Liina Kangur

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.

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.

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 β) 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 β-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.

Dissociation of the light-harvesting membrane protein complex I from Rhodobacter sphaeroides under high hydrostatic pressure

The light-harvesting complex 1 (LH1) from Rhodobacter sphaeroides is an excellent model system for investigating the stability of oligomeric membrane proteins under high hydrostatic pressure. The currently investigated LH1 forms a 16-meric ring structure of B825 subunits. B825 is a heterodimer of transmembrane α- and β-polypeptide chains, which non-covalently binds two bacteriochlorophyll a molecules. These pigment molecules were used as intrinsic spectroscopic sensors to follow the dissociation reaction. Our results demonstrate that the LH1 dissociates into B825 subunits through an intermediary tetrameric unit B845. The dissociation mechanism depends on pressure. At ∼200–500 MPa the dissociation corresponds to a pseudo-first-order reaction, characterised by the apparent reaction rate at atmospheric pressure k0 = 3·10−5 s−1, activation volume ΔV‡ = −4 mL/mol, and free energy of activation ΔG‡ = 26 kJ/mol. Below 200 MPa and above 500 MPa, the reaction is more complex, including further dissociation of B825 into monomers B777.

Estimating Hydrogen Bond Energy in Integral Membrane Chromoproteins by High Hydrostatic Pressure Optical Spectroscopy

Proteins are biological macromolecules that participate in virtually every process in live organisms. By nature they are divided into three categories: globular, membrane, and fibrous proteins. The globular proteins are present in the cytosol of cells and in body fluids such as blood; the membrane proteins are “solubilized” in two-dimensional lipid membranes that organize the macroscopic body space; the, usually, large fibrous proteins reinforce membranes and maintain the structure of cells and tissues. In the cell the membrane proteins are responsible for structural, catalytic, transport, signalling, control, and other crucial life-supporting functions (Palazzo 2006). Protein function is defined by its folded structure (Rose and Wolfenden 1993) and the self-assembly into the folded structure is largely governed by a manifold of generally weak, spatially oriented hydrogen (H) bonds that also confer distinct quantum properties (Li, Walker et al. 2011). Multiple H-bond interactions are responsible for binding the strands of RNA, DNA, and other biopolymers together, as well as for elasticity of skeletal and cardiac muscles. Cooperativity of the Hbond interactions appears to be a defining feature at all levels of biomolecular folding and function (Lu, Isralewitz et al. 1998; Finkelstein and Ptitsyn 2002; Lin, Mohammed et al. 2011). Proteins are only functional if associated with water, but unlike the dynamic network of H-bonds in the bulk solvent, the interactions with biomolecules are short-range, affecting only one or two layers of waters (Ball 2008). Although there is no doubt that H-bonds are extremely important structural elements of proteins, their role in stabilizing proteins is still a matter of debate. Studying H-bonds within proteins, particularly membrane bound proteins, therefore, permits insights into fundamental biological phenomena, indeed all of life on earth.

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.