In this project the structure and function of H(+)-ATPases from photosynthetic membrane systems were studied. Functional studies involved the efficiency and regulation of enzyme kinetics and proton flux, interactions between the subunits and with other functionally coupled proteins, and effects of membrane properties. Structural studies focussed on 2-D crystallization in solution and in lipid bilayers, using cryo-electron microscopy combined with image analysis, and atomic force microscopy. Protein (subunit) conformational transitions during physiological modulations were studied with fluorescent probes, dynamic fluorescence, Fourier transform infrared spectroscopy, and sensitive differential scanning calorimetry.
This network project was very productive. In particular much progress was made on functional and molecular genetic aspects of the H+-ATPases from various photosynthetic organisms. Moreover, the protein isolation procedures and especially the efficient incorporation of the various enzymes into liposomes were much improved, thus leading to formulation of generalized strategies. In many cases the reconstituted enzymes retained their native properties. A new isolation procedure for the cyanobacterial enzyme was devised, using biotin-ADP and binding to an avidin column. Alpha and beta subunits were purified from the Rhodobacter capsulatus enzyme for making monospecific antibodies. Reconstitution studies now also include planar supported lipid bilayers; a new type of support used is a micro-porous ceramic wafer which allows bulk transport studies. The new protein reconstitution methods also have positive impact on liposome technology for industrial and medical purposes.
The relation between the protonmotive force and the activity of the ATPases is twofold: on the one hand the protonmotive force is the major driving force for ATP synthesis, on the other hand a certain threshold level of the proton electrochemical potential difference is required to switch the enzyme on (activation). From two lines of research, on reconstituted proteoliposomes and on cyanobacterial vesicles and chloroplasts, respectively, it appears that the H+/ATP stoichiometry for ATP synthesis is 4. The H+/ATP varies with the growth conditions (light intensity, temperature) in cyanobacteria as an adaptation to stress. Cholesterol is an efficient stimulator of ATP synthesis in artificial membranes, mainly due to a lowering of the KM for ADP. The threshold for activation of cyanobacterial ATPases is relatively low and comparable to the chloroplast enzyme in its thiol-reduced form. When the thiol-regulated amino acid segment of the chloroplast F1 gamma subunit is incorporated into the cyanobacterial gamma subunit, the latter displays the activation characteristics of the chloroplast system. When the F0 b' subunit is truncated down to its hydrophobic N-terminus, the F1F0 ATP synthase still shows normal light- and proton jump-induced ATP synthesis. Other mutant enzymes are under study now in order to pin down the catalytically essential sectors. Various compounds which mimic the natural activation of the enzyme were also studied, e.g. sulfite effects. Special attention is given to the effects of Mg2+ on proton pumping and to the lack of proton transport in the presence of Ca-ATP.
Several new insights were obtained concerning the effects of nucleotides. ATP stimulates ATP synthesis (!) after its binding to a non-catalytic binding site. FTIR spectroscopy revealed that nucleotide binding induces an increase in the fraction of a-helix structure and reverse turns and a decrease in beta-structure, and that hydrogen-deuterium exchange is clearly affected by different nucleotide binding site occupancies. Differential scanning calorimetry was applied to study the effects of nucleotide binding on the thermal unfolding of the ATP synthase from the thermophilic bacterium PS3. The remarkable thermostability of this enzyme (Tm at 81.7 (C) was decreased upon tight binding of Mg-ATP to non-catalytic sites, whereas binding of Mg-ADP increased the temperature at which denaturation occurs.
It appears that ATP binding induces a more open or flexible conformation, while ADP binding induces a more compact structure of the protein. It was also found that the type of detergent used for isolation (or reconstitution) affects the nucleotide binding state of the protein; e.g. the relative ATP content is much higher in Triton X-100 than in octylglucoside.
Fluorescence spectroscopy was used to monitor local pH and polarity changes at the membrane surface of ATP synthase-containing membranes, using newly synthesized fluorescent probes. Also, intrinsic fluorescence of the holo-enzyme and of its e-subunit was studied. The new fluorescent probes and dynamic light scattering were applied to study membrane interfacial properties (surface charge density, differential cation association, shear layer thickness, membrane curvature) of (proteo)liposomes, in view of the role of lateral H+ communication between the DeltamicroH+ generators and consumers (such as the ATPases).
The molecular genetic work on the Rhodobacter capsulatus ATPase was also very successful. The entire operon of the F1 part has been cloned and sequenced. The operon contains the five genes in the usual deltaalphagammabetaepsilon order found in other bacteria. Downstream these genes a clear rho-independent terminator sequence has been detected. The sequenced fragment includes also a large 2 kb sequence upstream the transcription initiation point that should include the entire promoter structure. The F1 sequence reveals extensive homology with the proteins of other bacteria and eukaryotic ATPases. In particular, the sequences of the alpha and beta subunits are highly conserved; the gamma-subunit is conserved for extensive stretches, forming 3 long alpha-helices in contact with the alpha and beta subunits, in harmony with Walker's structural model. A plasmid vector was prepared containing the entire operon, to be used for future site-specific mutagenesis experiments.
Crystallization studies focussed on two-dimensional aggregates in solution and in membranes, and several new results were obtained. A new strategy was developed based on the removal of detergent by SM2 Bio-Beads. Crystals of Ca2+-ATPase and other proteins (melibiose permease, cytochrome b6/f) have thus been produced, with diffraction patterns of negatively stained samples of less than 20 Å resolution. Also, large bilayer sheets (about 1 mm diameter) with closely packed F1F0 ATPases have been obtained. This opens exiting new possibilities for obtaining large 2-D crystals of the ATPase proteins in the near future.
MAJOR SCIENTIFIC BREAKTHROUGHS:
-A new strategy for the 2-D crystallization of several membrane proteins, including the F1F0 ATPase was devised on the basis of detergent removal, and is highly promising for obtaining 2-D crystal lattices.
-Two-dimensional ordered arrays of the chloroplast enzyme display an asymmetric position of the F0 sector with respect to the F1 sector.
-New general methods were devised to purify and incorporate the ATPases into liposomes and several new types of planar supported membranes; the latter have some interesting applied features.
-Co-reconstitution of the ATPase with protonmotive force generating proteins was also optimized; in these systems strong evidence for direct proton communication between the proteins was obtained.
-The entire operon of the F1 part of the Rhodobacter capsulatus ATPase is now characterized.
-Binding of ATP and ADP to the ATPase differentially affect the gross conformation, as demonstrated by FTIR and DSC; ATP synthesis is stimulated by ATP binding to non-catalytic sites, and also by cholesterol.
-The H+/ATP stoichiometry of photosynthetic membrane ATPases ('energetic efficiency') is 4.
Funding SchemeCSC - Cost-sharing contracts
91191 Gif Sur Yvette
9712 CP Groningen