Uncoupling Proteins and Regulated Proton Leak in Mitochondria.

Higher concentration of protons in the mitochondrial intermembrane space compared to the matrix results in an electrochemical potential causing the back flux of protons to the matrix. This proton transport can take place through ATP synthase complex (leading to formation of ATP) or can occur via proton transporters of the mitochondrial carrier superfamily and/or membrane lipids. Some mitochondrial proton transporters, such as uncoupling proteins (UCPs), transport protons as their general regulating function; while others are symporters or antiporters, which use the proton gradient as a driving force to co-transport other substrates across the mitochondrial inner membrane (such as phosphate carrier, a symporter; or aspartate/glutamate transporter, an antiporter). Passage (or leakage) of protons across the inner membrane to matrix from any route other than ATP synthase negatively impacts ATP synthesis. The focus of this review is on regulated proton transport by UCPs. Recent findings on the structure and function of UCPs, and the related research methodologies, are also critically reviewed. Due to structural similarity of members of the mitochondrial carrier superfamily, several of the known structural features are potentially expandable to all members. Overall, this report provides a brief, yet comprehensive, overview of the current knowledge in the field.

New Insights into the Chloroplast Outer Membrane Proteome and Associated Targeting Pathways.

Plastids are a dynamic class of organelle in plant cells that arose from an ancient cyanobacterial endosymbiont. Over the course of evolution, most genes encoding plastid proteins were transferred to the nuclear genome. In parallel, eukaryotic cells evolved a series of targeting pathways and complex proteinaceous machinery at the plastid surface to direct these proteins back to their target organelle. Chloroplasts are the most well-characterized plastids, responsible for photosynthesis and other important metabolic functions. The biogenesis and function of chloroplasts rely heavily on the fidelity of intracellular protein trafficking pathways. Therefore, understanding these pathways and their regulation is essential. Furthermore, the chloroplast outer membrane proteome remains relatively uncharted territory in our understanding of protein targeting. Many key players in the cytosol, receptors at the organelle surface, and insertases that facilitate insertion into the chloroplast outer membrane remain elusive for this group of proteins. In this review, we summarize recent advances in the understanding of well-characterized chloroplast outer membrane protein targeting pathways as well as provide new insights into novel targeting signals and pathways more recently identified using a bioinformatic approach. As a result of our analyses, we expand the known number of chloroplast outer membrane proteins from 117 to 138.

Membrane Proteins: Structure, Function and Motion.

Cell membranes are intricate multicomponent supramolecular structures, with a complex variable morphology and chemical composition [...].

Biphasic Proton Transport Mechanism for Uncoupling Proteins.

It has been suggested that uncoupling proteins (UCPs) transport protons via interconversion between two conformational states: one in the "cytoplasmic state" and the other in the "matrix state". Matrix and cytoplasmic salt-bridge networks are key controllers of these states. This study proposes a mechanism for proton transport in tetrameric UCP2, with focus on the role of the matrix network. Eleven mutants were prepared to disrupt (K → Q or D → N mutations) or alter (K → D and D → K mutations) the salt-bridges in the matrix network. Proteins were recombinantly expressed in Escherichia coli membrane, reconstituted in model lipid membranes, and their structures and functions were analyzed by gel electrophoresis, circular dichroism spectroscopy, fluorescence assays, as well as molecular dynamics simulations. It is shown that the UCP2 matrix network contains five salt-bridges (rather than the previously reported three), and the matrix network can regulate the proton transport by holding the protein's transmembrane helices in close proximity, limiting the movement of the activator fatty acid(s). A biphasic two-state molecular model is proposed for proton transport in tetrameric (a dimer of stable dimers) UCP2, in which all the monomers are functional, and monomers in each dimer are in the same transport mode. Purine nucleotide (e.g., ATP) can occlude the internal pore of the monomeric units of UCP tetramers via interacting with positive residues at or in the proximity of the matrix network (K38, K141, K239, R88, R185, and R279) and prevent switching between cytoplasmic and matrix states, thus inhibiting the proton transport. This study provides new insights into the mechanism of proton transport and regulation in UCPs.

Systematic Design and Validation of Ion Channel Stabilization of Amphipathic α-Helical Peptides Incorporating Tryptophan Residues.

Aromatic interactions such as π-π interaction and cation-π interaction are present in membrane proteins and play important roles in both structure and function. To systematically investigate the effect of aromatic residues on the structural stability and ion permeability of peptide-formed ion channels, we designed several peptides with one or two tryptophan (Trp) residues incorporated at different positions in amphipathic α-helical peptides. Circular dichroism (CD) studies revealed the preferable position of Trp residues for self-association in these designed peptides. Systematically designed di-substituted peptides with two Trps at each helix termini demonstrated intermolecular Trp-Trp interactions caused by aggregation. In the presence of liposomes, Trp on the hydrophilic face of the peptide enhanced interaction with the lipid membrane to increase the amphipathic α-helical contents. Appropriate incorporation and positioning of Trp enabled peptides to form more stable channels and had notable effects with Trp di-substituted peptides. The ion channel forming capability of a series of these peptides showed that the cation-π interactions between Trp and Lys residues in adjacent transmembrane helices contribute to remarkable stabilization of the channel structure.

Functional Oligomeric Forms of Uncoupling Protein 2: Strong Evidence for Asymmetry in Protein and Lipid Bilayer Systems.

Stoichiometry of uncoupling proteins (UCPs) and their coexistence as functional monomeric and associated forms in lipid membranes remain intriguing open questions. In this study, tertiary and quaternary structures of UCP2 were analyzed experimentally and through molecular dynamics (MD) simulations. UCP2 was overexpressed in the inner membrane of Escherichia coli, then purified and reconstituted in lipid vesicles. Structure and proton transport function of UCP2 were characterized by circular dichroism (CD) spectroscopy and fluorescence methods. Findings suggest a tetrameric functional form for UCP2. MD simulations conclude that tetrameric UCP2 is a dimer of dimers, is more stable than its monomeric and dimeric forms, is asymmetrical and induces asymmetry in the membrane's lipid structure, and a biphasic on-off switch between the dimeric units is its possible mode of transport. MD simulations also show that the water density inside the UCP2 monomer is asymmetric, with the cytoplasmic side having a higher water density and a wider radius. In contrast, the structurally comparable adenosine 5'-diphosphate (ADP)/adenosine 5'-triphosphate (ATP) carrier (AAC1) did not form tetramers, implying that tetramerization cannot be generalized to all mitochondrial carriers.

Conformational Changes and Association of Membrane-Interacting Peptides in Myelin Membrane Models: A Case of the C-Terminal Peptide of Proteolipid Protein and the Antimicrobial Peptide Melittin.

Model membranes composed of various lipid mixtures can segregate into liquid-ordered (Lo) and liquid-disordered (Ld) phases. In this study, lipid vesicles composed of mainly Lo or Ld phases as well as complex lipid systems representing the cytosolic leaflet of the myelin membrane were characterized by fluorescence resonance energy transfer with a donor/acceptor pair that preferentially partitioned into Lo or Ld phases, respectively. The fluidity of the lipid systems containing >30% cholesterol was modulated in the presence of the amphipathic peptide melittin. With all the studied lipid systems, melittin attained an α-helical conformation as determined by CD spectroscopy and attained varying degrees of membrane association and penetration as determined by intrinsic Trp fluorescence. The other protein domain utilized was a putative amphipathic helical peptide derived from the cytosolic C-terminal sequence of proteolipid protein (PLP) which is the most abundant protein in the myelin membrane. The C-terminal PLP peptide transitioned from a random coil to an α-helix in the presence of trifluoroethanol. Upon interacting with each of lipid vesicle system, the PLP peptide also folded into a helix; however, at high concentrations of the peptide with fluid lipid systems, associated helices transmuted into a ß-sheet conformer. The membrane-associated aggregation of the cytosolic C-termini could be a mechanism by which the transmembrane PLP multimerizes in the myelin membrane.

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system.

Mitochondrial inner membrane uncoupling proteins (UCPs) facilitate transmembrane (TM) proton flux and consequently reduce the membrane potential and ATP production. It has been proposed that the three neuronal human UCPs (UCP2, UCP4 and UCP5) in the central nervous system (CNS) play significant roles in reducing cellular oxidative stress. However, the structure and ion transport mechanism of these proteins remain relatively unexplored. Recently, we reported a novel expression system for obtaining functionally folded UCP1 in bacterial membranes and applied this system to obtain highly pure neuronal UCPs in high yields. In the present study, we report on the structure and function of the three neuronal UCP homologues. Reconstituted neuronal UCPs were dominantly helical in lipid membranes and transported protons in the presence of physiologically-relevant fatty acid (FA) activators. Under similar conditions, all neuronal UCPs also exhibited chloride transport activities that were partially inhibited by FAs. CD, fluorescence and MS measurements and semi-native gel electrophoresis collectively suggest that the reconstituted proteins self-associate in the lipid membranes. Based on SDS titration experiments and other evidence, a general molecular model for the monomeric, dimeric and tetrameric functional forms of UCPs in lipid membranes is proposed. In addition to their shared structural and ion transport features, neuronal UCPs differ in their conformations and proton transport activities (and possibly mechanism) in the presence of different FA activators. The differences in FA-activated UCP-mediated proton transport could serve as an essential factor in understanding and differentiating the physiological roles of UCP homologues in the CNS.

Role of positively charged residues of the second transmembrane domain in the ion transport activity and conformation of human uncoupling protein-2.

Residing at the inner mitochondrial membrane, uncoupling protein-2 (UCP2) mediates proton transport from the intermembrane space (IMS) to the mitochondrial matrix and consequently reduces the rate of ATP synthesis in the mitochondria. The ubiquitous expression of UCP2 in humans can be attributed to the protein's multiple physiological roles in tissues, including its involvement in protective mechanisms against oxidative stress, as well as glucose and lipid metabolisms. Currently, the structural properties and ion transport mechanism of UCP2 and other UCP homologues remain poorly understood. UCP2-mediated proton transport is activated by fatty acids and inhibited by di- and triphosphate purine nucleotides. UCP2 also transports chloride and some other small anions. Identification of key amino acid residues of UCP2 in its ion transport pathway can shed light on the protein's ion transport function. On the basis of our previous studies, the second transmembrane helix segment (TM2) of UCP2 exhibited chloride channel activity. In addition, it was suggested that the positively charged residues on TM2 domains of UCPs 1 and 2 were important for their chloride transport activity. On this basis, to further understand the role of these positively charged residues on the ion transport activity of UCP2, we recombinantly expressed four TM2 mutants: R76Q, R88Q, R96Q, and K104Q. The wild type UCP2 and its mutants were purified and reconstituted into liposomes, and their conformation and ion (proton and chloride) transport activity were studied. TM2 Arg residues at the matrix interface of UCP2 proved to be crucial for the protein's anion transport function, and their absence resulted in highly diminished Cl(-) transport rates. On the other hand, the two other positively charged residues of TM2, located at the UCP2-IMS interface, could participate in the salt-bridge formation in the protein and promote the interhelical tight packing in the UCP2. Absence of these residues did not influence Cl(-) transport rates, but disturbed the dense packing in UCP2 and resulted in higher UCP2-mediated proton transport rates in the presence of long chain fatty acids. Overall, the outcome of this study provides a deeper and more detailed molecular image of UCP2's ion transport mechanism.

Folding and self-association of atTic20 in lipid membranes: implications for understanding protein transport across the inner envelope membrane of chloroplasts.

BACKGROUND: The Arabidopsis thaliana protein atTic20 is a key component of the protein import machinery at the inner envelope membrane of chloroplasts. As a component of the TIC complex, it is believed to form a preprotein-conducting channel across the inner membrane. RESULTS: We report a method for producing large amounts of recombinant atTic20 using a codon-optimized strain of E. coli coupled with an autoinduction method of protein expression. This method resulted in the recombinant protein being directed to the bacterial membrane without the addition of a bacterial targeting sequence. Using biochemical and biophysical approaches, we were able to demonstrate that atTic20 homo-oligomerizes in vitro when solubilized in detergents or reconstituted into liposomes. Furthermore, we present evidence that the extramembranous N-terminus of the mature protein displays characteristics that are consistent with it being an intrinsically disordered protein domain. CONCLUSION: Our work strengthens the hypothesis that atTic20 functions similarly to other small α-helical integral membrane proteins, such as Tim23, that are involved in protein transport across membranes.

Dynamic turn conformation of a short tryptophan-rich cationic antimicrobial peptide and its interaction with phospholipid membranes.

Cationic antimicrobial peptides are promising sources for novel therapeutic agents against multi-drug-resistant bacteria. HHC-36 (KRWWKWWRR) is a simple but effective antimicrobial peptide with similar or superior activity compared with several conventional antibiotics. In this biophysical study, unique conformational properties of this peptide and some of its analogs as well as its interaction with lipid membranes are investigated in detail. Circular dichroism (CD) and molecular dynamics modeling studies of HHC-36 in different environments reveal a dynamic amphipathic structure composed of competing turn conformations with free energies lower than that of the unfolded state, implying a strong influence of tryptophan interactions in formation of the turns. CD spectra and gel electrophoresis also show strong evidence of self-association of this peptide in aqueous milieu and interaction with both neutrally and negatively charged lipid membrane systems. Isothermal titration calorimetry and acrylamide fluorescence quenching experiments emphasize the preference of HHC-36 for negatively charged vesicles. In addition, dye leakage experiments suggest that this peptide functions through a surface-associated mechanism with weak lytic activity against bacterial model membranes.

Expression, folding, and proton transport activity of human uncoupling protein-1 (UCP1) in lipid membranes: evidence for associated functional forms.

Uncoupling protein-1 (UCP1) is abundantly expressed in the mitochondrial inner membrane of brown adipose tissues and has an important role in heat generation, mediated by its proton transport function. The structure and function of UCP1 are not fully understood, partially due to the difficulty in obtaining native-like folded proteins in vitro. In this study, using the auto-induction method, we have successfully expressed UCP1 in Escherichia coli membranes in high yield. Overexpressed UCP1 in bacterial membranes was extracted using mild detergents and reconstituted into phospholipid bilayers for biochemical studies. UCP1 was folded in octyl glucoside, as indicated by its high helical content and binding to ATP, a known UCP1 proton transport inhibitor. Reconstituted UCP1 in phospholipid vesicles also exhibited highly helical structures and proton transport that is activated by fatty acids and inhibited by purine nucleotides. Self-associated functional forms of UCP1 in lipid membranes were observed for the first time. The self-assembly of UCP1 into tetramers was unambiguously characterized by circular dichroism and fluorescence spectroscopy, analytical ultracentrifugation, and semi-native gel electrophoresis. In addition, the mitochondrial lipid cardiolipin stabilized the structure of associated UCP1 and enhanced the proton transport activity of the protein. The existence of the functional oligomeric states of UCP1 in the lipid membranes has important implications for understanding the structure and proton transport mechanism of this protein in brown adipose tissues as well as structure-function relationships of other mammalian UCPs in other tissues.

Tau-derived-hexapeptide 306VQIVYK311 aggregation inhibitors: nitrocatechol moiety as a pharmacophore in drug design.

The nitrocatechol derivatives tolcapone (1) and entacapone (2), used as adjunctive therapy in the treatment of Parkinson's disease, were investigated for their potential to inhibit the tau-derived-hexapeptide 306VQIVYK311. They were compared to small molecules that contain similar pharmacophores including the catechol derivatives (dopamine 3 and epinephrine 4), nitroderivatives (nifedipine 5 and chloramphenicol 6), nitrocatechol isomers (7 and 8), and a tolcapone derivative (13) lacking the nitrocatechol moiety. The aggregation kinetics by thioflavin S fluorescence assay indicates that both tolcapone (1) and entacapone (2) exhibit antiaggregation properties. These findings were supported by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy measurements which suggest that the nitrocatechol (3,4-dihydroxy-5-nitrophenyl) moiety is a suitable pharmacophore in the design of new tau-aggregation inhibitors. Furthermore, tolcapone (1) was identified as most active compound with antiaggregation activity (46% inhibition of fluorescence intensity at 50 µM), which was supported by TEM data. The in silico steric zipper model of the tau-derived-hexapeptide 306VQIVYK311 indicates that the 3,4-dihydroxy-substituent present in tolcapone (1) and entacapone (2) underwent polar contacts with lysine side chains (VQIVYK), whereas the charged 5-nitrosubstituent was in close contact with lysine side chain present in the steric zipper region suggesting the critical role of a nitrocatechol (3,4-dihydroxy-5-nitrophenyl) pharmacophore present in tolcapone (1) and entacapone (2) in tau-hexapeptide binding and prevention of ß-sheet assembly. Our results have significant implications in the design and development of tau-aggregation inhibitors.

pH-induced changes in intrinsically disordered proteins.

Intrinsically disordered proteins are typically enriched in amino acids that confer a relatively high net charge to the protein, which is an important factor leading to the lack of a compact structure. There are many different approaches that can be used to experimentally confirm whether a protein is intrinsically disordered. One such approach takes advantage of the distinctive amino acid composition to test whether a protein is a genuine IDP. In particular, the conformation of the protein can be monitored at different pHs; as opposed to globular or ordered proteins, IDPs will typically gain structure under highly acidic or basic conditions. Here, we describe circular dichroism and fluorescence spectroscopic experimental approaches in which the conformation of proteins is monitored as pH is altered as a way of testing whether the protein behaves as an intrinsically disordered protein.

Toward understanding the mechanism of ion transport activity of neuronal uncoupling proteins UCP2, UCP4, and UCP5.

Neuronal uncoupling proteins (UCP2, UCP4, and UCP5) have crucial roles in the function and protection of the central nervous system (CNS). Extensive biochemical studies of UCP2 have provided ample evidence of its participation in proton and anion transport. To date, functional studies of UCP4 and UCP5 are scarce. In this study, we show for the first time that, despite a low level of amino acid sequence identity with the previously characterized UCPs (UCP1-UCP3), UCP4 and UCP5 share their functional properties. Recombinantly expressed in Escherichia coli, UCP2, UCP4, and UCP5 were isolated and reconstituted into liposome systems, where their conformations and ion (proton and chloride) transport properties were examined. All three neuronal UCPs are able to transport protons across lipid membranes with characteristics similar to those of the archetypal protein UCP1, which is activated by fatty acids and inhibited by purine nucleotides. Neuronal UCPs also exhibit transmembrane chloride transport activity. Circular dichroism spectroscopy shows that these three transporters exist in different conformations. In addition, their structures and functions are differentially modulated by the mitochondrial lipid cardiolipin. In total, this study supports the existence of general conformational and ion transport features in neuronal UCPs. On the other hand, it also emphasizes the subtle structural and functional differences between UCPs that could distinguish their physiological roles. Differentiation between structure-function relationships of neuronal UCPs is essential for understanding their physiological functions in the CNS.

Ion channel activity of transmembrane segment 6 of Escherichia coli proton-dependent manganese transporter.

Synthetic peptides corresponding to the sixth transmembrane segment (TMS6) of secondary-active transporter MntH (Proton-dependent Manganese Transporter) from Escherichia coli and its two mutations in the functionally important conserved histidine residue were used as a model for structure-function study of MntH. The secondary structure of the peptides was estimated in different environments using circular dichroism spectroscopy. These peptides interacted with and adopted helical conformations in lipid membranes. Electrophysiological experiments demonstrated that TMS6 was able to form multi-state ion channels in model biological membranes. Electrophysiological properties of these weakly cation-selective ion channels were strongly dependent on the surrounding pH. Manganese ion, as a physiological substrate of MntH, enhanced the conductivity of TMS6 channels, influenced the transition between closed and open states, and affected the peptide conformations. Moreover, functional properties of peptides carrying two different mutations of His(211) were analogous to in vivo functional characteristics of Nramp/MntH proteins mutated at homologous residues. Hence, a single functionally important TMS can retain some of the functional properties of the full-length protein. These findings could contribute to understanding the structure-function relationship at the molecular level. However it remains unclear to what extent the peptide-specific channel activity represents a functional aspect of the full-length membrane carrier protein.

A comparative study on conformation and ligand binding of the neuronal uncoupling proteins.

Mitochondrial uncoupling proteins of the nervous system (UCPs 2, 4, and 5) have potential roles in the function and protection of the central nervous system (CNS). In the absence of structural information, conformations of the hexahistidine-tagged versions of all five human UCPs in liposomes were investigated for the first time, using far- and near-UV CD and fluorescence spectroscopy. Highly pure UCPs 1-5 were reconstituted in detergents and stable small unilamellar vesicles, appropriate for spectroscopic studies. All UCPs formed dominantly helical conformations in negatively charged phospholipid vesicles (palmitoyloleoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol, 7:3 molar ratio). UCPs 2 and 5 exhibited comparable helical conformations with possible association in lipid bilayers, whereas UCP4 had a different helical profile that can be related to its less associated form. Interaction of reconstituted UCPs with GDP and GTP, inhibitors of the prototypic UCP1, was detected by near-UV CD and fluorescence spectroscopy, utilizing the sensitivity of these techniques to microenvironments around Trp residues close to the nucleotide binding site. Binding of UCP4 to purine nucleotides was also different from other UCPs. Binding of fatty acids, activators of proton transport in UCPs, to UCPs could not be unambiguously detected, implying a nonbinding conformation/orientation of the proteoliposomes. Interaction of CoA with UCPs was comparable to nucleotide binding, suggesting a possible binding of this molecule at the nucleotide binding site. Despite dissimilar primary sequences, neuronal UCPs share common structural and functional properties with UCPs 1 and 3, supporting a common physiological role in addition to their specific roles in the CNS.

Chiral thiol-stabilized silver nanoclusters with well-resolved optical transitions synthesized by a facile etching procedure in aqueous solutions.

A novel approach of cyclic reduction in oxidative conditions has been developed to prepare a single dominant species of chiral thiol-stabilized silver nanoclusters (AgNCs). Such AgNCs, which are stable in solution for up to a few days, have been obtained for the first time. The generality of the established procedure is proven by using several enantiomeric water-soluble thiols, including glutathione, as protective ligands. The prepared AgNCs featured prominent optical properties including a single pattern of UV-vis absorption with well-resolved peaks. The chirality of the clusters has been investigated by circular dichroism (CD) spectroscopy. CD spectra displayed strong characteristic signatures in the visible range. Tentative identification of the cluster composition is discussed.