Protein Configuration Landscape Fluctuations Revealed by Exciton

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Protein Configuration Landscape Fluctuations Revealed by Exciton Transition Polarizations in Single Light Harvesting Complexes Sumera Tubasum, Magne Torbjörnsson, Dheerendra Yadav, Rafael Camacho, Gustaf Söderlind, Ivan G. Scheblykin, and Tönu Pullerits J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b12466 • Publication Date (Web): 07 Jan 2016 Downloaded from http://pubs.acs.org on January 10, 2016

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The Journal of Physical Chemistry

Protein Configuration Landscape Fluctuations Revealed by Exciton Transition Polarizations in Single Light Harvesting Complexes Sumera Tubasum,† Magne Torbjörnsson,† Dheerendra Yadav,† Rafael Camacho,† Gustaf Söderlind,‡ Ivan G. Scheblykin,† and Tõnu Pullerits*,† †

Division of Chemical Physics, Department of Chemistry, Lund University, Box 124, 22100 Lund, Sweden Division of Numerical Analysis, Centre for Mathematical Sciences, Lund University, Box 124, 22100 Lund, Sweden ‡

ABSTRACT: Protein is a flexible material with broad distribution of conformations forming an energy landscape of quasi-stationary states. Disentangling the system dynamics along this landscape is the key for understanding the functioning of the protein. Here we studied a photosynthetic antenna pigment-protein complex LH2 with single molecule 2dimensional polarization imaging. Modelling based on the Redfield relaxation theory well describes the observed polarization properties of LH2 fluorescence and fluorescence excitation strongly suggesting that at 77 K the conformational subspace of the LH2 is limited to about 3 configurations with relatively frequent switching among each other. At room temperature the next level of fluctuations determines the conformational dynamics. The results support the multi-tier model of the energy landscape of proteins and demonstrate the potential of the method for the studies of structural dynamics in proteins.

Excitation dynamics in condensed phase has been complex called reaction center (RC). In photosynthetic used for studies of conformations and changes in molecpurple bacteria the reaction center is surrounded by a so ular systems ever since Vavilov more than 90 years ago called core antenna LH1. Most of the purple bacteria explored fluorescence intensity dependence on the have an additional antenna LH2 which is in contact with quencher concentration in a dye molecule solution.1 The the LH1 and increases the total absorption cross section first quantitative theory capable of describing excitation of the whole system. Different parts of the whole antenna dynamics in such systems was developed by Förster.2 For are organized in such a way that they form an energy electronic excitations in molecular crystals a very differfunnel with the RC in the middle. This is the simplest ent physical model, molecular exciton theory, 3 was used. and most important optimization mechanism in the light While in Förster theory the transfer process can be visuharvesting process of photosynthesis.10,11 alized as excitation hopping between molecules, in the The atomic structures of the LH2 from two species are molecular exciton theory the elementary excitation is a known.12,13 LH2 consists of 8 or 9 pairs of helixes which delocalized exciton and the dynamics occurs via relaxaform a ring. Each pair of the helixes binds 3 tion between the exciton levels.4 The two dynamic probacteriochlorophyll a (BChl a) molecules. Two of the cesses, Förster hopping and exciton relaxation, can be BChls form a so called B850 ring and the remaining one, seen as limiting cases of excitation dynamics. 5,6 The relethe B800 ring. The names come from the characteristic vant measure which approach to use, is the ratio between Qy absorption wavelengths of the corresponding rings. the resonance interaction (often evaluated as the interacWhile B800 BChls are well separated from each other as tion between the transition dipole moments) of the molwell as from the B850s, the B850 BChls form a rather ecules and the electron-phonon interaction.7 The latter is densely packed ring where intermolecular resonance often called system-bath interaction. When the resointeractions are significant. The interaction is the main nance interaction is small and electron-phonon interacorigin of the shift of the absorption band to longer wavetion strong, Förster theory is used. The opposite case lengths compared to the B800 and leads to delocalizacorresponds to the molecular exciton theory. The two tion of the excitation over 3-4 BChl molecules.14,15,16 Early limiting cases are well understood, they are sensitive to time-resolved fluorescence studies showed that the B800 the structural details of the system and both have been to B850 transfer takes about 1 ps. This transfer step has extensively used to describe excitation dynamics in phobeen thoroughly investigated by transient absorption tosynthetic light harvesting.8,9 spectroscopy at various temperatures.17 Such experiIn photosynthesis the sun emission is collected by light ments have been carried out also in the LH2 complexes harvesting antenna complexes where absorption of a where the B800 BChls were replaced by spectrally simiphoton creates an electronic excitation. The excitation is lar molecules.18 Since the structure is known, the transfer very efficiently transferred to a special pigment-protein rate calculations were quantitative and it was concluded ACS Paragon Plus Environment

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that the simplest version of Förster theory leads to significantly slower transfer rates. It was suggested that the upper exciton levels can enhance the transfer process. 17 The quantitative modeling, where the effect was rigorously included, verified that the B850 exciton levels resonant to the B800 energy play major role in the B800 to B850 transfer process.19 Early time-resolved and low temperature high spectral resolution studies20,21 clearly showed that pigment protein complexes have significant spectral disorder due to the fluctuations of the environment. The energy landscape picture of proteins22 has direct relevance to the transition energy distribution of the pigment molecules. Understanding the energies and their fluctuation timescales is crucial for describing the dynamics of the system. The tier-like hierarchy of energies in protein configuration space means that some motions which are possible at room temperature will be frozen out at lower temperatures. The landscapes in the proximity of the chromophore can be different in the electronic ground and excited states of the chromophore. This means that electronic excitation may open pathways for conformation changes which would not occur in ground state. In a large ensemble there is a huge distribution of conformations which are all simultaneously measure in conventional bulk experiments. We need to be able to study one system at a time to draw conclusions about landscapes of conformations. Such experiments which are carried out at a single pigment-protein complex level have been performed by several groups.23,24,25,26,27 This brings us to an interesting question about timescales of changes and the relationship between the rates and the tier structure of the energy landscape. In ergodic systems the time average and the ensemble average give the same result.28 However, if the time average is short, the system can cover only a limited part of a phase space correlated by the tiers of the energy landscape hierarchy. In this work we apply two-dimensional (2D) fluorescence polarization imaging (2D-POLIM) 29,30 to individual LH2 complexes. The method can be seen as a further development of linear dichroism and fluorescence anisotropy techniques especially suited to single molecule studies. As distinct from the traditional fluorescence anisotropy experiment, 2D-POLIM detects fluorescence polarization properties of the sample while scanning the direction of the linearly polarized excitation light. 2DPOLIM thereby provides 2D fluorescence intensity plots where the polarization orientation angles in excitation and detection are the two dimensions. From the 2D plot not only the orientation parameters of the lightabsorbing and light emitting chromophore but also a quantitative measure of energy migration efficiency can be extracted.29,30,31 The method has previously been applied to conjugated polymers,29 chlorosomes32 and we have reported early room temperature results on LH2 complexes.33,34,35 Here, by combining experiments with exciton relaxation calculations, we show that at cryogenic temperatures an energy landscape appears with hierarchy of conformational disorder tiers with countable number of subspaces where different tiers are sampled during various characteristic timescales of the experiment.

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Experimental Methods The experimental setup and its characterization have been described in detail.30,33,34,35 The continuous-wave Ti:Sapphire laser (Spectra Physics, 3900S), pumped by Millennia Pro X, was used for excitation at 800 nm for B800, and at 850 nm for B850. The beam passed through a polarizer (wire-grid polarizer, Edmund Optics) and a rotating achromatic half-wave plate (Thorlabs, 600 - 1200 nm). The beam was directed to the microscope by a small aluminum mirror (aluminum coating of ~1mm diameter in the center of an optically transparent glass beam splitter).30 To compensate the phase shift introduced by the microscope optics, a Berek compensator was used. For room temperature measurements, oil immersion objective lens (60x, NA = 1.25) was used. A constant flow of nitrogen gas over sample was maintained to avoid photobleaching. An excitation spot of 30 m in diameter was achieved by using a de-collimating lens placed into the laser beam. The emission from the sample was collected by the same objective lens. The emission was passed through a set of long pass filters ([840LP, 830LP] for 800nm excitation, [860LP, 870LP] for 850nm excitation) that block the excitation beam. Later, the emission was split into two orthogonal polarization components by an analyzer (a rotating VLS-100NIR polarizer and a fixed Au mirror).29,30 Both polarization components were simultaneously imaged on the CCD camera (Princeton Acton, Photons max, 512x512pixels). Depolarization effects from the optics in the detection path were reduced by introducing a phase shift by slightly tilting of the long pass filters thereby no additional compensating devices were required. The speed of rotation of the half-wave plate and the analyzer were controlled electronically and provided real-time monitoring by LED triggers. All the optical elements had optimum range of their reflection or transmission efficiency in near infra-red region. For low temperature measurements a liquid-nitrogen cooled cryostat (Janis ST-500 microscopy) together with dry objective lens (40x, NA 0.65) was used. To ensure the uniformity of temperature inside the cryostat, a regular flow of liquid nitrogen was maintained throughout the experiment. Systematic polarization errors of the setup were characterized by a so-called ‘Artificial molecule’30 and were found to be less than 5 % for the modulation depths and ±4ᵒ for the polarization phase shift. The LH2 complexes from Rhodopseudomonas (Rps.) acidophila 10050 were received from our collaborators (University of Glasgow). A solution of LH2 complexes with pM concentration was prepared by diluting stock samples in Tris-HCl buffer (pH = 8. 0) containing 0.1% of lauryl dimethyl amine oxide, and oxygen scavengers (Glucose, Glucose oxidase, Catalase; Sigma-Aldrich). The diluted LH2 solution was mixed with polyvinyl alcohol (1.5%) in 1:1 ratio by volume. The final mixture was spin cast on surface oxidized silicon wafer (200 nm thermal oxide layer). We collected a series of images of individual LH2 complexes corresponding to many different combinations of excitation and analyzer angle. In that way a twodimensional map of the fluorescence intensity I (φex, φem), as a function of excitation polarization angle φex and analyzer rotation angle φem was constructed. Expo-

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sure time per single image was 0.2s. The frequency of rotation of excitation and analyzer was tuned in such a way that in total 1063 images were recorded to construct the full 2D polarization map. The power density at the sample plane was ~3kW/cm2. The resulting excitation frequency was about 1 excitation/17ns/LH2 – consequently the probability of singlet-singlet annihilation is very small. However some carotenoid triplet accumulation may occur.36 Within the excitation spot roughly 1020 single LH2 complexes were detected. LH2s appear as bright, diffraction-limited, isolated spots against the dark background. Unusually large bright spots, or the spots undergoing fluctuation of fluorescence intensity, were discarded. Most of the single LH2 complexes were found to be stable for more than 200 seconds at room temperature. At 77 K, the stability was further improved. No significant emission intensity variations were observed. The few LH2s which did change the emission intensity, were not analyzed. The number of analyzed single complexes was at 77K B800 excitation 289 and B850 excitation 588; at 300 K B800 excitation 267 and B850 excitation 311. Properties of polarization 2D maps A typical single molecule polarization 2D map is presented in Figure 1. By integrating over excitation or detection polarization angle, one-dimensional fluorescence angular dependencies are obtained with the following general functional form

where 0≤M≤1 is the modulation depth and 0≤ ≤π is the absorbing or emitting phase. In the case of single dipole absorption (emission) this angle is the same as the orientation of the dipole moment in the laboratory reference. The numerical value of modulation depth M for a perfect dipole is 1 and for an ideal circular absorber/emitter it is 0. For an absorber or emitter with MB850 Energy Transfer Mechanism in Bacterial LH2 Complexes Investigated by B800 Pigment Exchange. Biophys. J. 2000, 78, 2590-2596. Scholes, G. D.; Fleming, G. R. On the Mechanism of Light Harvesting in Photosynthetic Purple Bacteria: B800 to B850 Energy Transfer. J. Phys. Chem. B 2000, 104, 1854-1868. Pullerits, T.; Freiberg, A. Picosecond Fluorescence of Simple Photosynthetic Membranes: Evidence of Spectral Inhomogeneity and Directed Energy Transfer. Chem. Phys. 1991, 149, 409-418.

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Chem. Chem. Phys. 2015, DOI:10.1039/C5CP00295H. Walschaers, M.; Diaz, J.; Mulet, R.; Buchleitner, A. Optimally Designed Quantum Transport Across Disordered Networks. Phys. Rev. Lett. 2013, 111, 180601. Rebentrost, P.; Mohseni, M.; Kassal, I.; Lloyd, S.; Aspuru-Guzik, A. EnvironmentAssisted Quantum Transport. New J. Phys. 2009, 11, 033003. Strümpfer, J.; Schulten, K. Excited State Dynamics in Photosynthetic Reaction Center and Light Harvesting Complex 1. J. Chem. Phys. 2012, 137, 065101. van Grondelle, R.; Novoderezhkin, V. I. Energy Transfer in Photosynthesis: Experimental insights and Quantitative Models. Phys. Chem. Chem. Phys. 2006, 8, 793807. Pullerits, T. Exciton States and Relaxation in Molecular Aggregates: Numerical Study of Photosynthetic Light Harvesting. J. Chin. Chem. Soc. 2000, 47, 773-784. Spano, F. C. Absorption and Emission in Oligo-Phenylene Vinylene Nanoaggregates: The Role of Disorder and Structural Defects. J. Chem. Phys. 2002, 116, 5877-5891. Fowler, G. J. S.; Hess, S.; Pullerits, T.; Sundström V.; Hunter, C. N. The Role of βArg-10 in the B800 Bacteriochlorophyll and Carotenoid Pigment Environment within the Light-Harvesting LH2 Complex of Rhodobacter sphaeroides. Biochemistry 1997, 36, 11282-11291. He, Z.; Sundström, V.; Pullerits, T. Influence of the Protein Binding Site on the Excited States of Bacteriochlorophyll: DFT Calculations of B800 in LH2. J. Phys. Chem. B 2002, 106, 11606-11612. May, V.; Kühn, O. Charge and Energy Transfer Dynamics in Molecular Systems. Wiley-VCH, Weinheim, 2000. Polivka, T.; Pullerits, T.; Herek, J. L.; Sundström, V. Exciton Relaxation and Polaron Formation in LH2 at Low Temperature. J. Phys. Chem. B 2000, 104, 10881096. Freiberg, A.; Rätsep, M.; Timpmann, K.; Trinkunas, G.; Woodbury, N. W. SelfTrapped Excitons in LH2 Antenna Complexes between 5 K and Ambient Temperature. J. Phys. Chem. B 2003, 107, 1151011519. Pullerits, T.; Freiberg, A. Kinetic Model of Primary Energy Transfer and Trapping in Photosynthetic Membranes. Biophys. J. 1992, 63, 879-896. Polyutov, S.; Kühn, O.; Pullerits, T. ExcitonVibrational Coupling in Molecular Aggregates: Electronic versus Vibronic Dimer. Chem. Phys. 2012, 394, 21-28. Pullerits, T.; Monshouwer, R.; van Mourik, F.; van Grondelle, R. Temperature

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FIGURES

I(φex) I (φex) - Fitting X

I(φem)

180

I (φem) - Fitting

58.

Dependence of Electron-Vibronic Spectra of Photosynthetic Systems. Computer Simulations and Comparison with Experiment. Chem. Phys. 1995, 194, 395– 407. Schröter, M.; S. D. Ivanov; Schulze, J.; Polyutov, S. P.; Yan, Y.; Pullerits, T.; Kühn, O. Exciton–Vibrational Coupling in the Dynamics and Spectroscopy of Frenkel Excitons in Molecular Aggregates, Phys. Rep. 2015, 567, 1-78 Pullerits, T.; van Mourik, F.; Monshouwer, R. W.; Visschers, R. W.; van Grondelle, R. Electron-Phonon Coupling in the B820 Subunit form of LH1 Studied by Temperature Dependence of Optical Spectra. J. Luminesc. 1994, 58, 168-171. Peterman, E. J. G.; Pullerits, T.; van Grondelle, R.; van Amerongen, H. Electron−Phonon Coupling and Vibronic Fine Structure of Light-Harvesting Complex II of Green Plants: Temperature Dependent Absorption and High-Resolution Fluorescence Spectroscopy. J. Phys. Chem. B 1997, 101, 4448−4457. Christensson, N.; Dietzek, B.; Yartsev, A.; Pullerits, T. Probing the Strength of the System-Bath Interaction by Three-Pulse Photon Echoes. J. Chem. Phys. 2009, 130, 024510. Christensson, N.; Polívka, T.; Yartsev, A.; Pullerits, T. Photon Echo Spectroscopy Reveals Structure-Dynamics Relationships in Carotenoids. Phys. Rev. B: Condens. Matter Mater. Phys. 2009, 79, 2451181−245118-14. Freiberg, A.; Pajusalu, M.; Rätsep, M. Excitons in Intact Cells of Photosynthetic Bacteria. J. Phys. Chem. B 2013, 117, 11007-11014. Dahlbom, M.; Beenken, W.; Sundström, V.; Pullerits, T. Collective excitation dynamics and polaron formation in molecular aggregates. Chem. Phys. Lett. 2002, 364, 556561. Trinkunas, G.; Freiberg, A. Abrupt Exciton Self-Trapping in Finite and Disordered One-Dimensional Aggregates. J. Lumin. 2005, 112, 420-423. Rutkauskas, D.; Novoderezkhin, V.; Cogdell, R. J.; van Grondelle, R. Fluorescence Spectral Fluctuations of Single LH2 Complexes from Rhodopseudomonas acidophila Strain 10050. Biochemistry 2004, 43, 4431-4438. Rancova, O.; Abramavicius, D. Static and Dynamic Disorder in Bacterial LightHarvesting Complex LH2: A 2DES Simulation Study. J. Phys. Chem. B 2014, 118, 7533-7540. Meldaikis, J.; Zerlauskiene, O.; Abramavicius, D.; Valkunas, L. Manifesta-

90

φem (Deg)

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Δφ

0

90 φex (Deg) 0.3

0.4

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0 180

0.7

Figure 1 A typical 2D polarization plot of a single LH2 complex. In the left and top we show the onedimensional intensity curves giving the integrated excitation and detection angle dependent fluorescence intensities. Vertical shift of the position of the maximum of the 2D plot from the diagonal is defined as luminescence phase shift. It takes 212 s to collect the whole 2D data set.

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T = 77K

Expr() fguass

1,5

(a)

Expr() fguass

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sin()

P(θ)

P(θ)

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0,0

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Tilt angle θ (Deg)

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0,88177

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ExpThP

y0

ExpThP

xc

Standard Error 0

0

47,32794

1,27297

ExpThP

w

31,10782

2,55458

ExpThP

A

51,87995

3,6813

ExpThP

sigma

15,55391

ExpThP

FWHM

36,62666

ExpThP

Height

1,33067

90

Tilt angle θ (Deg)

y=y0 + (A/(w*sqrt(PI/2)))*exp(-2*((x-xc)/w)^2)

Adj. R-Square

0,86303

Figure 2 The experimental distribution (black) of the tilt angle (the angle between the principal axis of the cylindrical LH2 and the laser excitation beam) showing maximum at around 42 and 47 degree for 300k (a) and 77k (b), respectively. The distribution curves are welldescribed by the Gaussian function (red). The theoretical prediction of tilt angle distribution (blue) in case of perfectly random spatial orientation is also included. Value

Standard Error

ExpThP

y0

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ExpThP

xc

42,94388

0 0

ExpThP

w

33,77778

0

ExpThP

A

49,73184

2,7498

ExpThP

sigma

16,88889

ExpThP

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39,7703

ExpThP

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1,17474

Mex λex 800nm

Mem

Figure 4 Normalized distribution of modulation depths M at RT and at 77 K. 800nm excitation black columns and 850nm excitation grey columns. We have indicated the change in the maximum M probability while temperature is changed from 77 K to 300 K. The maxima are found using three highest values of the histogram. The change in Mex is minor while Mem change is large.

λex 850nm

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300K 0.4 0.2

(a)

(b)

0 300K λex 800nm

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∆Mem

77K

Equation

λex 850

y=y0 + (A/(w*sqrt(PI/2)))*exp(-2*((x-xc)/w)^2)

Adj. R-Square

Value

0,0

λex 800

∆Mex

(b)

sin()

1,0

Mem

0.6 0.4 77K λex 800nm

0.2

(c) 0 0

0.2

0.4

0.6

0.8

(d) 0

0.2

0.4

0.6

0.8

1

Mex

Figure 3 Experimental (blue points) modulation depths correlation plots for 77 K and RT, for excitation of B800 and B850 together with the corresponding calculated results. The theoretical density maps agree well with the density of experimental points. The number of analyzed single LH2s is at 300 K 800 nm 267 and 850 nm 311; at 77 K 800 nm 289 and 850 nm 588.

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300K λex 850nm

77K λex 850nm

Luminescence Phase Shift (∆φ)

Figure 5 Comparison of experimental (circles) and calculated (red solid line) luminescence phase shift distributions for 77 K and RT, for excitation of B800 and B850. The maxima of the theoretical distributions were normalized to one.

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The Journal of Physical Chemistry at 77 K with excitation conditions used in the present study.

AUTHOR INFORMATION Corresponding Author * Address correspondence to [email protected]

Present Addresses †[email protected] Institute of Cell and Molecular Biology Biomedicinskt Centrum (BMC), Uppsala Universitet, Uppsala, Sweden

Figure 6 Schematic illustration of the hierarchies in energy landscape of configuration space and the timescales of transitions between the quasi-stationary states

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Table of Contents artwork

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