear dependence on Q2 with Q being the momentum transfer. This linear dependence, which is the signature of the decreasing elastic intensity with increasing Q, is clearly visible in Fig. 7A for both, trimeric and monomeric LHC II. In turn, the natural logarithm of the scattering intensity at the inelastic peak position (2.4 meV) is shown to follow a linear increase with

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increasing Q2 in Fig. 7B. The latter observations prove that the INS spectra follow the expected Q dependence for harmonic vibrational motions (see Eq. 3).

Figure 6. INS spectra of trimeric LHC II measured at 80 K for different Q-values.

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Figure 7. Q dependence of the peak intensities of the elastic peak at 0 meV (A) and of the inelastic peak at 2.4 meV (B), respectively, derived from the INS spectra of LHC II trimers (circles) and monomers (squares) at a temperature of 80 K.

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DISCUSSION Conformational protein dynamics: Information on conformational (diffusive) protein motions can be gathered from the temperature dependence of the relative quasielastic intensity in terms of the QISF of monomeric (see open black symbols in Fig. 5) and trimeric LHC II (see full black symbols in Fig. 5). For both complexes, the data reveal a dynamical transition with a drastic increase of protein flexibility at about 240 K. As pointed out in the Introduction, a qualitatively similar behavior was reported for a number of globular and membrane proteins as well as for native membranes. The data of the trimeric complex resemble those shown by 25, 58. At 280 K, the QISF of monomeric LHC II appears to be larger than that of trimeric LHC II, which would be consistent with a larger flexibility due to a higher number of solvent-exposed protein residues at the surface of the protein. A similar tendency was reported based on spectral hole burning experiments, which revealed a larger structural hererogeneity due to a higher number of conformational substates for monomeric LHC II 62 than for trimeric LHC II 64, 71. Below 240 K, the flexibility of both complexes appears to be more restricted indicated by the generally much smaller QISFs. No quasielastic contribution can be detected below 80 K, i.e. conformational dynamics is frozen. However, rather small QISFs corresponding to some internal flexibility are observed at temperatures as low as 80 K as reported before by 25, 58. In contrast to the situation above 240 K, the low-temperature dynamics are rather similar for both monomeric and trimeric LHC II. This is consistent with earlier findings that protein dynamics in this temperature range is independent of solvent effects and mainly due to internal protein motions of e.g. methyl groups and other small protein residues, which is an intrinsic property of a given protein and not related to its particular environment or solvent 36, 72-74. In summary, we can distinguish three ranges of different protein dynamics: a) below about 80 K conformational protein dynamics is frozen and the protein is trapped in individual conformational substates, b) onset of motions of internal protein residues on the picosecond timescale at 80 K, and c) protein dynamics on the picosecond timescale fully unfolds above the dynamical transition at 240 K in monomeric and trimeric LHC II (for reviews see

32-34

and references therein). The

transition temperature of about 240 K is similar to that of fully hydrated PS II membrane 19



fragments

26, 42

Page 20 of 35

containing LHC II as one of the embedded proteins. However, the dynamical

transition was shown to be absent in dry PS II membrane fragments and at hydration levels below 45 % r.h.

75

. The latter findings underline the importance of the solvent in determining the

transition temperature. More recently, NMR studies of solubilized LHC II

76

and of whole

thylakoid membranes 77 emerged. The results indicate that NMR signals from the Chl macrocyles of LHC II disappear between 223 and 244 K76, i.e. they have become too flexible to be detected, which is in line with the dynamical transition observed in this study. Temperature-dependence of protein vibrations: In addition to the QISFs reflecting the protein dynamics of LHC II, Fig. 5 also shows the inelastic peak positions, which are indicative of the distribution of protein vibrational energies of monomeric and trimeric LHC II, respectively (blue symbols in Fig. 5). Interestingly, the inelastic peak position follows a complex dependence on temperature, which differs from that of the QISFs. The inelastic peak remains almost invariant within experimental uncertainty up to about 160 K, but shifts to lower energies above this temperature. Furthermore, there is a more pronounced shift along with the dynamical transition at ~240 K. However, at these temperatures we also observe a rather strong overlap with the enhanced quasielastic contribution so that the uncertainty of determining the peak position becomes higher. Overall, the temperature dependence of the inelastic peak position is rather similar for monomeric and trimeric LHC II. Similar effects were reported previously for a polymer glass 78, for other proteins 52, 79-81, and also for intact PS II membrane fragments 43. In some other cases, models imposing constant inelastic peak positions were used 42, 82. When comparing these observations with the temperature dependence of the corresponding QISFs, it appears that the above-mentioned shift of the inelastic peak is not correlated with the low-temperature onset of conformational protein dynamics at about 80 K, but may be related to the dynamical transition at 240 K. As discussed in ref. 25 before, the temperature dependent QISFs suggest that conformational protein dynamics in trimeric LHC II is frozen and the protein is trapped in individual conformational substates below a temperature of about 80 K. It was also shown by INS experiments on trimeric LHC II that the inelastic peak reflects harmonic 20


Page 21 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


vibrational motions in the temperature range between 5 and 120 K.43 The absence of conformational protein dynamics is also regularly assumed in simulations of EET, which are often restricted to the case of low (non-physiological) temperatures 8, 17, 18. As inferred before58, the inelastic peak shift above 240 K may be correlated with the dynamical transition in LHC II. It could be rationalized assuming that the onset of conformational motions during the dynamical transition leads to an average softening of the protein matrix, because e.g. hydrogen bonds are continuously broken and re-formed by moving protein residues. When identifying a delocalized protein vibration with a harmonic oscillator as the simplest possible model, its frequency is determined by its mass and by the spring constant. Then, a softening of the protein matrix would correspond to reducing the spring constant and result in a shift of the normal modes to lower energies. However, a similar argument would not hold in the case of the low-temperature shift of the inelastic peak observed above ~160 K, which seems to be uncorrelated with the onset of internal protein dynamics observed at temperatures as low as 80 K in the case of LHC II. A possible explanation for this finding can be provided in terms of vibrational anharmonicity. Assuming a Lennard-Jones potential as a good approximation of the potential energy surface of a representative protein normal mode, the harmonic approximation is only valid at sufficiently low temperatures, where the asymmetry of the potential energy surface can be neglected. At higher temperatures, however, the latter asymmetry leads to a deviation from the harmonic approximation which becomes visible as a shift of the respective normal mode to lower energy (see e.g.

83

and references therein. Therefore, vibrational anharmonicity provides a perfect

explanation for the temperature dependence of the inelastic peak position between ~160 K and 240 K. Our data demonstrate that INS is capable of providing information on vibrational dynamics and anharmonicity in pigment-protein complexes even at elevated or almost physiological temperatures. This is in stark contrast to optical line-narrowing techniques, which are usually restricted to liquid helium temperatures.2

21



Figure 8. Theoretical simulations of absorption spectra, where: 1) assuming a rigid protein matrix, the one-phonon profile taken from

53

and Huang-Rhys factor S=0.4425 are kept constant (open circles); 2) the one-phonon profile is varied

with temperature based on the INS data of this study (solid black lines) with a constant Huang-Rhys factor S of 0.44, and 3) in addition to the one-phonon profile, the S-factor is varied from 0.44 to 0.75 for temperatures 120 and 240 K taking into account the effect of protein dynamics according to ref. 25 (red lines).

22


Page 22 of 35

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Simulated absorption spectra: As a consequence of the dynamical properties of LHC II reported above, simulations of spectral lineshapes and/or EET kinetics in photosynthetic pigment-protein complexes at physiological temperatures should account for a temperaturedependent density of vibrational states. We have also shown previously that the positions of excited electronic states of LHC II and their electron-phonon coupling strengths depend on temperature and exhibit a different behavior in selected temperature regions.25 Our results confirmed that a pigment-protein complex switches between different conformational (and energetically inequivalent) substates on the picosecond timescale, i.e. on a timescale comparable to EET kinetics at physiological temperatures. The latter findings were in agreement with a number of scientific studies that revealed effects of the protein environment or the solvent on pigment transition energies in general, see e.g. refs.

84-86

in general and see e.g.

18, 20

specifically

for the case of LHC II. In order to investigate the effect of the temperature-dependent changes in the density of vibrational states reported here and the changes in electron-phonon coupling reported by

25

we

have simulated absorption spectra following Eq. 5 at three representative temperatures (80, 120 and 240 K). As outlined in detail in Methods and in ref. 53, we further assumed that the onephonon profile used in the calculation of the optical spectra is widely similar to the lineshape of the inelastic peak observed in INS experiments, see Table 1 for parameters. In physical terms this means that the shape of the one-phonon profile is mainly determined by the density of vibrational states. We add, that the one-phonon profile is essentially identical in its lineshape to the more commonly used term phonon spectral density. When simulating absorption spectra, we first assumed the absence of conformational protein dynamics consistent with a rigid protein matrix. Thus, spectra at all three temperatures were calculated based on the parameters determined for Chlorophyll a 612 of LHC II by lowtemperature site-selective optical spectroscopy, i.e. a Huang-Rhys factor S of 0.44,25 the onephonon profile determined in ref. 53 and an inhomogeneous width of 80 cm-1 64, 71. The absorption spectra obtained by this approach (Eq. 5) are shown as open circles in Fig. 8. This approach 23



Page 24 of 35

predicts that the shape of the absorption spectrum becomes more symmetric with increasing temperature, which is mainly due to thermal occupation of higher phonon levels and the concomitant increase in phonon annihilation processes which lead to an increase of the antiStokes contribution at the low-energy side of the spectra. We note that the theory used here was initially developed for impurity centers in crystals complexes

87

83

and later adapted to pigment-protein

. This means that effects of conformational protein dynamics cannot be properly

described within this approach, i.e. the protein is taken as a rigid entity that is capable of smallamplitude vibrational motions only. In a second step, we considered the effect of the changing density of phonon states as determined in the present study (see Table 1). The resulting absorption spectra are shown as full black lines in Fig. 8. A comparison of the two data sets shows rather subtle changes only. Except for a slightly smaller asymmetry at 80 and 120 K, the simulated spectra appear to be widely similar. This is especially true for the spectrum calculated at 240 K. Although an apparent shift of the inelastic peak is observed by INS, almost no difference is visible in the inhomogeneously broadened absorption spectrum. Finally, the correct S-factors determined by

25

were applied at

each temperature in addition to the varying one-phonon profile. The related spectra are shown as red lines in Fig. 8. In this approach, the spectra at 120 and 240 K exhibit significant differences compared to those calculated based on the low-temperature model. The peak position is shifted towards higher energies, i.e. the Stokes-shift is larger, and the broadening of the spectra is enhanced. The calculated spectra presented above cannot be directly compared to absorption spectra of trimeric LHC II, because they consist of unresolved absorption bands of 42 Chl molecules. However, the general shape and its temperature dependence known from mutant spectra25 are properly reproduced. The latter findings indicate that the spectral properties of Chlorophyll a 612 of LHC II change significantly with temperature due to different dynamical regimes of the protein matrix. We anticipate that this is a general effect in photosynthetic pigment-protein complexes. As pointed out in the Introduction, electron-phonon coupling in an antenna complex like LHC II permits EET between energetically inequivalent pigment energy levels (see Fig. 1) by dumping the 24


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energetic difference into vibrations. Therefore, proper simulations of EET have to account for the temperature-dependent change in excited state positions and coupling to vibrational modes reported above.

25



Conclusions In summary, the vibrational dynamics of monomeric and trimeric LHC II is characterized by three different temperature regions: i) harmonic vibrational motions below roughly 160 K, ii) appearance of vibrational anharmonicity along with a shift of protein vibrations to lower energies above about 160 K, and iii) a further downshift of protein vibration above the dynamical transition at 240 K, which may be attributed to a “softening” of the protein matrix along with the onset of conformational dynamics. Monomeric LHC II exhibits a larger degree of conformational mobility at physiological temperature at 285 K, most probably due to a higher number of solventexposed sidechains at the protein surface. In order to investigate the effect of the above changes in vibrational dynamics on spectroscopic properties and EET in LHC II, comparative model calculations were carried out, which indicate that vibrational and conformational protein dynamics affect the spectroscopic (absorption) properties of trimeric and monomeric LHC II at physiological temperatures.

Acknowledgement We gratefully acknowledge financial support by the Estonian Research Council (Grants ETF 9453, IUT 2-28 and SLOKT 12026 T). J.P. is thankful for financial support by the European Social Fund’s Doctoral Studies and Internationalisation Programme DoRa. L.R. would like to thank the support of Latvian National research program IMIS2 (2014-2017). We are also grateful to S. Kussin and M. Weß (TU Berlin) for their help in sample preparation and to HZB Berlin for allocation of neutron beam time.

26


Page 26 of 35

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Table 1 Parameters of calculations shown in Fig. 8, positions and widths are given in cm-1 as commonly used in calculations of optical absorption spectra. The values have to be divided by 8 to yield the parameters in meV.

80 K Phonon-

Peak 1

Peak 2

profile S-factor

0.44

0.33

0.11

Position Gaussian width Lorentzian width

21 30

21 32

52 56

75

64

86

Peak 1

Peak 2

0.44

0.18

0.26

[0.75]

[0.3]

[0.45]

Position

21

21

52

Gauss

31

32

58

Lorentz

100

64

126

Peak 1

Peak 2

0.44

0.2

0.24

[0.75]

[0.35]

[0.4]

Position

16.4

16.4

48

Gauss

32

32

32

Lorentz

156

64

240

120 K Phononprofile S-factor

240 K Phononprofile S-factor

27



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Light-Harvesting Complex II Exhibits a Temperature-Dependent

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