ESR spin-label studies of lipid-protein interactions in membranes

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.     

Studies on cytochrome c oxidase, X. Isolation and amino-acid sequence of polypeptide VIIIb

The isolation and sequence determination of the cytoplasmically synthesized polypeptide VIIIb from beef heart cytochrome c oxidase is described. Several methods for isolating polypeptide VIIIb with gelchromatographic technics are presented. The complete amino-acid sequence is deduced from a N-terminal sequencer run, overlapping tryptic peptides and peptides obtained after tryptophan specific cleavage with cyanogen bromide in heptafluorobutyric acid/formic acid. The small protein consists of 46 amino acids and has a molecular mass of 4 962 Da. The existence of a hydrophobic segment with a length of 20 residues characterizes it as a membrane penetrating protein. The stoichiometry of this polypeptide in the functional monomer of cytochrome c oxidase (complex IV) is 2 and is thus different from all the other polypeptides constituting the respiratory complex IV. The function of this component of the terminal oxidase is as yet unknown.     

A highly active, soluble mutant of the membrane-associated (S)-mandelate dehydrogenase from Pseudomonas putida.

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida, a member of the flavin mononucleotide-dependent alpha-hydroxy acid oxidase/dehydrogenase family, is a membrane-associated protein, in contrast to the more well-characterized members of this protein family including glycolate oxidase (GOX) from spinach. In a previous study [Mitra, B., et al. (1993) Biochemistry 32, 12959-12967], the membrane association of MDH was correlated to a 53 amino acid segment in the interior of the primary sequence by construction of a chimeric enzyme, MDH-GOX1, in which the membrane-binding segment in MDH was deleted and replaced with the corresponding 34 amino acid segment from the soluble GOX. Though MDH-GOX1 was soluble, it was an inefficient, nonspecific enzyme that involved a different transition state for the catalyzed reaction from that of the wild-type MDH. In the present study, it is shown that the membrane-binding segment in MDH is somewhat shorter, approximately 39 residues long. Partial or total deletion of this segment disrupts membrane localization of MDH. This segment is not important for substrate oxidation activity. A new chimera, MDH-GOX2, was created by replacing this shorter membrane-binding segment from MDH with the corresponding 20 amino acid segment from GOX. The soluble MDH-GOX2 is very similar to the wild-type membrane-bound enzyme in its spectroscopic properties, substrate specificity, catalytic activity, kinetic mechanism, and lack of reactivity toward oxygen. Therefore, it should prove to be a highly useful model for structural studies of MDH.     

Fusion protein approach to improve the crystal quality of cytochrome bo(3) ubiquinol oxidase from Escherichia coli

Crystals of cytochrome bo(3) ubiquinol oxidase from E. coli diffract X-rays to 3.5 A and the structure determination is in progress. The limiting factor to the elucidation of the structural detail is the quality of the crystals; the diffraction spots from the crystals are diffused which leads to difficulties in processing the data beyond 4.0 A. Weak protein-protein contacts within the crystal lattice is assumed to be the cause of this problem. To improve these contacts, we have introduced protein Z to the C-terminal end of the subunit IV of cytochrome bo(3) and expressed both proteins as a single fusion. We have successfully obtained crystals of this fusion protein. The spot shape problem has clearly been solved in the crystals of the fusion protein although further optimization is necessary to obtain higher resolution. We also discuss the potential applications of this approach to the crystallization of membrane proteins in general.     

Cox17 is functional when tethered to the mitochondrial inner membrane

Cox17 is an essential protein in the assembly of cytochrome c oxidase within the mitochondrion. Cox17 is implicated in providing copper ions for formation of CuA and CuB sites in the oxidase complex. To address whether Cox17 is functional in shuttling copper ions to the mitochondrion, Cox17 was tethered to the mitochondrial inner membrane by a fusion to the transmembrane domain of the inner membrane protein, Sco2. The copper-binding domain of Sco2 that projects into the inter-mitochondrial membrane space was replaced with Cox17. The Sco2/Cox17 fusion protein containing the mitochondrial import sequence and transmembrane segment of Sco2 is exclusively localized within the mitochondrion. The Sco2/Cox17 protein restores respiratory growth and normal cytochrome oxidase activity in cox17Delta cells. These studies suggest that the function of Cox17 is confined to the mitochondrial intermembrane space. Domain mapping of yeast Cox17 reveals that the carboxyl-terminal segment of the protein has a function within the intermembrane space that is independent of copper ion binding. The essential C-terminal function of Cox17 maps to a candidate amphipathic helix that is important for mitochondrial uptake and retention of the Cox17 protein. This motif can be spatially separated from the N-terminal copper-binding functional motif. Possible roles of the C-terminal motif are discussed.     

Disposition of polar and nonpolar residues on outer surfaces of transmembrane helical segments of proteins involved in proton translocation

"Helical wheel" projections of transmembrane helical segments of membrane proteins involved in proton translocation were constructed. The particular proteins studied were the uncF protein subunit of the Escherichia coli proton-ATPase, the uncE protein subunit of the E. coli proton-ATPase, and cytochrome oxidase subunit III. Clear demarcation of polar and nonpolar regions on surfaces of transmembrane helical segments was seen in the uncF protein and in uncE protein helical segment two, but not in uncE protein helical segment one. The transmembrane segment of cytochrome oxidase subunit III which includes the dicyclohexylcarbodiimide (DCCD)-reactive residue was very similar to E. coli uncE protein helical segment two. The DCCD-reactive residue in both was clearly located on a nonpolar surface.     

Studies on cytochrome c oxidase, V. Polypeptide IV: alignment and amino acid sequences on cyanogen bromide fragments.

The isolation and chemical characterization of polypeptide IV from beef heart cytochrome oxidase is described. The protein is one of the main (stoichiometric) components of the oxidase. It is the largest polypeptide of the enzyme synthezised in the cytoplasm and has, as such, also been identified in enzyme preparations from yeast and Neurospora. A partial sequence, consisting of 105 amino acid residues which give a frame work of the covalent structure of the polypeptide is obtained from N- anc C-terminal sequencing and from the cyanogen bromide fragments of the chain. The isolation and sequencing of the fragments of this membrane protein are discussed.     

Putting together a plasma membrane NADH oxidase: A tale of three laboratories

The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism.     

The conserved third transmembrane segment of YidC contacts nascent Escherichia coli inner membrane proteins

Escherichia coli YidC is a polytopic inner membrane protein that plays an essential and versatile role in the biogenesis of inner membrane proteins. YidC functions in Sec-dependent membrane insertion but acts also independently as a separate insertase for certain small membrane proteins. We have used a site-specific cross-linking approach to show that the conserved third transmembrane segment of YidC contacts the transmembrane domains of both nascent Sec-dependent and -independent substrates, indicating a generic recognition of insertion intermediates by YidC. Our data suggest that specific residues of the third YidC transmembrane segment alpha-helix is oriented toward the transmembrane domains of nascent inner membrane proteins that, in contrast, appear quite flexibly positioned at this stage in biogenesis.     

Absorption spectra and magnetic circular dichroism of heme-containing proteins in nonequilibrium states. VI. Cytochrome c-oxidase

Low temperature (77 degrees K) absorption spectra of nonequilibrium states of cytochrome c oxidase produced by reduction of oxidases form protein by thermolysed electrons at 77 degrees K was studied. During reduction of cytochrome oxidase water-glycerol solution by thermolysed electrons at 77 degrees K a nonequilibrium reduced protein is formed. Low temperature (77 degrees K) absorption spectra of the nonequilibrium cytochrome oxidase differs from those reduced by ditionite. It was shown that the oxidation state of cytochrome a3 or addition of cytochrom c have no influence on these spectral changes. It is assumed, that the observed effects are conditioned by structural differences of reduced and oxidased cytochrome oxidase active center. Similar spectral changes were observed for cytochrome oxidase, bound to the mitochondrial membrane. At temperature increasing the low temperature reduced protein is relaxed to a corresponding equilibrium state. The spectral properties of bacterial cytochrome oxidase M. lysodeicticus do not depend on the way of reduction (by dytionite or thermolysed electrons at 77 degrees K).     

Crystallographic structure of SurA, a molecular chaperone that facilitates folding of outer membrane porins

The SurA protein facilitates correct folding of outer membrane proteins in gram-negative bacteria. The sequence of Escherichia coli SurA presents four segments, two of which are peptidyl-prolyl isomerases (PPIases); the crystal structure reveals an asymmetric dumbbell, in which the amino-terminal, carboxy-terminal, and first PPIase segments of the sequence form a core structural module, and the second PPIase segment is a satellite domain tethered approximately 30 A from this module. The core module, which is implicated in membrane protein folding, has a novel fold that includes an extended crevice. Crystal contacts show that peptides bind within the crevice, suggesting a model for chaperone activity whereby segments of polypeptide may be repetitively sequestered and released during the membrane protein-folding process.     

Lipid-protein interaction in reconstituted cytochrome c oxidase/phospholipid membranes.

Deuterium and 31P nuclear magnetic resonance have been employed in an investigation of the effect of cytochrome c oxidase (EC 1.9.3.1) on the structure of lecithin bilayers. Cytochrome c oxidase was isolated from beef heart mitochondria in lipid-free form and reconstituted as a functional enzyme in bilayers composed of synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Two separate reconstitution experiments were performed in which the lipid was selectively deuterated either at the C-5' or at the C-14' segment of the palmitic acyl chain. The phospholipid-to-protein ratio of both reconstituted complexes was 0.74 (mg/mg), corresponding to about 200 molecules lipid per molecule cytochrome c oxidase. The deuterium quadrupole splitting deltanuQ, and the phosphorus chemical shielding anisotropy, deltasigma, of the cytochrome c oxidase-phospholipid recombinants were measured as a function of temperature and compared to the results obtained for the pure lipid membrane without protein for the pure lipid membrane without protein. deltanuQ and deltasigma are highly sensitive to the structural organization of the lipid membrane and these measurements demonstrate that the incorporation of cytochrome c oxidase into phosphatidylcholine bilayers leads to a more disordered conformational state of the lipids. This result can be explained by a rapid exchange between lipids in direct contact with hydrophobic protein and those further away from it (exchange rate greater than 10(4) Hz). The irregular protein surface is sensed by all lipid molecules and induces a more disordered bilayer structure. In contrast to previous interpretations, our measurements do not suggest a special type of boundary lipid.     

The active form of cytochrome c oxidase: effects of detergent, the intact membrane, and radiation inactivation.

Cytochrome oxidase is a multisubunit, intrinsic membrane protein with a complex function that includes oxidation of cytochrome c, reduction of oxygen and generation of a membrane potential. To clarify the relationship of its normal function to protein and membrane structure, we have examined the kinetic behavior of rat liver cytochrome oxidase in the intact inner mitochondrial membrane and in detergent solubilized states. Dissolution of rat liver mitochondrial membranes alters the kinetic parameters of the oxidase in a manner dependent in part on the dispersing agent, and characterized by a large increase in maximal activity which is not attributable to exposure of more oxidase or diminished affinity for cytochrome c. The most profound effect of solubilization of the membrane is seen on the low affinity reaction of cytochrome c, suggesting that the electron transfer pathway from this site to oxygen is sensitive to alterations in hydrophobic interactions within the oxidase. Purified rat liver and beef heart oxidase exists predominantly in a monodisperse, 300 kilodalton form in laurylmaltoside (Rosevear et al., 1980). However, a smaller, 130 kd species that exhibits high turnover rates equal to the 300 kd form is detected in some beef heart preparations, implying that the dimer may not be essential for high activity. Radiation inactivation studies on purified oxidase reveal a molecular weight for the functional unit of approximately 70 kd. It is concluded that less than a complete set of subunits may be sufficient for both normal binding of cytochrome c and rapid electron transfer to oxygen.     

Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane.

We have extended a previously developed photo-crosslinking approach to systematically probe the protein environment of the secretory protein preprolactin, trapped during its transfer through the endoplasmic reticulum membrane. Single photoreactive groups were placed at various positions of nascent polypeptide chains of various length, corresponding to different stages of the transport process, and photo-crosslinks to membrane proteins were analyzed. In all cases, the polypeptide segment extending from the ribosome was found to be located in a membrane environment that is formed almost exclusively from Sec61 alpha, the multi-spanning subunit of the Sec61p complex that is essential for translocation. At early stages of the translocation process, before cleavage of the signal sequence, almost the entire nascent chain emerged from the ribosome contacts Sec61 alpha. The 'translocating chain-associating membrane' protein interacts mainly with the region of the signal sequence preceding its hydrophobic core. Our results suggest that the nascent chain is transferred directly from the ribosome into a protein-conducting channel, the major constituent of which is Sec61 alpha.     

Cysteine-directed cross-linking demonstrates that helix 3 of SecE is close to helix 2 of SecY and helix 3 of a neighboring SecE.

Preprotein translocation in Escherichia coli is mediated by translocase, a multimeric membrane protein complex with SecA as the peripheral ATPase and SecYEG as the translocation pore. Unique cysteines were introduced into transmembrane segment (TMS) 2 of SecY and TMS 3 of SecE to probe possible sites of interaction between the integral membrane subunits. The SecY and SecE single-Cys mutants were cloned individually and in pairs into a secYEG expression vector and functionally overexpressed. Oxidation of the single-Cys pairs revealed periodic contacts between SecY and SecE that are confined to a specific alpha-helical face of TMS 2 and 3, respectively. A Cys at the opposite alpha-helical face of TMS 3 of SecE was found to interact with a neighboring SecE molecule. Formation of this SecE dimer did not affect the high-affinity binding of SecA to SecYEG and ATP hydrolysis, but blocked preprotein translocation and thus uncouples the SecA ATPase activity from translocation. Conditions that prevent membrane deinsertion of SecA markedly stimulated the interhelical contact between the SecE molecules. The latter demonstrates a SecA-mediated modulation of the protein translocation channel that is sensed by SecE.     

Reconstitution of Sec-dependent membrane protein insertion: nascent FtsQ interacts with YidC in a SecYEG-dependent manner

The inner membrane protein YidC is associated with the preprotein translocase of Escherichia coli and contacts transmembrane segments of nascent inner membrane proteins during membrane insertion. YidC was purified to homogeneity and co-reconstituted with the SecYEG complex. YidC had no effect on the SecA/SecYEG-mediated translocation of the secretory protein proOmpA; however, using a crosslinking approach, the transmembrane segment of nascent FtsQ was found to gain access to YidC via SecY. These data indicate the functional reconstitution of the initial stages of YidC-dependent membrane protein insertion via the SecYEG complex.     

Cytochrome oxidase assembly in yeast requires the product of COX11, a homolog of the P. denitrificans protein encoded by ORF3.

The synthesis of cytochrome oxidase in Saccharomyces cerevisiae was recently shown to require a protein encoded by the nuclear gene COX10. This protein was found to be homologous to the putative protein product of the open reading frame ORF1 reported in one of the cytochrome oxidase operons of Paracoccus denitrificans. In the present study we demonstrate the existence in yeast of a second nuclear gene, COX11, whose encoded protein is homologous to another open reading frame (ORF3) present in the same operon of P. denitrificans. Mutations in COX11 elicit a deficiency in cytochrome oxidase. In this and in other respects cox11 and cox10 mutants have very similar phenotypes. An antibody has been obtained against the yeast COX11 protein. The antibody recognizes a 28 kd protein in yeast mitochondria, consistent with the size of the protein predicted from the sequence of COX11. The COX11 protein is tightly associated with the mitochondrial membrane but is not a component of purified cytochrome oxidase. An analysis of cytochrome oxidase subunits in wild type and in a cox11 mutant suggests that the COX11 protein is not required either for synthesis or transport of the subunit polypeptides into mitochondria. It seems more probable that COX11 protein exerts its effect at some terminal stage of enzyme synthesis, perhaps in directing assembly of the subunits.     

Prediction of helix-helix contacts and interacting helices in polytopic membrane proteins using neural networks

Despite rapidly increasing numbers of available 3D structures, membrane proteins still account for less than 1% of all structures in the Protein Data Bank. Recent high-resolution structures indicate a clearly broader structural diversity of membrane proteins than initially anticipated, motivating the development of reliable structure prediction methods specifically tailored for this class of molecules. One important prediction target capturing all major aspects of a protein's 3D structure is its contact map. Our analysis shows that computational methods trained to predict residue contacts in globular proteins perform poorly when applied to membrane proteins. We have recently published a method to identify interacting alpha-helices in membrane proteins based on the analysis of coevolving residues in predicted transmembrane regions. Here, we present a substantially improved algorithm for the same problem, which uses a newly developed neural network approach to predict helix-helix contacts. In addition to the input features commonly used for contact prediction of soluble proteins, such as windowed residue profiles and residue distance in the sequence, our network also incorporates features that apply to membrane proteins only, such as residue position within the transmembrane segment and its orientation toward the lipophilic environment. The obtained neural network can predict contacts between residues in transmembrane segments with nearly 26% accuracy. It is therefore the first published contact predictor developed specifically for membrane proteins performing with equal accuracy to state-of-the-art contact predictors available for soluble proteins. The predicted helix-helix contacts were employed in a second step to identify interacting helices. For our dataset consisting of 62 membrane proteins of solved structure, we gained an accuracy of 78.1%. Because the reliable prediction of helix interaction patterns is an important step in the classification and prediction of membrane protein folds, our method will be a helpful tool in compiling a structural census of membrane proteins.     

Cox17 Protein Is an Auxiliary Factor Involved in the Control of the Mitochondrial Contact Site and Cristae Organizing System

The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex.     

Reconstitution of biological molecular generators of electric current. Cytochrome oxidase.

1. Direct measurement of the electric current generation by cytochrome oxidase has been carried out. To this end, two procedures were used. The simpler one consists in formation of planar artificial membrane from the mixture of decane solution of soya bean phospholipids and beef heart cytochrome oxidase. Addition of cytochrome c and ascorbate to one of the two compartments separated by the cytochrome oxidase-containing planar membrane was found to result in a transmembrane electric potential difference being formed (plus on cytochrome c side of the membrane). Maximal values of potential differences obtained by this method were about 40 mV. Much higher potentials were observed when another ("photeoliposome-planar membrane") method was applied. In this case cytochrome oxidase was reconstituted with phospholipid to form proteoliposomes which adhered to planar phospholipid membrane in the presence of Ca2+ ions. Addition of cytochrome c and ascorbate to the proteoliposome-containing compartment gives rise to generation of an electric potential difference across the planar membrane, which reached 100 mV at a current of about 1 X 10(-11) A (minus in the proteoliposome-free compartment). The electromotive force of this generator was estimated as being about 0.2 V. If ascorbate and proteoliposomes were added into different compartments, a penetrating hydrogen atom carrier (phenazine methosulfate, (PMS) or tetramethyl-p-phenylenediamine (TMPD)) was required for a membrane potential to be formed. Generation of an electric potential difference of the opposite direction (plus in the proteoliposome-free compartment) was revealed in experiments with cytochrome oxidase proteoliposome containing cytochrome c in their interior. In this case, addition of PMS or TMPD was necessary. 2. In the suspension of cytochrome oxidase proteoliposome the uptake of a cationic penetrant (tetraphenyl phosphonium cation) was found to be coupled with electron transfer via external cytochrome c. Electron transfer via intraproteoliposomal cytochrome c induced the uptake of anionic penetrants (tetraphenyl borate and phenyldicarbaundecaborane anions). 3. All the above effects were sensitive to cyanide and protonophorous uncouplers. 4. In proteoliposomes containing both cytochrome oxidase and bacteriorhodopsin, the light- and oxidation-dependent generations of membrane potential have been revealed. 5. The data obtained are in agreement with Mitchell's idea of transmembrane electron flow in the cytochrome oxidase segment of the respiratory chain.