The Activity of Native Vacuolar Proton-ATPase in an Oscillating Electric Field – Demystifying an Apparent Effect of Music on a Biomolecule

The effect of an oscillating electric field generated from music on yeast vacuolar proton-ATPase (V-ATPase) activity in its native environment is reported. An oscillating electric field is generated by electrodes that are immersed into a dispersion of yeast vacuolar membrane vesicles natively hosting a high concentration of active V-ATPase. The substantial difference in the ATP hydrolysing activity of V-ATPase under the most stimulating and inhibiting music is unprecedented. Since the topic, i.e., an effect of music on biomolecules, is very attractive for non-scientific, esoteric mystification, we provide a rational explanation for the observed new phenomenon. Good correlation is found between changes in the specific activity of the enzyme and the combined intensity of certain frequency bands of the Fourier spectra of the music clips. Most prominent identified frequencies are harmonically related to each other and to the estimated rotation rate of the enzyme. These results lead to the conclusion that the oscillating electric field interferes with periodic trans-membrane charge motions in the working enzyme

Fehérjék membránba ágyazódásának, szerveződésének és lipidekkel való kölcsönhatásának biofizikája = Biophysics of protein insertion and folding in membranes and their interaction with lipids

1. A vakuólum ATPáz (V-ATPáz). Ez a membránkötött molekuláris motor az ATP hidrolízisből nyert kémiai energia révén protonokat pumpál a membránon keresztül, ami miatt a csontritkulás potenciális terápiájában ez az egyik kulcs target enzim. Meghatároztuk szintetikus V-ATPáz gátlóanyagok membránbeli lokalizációját, ami segíti az enzimen található kötőhelyeik azonosítását. Kimutattuk, hogy a V-ATPáz c alegységét funkcionálisan helyettesíteni képes langusztából izolált membránfehérje kétértékű kation kötőhelyet tartalmaz, aminek valószínűleg szerepe van a V-ATPáz c alegységének hatos gyűrűbe való rendeződésében. 2. Fehérjék és polipeptidek membránlipidekkel való kölcsönhatása. Foszfolipid membránok összetételének és fizikai állapotának a hidrofób gramicidin A és vízoldékony lizozim antibiotikumok membrán-kötődésére, -orientációjára és termikus kitekeredésére gyakorolt hatását tanulmányoztuk. A kapott eredmények új adatokat szolgáltatnak a nem kovalens lipid-fehérje kölcsönhatások szerepére az antibiotikus folyamatokban. A membránfehérjékkel kölcsönható határfelületi és kofaktor lipidek konformációját és a kölcsönhatás sztöchiometriáját tanulmányoztuk atomi felbontásban, publikált kristályszerkezetek felhasználásával. Az eredmények fontosak a lipid-fehérje kölcsönhatás natív biomembránokban tapasztalható funkcionális jelentőségének megértése szempontjából. A munkaterv némileg módosult a szerződött pályázatból való utólagos elvonások miatt. | 1. The vacuolar proton-ATPase (V-ATPase). This membranous molecular motor uses energy from ATP hydrolysis to drive proton transfer across membranes, hence it is a key potential target enzyme in osteoporosis therapy. Synthetic V-ATPase inhibitors were located in model membranes aiding the identification of their binding sites on the enzyme. A divalent cation binding site has been identified on a membrane protein isolated from lobster that is able to functionally substitute the V-ATPase subunit c. This binding site is likely to be related to the hexameric assembly of the subunit c ring of V-ATPase. 2. Protein- and polypeptide-membrane lipid interactions. The effect of the composition and the physical state of the phospholipid bilayer on the membrane-binding, -orientation and thermal unfolding of the hydrophobic gramicidin A and the water-soluble lysozyme hydrolase antibiotics were studied. The results provide new data on the significance of non-covalent lipid-protein interactions in anti-microbial processes. The conformation and stoichiometry of both annular and co-factor lipids interacting with membrane proteins were studied at atomic resolution using published crystal structures. The results are essential for the understanding the functional significance of lipid-protein interactions in native biomembranes. The grant suffered from some post-contract fund with-drawals

Photobleaching of Chlorophyll in Light-Harvesting Complex II Increases in Lipid Environment

Excess light causes damage to the photosynthetic apparatus of plants and algae primarily via reactive oxygen species. Singlet oxygen can be formed by interaction of chlorophyll (Chl) triplet states, especially in the Photosystem II reaction center, with oxygen. Whether Chls in the light-harvesting antenna complexes play direct role in oxidative photodamage is less clear. In this work, light-induced photobleaching of Chls in the major trimeric light-harvesting complex II (LHCII) is investigated in different molecular environments - protein aggregates, embedded in detergent micelles or in reconstituted membranes (proteoliposomes). The effects of intense light treatment were analyzed by absorption and circular dichroism spectroscopy, steady-state and time-resolved fluorescence and EPR spectroscopy. The rate and quantum yield of photobleaching was estimated from the light-induced Chl absorption changes. Photobleaching occurred mainly in Chl a and was accompanied by strong fluorescence quenching of the remaining unbleached Chls. The rate of photobleaching increased by 140% when LHCII was embedded in lipid membranes, compared to detergent-solubilized LHCII. Removing oxygen from the medium or adding antioxidants largely suppressed the bleaching, confirming its oxidative mechanism. Singlet oxygen formation was monitored by EPR spectroscopy using spin traps and spin labels to detect singlet oxygen directly and indirectly, respectively. The quantum yield of Chlaphotobleaching in membranes and detergent was found to be 3.4 x 10(-5)and 1.4 x 10(-5), respectively. These values compare well with the yields of ROS production estimated from spin-trap EPR spectroscopy (around 4 x 10(-5)and 2 x 10(-5)). A kinetic model is proposed, quantifying the generation of Chl and carotenoid triplet states and singlet oxygen. The high quantum yield of photobleaching, especially in the lipid membrane, suggest that direct photodamage of the antenna occurs with rates relevant to photoinhibitionin vivo. The results represent further evidence that the molecular environment of LHCII has profound impact on its functional characteristics, including, among others, the susceptibility to photodamage

The Small Heat Shock Protein, HSPB1, Interacts with and Modulates the Physical Structure of Membranes

Small heat shock proteins (sHSPs) have been demonstrated to interact with lipids and modulate the physical state of membranes across species. Through these interactions, sHSPs contribute to the maintenance of membrane integrity. HSPB1 is a major sHSP in mammals, but its lipid interaction profile has so far been unexplored. In this study, we characterized the interaction between HSPB1 and phospholipids. HSPB1 not only associated with membranes via membrane-forming lipids, but also showed a strong affinity towards highly fluid membranes. It participated in the modulation of the physical properties of the interacting membranes by altering rotational and lateral lipid mobility. In addition, the in vivo expression of HSPB1 greatly affected the phase behavior of the plasma membrane under membrane fluidizing stress conditions. In light of our current findings, we propose a new function for HSPB1 as a membrane chaperone

Ion Channels and Pumps in Autophagy: A Reciprocal Relationship

Autophagy, the process of cellular self-degradation, is intrinsically tied to the degradative function of the lysosome. Several diseases have been linked to lysosomal degradative defects, including rare lysosomal storage disorders and neurodegenerative diseases. Ion channels and pumps play a major regulatory role in autophagy. Importantly, calcium signaling produced by TRPML1 (transient receptor potential cation channel, mucolipin subfamily) has been shown to regulate autophagic progression through biogenesis of autophagic-lysosomal organelles, activation of mTORC1 (mechanistic target of rapamycin complex 1) and degradation of autophagic cargo. ER calcium channels such as IP(3)Rs supply calcium for the lysosome, and lysosomal function is severely disrupted in the absence of lysosomal calcium replenishment by the ER. TRPML1 function is also regulated by LC3 (microtubule-associated protein light chain 3) and mTORC1, two critical components of the autophagic network. Here we provide an overview of the current knowledge about ion channels and pumps—including lysosomal V-ATPase (vacuolar proton-ATPase), which is required for acidification and hence proper enzymatic activity of lysosomal hydrolases—in the regulation of autophagy, and discuss how functional impairment of some of these leads to diseases

Tuning the coordination properties of multi-histidine peptides by using a tripodal scaffold: solution chemical study and catechol oxidase mimicking

Two new tripodal peptides containing non-protected N-terminal (L1, tren3his) and C-terminal (L2, nta3his) histidines have been synthesized in order to combine the structuring effect of tripodal scaffolds and the strong metal binding properties of histidine moieties. In the present work the copper(ii) complexes of these ligands have been studied by combined pH-metric, UV-Vis, CD, EPR and MS methods. At a 1 : 1 metal-to-ligand ratio the two ligands behave as the corresponding dipeptides containing N/C-terminal histidines, but above pH 9 the participation of the tertiary amine in the fused chelate rings results in unique binding modes in the case of both ligands. Besides, the formation of oligonuclear complexes also confirms the positive influence of tripodal platforms on metal coordination, and provides the potential to be efficient functional models of oxidase enzymes. Accordingly, the oligonuclear complexes of both ligands exhibit considerable catecholase-like activity. The oxidation of 3,5-di-tert-butyl-catechol proceeds with the participation of separated Cu2+ centers in the presence of L1 complexes. However, the proximity of the two metal ions in the dinuclear complexes of L2 allows their cooperation along the catalytic cycle. Substrate binding modes, effects of reactants, intermediate and side product formation have also been studied, allowing us to propose a plausible catalytic mechanism for each copper(ii)-ligand system