AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides.
AuthorsMasuda S, Bauer CE
JournalCell
PubMed ID12230978
Photosynthetic bacteria regulate photosystem synthesis in response to alterations in oxygen tension and light intensity. In this study we show that the PpsR repressor from Rhodobacter sphaeroides binds to DNA in a redox-dependent manner through the formation/breakage of an intramolecular disulfide bond. We also demonstrate that PpsR is antagonized by ... More
Protein-protein interactions between UDP-glucuronosyltransferase isozymes in rat hepatic microsomes.
AuthorsIkushiro S, Emi Y, Iyanagi T
JournalBiochemistry
PubMed ID9188715
'The interactions between UDP-glucuronosyltransferase (UGT) isozymes, UGT1s and UGT2B1, in rat hepatic microsomes were investigated using an immunopurification technique with anti-peptide antibodies and a chemical cross-linking strategy. A 50 kDa protein coimmunopurified with UGT1s was identified as UGT2B1 by amino-terminal sequencing and immunodetection with anti-peptide antibody against UGT2B1. Evidence for ... More
Extracellular DsbA-insensitive folding of Escherichia coli heat-stable enterotoxin STa in vitro.
AuthorsBatisson I, der Vartanian M
JournalJ Biol Chem
PubMed ID10744753
'To study the folding of human Escherichia coli heat-stable enterotoxin STh, we used the major protein subunit of CS31A fimbriae (ClpG) as a marker of STh secretion and a provider of a signal peptide. We established that STh genetically fused to the N or C terminus of ClpG was able ... More
Investigation of the extracellular accessibility of the connecting loop between membrane domains I and II of the bradykinin B2 receptor.
AuthorsQuitterer U, Zaki E, AbdAlla S
JournalJ Biol Chem
PubMed ID10329674
'In analogy to the structure of rhodopsin, the seven hydrophobic segments of G-protein-coupled receptors are supposed to form seven membrane-spanning alpha-helices. To analyze the topology of the bradykinin B2 receptor, we raised site-directed antibodies to peptides corresponding to the loop regions and the amino and carboxyl terminus of this receptor. ... More
Cysteine-scanning mutagenesis reveals a highly amphipathic, pore-lining membrane-spanning helix in the glutamate transporter GltT.
AuthorsSlotboom DJ, Konings WN, Lolkema JS
JournalJ Biol Chem
PubMed ID11148213
'The carboxyl-terminal membrane-spanning segment 8 of the glutamate transporter GltT of Bacillus stearothermophilus was studied by cysteine-scanning mutagenesis. 21 single cysteine mutants were constructed in a stretch ranging from Gly-374 to Gln-404. Two mutants were not expressed, four were inactive, and two showed severely reduced glutamate transport activity. Cysteine mutations ... More
DsbB catalyzes disulfide bond formation de novo.
AuthorsRegeimbal J, Bardwell JC
JournalJ Biol Chem
PubMed ID12072444
'DsbA and DsbB are responsible for disulfide bond formation. DsbA is the direct donor of disulfides, and DsbB oxidizes DsbA. DsbB has the unique ability to generate disulfides by quinone reduction. It is thought that DsbB oxidizes DsbA via thiol disulfide exchange. In this mechanism, a disulfide is formed across ... More
A novel topology and redox regulation of the rat brain K+-dependent Na+/Ca2+ exchanger, NCKX2.
AuthorsCai X, Zhang K, Lytton J
JournalJ Biol Chem
PubMed ID12377762
'In this study we have examined the roles of endogenous cysteine residues in the rat brain K(+)-dependent Na(+)/Ca(2+) exchanger protein, NCKX2, by site-directed mutagenesis. We found that mutation of Cys-614 or Cys-666 to Ala inhibited expression of the exchanger protein in HEK-293 cells, but not in an in vitro translation ... More
Transmembrane topography of the 100-kDa a subunit (Vph1p) of the yeast vacuolar proton-translocating ATPase.
AuthorsLeng XH, Nishi T, Forgac M
JournalJ Biol Chem
PubMed ID10329659
'The membrane topography of the yeast vacuolar proton-translocating ATPase a subunit (Vph1p) has been investigated using cysteine-scanning mutagenesis. A Cys-less form of Vph1p lacking the seven endogenous cysteines was constructed and shown to have 80% of wild type activity. Single cysteine residues were introduced at 13 sites within the Cys-less ... More
The first putative transmembrane segment of subunit c" (Vma16p) of the yeast V-ATPase is not necessary for function.
AuthorsNishi T, Kawasaki-Nishi S, Forgac M
JournalJ Biol Chem
PubMed ID12482875
'The yeast vacuolar ATPase (V-ATPase) contains three proteolipid subunits: c (Vma3p), c' (Vma11p), and c" (Vma16p). Each subunit contains a buried glutamate residue that is essential for function, and these subunits are not able to substitute for each other in supporting activity. Subunits c and c' each contain four putative ... More
Thioredoxin 2 is involved in the oxidative stress response in Escherichia coli.
AuthorsRitz D, Patel H, Doan B, Zheng M, Aslund F, Storz G, Beckwith J
JournalJ Biol Chem
PubMed ID10644706
'Two genes encoding thioredoxin are found on the Escherichia coli genome. Both of them are capable of reducing protein disulfide bonds in vivo and in vitro. The catalytic site contains a Cys-X(1)-X(2)-Cys motif in a so-called thioredoxin fold. Thioredoxin 2 has two additional pairs of cysteines in a non-conserved N-terminal ... More
Identification of amino acids in the binding pocket of the human KDEL receptor.
AuthorsScheel AA, Pelham HR
JournalJ Biol Chem
PubMed ID9442098
'Retention of soluble proteins in the endoplasmic reticulum is dependent on their interaction with the KDEL (Lys-Asp-Glu-Leu) receptor in the Golgi apparatus and their subsequent retrieval back to the endoplasmic reticulum. We have studied the three-dimensional organization of the human KDEL receptor using site-directed mutagenesis and sulfhydryl-specific labeling. We have ... More
Pegylation: a method for assessing topological accessibilities in Kv1.3.
AuthorsLu J, Deutsch C
JournalBiochemistry
PubMed ID11683639
'Each subunit of a voltage-gated potassium channel (Kv) contains six putative transmembrane segments, S1-S6, and a cytosolic N-terminal recognition domain, T1. Although it is well-established that Kv channels are tetrameric structures, the protein-protein, protein-lipid, and protein-aqueous interfaces are not precisely mapped. The topological accessibility of specific amino acids may help ... More
Glutathione directly reduces an oxidoreductase in the endoplasmic reticulum of mammalian cells.
AuthorsJessop CE, Bulleid NJ
JournalJ Biol Chem
PubMed ID15507438
'The formation of disulfide bonds is an essential step in the folding of many glycoproteins and secretory proteins. Non-native disulfide bonds are often formed between incorrect cysteine residues, and thus the cell has dedicated a family of oxidoreductases that are thought to isomerize non-native bonds. For an oxidoreductase to be ... More
Membrane insertion kinetics of a protein domain in vivo. The bacterioopsin n terminus inserts co-translationally.
AuthorsDale H, Krebs MP
JournalJ Biol Chem
PubMed ID10428851
'The pathway by which segments of a polytopic membrane protein are inserted into the membrane has not been resolved in vivo. We have developed an in vivo kinetic assay to examine the insertion pathway of the polytopic protein bacterioopsin, the apoprotein of Halobacterium salinarum bacteriorhodopsin. Strains were constructed that express ... More
Membrane topology of the transposon 10-encoded metal-tetracycline/H+ antiporter as studied by site-directed chemical labeling.
AuthorsKimura T, Ohnuma M, Sawai T, Yamaguchi A
JournalJ Biol Chem
PubMed ID8995300
'The transposon (Tn) 10-encoded metal-tetracycline/H+ antiporter (Tn10-TetA) is predicted to have a membrane topology involving 12 transmembrane domains on the basis of the hydropathy profile of its sequence and the results of limited proteolysis; however, the experimental results of limited proteolysis are not enough to confirm the topology because proteases ... More
Membrane topology of the di- and tripeptide transport protein of Lactococcus lactis.
AuthorsHagting A, vd Velde J, Poolman B, Konings WN
JournalBiochemistry
PubMed ID9184160
'Transport of hydrophilic di- and tripeptides into Lactococcus lactis is mediated by a proton motive force-driven peptide transport protein (DtpT) that shares similarity with eukaryotic peptide transporters, e.g., from kidney and small intestine of rabbit, man, and rat. Hydropathy profiling in combination with the "positive inside rule" predicts for most ... More
Analysis of the membrane topology for transmembrane domains 7-12 of the human reduced folate carrier by scanning cysteine accessibility methods.
AuthorsCao W, Matherly LH
JournalBiochem J
PubMed ID14602046
'The hRFC (human reduced folate carrier) is the major membrane transporter for both reduced folates and antifolates in human tissues and tumours. The primary amino acid sequence of hRFC predicts a membrane topology involving 12 TMDs (transmembrane domains) with cytosolic oriented N- and C-termini, and a large internal loop connecting ... More
The effects of glutaredoxin and copper activation pathways on the disulfide and stability of Cu,Zn superoxide dismutase.
'Mutations in Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS) through mechanisms proposed to involve SOD1 misfolding, but the intracellular factors that modulate folding and stability of SOD1 are largely unknown. By using yeast and mammalian expression systems, we demonstrate here that SOD1 stability is governed by post-translational ... More
Cysteine-scanning mutagenesis around transmembrane segments 1 and 11 and their flanking loop regions of Tn10-encoded metal-Tetracycline/H+ antiporter.
AuthorsKimura-Someya T, Iwaki S, Konishi S, Tamura N, Kubo Y, Yamaguchi A
JournalJ Biol Chem
PubMed ID10747900
'Putative transmembrane helices (TM) 1 and 11 in the metal-tetracycline/H(+) antiporter are predicted to be close to each other on the basis of disulfide cross-linking experiments of the double-cysteine mutants in the periplasmic loop regions (Kubo, Y., Konishi, S., Kawabe, T., Nada, S., and Yamaguchi, A. (2000) J. Biol. Chem. ... More
Topology of the region surrounding Glu681 of human AE1 protein, the erythrocyte anion exchanger.
AuthorsTang XB, Fujinaga J, Kopito R, Casey JR
JournalJ Biol Chem
PubMed ID9712881
'AE1 protein transports Cl- and HCO3- across the erythrocyte membrane by an electroneutral exchange mechanism. Glu681 of human AE1 may form part of the anion translocation apparatus and the permeability barrier. We have therefore studied the structure of the sequence surrounding Glu681, using scanning cysteine mutagenesis. Residues of the Ser643 ... More
Two pairs of conserved cysteines are required for the oxidative activity of Ero1p in protein disulfide bond formation in the endoplasmic reticulum.
AuthorsFrand AR, Kaiser CA
JournalMol Biol Cell
PubMed ID10982384
'In the major pathway for protein disulfide-bond formation in the endoplasmic reticulum (ER), oxidizing equivalents flow from the conserved ER-membrane protein Ero1p to secretory proteins via protein disulfide isomerase (PDI). Herein, a mutational analysis of the yeast ERO1 gene identifies two pairs of conserved cysteines likely to form redox-active disulfide ... More
The structure of precursor proteins during import into mitochondria.
AuthorsSchwartz MP, Huang S, Matouschek A
JournalJ Biol Chem
PubMed ID10212260
Precursor proteins must be at least partially unfolded during import into mitochondria, but their actual conformation during translocation is not known. Are proteins fully unfolded and threaded through the import machinery amino acid by amino acid, or do they retain some partial structure? The folding pathway of most proteins in ... More
Transmembrane electron transfer by the membrane protein DsbD occurs via a disulfide bond cascade.
AuthorsKatzen F, Beckwith J
JournalCell
PubMed ID11114333
The cytoplasmic membrane protein DsbD transfers electrons from the cytoplasm to the periplasm of E. coli, where its reducing power is used to maintain cysteines in certain proteins in the reduced state. We split DsbD into three structural domains, each containing two essential cysteines. Remarkably, when coexpressed, these truncated proteins ... More
DsbD-catalyzed transport of electrons across the membrane of Escherichia coli.
AuthorsKrupp R, Chan C, Missiakas D
JournalJ Biol Chem
PubMed ID11085993
Dsb proteins catalyze folding and oxidation of polypeptides in the periplasm of Escherichia coli. DsbC reduces wrongly paired disulfides by transferring electrons from its catalytic dithiol motif (98)CGYC. Genetic evidence suggests that recycling of this motif requires at least three proteins, the cytoplasmic thioredoxin reductase (TrxB) and thioredoxin (TrxA) as ... More
Mitochondria can import artificial precursor proteins containing a branched polypeptide chain or a carboxy-terminal stilbene disulfonate.
AuthorsVestweber D, Schatz G
JournalJ Cell Biol
PubMed ID2848848
A purified, artificial precursor protein was used as a transport vehicle to test the tolerance of the mitochondrial protein import system. The precursor was a fusion protein consisting of mouse dihydrofolate reductase linked to a yeast mitochondrial presequence; it contained a unique cysteine as its COOH-terminal residue. This COOH-terminal cysteine ... More
Human mitochondrial thioredoxin. Involvement in mitochondrial membrane potential and cell death.
AuthorsDamdimopoulos AE, Miranda-Vizuete A, Pelto-Huikko M, Gustafsson JA, Spyrou G.
JournalJ Biol Chem
PubMed ID12080052
Thioredoxins (Trx) are a class of small multifunctional redox-active proteins found in all organisms. Recently, we reported the cloning of a mitochondrial thioredoxin, Trx2, from rat heart. To investigate the biological role of Trx2 we have isolated the human homologue, hTrx2, and generated HEK-293 cells overexpressing Trx2 (HEK-Trx2). Here, we ... More
Complete pathway for protein disulfide bond formation encoded by poxviruses.
AuthorsSenkevich TG, White CL, Koonin EV, Moss B
JournalProc Natl Acad Sci U S A
PubMed ID11983854
We show that three cytoplasmic thiol oxidoreductases encoded by vaccinia virus comprise a complete pathway for formation of disulfide bonds in intracellular virion membrane proteins. The pathway was defined by analyzing conditional lethal mutants and effects of cysteine to serine substitutions and by trapping disulfide-bonded heterodimer intermediates for each consecutive ... More
Reconstitution of a protein disulfide catalytic system.
AuthorsBader M, Muse W, Zander T, Bardwell J
JournalJ Biol Chem
PubMed ID9553083
Disulfide bonds are important for the structure and stability of many proteins. In prokaryotes their formation is catalyzed by the Dsb proteins. The DsbA protein acts as a direct donor of disulfides to newly synthesized periplasmic proteins. Genetic evidence suggests that a second protein called DsbB acts to specifically reoxidize ... More
Functional differences in yeast protein disulfide isomerases.
PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of ... More
Interactions of a fluorescently labeled peptide with kringle domains in proteins.
AuthorsBalciunas A, Fless GM, Scanu AM, Copeland RA
JournalJ Protein Chem
PubMed ID8381284
The tripeptide Lys-Cys-Lys has been synthesized and covalently labeled at the cysteine sulfhydryl with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid to produce a fluorescent labeled peptide (FLP). When excited at 340 nm, the FLP fluorescence strongly with maximal intensity at 405 nm. Addition of proteins containing the kringle lysine-binding domain, such as human lipoprotein ... More
In vitro and in vivo redox states of the Escherichia coli periplasmic oxidoreductases DsbA and DsbC.
AuthorsJoly JC, Swartz JR
JournalBiochemistry
PubMed ID9254601
DsbC is a periplasmic protein of Escherichia coli that was previously identified by a genetic selection that rescued sensitivity to dithiothreitol in Tn10 mutagenized cells. The Erwinia chrysanthemi dsbC gene was identified in a previous genetic screen to restore motility in a dsbA null strain. In order to analyze the ... More
Characterization of the ERp57-Tapasin complex by rapid cellular acidification and thiol modification.
AuthorsAntoniou AN, Powis SJ
JournalAntioxid Redox Signal
PubMed ID13678524
Major histocompatibility complex (MHC) class I molecules bind and present short peptides to cells of the immune system. The oxidoreductase ERp57 is involved in the assembly of MHC class I molecules and is a component of the peptide loading complex, where it is found disulfide-bonded to tapasin. We have studied ... More
Membrane topology of the beta-subunit of the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae.
AuthorsJockel P, Di Berardino M, Dimroth P
JournalBiochemistry
PubMed ID10521253
The topology of the beta-subunit of the oxaloacetate Na+ pump (OadB) was probed with the alkaline phosphatase (PhoA) and beta-galactosidase (lacZ) fusion technique. Additional evidence for the topology was derived from amino acid alignments and comparative hydropathy profiles of OadB with related proteins. Consistent results were obtained for the three ... More
Membrane topology of subunit a of the F1F0 ATP synthase as determined by labeling of unique cysteine residues.
AuthorsLong JC, Wang S, Vik SB
JournalJ Biol Chem
PubMed ID9632682
The membrane topology of the a subunit of the F1F0 ATP synthase from Escherichia coli has been probed by surface labeling using 3-(N-maleimidylpropionyl) biocytin. Subunit a has no naturally occurring cysteine residues, allowing unique cysteines to be introduced at the following positions: 8, 24, 27, 69, 89, 128, 131, 172, ... More
Translocator proteins in the two-partner secretion family have multiple domains.
AuthorsSurana NK, Buscher AZ, Hardy GG, Grass S, Kehl-Fie T, St Geme JW
JournalJ Biol Chem
PubMed ID16648638
The two-partner secretion pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane translocator protein. Previous work has demonstrated that the TpsB protein forms a beta-barrel structure with pore forming activity and facilitates translocation of the TpsA protein across the outer membrane. In this study, ... More
Transmembrane topography of subunit a in the Escherichia coli F1F0 ATP synthase.
AuthorsValiyaveetil FI, Fillingame RH
JournalJ Biol Chem
PubMed ID9632683
Subunit a is the least understood of the three subunits that compose the F0 sector in the Escherichia coli F0F1 ATP synthase. In this study, we have substituted Cys into predicted extramembranous loops of the protein and used chemical modification to obtain topographical information on the folding of subunit a. ... More
Topology of the integral membrane form of Escherichia coli SecA protein reveals multiple periplasmically exposed regions and modulation by ATP binding.
AuthorsRamamurthy V, Oliver D
JournalJ Biol Chem
PubMed ID9287332
SecA insertion and integration into the Escherichia coli inner membrane is a critical step for the catalysis of protein translocation across this layer. To understand this step further, SecA topology was investigated. To determine which regions of SecA are periplasmically exposed, right-side out membrane vesicles were prepared from strains synthesizing ... More
In vitro reconstituted Dictyostelium discoideum early endosome fusion is regulated by Rab7 but proceeds in the absence of ATP-Mg2+ from the bulk solution.
AuthorsLaurent O, Bruckert F, Adessi C, Satre M
JournalJ Biol Chem
PubMed ID9422733
We characterized the in vitro fusion of endosomal compartments from Dictyostelium discoideum. Fusion activity was restricted to early compartments, was dependent on cytosolic proteins, and was activated by GTP and guanosine 5'-O(3-thio)triphosphate (GTPgammaS). This stimulation suggests the involvement of a small G protein, which we propose to be Rab7 on ... More
Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications.
AuthorsGhosh SS, Sappal BS, Kalpana GV, Lee SW, Chowdhury JR, Chowdhury NR
JournalJ Biol Chem
PubMed ID11546782
Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat ... More
Behavior in the eukaryotic secretory pathway of insulin-containing fusion proteins and single-chain insulins bearing various B-chain mutations.
AuthorsZhang BY, Liu M, Arvan P
JournalJ Biol Chem
PubMed ID12446709
In the secretory pathway, endoproteolytic cleavage of the insulin precursor protein promotes a change in the biophysical properties of the processed insulin product, and this may be relevant for its intracellular trafficking. We have now studied several independent point mutants contained within the insulin B-chain, S9D, H10D, V12E (called B9D, ... More
Remote conformational effects of the Gly-62 --> Leu mutation of the Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli and its second-site suppressor mutation.
AuthorsKimura T, Sawai T, Yamaguchi A
JournalBiochemistry
PubMed ID9188689
Substitution of Gly-62 of the Tn10-encoded metal-tetracycline/H+ antiporter caused a functional defect corresponding to the volume of the substituent [Yamaguchi, A., Someya, Y., and Sawai, T. (1992) J. Biol. Chem. 267, 19155-19162]. A spontaneous revertant exhibiting tetracycline resistance was isolated from Escherichia coli cells carrying the tetA(B) gene encoding the ... More
Reconstitution of drug transport by purified P-glycoprotein.
AuthorsShapiro AB, Ling V
JournalJ Biol Chem
PubMed ID7608182
P-glycoprotein confers multidrug resistance upon cells in which it is highly expressed, reducing the effectiveness of numerous cytotoxic drugs, including many of those used for chemotherapy of cancer. Although P-glycoprotein is widely believed to function as an ATP-dependent drug efflux pump, the unusually broad substrate specificity of P-glycoprotein has engendered ... More
Transmembrane segment (TMS) VIII of the Na(+)/Citrate transporter CitS requires downstream TMS IX for insertion in the Escherichia coli membrane.
Authorsvan Geest M, Lolkema JS
JournalJ Biol Chem
PubMed ID10514443
The amino acid sequence of the sodium ion-dependent citrate transporter CitS of K. pneumoniae contains 12 hydrophobic stretches that could form membrane-spanning segments. A previous analysis of the membrane topology in Escherichia coli using the PhoA gene fusion technique indicated that only nine of these hydrophobic segments span the membrane, ... More
Characterization of a cysteine-less human reduced folate carrier: localization of a substrate-binding domain by cysteine-scanning mutagenesis and cysteine accessibility methods.
AuthorsCao W, Matherly LH
JournalBiochem J
PubMed ID12749765
The human reduced folate carrier (hRFC) mediates the transport of reduced folates and classical anti-folates into mammalian cells. Whereas the functionally important domains in hRFC are poorly characterized, previous studies with anti-folate-resistant cells suggest critical roles for transmembrane domain (TMD) 1 and residues (Gly44, Glu45, Ser46 and Ile48) in or ... More
Glutaredoxin modulates platelet-derived growth factor-dependent cell signaling by regulating the redox status of low molecular weight protein-tyrosine phosphatase.
AuthorsKanda M, Ihara Y, Murata H, Urata Y, Kono T, Yodoi J, Seto S, Yano K, Kondo T
JournalJ Biol Chem
PubMed ID16893901
Glutaredoxin (GRX) is a glutathione-disulfide oxidoreductase involved in various cellular functions, including the redox-dependent regulation of certain integral proteins. Here we demonstrated that overexpression of GRX suppressed the proliferation of myocardiac H9c2 cells treated with platelet-derived growth factor (PDGF)-BB. After stimulation with PDGF-BB, the phosphorylation of PDGF receptor (PDGFR) beta ... More
Identification of two conformationally sensitive cysteine residues at the extracellular surface of the Na,K-ATPase alpha-subunit.
AuthorsLutsenko S, Daoud S, Kaplan JH
JournalJ Biol Chem
PubMed ID9030596
Na,K-ATPase in right-side-out oriented vesicles was stabilized in different conformations, and the location of intramembrane Cys residues of the alpha-subunit was assessed with membrane-permeable and membrane-impermeable Cys-directed reagents. In the presence of Mg2+ and Pi, Cys964 was the most accessible for both membrane-impermeable 4-acetamido-4'-maleimidylstilbene-2, 2'disulfonic acid (or stilbene disulfonate maleimide, ... More
Importance of the carboxyl terminus in the folding and function of alpha-hemolysin of Staphylococcus aureus.
AuthorsSangha N, Kaur S, Sharma V, Krishnasastry MV
JournalJ Biol Chem
PubMed ID10092591
The physical state of two model mutants of alpha-hemolysin (alphaHL), alphaHL(1-289), a carboxyl-terminal deletion mutant (CDM), and alphaHL(1-331), a carboxyl-terminal extension mutant (CEM), were examined in detail to identify the role of the carboxyl terminus in the folding and function of native alphaHL. Denatured alphaHL can be refolded efficiently with ... More
Membrane topology of a cysteine-less mutant of human P-glycoprotein.
AuthorsLoo TW, Clarke DM
JournalJ Biol Chem
PubMed ID7822320
A human P-glycoprotein devoid of cysteine residues was constructed by site-directed mutagenesis for studying its topology. The cDNA for human P-glycoprotein-A52 in which codons for cysteines 137, 431, 717, 956, 1074, 1125, 1227, 1288, and 1304 were changed to Ala, was transfected into NIH 3T3 cells and analyzed with respect ... More
Membrane topology prediction by hydropathy profile alignment: membrane topology of the Na(+)-glutamate transporter GltS.
AuthorsDobrowolski A, Sobczak-Elbourne I, Lolkema JS
JournalBiochemistry
PubMed ID17269795
Structural classification of families of membrane proteins by bioinformatics techniques has become a critical aspect of membrane protein research. We have proposed hydropathy profile alignment to identify structural homology between families of membrane proteins. Here, we demonstrate experimentally that two families of secondary transporters, the ESS and 2HCT families, indeed ... More
Kinetics of structural changes of reconstituted skeletal muscle thin filaments observed by fluorescence resonance energy transfer.
AuthorsMiki M, Iio T
JournalJ Biol Chem
PubMed ID8463245
Fluorescence resonance energy transfer was measured between probes attached to Troponin-I and actin in the reconstituted skeletal muscle thin filament. Cys-133 on TnI was labeled with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, and Lys-61 on actin was labeled with fluorescein 5-isothiocyanate. A large difference in the efficiency of resonance energy transfer was observed between ... More
Stilbene disulfonic acids inhibit synexin-mediated membrane aggregation and fusion.
AuthorsLiu L, Chander A
JournalBiochim Biophys Acta
PubMed ID7857967
Stilbene disulfonic acids inhibit surfactant secretion from lung epithelial type II cells by an undefined mechanism, and inhibit CD4 mediated cell-cell fusion. We have previously shown that lung synexin promotes in vitro fusion of lamellar bodies and plasma membranes, an obligatory process for surfactant secretion. This study investigates the effect ... More
Thiol-specific biotinylation of the insulin receptor in permeabilized cells enhances receptor function.
AuthorsBernier M, Nadiv O, Kole HK
JournalBiochemistry
PubMed ID7599127
We examined the reactivity of insulin receptor sulfhydryls to biotinylation in Chinese hamster ovary cells that express high levels of human insulin receptors (CHO/HIRc cells). Following the biotinylation reaction, the insulin receptor was purified by immunoprecipitation, and resolved by SDS-polyacrylamide gel electrophoresis before electrotransfer to membranes. The use of enzyme-linked ... More
Cysteine-scanning mutagenesis of transmembrane segments 4 and 5 of the Tn10-encoded metal-tetracycline/H+ antiporter reveals a permeability barrier in the middle of a transmembrane water-filled channel.
AuthorsIwaki S, Tamura N, Kimura-Someya T, Nada S, Yamaguchi A
JournalJ Biol Chem
PubMed ID10930423
Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, ... More