Characterization of an ascorbate-reducible cytochrome b561 by site-directed mutagenesis

Ascorbate(ASC)-reducible cytochrome b561 (Cyt-b561) proteins are present in both plants and animals and create a well-distinguished protein family amongst the two-heme containing b-type cytochromes. One isoform of the Cyts-b561 identified by genomic analysis of Arabidopsis thaliana has been localized in the tonoplast. We have expressed the tonoplastlocalized Cyt-b561 (TCyt-b561) in yeast (Saccharomyces serevisiae) cells and shown that the biophysical properties of the recombinant TCyt-b561 is very similar to those of the chromaffin granule Cyt-b561 (CGCyt-b561). Mutation of 4 well-conserved histidine residues (H50, H83, H117, H156) resulted in different expression levels and revealed the importance of these 4 His residues in heme binding and protein expression. Modification of the protein by FLAG-tag or His6-tag resulted in different degrees of reduced expression levels. When all lysine residues (K70, K76, K79, K80, and K159) in the vicinity of the putative ASC-binding motive were one-by-one replaced by alanine, no major changes in the expression levels were observed. Except in case of the K80A mutant, where the low-affinity ASC-binding constant increased significantly, there were no significant changes in either kinetic parameter characterizing the bi-phase ASC-dependent reduction of TCytb-b561. These observations are discussed in comparison to properties of the recombinant CGCyt-b561

Ascorbate-reducible b-type cytochrome in the plant plasma membrane

It has been know for 20 years that the plasma membrane (PM) in plants contains more than one kind of ABSTRACT btype cytochromes. One of them has rather high redox potential (can fully be reduced by ascorbate) and is capable of electron transport through the PM. Three b-type cytochromes have recently been predicted from the full genome of Arabidopsis thaliana. In order to identify and characterize the one located in the PM, first PM vesicles were purified from Arabidopsis leaves, then the PM vesicles were solubilized and the fully ascorbate-reducible b-type cytochrome was partially purified. Redox titration of the partially purified b-type cytochrome revealed the presence of two hemes with redox potentials higher than 100 mV. The major polypeptide band of this fraction on SDS-PAGE was at ~120 kDa. This value is much higher than the apparent molecular mass of either the fully ascorbate-reducible b-type cytochrome purified from Phaseolus hypocotyls or the cyt. b-561 proteins purified from chromaffin granule membranes or the calculated molecular masses for the three polypeptides predicted from the full genome of Arabidopsis

Lipid rafts in the plant plasma membrane?

In order to study the molecular characteristics of an integral membrane protein, first the protein should be solubilized. Ascorbate-reducible b-type cytochromes are highly hydrophobic integral membrane proteins with six trans-membrane a-helices. A fully ascorbate-reducible b-type cytochrome was easily solubilized by Triton X-100 from phase partition-purified plasma membranes of 5-day-old etiolated bean (Phaseolus vulgaris L.) hooks (Trost et al. 2000). However, under the very same conditions, a similar protein in the phase partition-purified plasma membrane of 9-week-old green Arabidopsis thaliana leaves seemed to be very resistant to solubilization with Triton X-100 (Bérczi et al. 2001). It was assumed that the composition of lipids in the plasma membrane of the two different tissues might influence the solubilization. Results obtained with thin layer chromatography revealed that both quan-titative and qualitative differences exist between the lipid composition of the two plant plasma membranes. The observations are discussed in the light of possible existence of “lipid rafts” in the plant plasma membranes

Expression and purification of the recombinant mouse tumor supressor cytochrome b561 protein

It has recently been recognized that ascorbate-reducible cytochrome b561 (Cyt-b561) proteins constitute a well-distinguished protein family amongst the two-heme containing b-type cytochromes, ubiquitously present in animals and plants. Of the six isoforms that have been identified in mammals, three isoformes (called CGCytb, DCytb, and LCytb) have been cloned and expressed in yeast and/or bacterial cells. The recombinant proteins have been characterized in some detail. A particular gene product of the 3p21.3 (human) and 9F1 (mouse) chromosomal region, a so-called tumor supressor protein (101F6, TSP10), was identified as a Cyt-b561 protein by sequence homology. We have cloned and expressed the mouse tumor supressor Cyt-b561 protein (TSCytb) in yeast (Saccharomyces serevisiae), without and with a His6-tag on either the N- or the C-terminus. The C-terminal His6-tagged recombinant protein was purified on Ni-NTA His•Bind resin to almost homogeneity. Using optical spectroscopy we show that TSCytb is indeed an ASC-reducible cytochrome b561 protein and that ASC-reducibility is not affected by the presence of a His6-tag on the C-terminus. Minor differences in the properties of TSCytb and the other three mammalian Cyts-b561 are discussed

Substrate-dependent reduction of a recombinant chromaffin granule Cyt-b561 and its R72A mutant

Cytochrome b561 (Cyt-b561) proteins constitute a family of integral membrane proteins, catalyzing ASC-driven trans-membrane electron transport. Numerous isoforms of Cytb561 are present in invertebrates, vertebrates, and plants. The only protein of this family, however, which has been characterized in details at both biophysical, biochemical and physiological levels so far, is the bovine chromaffin granule Cyt-b561 (CGCyt-b561). Recently, both the bovine and the mouse CGCyt-b561 has been expressed in yeast cells and the recombinant proteins were shown to have biophysical properties similar to the native bovine CGCyt-b561. We have expressed the mouse CGCyt-b561 with a His6 -tag at the C terminus (CGCyt-b561(C6H)) in yeast (Saccharomyces serevisiae) cells and studied the reduction of CGCyt-b561(C6H) in the presence of different natural reducing agents. Besides the well-known natural reductant ascorbate (ASC) and the often-used artificial reductant dithionate, NADH, GSH, and dihydrolipoic acid (DHLA), also reduced the fully oxidized protein. Interestingly however, NADPH was not effective at all. When the same reductants were tested with the R72A mutant of CGCyt-b561(C6H), a mutant with impaired ASC-dependent reducibility, neither pyridine-dinucleotides could reduce the R72A mutant. DHLA-dependent and ASC-dependent reduction kinetics were very similar in case of the R72A mutant but differed in case of CGCyt-b561. These results raise the question of how many natural reductants the CGCyt-b561 may utilize in vivo

A fehérjemátrix szerepe a redoxfehérjék működésében = Role of the protein matrix in the function of redox proteins

Rekombináns citokróm c mutánsokat állítottunk elő és a bevezetett ciszteineket TUPS fotoaktív redox jelölővel jelöltük. Ugyancsak jelöltünk felszíni lizineket. Lézerimpulzussal való gerjesztés után kinetikus spektroszkópiai módszerrel mértük és értelmeztük az elektrontranszfer sebességét a jelölő és a citokróm hem kofaktora között. Megállapítottuk, hogy a jelölő flexibilis kovalens kapcsolata miatt több pozíciót tud felvenni és ezért az elektrontranszfer több exponenciálisból álló kinetikát mutat. A kísérleti adatokat összehasonlítottuk modellszámításokkal és megállapítottuk, hogy mind a konkrét elektrontranszfer útvonalakkal, mind pedig a fehérjével, mint változó sűrűségű folytonos közeggel számoló modell az eredményekkel jól megegyező sebességeket jósol. A citokróm felszínét feltérképeztük elektrontranszfer hatékonyság szempontjából: a hem síkjához viszonyított poláros régiók valamivel hatékonyabbak az egyenlítői régióknál. A citokróm c és a citokróm oxidáz komplexében mérve az elektrontranszfert az eddigieknél nagyobb időfelbontással tudtuk megmérni a CuA (első) elektronakceptor redukciójának idejét, és azt 100 ns-nál gyorsabbnak találtuk. Megállapítottuk, hogy E. coli baktériumban érett holocitokróm c kis mennyiségben spontán módon, hem liáz segítsége nélkül is képződik. Sikerült a hem liáz fehérjét heterológ módon kifejezni és tisztítani, és így apocitokróm és hem felhasználásával in vitro holocitokrómot termelni. | We produced recombinant mutant cytochrome c proteins and labeled the introduced cysteines and native lysines with the photoactive redox label TUPS. Pulse laser excitation of TUPS and the ensuing electron transfer between the label and the heme of cytochrome c was followed by kinetic absorption spectroscopy. Due to the flexible covalent link between the label and the protein TUPS can assume multiple positions which result in multiexponential electron transfer kinetics. We compared the experimental data with model calculations and concluded that both the model assuming explicite electron transfer pathways and the model assuming a protein matrix with varying packing density can satisfactorily describe the experimental observations. We mapped the surface of cytochrome c in terms of electron transfer efficiency towards the heme. The polar regions were found to be slightly more efficient ('hotter') than the equatorial regions relative to the plane of the heme. By measuring the electron transfer in the complex of cytochrome c and cytochrome c oxidase at a higher time resolution than in previous studies, we obtained a reduction time of the primary electron acceptor, CuA, which is faster than 100 ns. We found that in E. coli a low efficiency spontaneous maturation of holocytochrome c can take place without the assistance of heme lyase. We expressed and purified yeast heme lyase from E. coli and demonstrated in vitro holocytochrome maturation upon addition of apocytochrome and heme

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