BRC Group Page
Institute of BiophysicsDescription of Projects
Proton transport across the membrane - the vacuolar proton-ATPase: proton pumping by a membranous molecular motor.
The non-uniform distribution of ions in living organism, which is one most important regulating factor of many life processes, is mostly due to the function of ATP-dependent transport enzymes. As an ATP-dependent proton pump, the vacuolar proton-ATPase (V-ATPase) is a distant relative of the F-ATPase with some structural similarities (at the quaternary structural level) but it has more complicated subunit arrangement and there are significant differences between the F0 and V0 proteolipid complexes too. The V-ATPase works in the opposite direction as the F-ATPase, since it hydrolyses ATP to pump protons across, e.g the vacuolar membrane. The structure and the mechanism of V-ATPase is not known but ATP-dependent proton translocation is almost certainly coupled with a rotary mechanism like in the F-ATPase although the ATP:proton:rotation cycle stoichiometry seems to be different. The 16kDa protein isolated from Nephrops (lobster) is a representative of the ductins family [Holzenburg et al., 1993] closely related to subunit c of the F-ATPase and is almost identical with the corresponding V0 subunit. In both ATPases, this protein is essential for proton translocation. Some members of this family are able to function as passive proton channel, gap junction as well as neurotransmitter release pore (in oligomeric form) in certain systems. We were able to locate a unique cystein (Cys54) and the essential Glu140 residue in the membrane which was found to face lipids [Pali et al., 1999]. We could also determine the oligomer number of this protein in native membranes from the hepatopancreas of Nephrops norvegicus. Current efforts are directed towards non-linear spin label electron paramagnetic resonance (EPR) spectroscopic structural studies and calorimetric, Fourier transform infrared (FTIR) and combined fluorescent-EPR spectroscopic characterization of non-covalent subunit-subunit and subunit-lipid interactions in native and reconstituted yeast systems. In addition, attempts are undertaken to uncouple passive proton translocation and ATP hydrolysis and to observe the rotary mechanism spectroscopically.
Electron transport across the membrane - cytochrome b-561: a two-heme 4-helix electron channel?
It has recently been shown that the plant plasma membrane may contain ascorbate-reducible b-type cytochrome(s) similar to that identified and characterized in chromaffin granule membranes of the mammalian adrenal medulla [Asard et al., 2001]. Both the mammalian cytochrome (called cyt. b-561) and the predicted plant proteins are highly hydrophobic and transport electrons through membrane bilayers, have high redox potential and are fully ascorbate-reducible. Electron transfer happens almost certainly with the involvement of the two heme centers [Trost et al., 2000], but even putative models for the electron transfer mechanism(s) are lacking. Molecular models are being built based on the available sequences of several mammalian and plant proteins and data on function and structure(s). All sequences tested contain 6 trans-membrane segments and a conserved region for a putative monodehydroascorbate binding site. Highly conserved His residues anchoring the two hemes on the periplasmic and cytoplasmic side of the membranes are located on the trans-membrane a helices 2-4 and 3-5. Efforts are going on to purify cytochrome b-561 from plants and to study its membrane location with spectroscopic techniques after functional reconstitution. This will provide data to further refine our molecular model and to assign function to the new members of this protein family.
Protein insertion, folding and assembly in membranes and on membrane surfaces:
An experimentally and computationally challenging problem in biophysics is how certain membrane proteins are able to insert, fold and assemble in the lipid bilayer, in some cases even pass it through, often in the absence of folding assistants (like chaperones). In this so-called "membrane-protein folding problem" specific interactions between the protein and lipids of the target membrane must clearly play crucial role yet to be explored in detail. We got involved in this problem because work on the above two target ion-transport proteins requires they be inserted and assembled properly in the bilayer to achieve functional reconstitution. In reconstituted lysozyme-lipid complexes, we are studying the influence of the lipid bilayer on activity, thermal (un)folding and stability by DSC, FTIR and spin label EPR methods. Lysozyme is a soluble protein but (i) its folding properties are well known in solutions, hence serving a perfect reference, (ii) it is exposed to bacterial membranes during its function as hydrolase and (iii) even closely related soluble proteins unfold or refold at the membrane-water interface very differently. Our preliminary results show that thermal (un)folding of lysozyme depends on the nature of the interactions (e.g. electrostatic or hydrophobic) with the bilayer, the pH and the primary and secondary structure of the protein segment involved in the interaction. Small membrane active antimicrobial peptides on the other side are able to insert and form channels or disrupt the bilayer integrity causing lysis without suffering major structural changes themselves. Since cell membrane selectivity is the key to develop new and effective antibiotics, the molecular interactions of these peptides with various classes of lipids has to be understood at atomic details. We are studying membrane-disrupting peripheral and channel-forming integral antimicrobial polypeptides (gramicidin S and gramicidin A, respectively) reconstituted in membranes of variety of lipids using the same techniques [Kiricsi et al., 2001]. The M13 bacteriophage is able to insert its coat proteins into the membrane of the host cell and then the coat proteins are reassembled and leave the host with the newly synthesized virus - all this without causing lysis of the host cell. We have modelled the coat protein together with the first-shell solvating lipids based on NMR, and spin label mobility and structural data [Bashtovyy et al., 2001]. The model stimulates new experiments and full scale molecular modelling. We have also adressed the membrane-protein folding problem at a more general theoretical level as we described the geometry (tilt, twist and coiling angles) of properly folded beta-barrel membrane proteins based on the analysis of all available X-ray structures of the porins superfamily and relatives [Pali and Marsh, 2001].
The protein-lipid interface in native membranes:
Uncoupling or perturbing the selective structural and dynamic balance, developed by nature to enhance the biological function and provide flexibility, between membrane proteins and lipids may either force the organism to adapt to the new conditions, by e.g. modifying its lipid composition, or die when unable to do so. The protein-lipid interface which takes several different forms, all of which are crucial to biology. For biological membranes the protein-lipid interface may be either polar in the case of surface-bound or absorbed peripheral proteins, or apolar in the case of integral transmembrane proteins. Membrane-active peptides of biological or synthetic origin may encompass either or both of these types. As the site of non-competitive inhibition by local anesthetics and probably other hydrophobic drugs, the protein-lipid interface also has direct functional and pharmacological implications. Focusing on the protein-lipid interface requires studies on structure, dynamics and function of both membrane proteins and lipids. One of our interest is the presence of free radicals in the membrane. Nitric oxide (NO) is membrane soluble gas that reacts readily with thiol and tyrosin amino acid side chains. We are studying the effect of NO on the activity, side-chain mobility and the lipid solvation shell of the sarco/endo-plasmic reticulum Ca2+-ATPase in native membranes using spin trapping [Nedeianu and Pali, 2002] and spin label EPR methods. Heavy metal poisoning in yeast [Belagyi et al., 1999] and thylakoid membranes [Szalontai et al., 1999] results in major changes in membrane fluidity, protein secondary structures and lipid-protein interactions. In thylakoid membranes, the temperature-induced uncoupling of light-harvesting-complex II proteins and non-bilayer forming lipids could be observed (Kota et al., 2002). In photosynthetic membranes, we could prove that carotenoids can have direct influence on local membrane dynamics, when performing their light-protective function [Zsiros et al., 2001]. We could also prove that temperature-dependent membrane dynamics is maintained only in functioning photosynthetic membranes, i.e. in the presence of light [Varkonyi et al., 2002]. We are focusing now on the role of protein-lipid interactions in maintaining and regulating overall membrane dynamics.