Picosecond Dynamical Response to a Pressure-Induced Break of the

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Picosecond Dynamical Response to a Pressure-Induced Break of the Tertiary Structure Hydrogen Bonds in a Membrane Chromoprotein Maksym Golub, Jorg Pieper, Judith Peters, Liina Kangur, Elizabeth C. Martin, Christopher Neil Hunter, and Arvi Freiberg J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b11196 • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 14, 2019

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The Journal of Physical Chemistry

Picosecond Dynamical Response to a Pressure-Induced Break of the Tertiary Structure Hydrogen Bonds in a Membrane Chromoprotein

Maksym Goluba, Jörg Piepera, Judith Petersb,c, Liina Kangura, Elizabeth C. Martind, C. Neil Hunterd, Arvi Freiberga, e*

aInstitute

of Physics, University of Tartu, W. Ostwald Str. 1, Tartu 50411, Estonia bInstitut

cUniv. dDepartment

Laue Langevin, F-38042 Grenoble Cedex 9, France

Grenoble Alpes, CNRS, LIPhy, 38000 Grenoble, France.

of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK

eInstitute

of Molecular and Cell Biology, University of Tartu, Riia 23, Tartu 51010, Estonia

*Author to whom correspondence should be addressed: Arvi Freiberg email: [email protected] Abstract We used elastic incoherent neutron scattering (EINS) to find out if structural changes accompanying local hydrogen bond rupture are also reflected in global dynamical response of the protein complex. Chromatophore membranes from LH2-only strains of the photosynthetic bacterium Rhodobacter sphaeroides, with spheroidenone or neurosporene as the major carotenoids, were subjected to high hydrostatic pressure at ambient temperature. Optical spectroscopy conducted at high pressure confirmed rupture of tertiary structure hydrogen bonds. In parallel, we used EINS to follow average motions of the hydrogen atoms in LH2, which reflect the flexibility of this complex. A decrease of the average atomic mean square displacements of hydrogen atoms was observed up to a pressure of 5 kbar in both carotenoid samples due to general stiffening of protein structures, while at higher pressures a slight increase of the displacements was detected in the neurosporene mutant LH2 sample only. These data show a correlation between the local pressure-induced breakage of H-bonds, observed in optical spectra, with the altered protein dynamics monitored by EINS. The somewhat higher compressibility of the neurosporene mutant sample shows that even subtle alterations of carotenoids are manifested on a larger scale, and emphasize a close connection between the local structure and global dynamics of this membrane protein complex.

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Introduction Light-harvesting (LH) pigment-protein complexes transfer solar excitation energy to the photosynthetic reaction center where it is transformed into chemical potential energy. Among all photosynthetic organisms, purple bacteria are considered to have the simplest photosynthetic apparatus, making them a favorite model system for photosynthetic as well as for photobiology studies (see 1 for a comprehensive review). High hydrostatic pressure has been used to selectively rupture hydrogen (H-) bonds in the purple bacterial LH 2, 3 and reaction center 4 complexes, which form part of the network that stabilizes the bacteriochlorophyll a (BChl) pigments locally in their protein binding pockets. In these experiments, absorption spectroscopy, making use of the BChl molecules as local optical probes, was applied. A collective rupture of multiple H-bonds in the peripheral LH2 complexes has been observed to take place between 5 and 8 kbar (1 bar equals to 0.1 MPa), depending on the species. 2-5. The local structural modifications that follow the break of local H-bonds in the LH2 complex are also expected to affect the motional degree of freedom of the involved hydrogens and, thus to influence the global dynamical response of the protein. This can be conveniently studied by elastic incoherent neutron scattering (EINS), which directly measures the mobility on the picosecond timescale of hydrogen atoms as well as of the molecular subgroups to which they are bound. EINS, typically displayed in terms of average atomic mean square displacements (MSD), is sensitive to the global dynamics of proteins because abundant hydrogen atoms are almost homogeneously distributed over the whole protein structure and the incoherent neutron scattering cross section for hydrogens is the highest 6. Taking advantage of the much smaller incoherent scattering cross section of deuterium D compared with hydrogen H, the proteins in EINS experiments are usually suspended in heavy water (D2O) to suppress the signal originating from the solvent water. EINS is also a rather gentle technique, which does not induce radiation damage as often observed in X-ray scattering 7, 8. Applications of EINS as well as quasielastic neutron scattering to study the dynamics of water soluble, globular proteins are rather common already, see 7, 8 for reviews. However, only in a few

2


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cases they have been used to investigate the much more complex photosynthetic membrane complexes

9-12.

The reviews

13, 14

provide a general overview of the neutron scattering studies of

lipid mesophases, model biomembranes and proteins in solution at high pressure. Here, we report the effects of various hydrostatic high pressures on intracytoplasmic membrane vesicles (chromatophores) from an LH2-only strain of Rhodobacter (Rba.) sphaeroides containing either the native carotenoid, spheroidenone, or an alien one, neurosporene. The spatial structure of the LH2 complex from Rba. sphaeroides Rhodoblastus acidophilus

16.

15

shows a fair amount of similarity with that from

The Rhodoblastus acidophilus complex forms a cylinder of C9

symmetry, where the monomer units are composed of two α-helical transmembrane proteins, noncovalently associated with three BChls and one carotenoid chromophore. In the complete assembly the BChls organize into two rings: 9 BChls of the B800 ring towards the cytoplasmic face of the complex and 18 BChls of the B850 ring in the hydrophobic part of the complex. In addition, 9 carotenoid pigment chromophores structurally interconnect the B850 and B800 BChl rings 16. These membrane chromoproteins, which contain protein, pigment, lipid, and solvent (water) components, are obviously much more complicated systems than most other proteins. Still, there are a number of factors in favor of selecting the LH2 membrane complex for this study. (i) The LH2 proteins enclosed in the native chromatophore membrane bilayer and suspended a physiological buffer solution are a good approximation of the in vivo cellular environment, avoiding further complexities met in studies of either whole bacterial cells or detergent-purified LH2 complexes. In the latter case, one has to consider an influence on the protein dynamics (and vice versa) of the detergent micelle that surrounds hydrophobic parts of the membrane protein. (ii) LH2 complexes protected within the native membrane are generally more robust against longlasting neutron scattering measurements (see Materials and Methods) compared with the detergentisolated proteins. (iii) The large size of the LH2-only chromatophore, with a diameter of 53 or 64 nm, determined, respectively, by atomic force microscopy 17 or dynamic light scattering 18, allows a focus on the internal dynamics of the proteins, circumventing the need to additionally consider translational and rotational motions of the whole chromophore.

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The data obtained showed monotonic decrease up to a pressure of 5 kbar of the hydrogen MSD in both samples, while in the neurosporene mutant sample a slight increase of MSD was detected at still higher pressures. This observation is in good qualitative agreement with the pressure-induced effects observed in optical spectra, suggesting a change of global dynamics of the protein in response to breakage of local H-bonds coordinating the B850 BChl pigments in their protein binding pockets 19.

Materials and Methods Sample preparation. The suspensions of chromatophore vesicles from Rba. sphaeroides embedding native and neurosporene mutant LH2 complexes were prepared as described in 20. In order to highlight protein signal and to suppress solvent scattering, membranes were suspended in 0.1 mM HEPES pH 8 and vacuum dried in an Eppendorf Concentrator Plus, then resuspended in D2O buffered with 20 mM HEPES pH 8 to a concentration of 10-20 mg/ml protein. Temperature was maintained constant at 300 K during all measurements. EINS measurements. The EINS measurements as a function of pressure were carried out on the backscattering spectrometer IN13 at the Institute Laue-Langevin (ILL) in Grenoble, France

21.

Using an incident neutron wavelength of 2.23 Å (1 Å = 0.1 nm) corresponding to a particle energy of ~16 meV, the instrument provides an almost Q-independent energy resolution of 8 μeV in the accessible momentum transfer range of 0.19 – 4.9 Å-1. Let us note that while the 8 μeV energy resolution ensures recording dynamical movements of hydrogen atoms faster than ~100 ps 21, the actual Q range corresponds to a spatial resolution between 33 and 1.3 Å, respectively. The measured data were normalized to the incoming flux and corrected for the contribution of the empty high-pressure cell. A correction for the sensitivity of detectors was done using the purely incoherent scattering of a vanadium sample. The Large Array Manipulation Program (LAMP) developed at the ILL

22

was applied for data evaluation. In order to account for the pressure effect on buffer 4


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solution, the buffer was probed at 50 bar and 6 kbar, then a linear interpolation was applied for intermediate pressure points and the buffer contribution subtracted from the sample. The EINS data are commonly presented in terms of the incoherent dynamical structure factor, S(Q,0 ± E), where Δ 8 μeV) is the instrumental energy resolution determined as full width at half maximum. Assuming a Gaussian distribution of the atoms around their average position the structure factor reduces to 7:  1  S(Q,0 ± ΔE)  S 0 exp  Q 2 u 2  .  3 

(1)

Here, S0 is the incident neutron intensity and is the MSD. The MSD values are obtained for each pressure point from the slope of the natural logarithm of the scattered intensities S(Q,0 ± E) plotted versus Q2 according to Eq. (2):

u 2  3

dlnS(Q,0 ± ΔE) dQ 2

(2)

This approximation is strictly speaking valid only at Q → 0, but can be extended to Q2 ≤1 7. In this study, as shown below, the fit range was restricted to the Q values between 0.19 and 2.27 Å-1, where the ln[S(Q,0 ± E)] dependence on Q2 was found to be linear, within the experimental uncertainty independent of the pressure applied. Pressure setup for EINS. A high pressure sample cell of cylindrical geometry allowing 6 kbar of load was made from a high tensile strength aluminum alloy (7075-T6)

23.

An additional bulk

aluminum cylinder was inserted into the center of the cell to reduce its absorption, thus the multiple scattering effect, and also the sample volume (to ~1 ml). The pressure was transmitted through a specific high pressure stick 24 using Fluorinert (3M) that was isolated from the sample solution by a piston. Pressure was increased stepwise in the hydrostatic pressure range from 50 bar to 6 kbar, adjusted and permanently controlled by an automatic pressure pump with an accuracy of about 30 bar. Due to geometrical limitations set by the high-pressure cell (a rectangle of about 20x6 mm2) 5



and the related decrease of the neutron flux applied to the sample, the data acquisition time at each pressure point was considerably long, 7-8 hours. Optical measurements under high pressure. A range of supporting optical absorption measurements was performed at the Institute of Physics, Tartu University, to validate the sample condition before and after the neutron scattering measurements. The instrumentation used in these measurements has been described in detail 25.

Results and Discussion Pressure-dependent optical absorption spectra. As described in the Introduction, optical spectroscopy under high hydrostatic pressure revealed significant differences in the stability of native and neurosporene mutant LH2 complexes embedded into native photosynthetic membranes 5.

Shown in Fig. 1A are absorption spectra of the two samples studied in this work and measured

at ambient pressure. The spectra are practically identical, except in the carotenoid absorption range between 400 and 600 nm, where they deviate significantly because of the different carotenoid contents: mainly spheroidenone in the native complex and neurosporene in the mutant LH2 complex. We note that the latter sample is the only membrane where a pressure-induced H-bond break has been observed within the accessible pressure range of 6 kbar of the high pressure cell available at IN13. Upon the sample compression the spectral bands seen in Fig. 1A universally shift toward longer wavelengths (red shift), albeit with different rates, as shown in 26, 27. Figure 1B compares the B850 exciton absorption band shift in the two membrane samples studied. At pressures ≤ 4 kbar, the initially overlapping spectra red shift coincidently and almost linearly with the rise in pressure. Subsequently, the two paths begin to separate, so that, while the spectrum of native sample continues its linear course with a constant speed/slope, the mutant sample increasingly lags behind. 6


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Following an abrupt separation at about 7 kbar, the two spectra continue red shifting almost in parallel. This routine, observed in a number of detergent-isolated LH2 complexes 2, 5, 25, was related to a collective rupture of all the 18 H-bonds between the acetyl carbonyl side groups of the B850 BChl pigments and tyrosine (or alternatingly tyrosine and tryptophan as in Rhodoblastus acidophilus

16)

residues of the surrounding protein scaffold

28.

The fact that the rupture in the

mutant sample takes place at considerably lower pressure compared with that in the native sample emphasizes an essential role played by carotenoids in structural stabilization of the LH2 complex.

7



Fig. 1. (A) Overview absorption spectra of the LH2 membrane samples dissolved in water buffer. The spectra are recorded at ambient pressure of ~1 bar. Indicated is the molecular origin of the main absorption bands. (B) Transition energy of the B850 exciton absorption band as a function of applied pressure. The dashed line designates the maximum pressure achieved in the present neutron scattering experiments. Concerns have been raised about possible effects the H2O/D2O exchange might have on protein stability as well as other properties

7, 29.

To ensure that this is not the case with

respect to the H-bond breakage in our samples, absorption spectra of native and mutant LH2-only chromatophores dissolved either in H2O- or D2O-based buffer were studied in parallel at different pressures. The results, as demonstrated in Fig. 2 in the case of the mutant sample, show only minor variations between the B850 exciton spectra. Similar measurements on native samples led to the conclusion that in our experiments any effects of the H2O/D2O exchange can most probably be ignored. EINS experiments. The EINS experiments were carried out using similar two types of LH2 membrane samples as in optical measurements, except this time we considered only the D2O-buffered samples. The MSD of hydrogens extracted from the EINS signal at fixed temperature and pressure are a measure of flexibility of the biological system on the picosecond timescale under the given thermodynamic conditions. 8


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Shown in Fig. 3 is a characteristic example of EINS data of mutant sample at 20 bar pressure, obtained over the whole accessible momentum transfer range. The data are presented in terms of the natural logarithm of the neutron scattering intensity versus the momentum transfer squared, convenient for extracting the MSD data according to Eq. 2. The signal in Fig. 3 (similarly for other pressures and samples) generally decreases with the square of the momentum transfer. Notably, at small Q values, a range could always be found where the decrease was nearly linear (highlighted in Fig. 3 by red data points). Accordingly, as follows, data within this limited Q range (Q ~ 0.19 - 2.27 Å-1 or Q ~ 0.19 2.06 Å-1, correspondingly, in native or mutant samples) will only be analyzed in order to determine the pressure dependence of the slopes, i.e., MSD values. Let us note that the above Q ranges correspond, respectively, to approximate spatial resolution between 33 and 2.8 Å or 33 and 3.1 Å, well within the diameter of the LH2 complex in the membrane plane, which is ~65 Å 15, 16.

9



Fig. 2. Comparison at different pressures of the absorption spectra of mutant chromatophores solvated either in H2O -based (blue line) or D2O -based (black line) buffer solutions. Only the near-infrared B800-850 part of the spectrum is shown. The two spectra measured at each indicated pressure are normalized with respect to their B850 peak intensity. Open rings related to the pressure axis on the right show the B850 peak positions for all performed measurements.

10


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Figure 4 shows the limited Q2 plots of the logarithm of the neutron scattering intensity in the case of native (A) and mutant (B, C) LH2 membranes. Linear approximation appears to work reasonably well for both samples and at all the pressures applied. In the case of native sample, one can also notice a systematic rise of the elastic scattering signal amplitude and a decrease of the slope with increasing pressure. The former effect was observed earlier in studies of globular proteins

30,

being explained by a reduction in

the rate and amplitude of the fluctuations of hydrogen atoms. The decrease of the slope, i.e., stiffening of the protein upon compression, has also been described previously for globular proteins 31, 32.

11



Fig. 3. Logarithm of the neutron scattering intensity as a function of the square of the momentum transfer Q in the case of mutant LH2 membranes at the pressure of 20 bar. Straight line designates linear fit of the data in the Q-range up to 2.27 Å-1.

The slope dependence on pressure in mutant sample (Fig. 4B) is notably different from that in native sample (Fig. 4A). In this former case, the initial decrease of the slope with increasing pressure is at about 5 kbar replaced by an increase of the slope (Fig. 4C).

12


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Fig. 4. Dependence of the neutron scattering intensity on the square of the momentum transfer measured in the case of native (A) and mutant (B) LH2 membranes at different

13



pressures indicated. Straight lines designate linear fits of the data. Panel C highlights the data for mutant sample from panel B corresponding to the highest pressures starting from 5 kbar.

The pressure dependence of the average atomic MSD deduced from the neutron scattering data for both native and mutant samples is shown in Fig. 5. At first sight at least, the performance of the two samples appears similar. (Apparent miss of data points between 0.8 and 3.2 kbar in case of native sample is due to temporary leak in the highpressure system.) At low pressures (1 bar to ~1.5 kbar), the MSD are equal as well as stable within the experimental uncertainty, smoothly decreasing thereafter. The steady initial phase of MSD, also observed in globular proteins 31, 32, implies that flexibility of the protein is not appreciably modified in this pressure range. It is most probably related to initial compression of voids and other packing defects. The subsequent decrease of MSD corroborates Le Chatelier’s principle

33,

which with respect to pressure states that a

compression of the sample will favor a volume reduction, thus a decreased flexibility and/or dynamics of the protein samples with increasing pressure.

14


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A closer look, however, reveals important differences between the pressure dependences of the two samples. Firstly, the MSD for the mutant sample decreases faster upon compression than for the native sample. The higher compressibility of the mutant sample is a likely consequence of replacing the native carotenoid with neurosporene, similar to the decreased structural stability of the mutant complex (see Fig. 1B). Both effects might be caused by a distinct molecular structure of neurosporene, the carotenoid exchanged. Indeed, while native carotenoids, mainly spheroidenone

34,

possess a bulky head group

that may effectively obstruct the surrounding protein movements, neurosporene has a simple rod-like structure, presumably less resistant to the compression of the protein. Furthermore, if to look at neurosporene and spheroidenone structures provided in Fig. 6, one can see the difference in size at one end of the molecule. With nine of these per complex, there could be a difference in packing of the cyclic LH2 complex. Another example of a significant role of carotenoids in stabilizing membrane protein structures via packing mechanism can be found in 35.

15



Fig. 5. Average atomic MSD as a function of pressure for the native membranes containing only wild type LH2 complexes with spheroidenone (purple balls) and the membranes comprising mutant LH2 complexes with neurosporene (green balls). The rather large error bars are due to the high background mostly produced by the screening effect of the high-pressure cell and the related decrease of the neutron flux applied to the sample, see the Materials and Method section.

Secondly, and most remarkably, while in the native sample the decrease of MSD, which is consistent with a simple compression of the sample, continues until at least 5.5 kbar 16


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(the maximum pressure applied in this experiment), this trend in the mutant sample stops at 5 kbar, followed by an increase of MSD. This qualitative change in the flexibility parallels with the breakage of H-bonds in the same sample inferred from optical absorption spectroscopy (Fig. 1B). This is reasonable because the breakage of H-bonds would lead to a weakening of the LH2 structure, concomitantly to a higher degree of motional freedom (dynamics) of the protein residues involved in H-bonding. The accompanying bigger exposure of the protein to hydration water is also known to increase protein flexibility and dynamics 36, 37.

Fig. 6. Chemical structure of neurosporene (top) and spheroidenone (bottom) adapted from 34.

Despite this nice harmony between the optical and neutron scattering observations, caution must be exercised not to over-interpret the data; firstly, because of the limited pressure range that was

17



available in the present measurements, and secondly, because of considerable experimental uncertainty due mostly to a low neutron flux obtainable in the high pressure setting of the IN13 spectrometer. It should be noticed, though, that although the data presented in Fig. 5 represent studies of a single sample pair (native and mutant), three independent sample sets were measured over three separate measurement sessions. Qualitatively similar results in each case were obtained, which supports the conclusions drawn on the basis of the data in detail analyzed herein. A separate issue relates to principally indiscriminate nature of EINS regarding the origin and location of scattering hydrogens. LH2-only membranes inevitably contain to a limited extent several other complexes distinct from LH2. According to 38, these vital complexes include ATP synthase, cytochrome bc1 complex, NADH oxidoreductase, and some 34 chromophore-specific proteins, although their stoichiometry with respect to LH2 will be rather low, with according to 39, LH2 dominating by a factor of twenty-fold or more. Future measurements involving detergentisolated LH2 proteins and/or the proteins reconstituted into artificial membranes might shed more light on this question.

Summary and Conclusions We have used complementary optical and elastic neutron scattering spectroscopy at high hydrostatic pressure to study native and mutant LH2 protein complexes embedded in chromatophore membrane vesicles. The results obtained show that replacing the native carotenoid spheroidenone with neurosporene makes it more susceptible to hydrostatic pressure. Destabilization of the protein structure leads disrupting under high pressure the local H-bond network that binds the B850 BChl pigments within their protein binding pockets and to associated change of the protein dynamics. Shown in Graphical Abstract from left and right side of the Hbond breakage transition at around 6.5 kbar is a schematic arrangement for C-terminal domain of the LH2 complex viewed from above the complex, which comprises three of the nine αβ polypeptide pairs (the α polypeptide in yellow, the β polypeptide in magenta) of the LH2 ring and 18


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their bound BChls in green. The H-bonds denoted by red arrows between adjacent αβ protomers are present only at pressures below about 6 kbar. We have not observed any characteristic changes due to the lipid component of the membrane samples, which are likely to be seen around 1-2 kbar according to model membranes 13, 14. This is probably to be expected in membranes from a Rba. sphaeroides mutant with LH2 as the sole photosynthetic complex, since atomic force microscopy of these membranes shows tightly packed arrays of LH2 complexes 17.

Acknowledgements This work was supported by the Estonian-France bilateral research cooperation program “G. F. PARROT” 2017-2018 and, partly, by the Estonian Research Council (grants IUT02-28 and PRG539) and the H2020-MSCA-RISE-2015 program (grant 690853). ECM and CNH gratefully acknowledge financial support from the Biotechnology and Biological Sciences Research Council (BBSRC UK), award number BB/M000265/1, and as part of the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Award Number DE-SC 0001035. CNH was also supported by Advanced Award 338895 from the European Research Council.

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