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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.

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.

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.

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.

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

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.

Uncoupling proteins (UCPs) are members of the mitochondrial carrier family (MCF) that transport protons across the inner mitochondrial membrane, thereby uncoupling electron transport from ATP synthesis. The stoichiometry of UCPs, and the possibility of co-existence of this protein as mono-meric and associated forms in lipid membranes remain an intriguing open question. In the current study, the tertiary structure of UCP2 was analyzed both experimentally and through molecular dynamics (MD) simulations. After recombinant expression of UCP2 in the inner membrane of E. coli, the protein was directly extracted from the bacterial membranes with a non-denaturing detergent and purified both as a pure monomer and as a mixture of monomers, dimers and tetramers. Both protein preparations were re-constituted in egg yolk lipid vesicles. Gel electrophoresis, circular dichroism spectroscopy and fluorescence methods were used to characterize the structure and the proton transport function of protein. UCP2 showed unique stable tetrameric forms in lipid bilayers. MD simulations using membrane lipids and principal component analysis support the experimental results and provided new molecular insights into the nature of noncovalent interactions in oligomeric UCP2. MD simulations indicate that UCP2 tetramers are asymmetric dimers of dimers, in which the interactions between the monomers forming the dimer are stronger than the interactions between the dimers within the tetramer. It is also shown that UCP2 has a specific tendency to form functional tetramers in lipid bilayers, capable of proton transport. The asymmetric nature of the UCP2 tetramer could act as a scaffold for regulating the activity of the monomeric units through cooperative intercommunication between these subunits. Under similar experimental conditions, the structurally comparable ADP/ATP carrier protein did not form tetramers in vesicles, implying that spontaneous tetramerization cannot be generalized to all MCF members.STATEMENT OF SIGNIFICANCESelf-assembly of membrane proteins plays a significant role in their biological function. In this article, both experimental and computational evidence are provided for spontaneous tetramerization of one of the mitochondrial uncoupling proteins (UCP2) in model lipid membranes. It is also shown that the tetrameric form of UCP2 is capable of proton transport, which leads to regulation of ATP synthesis in mitochondrion. Molecular dynamics simulations confirm the presence of asymmetric UCP2 tetramers as a potential scaffold for regulating the activity of the monomeric units through mutual intercommunication. The outcome of this study provides a solid ground for potential co-existence of monomeric and multimeric functional forms of UCPs that contributes to a deeper molecular insight into their structure and function.

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 β-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.

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.

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.

The mechanisms underlying protein misfolding and aggregation are of significant interest considering their role in the pathophysiology of numerous amyloid-based diseases.1−4 Neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) are characterized by the accumulation of protein aggregates including amyloid-β (Aβ), tau (τ), and alpha-synuclein (α-syn) respectively.1,2,5−7 These amyloid deposits (plaques, neurofibrillary tangles or NFTs and Lewy bodies) form as a result of misfolding proteins and the progressive maturation of the soluble monomeric peptides into larger and insoluble cross-β-sheet assemblies, suggestive of shared aggregation dynamics.7−11 While these extra/intracellular proteinaceous deposits are mainly considered an end-result of the aggregation pathway, they do play a significant role in neurotoxicity, disease pathology, and diagnosis. These rolling toxicity cascades impair cellular metabolism, gene regulation, and, consequently, widespread neuronal cell death that leads to dementia.12−17 When examining the tauopathy disease mechanism in AD, it has been established that the downstream consequences of hyper-phosphorylated tau (hp-tau) lead to its dissociation from neuronal microtubule assemblies. This dissociation impacts the integrity of the neuroskeletal structure, and the hp-tau monomers initiate the rapid aggregation cascade to form intracellular NFTs resulting in metabolic impairment and cellular toxicity.18,19 Recently, tauopathy has gained significant research interest, as multiple trials on targeting Aβ alone as a potential therapeutic target seem to fail at the clinical level.20 Tau proteins are known to form aggregates known as paired helical filaments (PHFs) which can further grow to form NFTs. The molecular mechanisms of tau-protein aggregation and misfolding have been investigated by studying critical fragments of the protein sequence. One such sequence is the hexapeptide 306VQIVYK311 segment from the microtubule binding region of tau protein, which is known to promote nucleation dependent tau-aggregation. In this regard, the hexapeptide Ac-VQIVYK-NH2 (AcPHF6) is considered as a model peptide to investigate tau-aggregation. It is a well characterized hexapeptide that forms β-sheets and undergoes fibrillation efficiently in vitro to form cross-β-sheet structure similar to Aβ. This short peptide serves as a suitable model to study small molecule inhibitors of tau-aggregation.13,14,21−25 From a therapeutics research perspective, a number of drug classes have been discovered and proposed as suitable inhibitors of tau aggregation.26,27 A recent study showed that the small molecules, tolcapone (1) and entacapone (2) shown in Figure ​Figure1,1, exhibit antiaggregation properties against Aβ and α-synuclein.28 It should be noted that both tolcapone and entacapone are known to act as catechol-O-methyltransferase (COMT) inhibitors and are currently FDA approved as adjunctive therapies in PD.29 Both of these molecules contain a nitrocatechol (3,4-dihydroxy-5-nitrophenyl) pharmacophore. In our study, we investigated the ability of nitrocatechol, aromatic-nitro, and catechol containing small molecules such as tolcapone (1), entacapone (2), dopamine (3), epinephrine (4), chloramphenicol (5), nifedipine (6), and other small molecules 7–13 (Figure ​(Figure1)1) to prevent the tau hexapeptide Ac-VQIVYK-NH2 (AcPHF6) aggregation by in vitro kinetics using thioflavin S (ThS) based fluorescence measurements, transmission electron microscopy (TEM), circular dichroism (CD) spectroscopy, and molecular modeling investigations. These studies demonstrate for the first time that the nitrocatechol moiety can be a useful pharmacophore in the development of tau-aggregation inhibitors. Figure 1 Chemical structures of small molecules used (1–13).

Green chemistry of industrially important zein protein was explored in aqueous phase toward the synthesis of bioconjugated gold (Au) nanoparticles (NPs), which allowed us to simultaneously understand the unfolding behavior of zein with respect to temperature and time. Synthesis of Au NPs was monitored with simultaneous measurements of UV–visible absorbance due to the surface plasmon resonance (SPR) of Au NPs that triggered the adsorption of zein on the NP surface and thus resulted in its unfolding. Surface adsorption of zein further controlled the crystal growth of Au NPs, which relied on the degree of unfolding and fusogenic behavior of zein due to its predominant hydrophobic nature. The latter property induced a marked blue shift in the SPR rarely observed in the growing NPs during the nucleation process. A greater unfolding of zein in fact was instrumental in generating zein-coated faceted NPs that were subjected to their hemolytic response for their possible use as drug release vehicles. Zein coating ...

Several ion channel forming cyclic peptides have been reported over the past two decades and various ion conducting mechanisms have been proposed. In this article, we report on amino acid substitut...

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.

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic beta-sheet-beta-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar identical with Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic-exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (K(b)). The K(b1) and K(b2) values for endothermic two-step binding processes corresponded to high and low binding affinities (K(b1) >or= 100 K(b2)). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis.

An ultimate goal of synthetic ion-channel peptide design is to construct stable and functional ion-conducting pores. It is expected that specific interhelical interactions would facilitate the association of helices in phospholipid membranes and the successive helix-bundle formation. In the present study, we rationally designed helix-bundle ion channels using the synthetic hybrid peptide K20E20, a disulfide dimer of cationic- and anionic-amphiphilic helices Ac-CGG-(BKBA) 5-NH 2 and Ac-CGG-(BEBA) 5-NH 2. Circular dichroism (CD) measurements in aqueous media implied helix stabilization in the peptide caused by the interhelical electrostatic interactions. In addition, CD spectra recorded in the presence of DPPC liposomes and dye-leakage measurements suggested a high degree of association of peptide monomers in phospholipid membranes as well as high affinities between peptide and lipid bilayers. These features allowed ion-channel formation at extremely low peptide concentrations (as low as 1 nM). According to electrophysiological analyses, stable helix bundles were constructed of six peptide helices by association of three K20E20 molecules. Helix-helix association in lipid membranes, peptide-membrane interactions, and ion-channel formation of K20E20 peptides were all facilitated by intramolecular electrostatic interactions between the helices of the hybrid peptide and were pH-dependent. Conductance through K20E20 ion channels decreased under acidic conditions because of the interruption of the salt bridges.

Uncoupling proteins (UCP)2, UCP4 and UCP5 transport protons across the inner membrane of mitochondria in the central nervous system (CNS). Novel recombinant protein expression allowed expression of UCPs in Escherichia coli membranes. Functional neuronal UCPs formed multimers in membranes and interacted with various fatty acids (FAs) to transport protons. Self-association and unique ion transport properties of UCPs distinguish their physiological roles in the CNS., 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.

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.

Uncoupling proteins (UCPs), located in the mitochondrial inner membrane, facilitate the transmembrane proton flux and, consequently, reduce the membrane potential and ATP production. Found in the central nervous system (CNS), based on several studies on animal models and clinical investigations, three human UCP homologs (UCP2, UCP4, and UCP5) could have crucial physiological functions. Despite these studies, the detailed structural features and molecular physiology landscape of these proteins remain relatively unexplored. Recently, we reported a novel expression system for obtaining functionally folded UCP1 in bacterial membranes (Hoang et al., 2013). In the current study, employing similar expression and reconstitution methods, we will report our new findings for the three human neuronal UCP homologs. The reconstituted CNS proteins display high helical contents and transport protons in the presence of several physiologically-relevant fatty acid activators. In addition, experimental results from CD, fluorescence spectroscopy, mass spectrometry and semi-native electrophoresis suggest self-association of these proteins in the membranes. While sharing comparable secondary structures in the liposomes, neuronal UCPs differ in their proton transport rates (and possibly mechanism) in the presence of different fatty acid activators. The protein-fatty acids interaction is further investigated using near-UV CD spectroscopy. The differences in fatty-acid activated UCP-mediated proton transport could serve as an essential clue in understanding and differentiating the physiological roles of UCP homologs in the CNS.Hoang T, Smith MD, Jelokhani-Niaraki M (2013) Expression, folding, and proton transport activity of human uncoupling protein-1 (UCP1) in lipid membranes: evidence for associated functional forms. J. Biol. Chem. 288, 36244-36258

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. Electronic supplementary material The online version of this article (doi:10.1186/s12858-014-0029-y) contains supplementary material, which is available to authorized users.

Human uncoupling protein-1 (UCP1) is highly expressed in the inner mitochondrial membrane of brown adipose tissue (BAT). The physiologically important proton transport function of UCP1 is tightly linked to its vital thermogenic role, which has been shown to reduce obesity and improve insulin sensitivity. Nevertheless, the structure and mechanism of ion transport for UCP1 are not fully understood. In this study, using the auto-induction method, we have successfully expressed UCP1 in E. coli membranes in high yield. Folding and ion transport of UCP1, extracted from bacterial membranes, were studied after its reconstitution into lipid bilayers. The self-assembly of UCP1 into tetramers in lipid membranes was verified for the first time by circular dichroism and fluorescence spectroscopy, and semi-native gel electrophoresis. UCP1 tetramers in different phospholipid vesicles exhibited highly helical structures, as well as proton transport that was activated by fatty acids and inhibited by purine nucleotides. In addition, the mitochondrial lipid cardiolipin modulated both folding and ion transport function of UCP1 tetramers. Overall, the existence of functional oligomeric forms of UCP1 in the lipid membranes, found in this study, provides important implications for understanding the structure and proton transport mechanism of this protein in BAT, as well as structure-function relationships of other mammalian UCPs in other tissues. Common structural and functional features of human UCPs were explored in our previous studies(1).1. Hoang, T., Smith, M. D., and Jelokhani-Niaraki, M. 2012. Toward understanding the mechanism of ion transport activity of neuronal uncoupling proteins UCP2, UCP4, and UCP5. Biochemistry 51: 4004-4014.

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.

Molecular properties and physiological roles of the uncoupling proteins (UCPs) in the Central Nervous System (CNS) is an open question. In general, UCPs reduce the proton motive force across the mitochondrial inner membrane and uncouple the electron transfer process from ATP synthesis. Three of the five identified human UCPs (UCPs 2, 4 and 5) have been discovered in the CNS tissues. It has been widely suggested that the neuronal UCPs share common conformational and physiological properties with the prototypic UCP1, and have essential roles in the function and protection of the CNS. In addition to its uncoupling property, UCP1 has a distinct thermogenic role in brown adipose tissues. Important roles of neuronal UCPs may include thermal enhancement of synaptic neurotransmission and plasticity, and reduction of reactive oxygen species as one of the causes of neurodegenerative diseases. Despite extensive biological studies on UCPs, the structural properties and molecular details of the mechanisms of their functions are not clearly understood. In the past decade, our research group has been involved in comparative studies of the neuronal uncoupling proteins. Using CD and fluorescence spectroscopies and other biophysical techniques, we have shown that, despite their low sequence identity with each other and with UCP1, neuronal UCPs share common (dominantly helical) conformational features. Detailed studies in our laboratory also revealed the existence of common ion transport (proton and chloride) features in neuronal UCPs. To further clarify the molecular details of the physiological function of neuronal UCPs, we have proposed a simple molecular model for the coexistence of monomeric, dimeric and tetrameric functional forms of UCPs. These comparative studies emphasize on the subtle structural and functional differences between neuronal UCPs and their complex self-association that can be crucial in differentiating their physiological roles.

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.

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.

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.

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 His211 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. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 718–726, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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.

Background The Toc159 family of proteins serve as receptors for chloroplast-destined preproteins. They directly bind to transit peptides, and exhibit preprotein substrate selectivity conferred by an unknown mechanism. The Toc159 receptors each include three domains: C-terminal membrane, central GTPase, and N-terminal acidic (A-) domains. Although the function(s) of the A-domain remains largely unknown, the amino acid sequences are most variable within these domains, suggesting they may contribute to the functional specificity of the receptors. Results The physicochemical properties of the A-domains are characteristic of intrinsically disordered proteins (IDPs). Using CD spectroscopy we show that the A-domains of two Arabidopsis Toc159 family members (atToc132 and atToc159) are disordered at physiological pH and temperature and undergo conformational changes at temperature and pH extremes that are characteristic of IDPs. Conclusions Identification of the A-domains as IDPs will be important for determining their precise function(s), and suggests a role in protein-protein interactions, which may explain how these proteins serve as receptors for such a wide variety of preprotein substrates.

In a series of cyclic peptides based on GS10, an analogue of gramicidin S (GS), the ring size was varied from 10 to 16 amino acids. Alternative addition of basic and hydrophobic amino acids to the original GS10 construct generated a variety of even-numbered rings, i.e., GS10 [cyclo-(VKLdYPVKLdYP)], GS12 [cyclo-(VKLKdYPKVKLdYP)], GS14 [cyclo-(VKLKVdYPLKVKLdYP), and GS16 [cyclo-(VKLKVKdYPKLKVKLdYP)] (d stands for d-enantiomers). The odd-numbered analogues (11-, 13-, and 15-mers) were derived from these four peptides by either addition or deletion of single basic (Lys) or hydrophobic (Leu or Val) amino acids. The resulting peptides, divided into three groups on the basis of peptide ring size (10- to 12-meric, 13- and 14-meric, and 15- and 16-meric), illustrated a diverse spectrum of biological activity correlated to their ring size, degree of beta-structure disruption, charge, hydrophobicity, amphipathicity, and affinity for lipid membranes. Two of these peptides with potent antimicrobial activities and high therapeutic indexes (4.5- to 10-fold compared with GS) are promising candidates for development of broad-spectrum antibiotics.

Uncoupling proteins (UCPs), located in the inner membrane of the mitochondria, uncouple ATP-synthesis from the respiratory chain by transporting protons across the inner membrane into the matrix, hence dissipating the proton-motive force and releasing heat. The neuronal UCPs (nUCPs), UCP4 and UCP5, were discovered recently (in 1998) and little is known about their structure and function. To gain further insight into the potential importance of these two proteins in the neuroprotection and neuromodulation of neurodegenerative diseases, this study will focus on the structure, function and interaction of the nUCPs with nucleotides (inhibitors) and fatty acids (activators). A recombinant version of the proteins, utilizing a hexa-histidine tag and a TEV protease site (for subsequent His-tag cleavage) has been designed, expressed as insoluble inclusion bodies, and isolated and purified using immobilized metal affinity chromatography. Subsequent reconstitution of the proteins in mild detergent (DDM and digitonin) allowed for biophysical studies by circular dichroism and fluorescence spectroscopy. Circular dichroism spectroscopy has shown that, similar to the recombinant UCP1, nUCPs possess dominantly helical structures in digitonin and DDM [1]. Furthermore, detergent-mediated reconstitution of the proteins into preformed liposomes can give more physiologically relevant structural and functional information. Comparison of the structure and function of human UCP1 (thermogenin) to nUCPs, in lipid membranes and membrane-like environments, will eventually show whether these proteins have any similarity in conformation and functional behaviour.[1] Jelokhani-Niaraki, M., Ivanova, M.V., McIntyre, B.L., Newman, C.L., McSorley, F.R., Young, E.K. and Smith, M.D. (2008) Biochem. J., 411, 593-603.

Conformations of the prototypic UCP-1 (uncoupling protein-1) and its TM (transmembrane) and ML (matrix-loop) domains were studied by CD spectroscopy. Recombinant, untagged mouse UCP-1 and a hexahistidine-tagged version of the protein were obtained in high purity following their overexpression in Escherichia coli. The TM and ML domains of hamster UCP-1 were chemically synthesized. Conformations of both recombinant UCP-1 proteins were dominantly helical (40–50%) in digitonin micelles. Binding of the purine nucleotides GDP and GTP to UCP-1, detected in the near-UV CD region, supported the existence of the functional form of the protein in digitonin micelles. All individual TM and ML peptides, except the third ML domain, adopted helical structures in aqueous trifluoroethanol, which implies that, in addition to six TM segments, at least two of the ML domains of the UCP-1 can form helical structures in membrane interface regions. TM and ML domains interacted with vesicles composed of the main phospholipids of the inner membrane of mitochondria, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, to adopt dominantly β- and/or unordered conformations. Mixtures of UCP-1 peptide domains spontaneously associated in aqueous, phospholipid vesicles and digitonin micelle environments to form ordered conformations, which exhibited common features with the conformations of the full-length proteins. Thermal denaturations of UCP-1 and its nine-peptide-domain assembly in digitonin were co-operative but not reversible. Assembly of six TM domains in lipid bilayers formed ion-conducting units with possible helical bundle conformations. Consequently, covalent connection between peptide domains, tight domain interactions and TM potential are essential for the formation of the functional conformation of UCP-1.

Uncoupling proteins (UCP) are known to transport anions, such as Cl−, in addition to H+ transport. Although H+ transport by UCP is clearly involved in thermogenesis, the mechanism of its anion transport is not clearly understood. In this study, we examined the anion channel characteristics of the six individual helical transmembrane (TM) domains of the human UCP2. The second TM domain peptide (TM2) forms multi-state channels by assemblies of conductive oligomers. Furthermore, the TM2 exhibited voltage-dependent anion channels with properties comparable to those of UCP1 chloride channel. However, the other five TM peptides did not form UCP1-like channels. Moreover, an analog of TM2 in which two Arg residues were substituted by Ala residues did not form stable channels, implying the significance of Arg residues for anion transport. These results suggest that the anion channel structure of UCP2 protein is oligomeric and the second TM domain is essential for the voltage-dependence of this anion channel.

HHC-36 is an antimicrobial peptide, designed through neural network algorithms. It has been tested in vivo and in vitro, and has proved to be strongly effective against strains of multidrug-resistant P. aeruginosa, methicillin-resistant Staphylococcus aureus, and a few other ‘superbugs’ (Cherkasov et al., ACS Chem. Biol., 2009, 4 (1), pp 65-74). The peptide has also been observed through in vivo tests to be greatly pathogen-specific, hence proving to be a great candidate for developing future antibiotics.To understand the mechanism of activity of this peptide against bacterial membranes, we have performed a number of all-atom simulations, together with a series of circular dichroism spectroscopy (CD) and isothermal titration calorimetry (ITC) experiments. The small size (9 amino acids) and great charge density of HHC-36 make it problematic (if not unreliable) to find the structure of HHC-36 through conventional spectroscopy and/or crystallography methods. We have thus performed microsecond-scale molecular dynamics simulations, starting from an unfolded structure of the peptide, to find its folded structure. An amphipathic turn structure has been obtained, which was observed to be very stable over few hundred nanosecond timescales of simulation. This result has been compared to circular dichroism spectroscopy results, and the presence of the turn structure has been verified. To assess the stability of the observed structure, we have also performed temperature-dependent simulations and CD measurements, which have shown the stability of the turn structure at close-to-physiological temperatures.The obtained structure is then used in peptide-membrane simulations with a few different membrane compositions mimicking both bacterial and animal cell membranes. Profiles of the potential of mean force have been obtained, and the relevant binding parameters extracted from these simulations are then compared with the binding free energies obtained from ITC experiments.

Located in the inner mitochondrial membrane, uncoupling proteins (UCPs) dissipate the proton electrochemical gradient causing reduction in the rate of ATP synthesis. Among five human UCP homologues, UCP2 is unique with its ubiquitous expression in various tissues. This important feature has been attributed to UCP2's multiple physiological roles in tissues, including its involvement in protective mechanisms against oxidative stress, glucose and lipid metabolisms. Despite numerous physiological studies, UCP2 function in cell remains poorly understood. UCP2 proton transport is regulated by purine nucleotides such as ATP, ADP, GTP and GDP. In addition, UCP2 has also been observed to transport chlorides and other small anions. Identification of the key amino acid residues in UCP2 in proton, anion transport and regulation will help determine the protein's mechanism of action in cells. It has been established that positively charged residues on transmembrane helix 2 (TM2) of UCP1 and UCP2 are crucial for chloride transport. However, a full understanding of the transport mechanism is yet to be achieved. More importantly, some of these residues are also involved in the UCP2 proton transport regulation. To further understand the ion transport of UCP2, four TM2 mutants have been made (R76Q, R88Q, R96Q, and K104Q). Over-expressed proteins were purified and reconstituted into liposomes for structural and functional studies. All mutants share an overall helical conformation with wtUCP2. using anion-sensitive fluorescent probes, proton and chloride transport of UCP2 mutants are examined to determine the effect of each mutation on the ion transport of UCP2. In addition, Mant-modified purine nucleotide will be used to study the binding of UCP2 and its mutants to purine nucleotides. Overall, the outcome of this study will provide a more detailed molecular image of UCP2 ion transport mechanism.

The increasing demand for antibiotics has contributed to the investigation of possible novel antibiotics by many researchers. For this purpose, experimental and theoretical studies have been carried out to draw scientists' attention to antimicrobial peptides and their interaction with the surface of bacterial membranes. Their ability to disrupt the functioning of bacterial membranes has been probed from different perspectives. The best possible choice of antimicrobial peptides are those which do not harm plant or animals' membranes but which disrupt bacterial membranes. It has been found that some cationic antimicrobial peptides (CAPs) satisfy these requirements. CAPs interacting with the outer membrane of gram-negative bacteria and the membrane of gram-positive bacteria has been studied recently.We conduct a MD simulation study of peptide-peptide interactions in the physiological solutions and investigate the mechanism of CAPs aggregation, since aggregation of the peptides usually precedes formation of a pore in the membrane. Different algorithms will be applied to calculate the potential mean force of the aggregation process of peptides to select the most efficient one. Also, we have run CD spectroscopy and Calorimetry experiments to predict the structure of the peptide and measure the peptide-peptide binding enthalpy, and we compare the results with our simulation data.The particular CAP studied is HHC-10, a peptide designed by the Hancock group, which has 9 amino acid residues and charge +4.