Light-Harvesting Complex II Exhibits a Temperature-Dependent

Article Cite This: J. Phys. Chem. B 2018, 122, 7111−7121

pubs.acs.org/JPCB

Rigid versus Flexible Protein Matrix: Light-Harvesting Complex II Exhibits a Temperature-Dependent Phonon Spectral Density Maksym Golub,† Leonid Rusevich,‡,§ Klaus-Dieter Irrgang,∥ and Jörg Pieper*,† †

Institute of Physics, University of Tartu, W. Ostwaldi 1, 50411 Tartu, Estonia Institute of Physical Energetics, Krivu 11, LV-1006 Riga, Latvia § Institute of Solid State Physics, University of Latvia, Kengaraga 8, LV-1063 Riga, Latvia ∥ Department of Life Science & Technology, Laboratory of Biochemistry, University for Applied Sciences, 10318 Berlin, Germany Downloaded via NAGOYA UNIV on July 30, 2018 at 19:21:15 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

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 LHC II from spinach using inelastic neutron scattering (INS) in the temperature range of 20−305 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 toward 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 higher degree of conformational mobility at physiological temperatures, which can be attributed to a higher number of solvent-exposed side chains 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 line shapes of a pigment molecule embedded into LHC II are simulated for the cases of (i) a rigid protein matrix, (ii) a protein matrix with temperaturedependent 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 LHC II at physiological temperatures.

1. INTRODUCTION

The specific structural arrangement of pigment molecules in LHC II provides the framework for efficient, ultrafast excitation energy transfer (EET). A number of experimental time-resolved absorption and two-dimensional spectroscopy studies have directly revealed time constants in the order of femtoseconds to picoseconds for Chl-to-Chl11−14 and Car-toChl 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 nonphotochemical quenching (NPQ) and thus in protection against excess energy (see ref 21 and references therein). 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

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 refs 1−3). The trimeric light-harvesting complex of photosystem (PS) II (LHC II) is the major antenna complex of green plants (see Figure 1). Its structure is well characterized by X-ray crystallography4,5 revealing the presence of 14 chlorophyll (Chl) and 4 carotenoid (Car) molecules bound by each protein monomer. The center-to-center distances between Chls in the order of only 8−10 Å suggest sizeable excitonic interactions, whereas 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 © 2018 American Chemical Society

Received: March 28, 2018 Revised: June 14, 2018 Published: June 16, 2018 7111

DOI: 10.1021/acs.jpcb.8b02948 J. Phys. Chem. B 2018, 122, 7111−7121

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The Journal of Physical Chemistry B

Figure 1. Left panel: crystal structure of the LHC II monomer according to ref 4 (pdb 1RWT). Chl a molecules are shown in orange, Chl b in green, and carotenoids in purple. 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, and the higher-frequency pigment vibrations are shown by dashed lines. Light absorption, i.e., the transition from the ground state of both pigments P1P2 to the excited state of the first pigment P1*P2 is illustrated by the blue arrow. Due to electron-vibrational coupling, light absorption cannot only occur in the excited electronic state S1, but also in 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 deexcitation 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.

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 has been shown that excited-state positions and/or electron−phonon coupling are affected by temperature-dependent onset of protein dynamics.25 The latter finding is surprising 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 In addition to 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 ref 2). This is illustrated schematically in Figure 1 (see figure caption for details). Briefly, electron−phonon coupling broadens the spectral range of light absorption (see blue arrow in Figure 1) because transitions are allowed not only 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 Figure 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 line shapes and EET between pigments. However, detailed information on the temperature dependence of protein vibrations is so far 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 ((Δ)FLN) (for a review, see ref 2). The electron−phonon coupling strengths reported for antenna complexes are often low 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 of about 2.5 meV equivalent to 20 cm−1, but they 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 water-soluble chlorophyll-binding protein.30,31 However, experimental techniques like SHB and FLN are technically restricted to temperatures below 80 K2 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, see refs 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 proteins35−39 and biological membranes40−43 display complex hydration-dependent conformational (diffusive) dynamics at physiological temperatures above a dynamical transition. The latter transition occurs in the temperature range of 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 membrane lipids.44,45 Depending on the spectral resolution employed, QENS experiments probe different time scales of protein motions,46,47 which in turn affects the observed dynamical transition.48,49 Hydration water dynamics can also be studied by QENS using contrast variation.50,51 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 7112


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tion 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. 2.3. Data Analysis. 2.3.1. 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 experiment is given by the double-

to the Debye-like density of states (see, e.g., refs 52, 53). More recently, collective water dynamics has also been studied in whole cells of green algae using inelastic coherent neutron scattering.54 Furthermore, QENS data are complementary to molecular dynamics 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 experiments.22,33,53 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 the 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.

differential cross section 65)

δ 2σ δ Ωδω

(for an overview, see, e.g., ref

|k | 2 δ 2σ = 1 binc Sinc(Q, ω) δ Ωδω |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 i ℏω yz zzR(Q, ω) ⊗ Stheo(Q, ω) Sexp(Q, ω) = FN expjjj− k 2kT {

(2)

which is the product of a normalization factor FN, the detailed

(

2. MATERIALS AND METHODS 2.1. Sample Preparation. Solubilized LHC II was isolated and prepared for neutron scattering experiments, as reported earlier.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.59,60 Monomerization of trimeric LHC II was induced as described by Nussberger et al.61 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, Euriso-Top, CEA Group, Saclay, France), resulting in a final concentration of about 25 mg Chl/mL. This is equivalent to a protein mass of approximately 100 mg in a 1 mL sample cell. The buffer solution is composed of D2O, 25 mM Mes-NaOD, 10 mM CaCl2, 0.025% w/v ß-dodecylmaltoside, and 30% w/v sucrose at a pD value of 6.7 (under the assumption that pD = pH + 0.4). 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, and the analyses yielded no indication for radiation damage. 2.2. Experimental Methods. 2.2.1. 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 ΔE = 0.117 meV was determined by vanadium standard runs. The samples were kept in cylindrical aluminum 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 geometrydependent attenuation, normalized, and transferred to energy scale using the program package FITMO-4. Solubilized LHC II complexes and the D2O-containing buffer solution were studied separately to independently investigate the contribu-

ℏω

)

balance factor exp − 2kT , and 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 l 2 2o o Stheo(Q, ω) = e−⟨u ⟩Q m oA 0(Q)δ(ω) o n +

| o

∑ A n(Q)Ln(Hn , ω) + Sin(Q, ω)o}oo n

~ (3)

where the scattered intensity is the product of the Debye− 2 2 Waller factor e−⟨u ⟩Q , characterized by the “global” vibrational mean-square displacement ⟨u2⟩ and the sum of three contributions: (i) the elastic component A0(Q)δ(ω), (ii) the quasielastic component ∑nAn(Q)Ln(Hn,ω), and (iii) the inelastic contribution Sin(Q,ω) describing low-energy vibrational motions. Hn is the half-width at half-maximum. In the simplest case of an exponential protein relaxation, the line shape function Ln(Hn,ω) is a Lorentzian, whose width is related to the characteristic decay time τR of the relaxation process. A0(Q) and An(Q) are the elastic and quasielastic incoherent structure factors (QISF), respectively, which are related to each other as

∑ A n(Q) = 1 − A 0(Q) n

(4)

For proper comparison to optical spectra (see below), we will model the inelastic scattering contribution Sin(Q,ω) by an asymmetric line shape composed of a Gaussian at its lowenergy side and a Lorentzian at its high-energy side. Similar asymmetric line shapes can be achieved by employing the shape of a damped harmonic oscillator,66 which was also 7113


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The Journal of Physical Chemistry B successfully used for biological systems.42,67 INS spectra were simulated using the built-in fitting routines of Origin. The fit quality achieved was evaluated by the χ2 values obtained. 2.3.2. Calculation of Optical Spectra. Within the Franck− Condon and mean frequency approximations, the lowtemperature single-site absorption and fluorescence spectra of a chromophore embedded into an amorphous protein matrix are given by68 i

∑ jjjjjS R e ∞

L(ω) =

R=0

k

−S y

zz z R! zz{

∫ dΩ0N(Ω0 − ωC)lR

(ω − Ω 0 ∓ Rωm)

(5)

where − and + correspond to absorption and fluorescence, respectively. The first term describes the zero-phonon line, i.e., a transition without a net change in phonon quanta, having a Lorentzian line shape l0 at frequency Ω. The phonon side band (PSB) consists of all lR terms with R = 1, 2, ... corresponding to the one-phonon (R = 1) peaking at ωm and further multiphonon (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 of the linear electron− phonon coupling strength and characterizes the average number of phonons accompanying a particular electronic transition. The one-phonon profile is composed of an asymmetric line shape 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

Figure 2. INS spectra of LHC II and the buffer solution at 120 K. (A) INS spectra of trimeric LHC II (green circles) and 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. The black arrows indicate inelastic peaks at about 2.4 and 6.5 meV. (B) 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 are the same as those in (A).

(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 previously53 that the shape of l1 is widely similar to the shape of the inelastic peak in LHC II INS spectra.

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, as reported in refs 53, 58. The buffer contribution was subtracted in the same way at all temperatures. 3.2. Temperature-Dependent INS Spectra. INS spectra of monomeric and trimeric LHC II were measured over a wide temperature range of 20−305 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 Figure 3. The data for T < 80 K (not shown) are very similar to those reported in ref 53. 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 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 sides, respectively. The fits are shown in Figure 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 (full width at half-maximum) 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.

3. RESULTS 3.1. 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 Figure 2A,B, respectively (green symbols). 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 Figure 2A,B) 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 2.4 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 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 7114


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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 shown as red lines. Data of trimeric LHC II are taken from ref 25. Copyright 2015 American Chemical Society.

The latter peak positions remain virtually invariant for temperatures below ∼160 K, but shift to lower energies at higher temperatures. This effect is illustrated for two selected temperatures in Figure 4. Above 240 K, however, the inelastic peak merges with the more pronounced quasielastic contribution visible around the elastic peak. The inelastic peak positions determined by fitting are shown in Figure 5 as blue squares and circles, representing trimeric and monomeric LHC II complexes, respectively. The fit results for monomeric LHC II are rather similar, whereas 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 to the temperature-dependent positions of the inelastic peaks of trimeric LHC II. The corresponding quasielastic incoherent structure factors (QISFs) obtained from the fits of Figure 4 are also shown in Figure 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

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 (gray circles). The red line is the fit function according to eq 2, whereas the blue line shows the lowest-energy inelastic contribution. The black arrows indicate inelastic peaks at about 2.4 and 2.1 meV. Right: the equivalent data are shown for the case of monomeric LHC II using the same color code. The black arrows indicate inelastic peaks at about 2.6 and 1.5 meV, respectively.

transition at about 240 K is consistent with refs 25, 57. As discussed in the latter references, the QISFs characterize the 7115


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intensity with increasing Q, is clearly visible in Figure 7A for both trimeric and monomeric LHC II. In turn, the natural

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

temperature dependence of the conformational protein flexibility, i.e., the large-amplitude motions of protein backbone and side chains on the picosecond time scale. At first glance, the temperature dependence of the QISFs and inelastic peak positions appears to be only partly correlated (see Discussion for more details). 3.3. Dependence of INS Spectra on Scattering Angle. INS spectra of trimeric LHC II obtained at different scattering angles are shown in Figure 6. The data were measured at a

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

logarithm of the scattering intensity at the inelastic peak position (2.4 meV) is shown to follow a linear increase with increasing Q2 in Figure 7B. The latter observations prove that the INS spectra follow the expected Q dependence for harmonic vibrational motions (see eq 3).

4. DISCUSSION 4.1. 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 Figure 5) and trimeric LHC II (see full black symbols in Figure 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 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 in refs 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 II62 than for trimeric LHC II.64,71 Below 240 K, the flexibility of both complexes appears to be more restricted, as indicated by the generally much smaller

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

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 at 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 7116


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experiments on trimeric LHC II that the inelastic peak reflects harmonic vibrational motions in the temperature range of 5− 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 (nonphysiological) temperatures.8,17,18 As inferred in ref 58, the inelastic peak shift above 240 K may be correlated to 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, for example, 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., ref 83 and references therein). Therefore, vibrational anharmonicity provides a perfect explanation for the temperature dependence of the inelastic peak position between ∼160 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 4.3. Simulated Absorption Spectra. As a consequence of the dynamical properties of LHC II reported above, simulations of spectral line shapes and/or EET kinetics in photosynthetic pigment−protein complexes at physiological temperatures should account for a temperature-dependent 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 time scale, i.e., on a time scale 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 refs 18, 20 specifically for the case of LHC II). 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 in ref 25, we have simulated absorption spectra following eq 5 at three

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 in refs 25, 58. In contrast to the situation above 240 K, the lowtemperature dynamics are rather similar for both monomeric and trimeric LHC II. This is consistent with previous 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 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 time scale at 80 K; and (c) protein dynamics on the picosecond time scale fully unfolds above the dynamical transition at 240 K in monomeric and trimeric LHC II (for review, see32−34 and references therein). The transition temperature of about 240 K is similar to that of fully hydrated PS II membrane fragments26,42 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% relative humidity.75 The latter findings underline the importance of the solvent in determining the transition temperature. More recently, NMR studies of solubilized LHC II76 and of whole thylakoid membranes77 emerged. The results indicate that NMR signals from the Chl macrocyles of LHC II disappear between 223 and 244 K,76 i.e., they have become too flexible to be detected, which is in line with the dynamical transition observed in this study. 4.2. Temperature Dependence of Protein Vibrations. In addition to the QISFs reflecting the protein dynamics of LHC II, Figure 5 also shows the inelastic peak positions, which are indicative of the distribution of protein vibrational energies of monomeric and trimeric LHC II (blue symbols). 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 other proteins,52,79−81 and also 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 above 160 K is not correlated with the low-temperature onset of conformational protein dynamics at about 80 K, while the shift at 240 K may be related to the dynamical transition. As discussed in ref 25, 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 7117


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The Journal of Physical Chemistry B representative temperatures (80, 120, and 240 K). As outlined in detail in Materials and Methods and in ref 53, we further assumed that the one-phonon profile used in the calculation of the optical spectra is widely similar to the line shape of the inelastic peak observed in INS experiments (see Table 1 for Table 1. Parameters of Calculations shown in Figure 8; Positions and Widths are Given in cm−1 as Commonly Used in Calculations of Optical Absorption Spectraa phonon profile

peak 1

peak 2

80 K S-factor position Gaussian width Lorentzian width S-factor position Gaussian width Lorentzian width S-factor position Gauss width Lorentz width

0.44 21 30 75 120 K 0.44 (0.75) 21 31 100 240 K 0.44 (0.75) 16.4 32 156

0.33 21 32 64

0.11 52 56 86

0.18 (0.3) 21 32 64

0.26 (0.45) 52 58 126

0.2 (0.35) 16.4 32 64

0.24 (0.4) 48 32 240

Figure 8. Theoretical simulations of absorption spectra, where assuming a rigid protein matrix, the one-phonon profile taken from ref 53 and Huang−Rhys factor S = 0.4425 are kept constant (open circles); 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 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).

a

The values have to be divided by 8 to yield the parameters in meV.

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 line shape 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 low-temperature site-selective optical spectroscopy, i.e., a Huang−Rhys factor S of 0.44,25 the one-phonon 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 Figure 8. This approach 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 crystals83 and later adapted to pigment− protein complexes.87 This means that effects of conformational protein dynamics cannot be properly described within this model, i.e., the protein is taken as a rigid entity that is capable of small-amplitude 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 Figure 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 in ref 25 were applied at each temperature in addition to the varying one-phonon profile. The related spectra are shown as red lines in Figure 8. In this approach, the spectra at 120 and 240 K exhibit significant differences compared to those calculated based on the lowtemperature model. The peak position is shifted toward higher energies, i.e., the Stokes shift is larger, and the broadening of the spectra is enhanced. The latter findings indicate that the spectral properties 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 Introduction, electron−phonon coupling in an antenna complex like LHC II permits EET between energetically inequivalent pigment energy levels (see Figure 1) by dumping the 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.

5. CONCLUSIONS In summary, the vibrational dynamics of monomeric and trimeric LHC II is characterized by three different temperature regions: (i) harmonic vibrational motions below approximately 160 K; (ii) appearance of vibrational anharmonicity along with a shift of protein vibrations to lower energies above 160 K; and (iii) a further downshift of protein vibrations 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 7118


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(285 K), most probably due to a larger number of solventexposed side chains at the protein surface. 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 LHC II at physiological temperatures.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +(372) 737 4627. Fax: +(372) 738 3033. ORCID

Jörg Pieper: 0000-0001-5995-9660 Notes

The authors declare no competing financial interest.

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

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REFERENCES

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