Hysteretic Pressure Dependence of Ca2+ Binding in LH1 Bacterial Membrane Chromoproteins.

Much of the thermodynamic parameter values that support life are set by the properties of proteins. While the denaturing effects of pressure and temperature on proteins are well documented, their precise structural nature is rarely revealed. This work investigates the destabilization of multiple Ca2+ binding sites in the cyclic LH1 light-harvesting membrane chromoprotein complexes from two Ca-containing sulfur purple bacteria by hydrostatic high-pressure perturbation spectroscopy. The native (Ca-saturated) and denatured (Ca-depleted) phases of these complexes are well distinguishable by much-shifted bacteriochlorophyll a exciton absorption bands serving as innate optical probes in this study. The pressure-induced denaturation of the complexes related to the failure of the protein Ca-binding pockets and the concomitant breakage of hydrogen bonds between the pigment chromophores and protein environment were found cooperative, involving all or most of the Ca2+ binding sites, but irreversible. The strong hysteresis observed in the spectral and kinetic characteristics of phase transitions along the compression and decompression pathways implies asymmetry in the relevant free energy landscapes and activation free energy distributions. A phase transition pressure equal to about 1.9 kbar was evaluated for the complexes from Thiorhodovibrio strain 970 from the pressure dependence of biphasic kinetics observed in the minutes to 100 h time range.

Hydrostatic High-Pressure-Induced Denaturation of LH2 Membrane Proteins.

The denaturation of globular proteins by high pressure is frequently associated with the release of internal voids and/or the exposure of the hydrophobic protein interior to a polar aqueous solvent. Similar evidence with respect to membrane proteins is not available. Here, we investigate the impact of hydrostatic pressures reaching 12 kbar on light-harvesting 2 integral membrane complexes of purple photosynthetic bacteria using two types of innate chromophores in separate strategic locations: bacteriochlorophyll-a in the hydrophobic interior and tryptophan at both protein-solvent interfacial gateways to internal voids. The complexes from mutant Rhodobacter sphaeroides with low resilience against pressure were considered in parallel with the naturally robust complexes of Thermochromatium tepidum. In the former case, a firm correlation was established between the abrupt blue shift of the bacteriochlorophyll-a exciton absorption, a known indicator of the breakage of tertiary structure pigment-protein hydrogen bonds, and the quenching of tryptophan fluorescence, a supposed result of further protein solvation. No such effects were observed in the reference complex. While these data may be naively taken as supporting evidence of the governing role of hydration, the analysis of atomistic model structures of the complexes confirmed the critical part of the structure in the pressure-induced denaturation of the membrane proteins studied.

Exciton Origin of Color-Tuning in Ca2+-Binding Photosynthetic Bacteria.

Flexible color adaptation to available ecological niches is vital for the photosynthetic organisms to thrive. Hence, most purple bacteria living in the shade of green plants and algae apply bacteriochlorophyll a pigments to harvest near infra-red light around 850-875 nm. Exceptions are some Ca2+-containing species fit to utilize much redder quanta. The physical basis of such anomalous absorbance shift equivalent to ~5.5 kT at ambient temperature remains unsettled so far. Here, by applying several sophisticated spectroscopic techniques, we show that the Ca2+ ions bound to the structure of LH1 core light-harvesting pigment-protein complex significantly increase the couplings between the bacteriochlorophyll pigments. We thus establish the Ca-facilitated enhancement of exciton couplings as the main mechanism of the record spectral red-shift. The changes in specific interactions such as pigment-protein hydrogen bonding, although present, turned out to be secondary in this regard. Apart from solving the two-decade-old conundrum, these results complement the list of physical principles applicable for efficient spectral tuning of photo-sensitive molecular nano-systems, native or synthetic.

The two light-harvesting membrane chromoproteins of Thermochromatium tepidum expose distinct robustness against temperature and pressure.

An increased robustness against high temperature and the much red-shifted near-infrared absorption spectrum of excitons in the LH1-RC core pigment-protein complex from the thermophilic photosynthetic purple sulfur bacterium Thermochromatium tepidum has recently attracted much interest. In the present work, thermal and hydrostatic pressure stability of the peripheral LH2 and core LH1-RC complexes from this bacterium were in parallel investigated by various optical spectroscopy techniques applied over a wide spectral range from far-ultraviolet to near-infrared. In contrast to expectations, very distinct robustness of the complexes was established, while the sturdiness of LH2 surpassed that of LH1-RC both with respect to temperatures between 288 and 360 K, and pressures between 1 bar and 14 kbar. Subtle structural variances related to the hydrogen bond network are likely responsible for the extra stability of LH2.

High-pressure tuning of primary photochemistry in bacterial photosynthesis: membrane-bound versus detergent-isolated reaction centers.

While photosynthesis thrives at close to normal pressures and temperatures, it is presently well known that life is similarly commonplace in the hostile environments of the deep seas as well as around hydrothermal vents. It is thus imperative to understand how key biological processes perform under extreme conditions of high pressures and temperatures. Herein, comparative steady-state and picosecond time-resolved spectroscopic studies were performed on membrane-bound and detergent-purified forms of a YM210W mutant reaction center (RC) from Rhodobacter sphaeroides under modulating conditions of high hydrostatic pressure applied at ambient temperature. A previously established breakage of the lone hydrogen bond formed between the RC primary donor and the protein scaffold was shown to take place in the membrane-bound RC at an almost 3 kbar higher pressure than in the purified RC, confirming the stabilizing role of the lipid environment for membrane proteins. The main change in the multi-exponential decay of excited primary donor emission across the experimental 10 kbar pressure range involved an over two-fold continuous acceleration, the kinetics becoming increasingly mono-exponential. The fastest component of the emission decay, thought to be largely governed by the rate of primary charge separation, was distinctly slower in the membrane-bound RC than in the purified RC. The change in character of the emission decay with pressure was explained by the contribution of charge recombination to emission decreasing with pressure as a result of an increasing free energy gap between the charge-separated and excited primary donor states. Finally, it was demonstrated that, in contrast to a long-term experimental paradigm, adding a combination of sodium ascorbate and phenazine methosulfate to the protein solution potentially distorts natural photochemistry in bacterial RCs.

High-Pressure Modulation of Primary Photosynthetic Reactions.

Photochemical charge separation is key to biological solar energy conversion. Although many features of this highly quantum-efficient process have been described, others remain poorly understood. Herein, ultrafast fluorescence barospectroscopy is used for the first time to obtain insights into the mechanism of primary charge separation in a YM210W mutant bacterial reaction center under novel surrounding modulating conditions. Over a range of applied hydrostatic pressures reaching 10 kbar, the rate of primary charge separation monotonously increased and that of the electron transfer to secondary acceptor decreased. While the inferred free energy gap for charge separation generally narrowed with increasing pressure, a pressure-induced break of a protein-cofactor hydrogen bond observed at ∼2 kbar significantly (by 219 cm-1 or 27 meV) increased this gap, resulting in a drop in fluorescence. The findings strongly favor a model for primary charge separation that incorporates charge recombination and restoration of the excited primary pair state, over a purely sequential model. We show that the main reason for the almost threefold acceleration of the primary electron transfer rate is the pressure-induced increase of the electronic coupling energy, rather than a change of activation energy. We also conclude that across all applied pressures, the primary electron transfer in the mutant reaction center studied can be considered nonadiabatic, normal region, and thermally activated.

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

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 slightly 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.

Structural stability of human butyrylcholinesterase under high hydrostatic pressure.

Human butyrylcholinesterase is a nonspecific enzyme of clinical, pharmacological and toxicological significance. Although the enzyme is relatively stable, its activity is affected by numerous factors, including pressure. In this work, hydrostatic pressure dependence of the intrinsic tryptophan fluorescence in native and salted human butyrylcholinesterase was studied up to the maximum pressure at ambient temperature of about 1200 MPa. A correlated large shift toward long wavelengths and broadening observed at pressures between 200 and 700 MPa was interpreted as due to high pressure-induced denaturation of the protein, leading to an enhanced exposure of tryptophan residues into polar solvent environment. This transient process in native butyrylcholinesterase presumably involves conformational changes of the enzyme at both tertiary and secondary structure levels. Pressure-induced mixing of emitting local indole electronic transitions with quenching charge transfer states likely describes the accompanying fluorescence quenching that reveals different course from spectral changes. All the pressure-induced changes turned irreversible after passing a mid-point pressure of about 400 ±â€¯50 MPa. Addition of either 0.1 M ammonium sulphate (a kosmotropic salt) or 0.1 M lithium thiocyanate (a chaotropic salt) to native enzyme similarly destabilized its structure.

Hydrogen bonds in the vicinity of the special pair of the bacterial reaction center probed by hydrostatic high-pressure absorption spectroscopy.

Using the native bacteriochlorophyll a pigment cofactors as local probes, we investigated the response to external hydrostatic high pressure of reaction center membrane protein complexes from the photosynthetic bacterium Rhodobacter sphaeroides. Wild-type and engineered complexes were used with a varied number (0, 1 or 2) of hydrogen bonds that bind the reaction center primary donor bacteriochlorophyll cofactors to the surrounding protein scaffold. A pressure-induced breakage of hydrogen bonds was established for both detergent-purified and membrane-embedded reaction centers, but at rather different pressures: between 0.2 and 0.3GPa and at about 0.55GPa, respectively. The free energy change associated with the rupture of the single hydrogen bond present in wild-type reaction centers was estimated to be equal to 13-14kJ/mol. In the mutant with two symmetrical hydrogen bonds (FM197H) a single cooperative rupture of the two bonds was observed corresponding to an about twice stronger bond, rather than a sequential rupture of two individual bonds.

Structural implications of hydrogen-bond energetics in membrane proteins revealed by high-pressure spectroscopy.

The light-harvesting 1 (LH1) integral membrane complex of Rhodobacter sphaeroides provides a convenient model system in which to examine the poorly understood role of hydrogen bonds (H-bonds) as stabilizing factors in membrane protein complexes. We used noncovalently bound arrays of bacteriochlorophyll chromophores within native and genetically modified variants of LH1 complexes to monitor local changes in the chromophore binding sites induced by externally applied hydrostatic pressure. Whereas membrane-bound complexes demonstrated very high resilience to pressures reaching 2.1 GPa, characteristic discontinuous shifts and broadenings of the absorption spectra were observed around 1 GPa for detergent-solubilized proteins, in similarity to those observed when specific (α or ß) H-bonds between the chromophores and the surrounding protein were selectively removed by mutagenesis. These pressure effects, which were reversible upon decompression, allowed us to estimate the rupture energies of H-bonds to the chromophores in LH1 complexes. A quasi-independent, additive role of H-bonds in the α- and ß-sublattices in reinforcing the wild-type LH1 complex was established. A comparison of a reaction-center-deficient LH1 complex with complexes containing reaction centers also demonstrated a stabilizing effect of the reaction center. This study thus provides important insights into the design principles of natural photosynthetic complexes.

Stability of integral membrane proteins under high hydrostatic pressure: the LH2 and LH3 antenna pigment-protein complexes from photosynthetic bacteria.

The bacteriochlorophyll a-containing LH2 and LH3 antenna complexes are the integral membrane proteins that catalyze the photosynthetic process in purple photosynthetic bacteria. The LH2 complex from Rhodobacter sphaeroides shows characteristic strong absorbance at 800 and 850 nm due to the pigment molecules confined in two separate areas of the protein. In the LH3 complex from Rhodopesudomonas acidophila the corresponding bands peak at 800 and 820 nm. Using the bacteriochlorophyll a cofactors as intrinsic probes to monitor local changes in the protein structure, we investigate spectral responses of the antenna complexes to very high hydrostatic pressures up to 2.5 GPa when embedded into natural membrane environment or extracted with detergent. We first demonstrate that high pressure does induce significant alterations to the tertiary structure of the proteins not only in proximity of the 800 nm-absorbing bacteriochlorophyll a molecules known previously (Gall, A.; et al. Biochemistry 2003, 42, 13019) but also of the 850 nm- and 820 nm-absorbing molecules, including breakage of the hydrogen bond they are involved in. The membrane-protected complexes appear more resilient to damaging effects of the compression compared with the complexes extracted into mixed detergent-buffer environment. Increased resistance of the isolated complexes is observed at high protein concentration resulting aggregation as well as when cosolvent (glycerol) is added into the solution. These stability variations correlate with ability of penetration of the surrounding polar solvent (water) into the hydrophobic protein interiors, being thus the principal reason of the pressure-induced denaturation of the proteins. Considerable variability of elastic properties of the isolated complexes was also observed, tentatively assigned to heterogeneous protein packing in detergent micelles. While a number of the isolated complexes release most of their bacteriochlorophyll a content under high pressure, quite some of them remain apparently intact. The pigmented photosynthetic antenna complexes thus constitute a suitable model system for studying in detail the stability of integral membrane proteins.

Metal binding of metallothionein-3 versus metallothionein-2: lower affinity and higher plasticity.

Mammalian metallothioneins (MTs) are involved in cellular metabolism of zinc and copper and in cytoprotection against toxic metals and reactive oxygen species. MT-3 plays a specific role in the brain and is down-regulated in Alzheimer's disease. To evaluate differences in metal binding, we conducted direct metal competition experiments with MT-3 and MT-2 using electrospray ionization mass spectroscopy (ESI-MS). Results demonstrate that MT-3 binds Zn2+ and Cd2+ ions more weakly than MT-2 but exposes higher metal-binding capacity and plasticity. Titration with Cd2+ ions demonstrates that metal-binding affinities of individual clusters of MT-2 and MT-3 are decreasing in the following order: four-metal cluster of MT-2>three-metal cluster of MT-2 approximately four-metal cluster of MT-3>three-metal cluster of MT-3>extra metal-binding sites of MT-3. To evaluate the reasons for weaker metal-binding affinity of MT-3 and the enhanced resistance of MT-3 towards proteolysis under zinc-depleted cellular conditions, we studied the secondary structures of apo-MT-3 and apo-MT-2 by CD spectroscopy. Results showed that apo-MT-3 and apo-MT-2 have almost equal helical content (approximately 10%) in aqueous buffer, but that MT-3 had slightly higher tendency to form alpha-helical secondary structure in TFE-water mixtures. Secondary structure predictions also indicated some differences between MT-3 and MT-2, by predicting random coil for common MTs, but 22% alpha-helical structure for MT-3. Combined, all results highlight further differences between MT-3 and common MTs, which may be related with their functional specificities.

Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase.

Cox17, a copper chaperone for cytochrome c oxidase, is an essential and highly conserved protein. The structure and mechanism of functioning of Cox17 are unknown, and even its metalbinding stoichiometry is elusive. In the present study, we demonstrate, using electrospray ionization-MS, that porcine Cox17 binds co-operatively four Cu+ ions. Cu4Cox17 is stable at pH values above 3 and fluorescence spectra indicate the presence of a solvent-shielded multinuclear Cu(I) cluster. Combining our results with earlier EXAFS results on yeast CuCox17, we suggest that Cu4Cox17 contains a Cu4S6-type cluster. At supramillimolar concentrations, dithiothreitol extracts metals from Cu4Cox17, and an apparent copper dissociation constant KCu=13 fM was calculated from these results. Charge-state distributions of different Cox17 forms suggest that binding of the first Cu+ ion to Cox17 causes a conformational change from an open to a compact state, which may be the rate-limiting step in the formation of Cu4Cox17. Cox17 binds non-co-operatively two Zn2+ ions, but does not bind Ag+ ions, which highlights its extremely high metal-binding specificity. We further demonstrate that porcine Cox17 can also exist in partly oxidized (two disulphide bridges) and fully oxidized (three disulphide bridges) forms. Partly oxidized Cox17 can bind one Cu+ or Zn2+ ion, whereas fully oxidized Cox17 does not bind metals. The metal-binding properties of Cox17 imply that, in contrast with other copper chaperones, Cox17 is designed for the simultaneous transfer of up to four copper ions to partner proteins. Metals can be released from Cox17 by non-oxidative as well as oxidative mechanisms.