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BMC Biotechnology May 2013The reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses....
BACKGROUND
The reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Current methods for performing such reconstitutions entail an initial detergent-mediated step to solubilize and isolate membrane proteins. Exposure to detergents, however, can destabilize many membrane proteins and result in a loss of function. Amphipathic copolymers have recently been used to stabilize membrane proteins and complexes following suitable detergent extraction. However, the ability of these copolymers to extract proteins directly from native lipid bilayers for subsequent reconstitution and characterization has not been explored.
RESULTS
The styrene-maleic acid (SMA) copolymer effectively solubilized membranes of isolated mitochondria and extracted protein complexes. Membrane complexes were reconstituted into polymer-bound nanoscale discs along with endogenous lipids. Using respiratory Complex IV as a model, these particles were shown to maintain the enzymatic activity of multicomponent electron transporting complexes.
CONCLUSIONS
We report a novel process for reconstituting fully operational protein complexes directly from cellular membranes into nanoscale lipid bilayers using the SMA copolymer. This facile, single-step strategy obviates the requirement for detergents and yields membrane complexes suitable for structural and functional studies.
Topics: Biotechnology; Maleates; Membrane Proteins; Mitochondrial Membranes; Models, Biological; Multienzyme Complexes; Nanoparticles; Polystyrenes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 23663692
DOI: 10.1186/1472-6750-13-41 -
The EMBO Journal Sep 1997The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we...
The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of alpha, precursor beta and processed beta subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of alpha and precursor beta but no processed beta subunits. These complexes possess short half-lives, are enzymatically inactive and their molecular weight is approximately 300 kDa. Radioactivity chases from these complexes into mature, long-lived approximately 700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing alpha subunits and the other, precursor beta subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor beta subunits PSMB2 (C7-I), PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the beta subunits and juxtaposition of the two beta rings) is accompanied by conformational changes in the (outer) alpha rings, and may be inefficient. Finally, interferon-gamma had no significant effect on the half-lives or total amounts of precursor or mature proteasomes.
Topics: Animals; Archaeal Proteins; Cells, Cultured; Cysteine Endopeptidases; Interferon-gamma; Liver; Macrophages; Mice; Models, Biological; Molecular Weight; Multienzyme Complexes; Precipitin Tests; Proteasome Endopeptidase Complex; Protein Biosynthesis; Protein Precursors; Protein Processing, Post-Translational; Proteins; Spleen; Tissue Distribution; Tumor Cells, Cultured
PubMed: 9311996
DOI: 10.1093/emboj/16.17.5363 -
Journal of Bacteriology May 2017Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA)...
Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from in and purification over His and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted with crude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive β-lactone intermediate produced by the OleC-catalyzed reaction. Bacteria biosynthesize hydrophobic molecules to maintain a membrane, store carbon, and for antibiotics that help them survive in their niche. The hydrophobic compounds are often synthesized by a multidomain protein or by large multienzyme assemblies. The present study reports on the discovery that long-chain olefinic hydrocarbons made by bacteria from different phyla are produced by multienzyme assemblies in The OleBCD multienzyme assemblies are thought to compartmentalize and sequester olefin biosynthesis from the rest of the cell. This system provides additional insights into how bacteria control specific biosynthetic pathways.
Topics: Alkenes; Bacterial Proteins; Biosynthetic Pathways; Catalytic Domain; Escherichia coli; Hydrocarbons; Multienzyme Complexes; Substrate Specificity; Xanthomonas campestris
PubMed: 28223313
DOI: 10.1128/JB.00890-16 -
American Journal of Human Genetics Jun 2005Single-gene disorders with "simple" Mendelian inheritance do not always imply that there will be an easy prediction of the phenotype from the genotype, which has been... (Review)
Review
Single-gene disorders with "simple" Mendelian inheritance do not always imply that there will be an easy prediction of the phenotype from the genotype, which has been shown for a number of metabolic disorders. We propose that moonlighting enzymes (i.e., metabolic enzymes with additional functional activities) could contribute to the complexity of such disorders. The lack of knowledge about the additional functional activities of proteins could result in a lack of correlation between genotype and phenotype. In this review, we highlight some notable and recent examples of moonlighting enzymes and their possible contributions to human disease. Because knowledge and cataloging of the moonlighting activities of proteins are essential for the study of cellular function and human physiology, we also review recently reported and recommended methods for the discovery of moonlighting activities.
Topics: Animals; Apoptosis; Gene Expression Regulation; Genetic Diseases, Inborn; Humans; Molecular Biology; Multienzyme Complexes; Proteins; Signal Transduction; Structure-Activity Relationship; Transcription, Genetic
PubMed: 15877277
DOI: 10.1086/430799 -
Microbiology and Molecular Biology... Mar 2008Many enzymes and other proteins are difficult subjects for bioinformatic analysis because they exhibit variant catalytic, structural, regulatory, and fusion mode... (Review)
Review
Many enzymes and other proteins are difficult subjects for bioinformatic analysis because they exhibit variant catalytic, structural, regulatory, and fusion mode features within a protein family whose sequences are not highly conserved. However, such features reflect dynamic and interesting scenarios of evolutionary importance. The value of experimental data obtained from individual organisms is instantly magnified to the extent that given features of the experimental organism can be projected upon related organisms. But how can one decide how far along the similarity scale it is reasonable to go before such inferences become doubtful? How can a credible picture of evolutionary events be deduced within the vertical trace of inheritance in combination with intervening events of lateral gene transfer (LGT)? We present a comprehensive analysis of a dehydrogenase protein family (TyrA) as a prototype example of how these goals can be accomplished through the use of cohesion group analysis. With this approach, the full collection of homologs is sorted into groups by a method that eliminates bias caused by an uneven representation of sequences from organisms whose phylogenetic spacing is not optimal. Each sufficiently populated cohesion group is phylogenetically coherent and defined by an overall congruence with a distinct section of the 16S rRNA gene tree. Exceptions that occasionally are found implicate a clearly defined LGT scenario whereby the recipient lineage is apparent and the donor lineage of the gene transferred is localized to those organisms that define the cohesion group. Systematic procedures to manage and organize otherwise overwhelming amounts of data are demonstrated.
Topics: Amino Acid Sequence; Bacteria, Anaerobic; Bacterial Proteins; Coenzymes; Computational Biology; Evolution, Molecular; Gene Transfer, Horizontal; Molecular Sequence Data; Multienzyme Complexes; Phylogeny; Substrate Specificity; Tyrosine
PubMed: 18322033
DOI: 10.1128/MMBR.00026-07 -
FEBS Letters Jan 1997The 20S proteasome, isolated from the nocardioform actinomycete Rhodococcus erythropolis strain NI86/21, is built from two alpha-type and two beta-type subunits. In...
The 20S proteasome, isolated from the nocardioform actinomycete Rhodococcus erythropolis strain NI86/21, is built from two alpha-type and two beta-type subunits. In order to probe the subunit topology, we have set up an expression system which allows coexpression of the genes encoding the alpha- and beta-subunits in all possible combinations. The four respective constructs obtained yielded fully assembled and proteolytically active proteasomes. Biochemical, kinetic and electron microscopy analysis allow us to rule out several of the models which were originally envisaged for the subunit topology of the Rhodococcus proteasome. The experiments further indicate that the assembly pathways of the Rhodococcus and of the Thermoplasma proteasome differ in some important details.
Topics: Cloning, Molecular; Cysteine Endopeptidases; Gene Expression; Kinetics; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Conformation; Recombinant Proteins; Rhodococcus; Substrate Specificity
PubMed: 9000518
DOI: 10.1016/s0014-5793(96)01403-2 -
Human Molecular Genetics Oct 2019Human dihydrolipoamide dehydrogenase (hLADH, hE3) deficiency (OMIM# 246900) is an often prematurely lethal genetic disease usually caused by inactive or partially...
Human dihydrolipoamide dehydrogenase (hLADH, hE3) deficiency (OMIM# 246900) is an often prematurely lethal genetic disease usually caused by inactive or partially inactive hE3 variants. Here we report the crystal structure of wild-type hE3 at an unprecedented high resolution of 1.75 Å and the structures of six disease-causing hE3 variants at resolutions ranging from 1.44 to 2.34 Å. P453L proved to be the most deleterious substitution in structure as aberrations extensively compromised the active site. The most prevalent G194C-hE3 variant primarily exhibited structural alterations close to the substitution site, whereas the nearby cofactor-binding residues were left unperturbed. The G426E substitution mainly interfered with the local charge distribution introducing dynamics to the substitution site in the dimer interface; G194C and G426E both led to minor structural changes. The R460G, R447G and I445M substitutions all perturbed a solvent accessible channel, the so-called H+/H2O channel, leading to the active site. Molecular pathomechanisms of enhanced reactive oxygen species (ROS) generation and impaired binding to multienzyme complexes were also addressed according to the structural data for the relevant mutations. In summary, we present here for the first time a comprehensive study that links three-dimensional structures of disease-causing hE3 variants to residual hLADH activities, altered capacities for ROS generation, compromised affinities for multienzyme complexes and eventually clinical symptoms. Our results may serve as useful starting points for future therapeutic intervention approaches.
Topics: Catalytic Domain; Dihydrolipoamide Dehydrogenase; Humans; Multienzyme Complexes; Mutagenesis, Site-Directed; Mutation; Protein Conformation; Reactive Oxygen Species
PubMed: 31334547
DOI: 10.1093/hmg/ddz177 -
Parasitology Research Feb 2020The microaerophilic human parasite Trichomonas vaginalis causes infections in the urogenital tract and is one of the most often sexually transmitted pathogens worldwide....
The microaerophilic human parasite Trichomonas vaginalis causes infections in the urogenital tract and is one of the most often sexually transmitted pathogens worldwide. Due to its anaerobic metabolism, it has to quickly remove intracellular oxygen in order to avoid deactivation of essential metabolic enzymes such as oxygen-sensitive pyruvate:ferredoxin oxidoreductase (PFOR). Two major enzyme activities which are responsible for the removal, i.e. reduction, of molecular oxygen have been identified in T. vaginalis flavin reductase, formerly designated NADPH oxidase, which indirectly reduces oxygen to hydrogen peroxide via flavin mononucleotide (FMN), and NADH oxidase which reduces oxygen to water. Flavin reductase has been identified and characterized at the gene level as well as enzymatically, but NADH oxidase has so far only been characterized enzymatically with enzyme isolated from T. vaginalis cell extracts. In this study, we identified NADH oxidase by mass spectrometry after isolation of the enzyme from gel bands positively staining for NADH oxidase activity. In strain C1 (ATCC 30001) which is known to lack NADH oxidase activity completely, the NADH oxidase gene has a deletion at position 1540 of the open reading frame leading to a frame shift and, as a consequence, to premature termination of the encoded polypeptide.
Topics: Mass Spectrometry; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Open Reading Frames; Sequence Deletion; Trichomonas vaginalis
PubMed: 31853623
DOI: 10.1007/s00436-019-06572-8 -
Chembiochem : a European Journal of... Nov 2019The αββα tryptophan synthase (TS), which is part of primary metabolism, is a paradigm for allosteric communication in multienzyme complexes. In particular, the...
The αββα tryptophan synthase (TS), which is part of primary metabolism, is a paradigm for allosteric communication in multienzyme complexes. In particular, the intrinsically low catalytic activity of the α-subunit TrpA is stimulated several hundredfold through the interaction with the β-subunit TrpB1. The BX1 protein from Zea mays (zmBX1), which is part of secondary metabolism, catalyzes the same reaction as that of its homologue TrpA, but with high activity in the absence of an interaction partner. The intrinsic activity of TrpA can be significantly increased through the exchange of several active-site loop residues, which mimic the corresponding loop in zmBX1. The subsequent identification of activating amino acids in the generated "stand-alone" TrpA contributes to an understanding of allostery in TS. Moreover, findings suggest an evolutionary trajectory that describes the transition from a primary metabolic enzyme regulated by an interaction partner to a self-reliant, stand-alone, secondary metabolic enzyme.
Topics: Amino Acid Sequence; Biocatalysis; Biological Evolution; Catalytic Domain; Kinetics; Models, Molecular; Multienzyme Complexes; Plant Proteins; Protein Subunits; Sequence Homology, Amino Acid; Tryptophan Synthase; Zea mays
PubMed: 31090986
DOI: 10.1002/cbic.201900323 -
Nucleic Acids Research Feb 2016Archaeosine (G(+)), which is found only at position 15 in many archaeal tRNA, is formed by two steps, the replacement of the guanine base with preQ0 by archaeosine...
Archaeosine (G(+)), which is found only at position 15 in many archaeal tRNA, is formed by two steps, the replacement of the guanine base with preQ0 by archaeosine tRNA-guanine transglycosylase (ArcTGT) and the subsequent modification of preQ0 to G(+) by archaeosine synthase. However, tRNA(Leu) from Thermoplasma acidophilum, a thermo-acidophilic archaeon, exceptionally has two G(+)13 and G(+)15 modifications. In this study, we focused on the biosynthesis mechanism of G(+)13 and G(+)15 modifications in this tRNA(Leu). Purified ArcTGT from Pyrococcus horikoshii, for which the tRNA recognition mechanism and structure were previously characterized, exchanged only the G15 base in a tRNA(Leu) transcript with (14)C-guanine. In contrast, T. acidophilum cell extract exchanged both G13 and G15 bases. Because T. acidophilum ArcTGT could not be expressed as a soluble protein in Escherichia coli, we employed an expression system using another thermophilic archaeon, Thermococcus kodakarensis. The arcTGT gene in T. kodakarensis was disrupted, complemented with the T. acidophilum arcTGT gene, and tRNA(Leu) variants were expressed. Mass spectrometry analysis of purified tRNA(Leu) variants revealed the modifications of G(+)13 and G(+)15 in the wild-type tRNA(Leu). Thus, T. acidophilum ArcTGT has a multisite specificity and is responsible for the formation of both G(+)13 and G(+)15 modifications.
Topics: Gene Expression Regulation, Enzymologic; Glycoside Hydrolases; Multienzyme Complexes; Pyrococcus horikoshii; RNA, Transfer; Thermoplasma; Transferases
PubMed: 26721388
DOI: 10.1093/nar/gkv1522