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The Journal of Biological Chemistry Apr 2018Voltage-gated, sodium ion-selective channels (Na) generate electrical signals contributing to the upstroke of the action potential in animals. Nas are also found in...
Voltage-gated, sodium ion-selective channels (Na) generate electrical signals contributing to the upstroke of the action potential in animals. Nas are also found in bacteria and are members of a larger family of tetrameric voltage-gated channels that includes Cas, Ks, and Nas. Prokaryotic Nas likely emerged from a homotetrameric Ca-selective voltage-gated progenerator, and later developed Na selectivity independently. The Na signaling complex in eukaryotes contains auxiliary proteins, termed beta (β) subunits, which are potent modulators of the expression profiles and voltage-gated properties of the Na pore, but it is unknown whether they can functionally interact with prokaryotic Na channels. Herein, we report that the eukaryotic Naβ1-subunit isoform interacts with and enhances the surface expression as well as the voltage-dependent gating properties of the bacterial Na, NaChBac in oocytes. A phylogenetic analysis of the β-subunit gene family proteins confirms that these proteins appeared roughly 420 million years ago and that they have no clear homologues in bacterial phyla. However, a comparison between eukaryotic and bacterial Na structures highlighted the presence of a conserved fold, which could support interactions with the β-subunit. Our electrophysiological, biochemical, structural, and bioinformatics results suggests that the prerequisites for β-subunit regulation are an evolutionarily stable and intrinsic property of some voltage-gated channels.
Topics: Amino Acid Sequence; Animals; Bacteria; Bacterial Proteins; Evolution, Molecular; HEK293 Cells; Humans; Models, Molecular; Phylogeny; Protein Conformation; Protein Subunits; Rats; Voltage-Gated Sodium Channels; Xenopus
PubMed: 29371400
DOI: 10.1074/jbc.RA117.000852 -
Nucleic Acids Research Dec 2015Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein...
Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1-2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qβ replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.
Topics: Allolevivirus; Dimerization; Escherichia coli Proteins; Genome, Viral; Mutation; Protein Binding; Protein Structure, Tertiary; Protein Subunits; Q beta Replicase; RNA, Viral; Ribosomal Proteins; Virus Replication
PubMed: 26578560
DOI: 10.1093/nar/gkv1212 -
The Journal of Biological Chemistry Jun 2017Sucrase-isomaltase (SI) is an intestinal membrane-associated α-glucosidase that breaks down di- and oligosaccharides to absorbable monosaccharides. SI has two...
Sucrase-isomaltase (SI) is an intestinal membrane-associated α-glucosidase that breaks down di- and oligosaccharides to absorbable monosaccharides. SI has two homologous functional subunits (sucrase and isomaltase) that both belong to the glycoside hydrolase family 31 (GH31) and differ in substrate specificity. All GH31 enzymes share a consensus sequence harboring an aspartic acid residue as a catalytic nucleophile. Moreover, crystallographic structural analysis of isomaltase predicts that another aspartic acid residue functions as a proton donor in hydrolysis. Here, we mutagenized the predicted proton donor residues and the nucleophilic catalyst residues in each SI subunit. We expressed these SI variants in COS-1 cells and analyzed their structural, transport, and functional characteristics. All of the mutants revealed expression levels and maturation rates comparable with those of the wild-type species and the corresponding nonmutated subunits were functionally active. Thereby we determined rate and substrate specificity for each single subunit without influence from the other subunit. This approach provides a model for functional analysis of the single subunits within a multidomain protein, achieved without the necessity to express the individual subunits separately. Of note, we also found that glucose product inhibition regulates the activities of both SI subunits. We experimentally confirmed the catalytic function of the predicted proton donor residues, and sequence analysis suggested that these residues are located in a consensus region in many GH31 family members. In summary, these findings reveal the kinetic features specific for each human SI subunit and demonstrate that the activities of these subunits are regulated via product inhibition.
Topics: Animals; COS Cells; Chlorocebus aethiops; Humans; Models, Molecular; Protein Subunits; Structure-Activity Relationship; Sucrase-Isomaltase Complex
PubMed: 28522605
DOI: 10.1074/jbc.M117.791939 -
Cell Cycle (Georgetown, Tex.) Jan 2009Septins are guanine nucleotide-binding proteins that form hetero-oligomeric complexes, which assemble into filaments and higher-order structures at sites of cell... (Review)
Review
Septins are guanine nucleotide-binding proteins that form hetero-oligomeric complexes, which assemble into filaments and higher-order structures at sites of cell division and morphogenesis in eukaryotes. Dynamic changes in the organization of septin-containing structures occur concomitantly with progression through the mitotic cell cycle and during cell differentiation. Septins also undergo stage-specific post-translational modifications, which have been implicated in regulating their dynamics, in some cases via purported effects on septin turnover. In our recent study, the fate of two of the five septins expressed in mitotic cells of budding yeast (Saccharomyces cerevisiae) was tracked using two complementary fluorescence-based methods for pulse-chase analysis. During mitotic growth, previously-made molecules of both septins (Cdc10 and Cdc12) persisted through multiple successive divisions and were incorporated equivalently with newly synthesized molecules into hetero-oligomers and higher-order structures. Similarly, in cells undergoing meiosis and the developmental program of sporulation, pre-existing copies of Cdc10 were incorporated into new structures. In marked contrast, Cdc12 was irreversibly excluded from septin complexes and replaced by another septin, Spr3. Here, we discuss the broader implications of these results and related findings with regard to how septin dynamics is coordinated with the mitotic cell cycle and in the yeast life cycle, and how these observations may relate to control of the dynamics of other complex multi-subunit assemblies.
Topics: Cytoskeletal Proteins; GTP-Binding Proteins; Meiosis; Mitosis; Models, Biological; Protein Subunits; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 19164941
DOI: 10.4161/cc.8.2.7381 -
The Analyst Jul 2020Native electrospray mass spectrometry is a powerful method for determining the native stoichiometry of many polydisperse multi-subunit biological complexes, including...
Native electrospray mass spectrometry is a powerful method for determining the native stoichiometry of many polydisperse multi-subunit biological complexes, including multi-subunit protein complexes and lipid-bound transmembrane proteins. However, when polydispersity results from incorporation of multiple copies of two or more different subunits, it can be difficult to analyze subunit stoichiometry using conventional mass spectrometry analysis methods, especially when m/z distributions for different charge states overlap in the mass spectrum. It was recently demonstrated by Marty and co-workers (K. K. Hoi, et al., Anal. Chem., 2016, 88, 6199-6204) that Fourier Transform (FT)-based methods can determine the bulk average lipid composition of protein-lipid Nanodiscs assembled with two different lipids, but a detailed statistical description of the composition of more general polydisperse two-subunit populations is still difficult to achieve. This results from the vast number of ways in which the two types of subunit can be distributed within the analyte ensemble. Here, we present a theoretical description of three common classes of heterogeneity for mixed-subunit analytes and demonstrate how to differentiate and analyze them using mass spectrometry and FT methods. First, we first describe FT-based analysis of mass spectra corresponding to simple superpositions, convolutions, and multinomial distributions for two or more different subunit types using model data sets. We then apply these principles with real samples, including mixtures of single-lipid Nanodiscs in the same solution (superposition), mixed-lipid Nanodiscs and copolymers (convolutions), and isotope distribution for ubiquitin (multinomial distribution). This classification scheme and the FT method used to study these analyte classes should be broadly useful in mass spectrometry as well as other techniques where overlapping, periodic signals arising from analyte mixtures are common.
Topics: 1,2-Dipalmitoylphosphatidylcholine; Fourier Analysis; Mass Spectrometry; Membrane Proteins; Nanostructures; Phosphatidylcholines; Polyethylene Glycols; Propylene Glycols; Protein Subunits
PubMed: 32459233
DOI: 10.1039/d0an00726a -
Molecules (Basel, Switzerland) Oct 2021Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria that is widely used as a fluorescent probe or as a colorant in the food and cosmetic...
Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria that is widely used as a fluorescent probe or as a colorant in the food and cosmetic industries. In this study, phycoerythrin was extracted from the red algae and purified by ammonium sulfate precipitation and various chromatography methods. The purified phycoerythrin was analyzed by UV-visible and fluorescence spectroscopy. The isolated pigment had the typical spectrum of R-phycoerythrin, with a trimmer state with absorbance maxima at 497, 536, and 565 nm. It was further purified and identified by LC-MS/MS and Mascot search. It showed a 100% sequence similarity with the R-phycoerythrin alpha subunit of . The molecular mass was 17.97 kDa. The antioxidant activity of the purified R-phycoerythrin alpha subunit was analyzed. It showed significant antioxidant activity in ABTS and FRAP assays and had significant cytotoxicity against HepG2 cells.
Topics: Amino Acid Sequence; Antineoplastic Agents; Antioxidants; Aquatic Organisms; Biological Products; Cell Line, Tumor; Cell Survival; Chemical Fractionation; Chromatography, Liquid; Dose-Response Relationship, Drug; Humans; Peptide Fragments; Phycoerythrin; Protein Subunits; Rhodophyta; Tandem Mass Spectrometry
PubMed: 34770894
DOI: 10.3390/molecules26216479 -
BMB Reports Nov 2011The Sec61α subunit is the core subunit of the protein conducting channel which is required for protein translocation in eukaryotes and prokaryotes. In this study, we...
The Sec61α subunit is the core subunit of the protein conducting channel which is required for protein translocation in eukaryotes and prokaryotes. In this study, we cloned a Sec61α subunit from Penicillium ochrochloron (PoSec61α). Sequence and 3D structural model analysis showed that PoSec61α conserved the typical characteristics of eukaryotic and prokaryotic Sec61α subunit homologues. The pore ring known as the constriction point of the channel is formed by seven hydrophobic amino acids. Two methionine residues from transmembrane α-helice 7 (TM7) contribute to the pore ring formation and projected notably to the pore area and narrowed the pore compared with the superposed residues at the corresponding positions in the crystal structures or the 3D models of the Sec61α subunit homologues in archaea or other eukaryotes, respectively. Results reported herein indicate that the pore ring residues differ among Sec61α subunit homologues and two hydrophobic residues in the TM7 contribute to the pore ring formation.
Topics: Amino Acid Sequence; Cloning, Molecular; Fungal Proteins; Genes, Fungal; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Penicillium; Phylogeny; Protein Subunits; Protein Transport; SEC Translocation Channels; Sequence Alignment; Sequence Analysis, Protein; Sequence Homology, Amino Acid; Structural Homology, Protein
PubMed: 22118537
DOI: 10.5483/BMBRep.2011.44.11.719 -
Cell Stress & Chaperones Nov 2019The eukaryotic cytosolic chaperonin, t-complex polypeptide 1 (TCP-1) ring complex or TRiC, is responsible for folding a tenth of the proteins in the cell. TRiC is a...
The eukaryotic cytosolic chaperonin, t-complex polypeptide 1 (TCP-1) ring complex or TRiC, is responsible for folding a tenth of the proteins in the cell. TRiC is a double-ringed barrel with each ring composed of eight different CCT (chaperonin containing TCP-1) subunits. In order for the subunits to assemble together into mature TRiC, which is believed to contain one and only one of each of these subunits per ring, they must be translated from different chromosomes, correctly folded and assembled. When expressed alone in Escherichia coli, the subunits CCT4 and CCT5, interestingly, form TRiC-like homo-oligomeric rings. To explore potential subunit-subunit interactions, we co-expressed these homo-oligomerizing CCT4 and CCT5 subunits or the archaeal chaperonin Mm-Cpn (Methanococcus maripaludis chaperonin) with CCT1-8, one at a time. We found that CCT5 shifted all of the CCT subunits, with the exception of CCT6, into double-barrel TRiC-like complexes, while CCT4 only interacted with CCT5 and CCT8 to form chaperonin rings. We hypothesize that these specific interactions may be due to the formation of hetero-oligomers in E. coli, although more work is needed for validation. We also observed the interaction of CCT5 and Mm-Cpn with smaller fragments of the CCT subunits, confirming their intrinsic chaperone activity. Based on this hetero-oligomer data, we propose that TRiC assembly relies on subunit exchange with some stable homo-oligomers, possibly CCT5, as base assembly units. Eventually, analysis of CCT arrangement in various tissues and at different developmental times is anticipated to provide additional insight on TRiC assembly and CCT subunit composition.
Topics: Chaperonin Containing TCP-1; Cloning, Molecular; Escherichia coli; Protein Folding; Protein Multimerization; Protein Subunits
PubMed: 31410727
DOI: 10.1007/s12192-019-01028-5 -
PLoS Biology May 2014The intrinsic flexibility of proteins allows them to undergo large conformational fluctuations in solution or upon interaction with other molecules. Proteins also...
The intrinsic flexibility of proteins allows them to undergo large conformational fluctuations in solution or upon interaction with other molecules. Proteins also commonly assemble into complexes with diverse quaternary structure arrangements. Here we investigate how the flexibility of individual protein chains influences the assembly and evolution of protein complexes. We find that flexibility appears to be particularly conducive to the formation of heterologous (i.e., asymmetric) intersubunit interfaces. This leads to a strong association between subunit flexibility and homomeric complexes with cyclic and asymmetric quaternary structure topologies. Similarly, we also observe that the more nonhomologous subunits that assemble together within a complex, the more flexible those subunits tend to be. Importantly, these findings suggest that subunit flexibility should be closely related to the evolutionary history of a complex. We confirm this by showing that evolutionarily more recent subunits are generally more flexible than evolutionarily older subunits. Finally, we investigate the very different explorations of quaternary structure space that have occurred in different evolutionary lineages. In particular, the increased flexibility of eukaryotic proteins appears to enable the assembly of heteromeric complexes with more unique components.
Topics: Animals; Apicomplexa; Arabidopsis; Bacteria; Evolution, Molecular; Fungi; Models, Molecular; Protein Multimerization; Protein Structure, Quaternary; Protein Subunits; Proteins
PubMed: 24866000
DOI: 10.1371/journal.pbio.1001870 -
Biochimica Et Biophysica Acta 2013Early structures of the cytochrome bc1 complex revealed heterogeneity in the position of the soluble portion of the Rieske iron sulfur protein subunit, implicating a... (Review)
Review
Early structures of the cytochrome bc1 complex revealed heterogeneity in the position of the soluble portion of the Rieske iron sulfur protein subunit, implicating a movement of this domain during function. Subsequent biochemical and biophysical works have firmly established that the motion of this subunit acts in the capacity of a conformationally assisted electron transfer step during the already complicated catalytic mechanism described within the modified version of Peter Mitchells Q cycle. How the movement of this subunit is initiated or how the frequency of its motion is controlled as a function of other steps during the catalysis remain topics of debate within the active research communities. This review addresses the historical aspects of the discovery and description of this movement, while attempting to provide a context for the involvement of conformational motion in the catalysis and efficiency of the enzyme. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.
Topics: Biocatalysis; Electron Transport; Electron Transport Complex III; Heme; Iron-Sulfur Proteins; Models, Chemical; Models, Molecular; Protein Conformation; Protein Subunits
PubMed: 23876289
DOI: 10.1016/j.bbabio.2013.07.007