stilbenes and 4-acetamido-4--maleimidylstilbene-2-2--disulfonic-acid

Hv1 (also named, voltage-sensor only protein, VSOP) lacks an authentic pore domain, and its voltage sensor domain plays both roles in voltage sensing and proton permeation. The activities of a proton channel are intrinsic to protomers of Hv1, while Hv1 is dimeric in biological membranes; cooperative gating is exerted by interaction between two protomers. As the signature pattern conserved among voltage-gated channels and voltage-sensing phosphatase, Hv1 has multiple arginines intervened by two hydrophobic residues on the fourth transmembrane segment, S4. S4 moves upward relative to other helices upon depolarization, causing conformational change possibly leading to the formation of a proton-selective conduction pathway. However, detailed mechanisms of proton-selectivity and gating of Hv1 are unknown. Here we took an approach of PEGylation protection assay to define residues facing the aqueous environment of mouse Hv1 (mHv1). Accessibilities of two maleimide molecules, N-ethylmaleimide (NEM) and 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), were examined on cysteine introduced into individual sites. Only the first arginine on S4 (R1: R201) was inaccessible by NEM and AMS in mHv1. This is consistent with previous results of electrophysiology on the resting state channel, suggesting that the accessibility profile represents the resting state of mHv1. D108, critical for proton selectivity, was accessible by AMS and NEM, suggesting that D108 faces the vestibule. F146, a site critical for blocking by a guanidinium-reagent, was accessible by NEM, suggesting that F146 also faces the inner vestibule. These findings suggest an inner vestibule lined by several residues on S2 including F146, D108 on S1, and the C-terminal half of S4.

Topics: Amino Acid Sequence; Animals; Ethylmaleimide; HEK293 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Ion Channel Gating; Ion Channels; Mice; Molecular Sequence Data; Point Mutation; Polyethylene Glycols; Protein Interaction Domains and Motifs; Protein Structure, Secondary; Protons; Species Specificity; Stilbenes; Structure-Activity Relationship; Sulfonic Acids; Water

C(4)-Dicarboxylate uptake transporter B (DcuB) of Escherichia coli is a bifunctional transporter that catalyzes fumarate/succinate antiport and serves as a cosensor of the sensor kinase DcuS. Sites and domains of DcuB were analyzed for their topology relative to the cytoplasmic or periplasmic side of the membrane and their accessibility to the water space. For the topology studies, DcuB was fused at 33 sites to the reporter enzymes PhoA and LacZ that are only active when located in the periplasm or the cytoplasm, respectively. The ratios of the PhoA and LacZ activities suggested the presence of 10 or 11 hydrophilic loops, and 11 or 12 α-helical transmembrane domains (TMDs). The central part of DcuB allowed no clear topology prediction with LacZ/PhoA fusions. The sites of DcuB accessible to the hydrophilic thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate (AMS) were determined with variants of DcuB that carried single Cys residues. After intact cells were labeled with the membrane-impermeable AMS, denatured cells were differentially labeled with the thiol reagent polyethylene-glycol-maleimide (PEGmal) and analyzed for a mass shift. From 35 positions 17 were accessible to AMS in intact bacteria. The model derived from topology and accessibility suggests 12 TMDs for DcuB and a waterfilled cavity in its central part. The cavity ends with a cytoplasmic lid accessible to AMS from the periplasmic side. The sensory domain of DcuB is composed of cytoplasmic loop XI/XII and a membrane integral region with the regulatory residues Thr396/Asp398 and Lys353.

Topics: Alkaline Phosphatase; Amino Acid Sequence; Bacterial Proteins; Catalytic Domain; Cell Membrane; Cysteine; Dicarboxylic Acid Transporters; Escherichia coli K12; Escherichia coli Proteins; Ethylmaleimide; Hydrophobic and Hydrophilic Interactions; Lac Operon; Models, Molecular; Molecular Sequence Data; Polyethylene Glycols; Protein Structure, Secondary; Recombinant Fusion Proteins; Stilbenes; Sulfonic Acids

Lactococcus lactis can decrease the redox potential at pH 7 (E(h7)) from 200 to -200 mV in oxygen free Man-Rogosa-Sharpe media. Neither the consumption of oxidizing compounds or the release of reducing compounds during lactic acid fermentation were involved in the decrease in E(h7) by the bacteria. Thiol groups located on the bacterial cell surface appear to be the main components that are able to establish a greater exchange current between the Pt electrode and the bacteria. After the final E(h7) (-200 mV) was reached, only thiol-reactive reagents could restore the initial E(h7) value. Inhibition of the proton motive force showed no effect on maintaining the final E(h7) value. These results suggest that maintaining the exofacial thiol (-SH) groups in a reduced state does not depend on an active mechanism. Thiol groups appear to be displayed by membrane proteins or cell wall-bound proteins and may participate in protecting cells against oxidative stress.

Topics: Carbonyl Cyanide m-Chlorophenyl Hydrazone; Culture Media, Conditioned; Dicyclohexylcarbodiimide; Electrochemistry; Ethylmaleimide; Fermentation; Hydrogen-Ion Concentration; Lactococcus lactis; Membrane Proteins; Nigericin; Oxidation-Reduction; Proton-Motive Force; Stilbenes; Sulfhydryl Compounds; Sulfhydryl Reagents; Sulfonic Acids; Valinomycin

The technique of rapid acidification and alkylation can be used to characterise the redox status of oxidoreductases, and to determine numbers of free cysteine residues within substrate proteins. We have previously used this method to analyse interacting components of the MHC class I pathway, namely ERp57 and tapasin. Here, we have applied rapid acidification/alkylation as a novel approach to analysing the redox status of MHC class I molecules. This analysis of the redox status of the MHC class I molecules HLA-A2 and HLA-B27, which is strongly associated with a group of inflammatory arthritic disorders referred to as Spondyloarthropathies, revealed structural and conformational information. We propose that this assay provides a useful tool in the study of in vivo MHC class I structure.

Topics: Alkylation; Cysteine; HLA-A2 Antigen; HLA-B27 Antigen; Humans; Hydrogen-Ion Concentration; Oxidation-Reduction; Oxidoreductases; Stilbenes; Sulfonic Acids; Trichloroacetic Acid

Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1.. Using yeast two-hybrid screen, we identified alpha (1,4)-glucan branching enzyme (GlgB) of M. tuberculosis as a interaction partner of WhiB1. In vitro GST pull down assay confirmed the direct physical interaction between GlgB and WhiB1. Both mass spectrometry data of tryptic digests and in vitro labeling of cysteine residues with 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid showed that in GlgB, C95 and C658 are free but C193 and C617 form an intra-molecular disulfide bond. WhiB1 has a C37XXC40 motif thus a C40S mutation renders C37 to exist as a free thiol to form a hetero-disulfide bond with the cysteine residue of substrate protein. A disulfide mediated binary complex formation between GlgB and WhiB1C40S was shown by both in-solution protein-protein interaction and thioredoxin affinity chromatography. Finally, transfer of reducing equivalent from WhiB1 to GlgB disulfide was confirmed by 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid trapping by the reduced disulfide of GlgB. Two different thioredoxins, TrxB/Rv1471 and TrxC/Rv3914 of M. tuberculosis could not perform this reaction suggesting that the reduction of GlgB by WhiB1 is specific.. We conclude that M. tuberculosis GlgB has one intra-molecular disulfide bond which is formed between C193 and C617. WhiB1, a thioredoxin like protein interacts with GlgB and transfers its electrons to the disulfide thus reduces the intra-molecular disulfide bond of GlgB. For the first time, we report that GlgB is one of the in vivo substrate of M. tuberculosis WhiB1.

Topics: 1,4-alpha-Glucan Branching Enzyme; Amino Acid Sequence; Bacterial Proteins; Chromatography, Affinity; Disulfides; Mass Spectrometry; Molecular Sequence Data; Mycobacterium tuberculosis; Oxidation-Reduction; Protein Interaction Domains and Motifs; Stilbenes; Sulfonic Acids; Thioredoxins; Two-Hybrid System Techniques

Redox-based regulation plays important roles in many cellular activities. Thioredoxins, one of the best characterized class of proteins involved in cellular redox regulation, are conserved components of eukaryotic ciliary/flagellar axonemal dyneins. Studies with Chlamydomonas showed that, under varying redox conditions, dynein-associated thioredoxins interact with different proteins through disulfide bonds and, as a consequence, flagella change their manner of beating. This chapter provides an overview of techniques for estimating and modulating the redox state of axonemal proteins, as well as for searching for redox-regulated proteins in the axoneme.

Topics: Algal Proteins; Axoneme; Biochemistry; Buffers; Chlamydomonas; Dyneins; Electrophoresis, Gel, Two-Dimensional; Flagella; Models, Biological; Mutation; Oxidation-Reduction; Protein Subunits; Stilbenes; Sulfonic Acids

The recently determined crystal structure of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, showed that the previously constructed series of NhaA-alkaline phosphatase (PhoA) fusions correctly predicted the topology of NhaA's 12 transmembrane segments (TMS), with the C- and N-termini pointing to the cytoplasm. Here, we show that these NhaA-PhoA fusions provide an excellent tool for mapping the epitopes of three NhaA-specific conformational monoclonal antibodies (mAbs), of which two drastically inhibit the antiporter. By identifying which of the NhaA fusions is bound by the respective mAb, the epitopes were localized to small stretches of NhaA. Then precise mapping was conducted by targeted Cys scanning mutagenesis combined with chemical modifications. Most interestingly, the epitopes of the inhibitory mAbs, 5H4 and 2C5, were identified in loop X-XI (cytoplasmic) and loop XI-XII (periplasmic), which are connected by TMS XI on the cytoplasmic and periplasmic sides of the membrane, respectively. The revealed location of the mAbs suggests that mAb binding distorts the unique NhaA TMS IV/XI assembly and thus inhibits the activity of NhaA. The noninhibitory mAb 6F9 binds to the functionally dispensable C-terminus of NhaA.

Topics: Alkaline Phosphatase; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Binding Sites; Cell Membrane; Cysteine; Epitope Mapping; Epitopes; Escherichia coli Proteins; Models, Molecular; Molecular Sequence Data; Mutagenesis; Protein Conformation; Recombinant Fusion Proteins; Sodium-Hydrogen Exchangers; Stilbenes; Sulfonic Acids

The central protein of the four component sulfur oxidizing (Sox) enzyme system of Paracoccus pantotrophus, SoxYZ, carries at the SoxY subunit the covalently bound sulfur substrate which the other three proteins bind, oxidize, and release as sulfate. SoxYZ of different preparations resulted in different specific thiosulfate-oxidizing activities of the reconstituted Sox enzyme system. From these preparations SoxYZ was activated up to 24-fold by different reductants with disodium sulfide being the most effective and yielded a uniform specific activity of the Sox system. The activation comprised the activities with hydrogen sulfide, thiosulfate, and sulfite. Sulfide-activation decreased the predominant beta-sheet character of SoxYZ by 4%, which caused a change in its conformation as determined by infrared spectroscopy. Activation of SoxYZ by sulfide exposed the thiol of the C-terminal Cys-138 of SoxY as evident from alkylation by 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. Also, SoxYZ activation enhanced the formation of the Sox(YZ)2 heterotetramer as evident from density gradient gel electrophoresis. The tetramer was formed due to an interprotein disulfide between SoxY to yield a SoxY-Y dimer as determined by combined high pressure liquid chromatography and mass spectrometry. The significance of the conformational change of SoxYZ and the interprotein disulfide between SoxY-Y is discussed.

Topics: Bacterial Proteins; Carrier Proteins; Cysteine; Dimerization; Disulfides; Enzyme Activation; Models, Biological; Molecular Conformation; Multienzyme Complexes; Oxidation-Reduction; Oxidoreductases Acting on Sulfur Group Donors; Paracoccus pantotrophus; Protein Conformation; Protein Disulfide-Isomerases; Protein Structure, Tertiary; Protein Subunits; Spectroscopy, Fourier Transform Infrared; Stilbenes; Sulfonic Acids; Sulfur; Sulfur Group Transferases; Thiosulfates

ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.

Topics: Algorithms; Amino Acid Sequence; Animals; Axons; Carrier Proteins; Chloride Channels; Cross-Linking Reagents; Cysteine; Disulfides; Indicators and Reagents; Ion Channel Gating; Membrane Potentials; Mesylates; Molecular Sequence Data; Mutagenesis; Mutagens; Oocytes; Patch-Clamp Techniques; Protein Conformation; Reverse Transcriptase Polymerase Chain Reaction; Stilbenes; Sulfonic Acids; Xenopus

The role of cytosolic ATPases such as N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) in membrane fusion is controversial. We examined the physiology and biochemistry of ATP and NSF in the cortical system of the echinoderm egg to determine if NSF is an essential factor in membrane fusion during Ca(2+)-triggered exocytosis. Neither exocytosis in vitro, nor homotypic cortical vesicle (CV) fusion required soluble proteins or nucleotides, and both occurred in the presence of non-hydrolyzable analogs of ATP. While sensitive to thiol-specific reagents, CV exocytosis is not restored by the addition of cytosolic NSF, and fusion and NSF function are differentially sensitive to thiol-specific agents. To test participation of tightly bound, non-exchangeable NSF in CV-CV fusion, we cloned the sea urchin homolog and developed a species-specific antibody for western blots and physiological analysis. This antibody was without effect on CV exocytosis or homotypic fusion, despite being functionally inhibitory. NSF is detectable in intact cortices, cortices from which CVs had been removed and isolated CVs treated with ATP-gamma-S and egg cytosol to reveal NSF binding sites. In contrast, isolated CVs, though all capable of Ca(2+)-triggered homotypic fusion, contain less than one hexamer of NSF per CV. Thus NSF is not a required component of the CV fusion machinery.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Amino Acid Sequence; Animals; Antibodies; Cell Membrane; Cloning, Molecular; Cytosol; Dextrans; Ethylmaleimide; Exocytosis; Female; Membrane Fusion; Molecular Sequence Data; N-Ethylmaleimide-Sensitive Proteins; Ovum; Protein Binding; Sea Urchins; Secretory Vesicles; Sequence Alignment; Sequence Analysis, Protein; Stilbenes; Sulfonic Acids; Vesicular Transport Proteins

The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to gamma-carboxy-glutamic acid by the integral membrane enzyme, gamma-glutamyl carboxylase. Whether or not cysteine residues are important for carboxylase activity has been the subject of a number of studies. In the present study we used carboxylase with point mutations at cysteines, chemical modification, and mass spectrometry to examine this question. Mutation of any of the free cysteine residues to alanine or serine had little effect on carboxylase activity, although C343A mutant carboxylase had only 38% activity compared with that of wild type. In contrast, treatment with either thiol-reactive reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, disodium salt, or sodium tetrathionate, caused complete loss of activity. We identified the residues modified, using matrix-assisted laser desorption/ionization time of flight mass spectrometry, as Cys(323) and Cys(343). According to our results, these residues are on the cytoplasmic side of the microsomal membrane, whereas catalytic residues are expected to be on the lumenal side of the membrane. Carboxylase was partially protected from chemical modification by factor IXs propeptide. Although all mutant carboxylases bound propeptide with normal affinity, chemical modification caused a >100-fold decrease in carboxylase affinity for the consensus propeptide. We conclude that cysteine residues are not directly involved in carboxylase catalysis, but chemical modification of Cys(323) and Cys(343) may disrupt the three-dimensional structure, resulting in inactivation.

Topics: Amino Acid Sequence; Binding Sites; Carbon-Carbon Ligases; Cysteine; Enzyme Inhibitors; Maleimides; Molecular Sequence Data; Molecular Structure; Mutagenesis, Site-Directed; Peptide Fragments; Point Mutation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stilbenes; Structure-Activity Relationship; Sulfates; Sulfhydryl Reagents; Sulfonic Acids; Trypsin; Vitamin K

SecG stimulates protein translocation in Escherichia coli by facilitating the membrane insertion-deinsertion cycle of SecA. SecG was previously shown to undergo membrane topology inversion, since SecA-dependent protein translocation renders the membrane-protected region of SecG sensitive to external proteases. To examine this topology inversion in more detail without protease-treatment, SecG derivatives with a single cysteine residue at various positions were labeled in the presence and absence of protein translocation with a membrane impermeable SH reagent, 4-acetamido-4'-maleimidylstilbene-2-2'-disulfonic acid (AMS). Treatment of spheroplasts with AMS revealed that a cysteine residue in the cytoplasmic region of SecG could be labeled from the periplasm side only in the presence of protein translocation, whereas a cytoplasmic protein, elongation factor, Tu, remained unlabeled. Treatment of inverted membrane vesicles with AMS also revealed that cysteine residues in the periplasmic region were labeled from the cytoplasmic side of membranes only when protein translocation was in progress. This labeling required ATP, SecA and a precursor protein, and became more efficient as the position of the cysteine residue became closer to the C-terminus. Crosslinking analyses revealed that the interaction between SecG and SecA in membranes markedly increases when SecA and SecG undergo membrane-insertion and topology inversion, respectively. Thus, the two most dynamic components of the translocation machinery were found for the first time to interact with each other when both undergo conformational changes.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Amino Acid Sequence; Bacterial Outer Membrane Proteins; Bacterial Proteins; Cell Membrane; Cell Membrane Permeability; Cross-Linking Reagents; Cysteine; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Immunoblotting; Membrane Proteins; Membrane Transport Proteins; Molecular Sequence Data; Peptide Elongation Factor Tu; Protein Precursors; Protein Transport; SEC Translocation Channels; SecA Proteins; Spheroplasts; Stilbenes; Sulfonic Acids

AcrA/B in Escherichia coli is a multicomponent system responsible for intrinsic resistance to a wide range of toxic compounds, and probably cooperates with the outer membrane protein TolC. In this study, acrAB genes were cloned from the E. coli W3104 chromosome. To determine the topology of the inner membrane component AcrB, we employed a chemical labeling approach to analyse mutants of AcrB in which a single cysteine residue had been introduced. The cysteine-free AcrB mutant, in which the two intrinsic Cys residues were replaced by Ala, retained full drug resistance. We constructed 33 cysteine mutants in which a single cysteine was introduced into each putative hydrophilic loop region of the cysteine-free AcrB. The binding of [(14)C]N-ethylmaleimide (NEM) to the Cys residue and the competition of NEM binding with the binding of a membrane-impermeant maleimide, 4-acetamide-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), in intact cells were investigated. The results revealed that the N- and C-terminals are localized on the cytoplasmic surface of the membrane and the two large loops are localized on the periplasmic surface of the membrane. The results supported the 12-membrane-spanning structure of AcrB. Three of the four short periplasmic loop regions were covered by the two large periplasmic loop domains and were not exposed to the water phase until one of the two large periplasmic loops was removed.

Topics: Amino Acid Sequence; Bacterial Proteins; Binding, Competitive; Carbon Radioisotopes; Carrier Proteins; Cloning, Molecular; Cysteine; Cytoplasm; Drug Resistance, Multiple; Escherichia coli; Escherichia coli Proteins; Ethylmaleimide; Membrane Proteins; Molecular Sequence Data; Multidrug Resistance-Associated Proteins; Mutagenesis, Site-Directed; Periplasm; Protein Conformation; Radioligand Assay; Stilbenes; Sulfonic Acids

Topics: Alkylating Agents; Cytoplasm; Escherichia coli; Escherichia coli Proteins; Oxidation-Reduction; Stilbenes; Sulfhydryl Compounds; Sulfonic Acids; Thioredoxins

The role of transducin sulfhydryl groups was examined by chemical modification with four different reagents: 4-acetamido-4'-maleimidyl-stilbene-2, 2' disulfonic acid (AMDA); 4-vinyl pyridine (VP); 2-nitro-5-thiocyano benzoic acid (NTCBA); and 2, 5-dimethoxystilbene-4'-maleimide (DM). All these compounds rapidly inhibited the [3H]GMPpNp-binding activity of transducin stimulated by photoexcited rhodopsin (R*). Sedimentation experiments showed that the labeling of transducin with AMDA or VP hindered its binding to R* while NTCBA-modified transducin was capable of interacting with the photoreceptor protein. In contrast, DM-labeled transducin precipitated even in the absence of R*. Photoactivated rhodopsin was capable of protecting against the observed AMDA and NTCBA inhibition in transducin function, but not against the inactivation caused by VP or DM. These results suggest the existence of different functional cysteines on transducin that are located in the proximity of the interaction site with the photoreceptor protein, near the guanine nucleotide binding site, or in regions involved in the structural changes taking place upon protein activation. With the use of these reagents, transducin appears to be "frozen" in various conformational stages of its cycle, providing conditions for studying two of the initial steps of the visual process: the light-dependent binding of transducin to rhodopsin and the transducin guanine nucleotide exchange reaction.

Topics: Animals; Binding Sites; Cattle; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Guanine Nucleotides; Guanylyl Imidodiphosphate; Light; Protein Binding; Protein Conformation; Pyridines; Reducing Agents; Rhodopsin; Rod Cell Outer Segment; Signal Transduction; Stilbenes; Sulfhydryl Compounds; Sulfhydryl Reagents; Sulfonic Acids; Thiocyanates; Transducin

SecG, a membrane component of the protein translocation apparatus of Escherichia coli, undergoes membrane topology inversion, which is coupled to the membrane insertion and deinsertion cycle of SecA. Eighteen SecG derivatives possessing a single cysteine residue at various positions were constructed and expressed in a secG null mutant. All the SecG-Cys derivatives retained the SecG function, and stimulated protein translocation both in vivo and in vitro. Inverted membrane vesicles containing a SecG-Cys derivative were labeled with a membrane-permeable or -impermeable sulfhydryl reagent before or after solubilization with a detergent. The accessibility of these reagents to the cysteine residue of each derivative determined the topological arrangement of SecG in the membrane. Derivatives having the cysteine residue in the periplasmic region each existed as a homodimer crosslinked through disulfide bonds, indicating that two SecG molecules closely co-exist in a single translocation machinery. The crosslinking did not abolish the SecG function and the crosslinked SecG dimer underwent topology inversion upon protein translocation.

Topics: Amino Acid Sequence; Bacterial Proteins; Biological Transport; Cell Membrane; Cell Membrane Permeability; Cross-Linking Reagents; Cysteine; Dimerization; Disulfides; Escherichia coli Proteins; Ethylmaleimide; Membrane Proteins; Molecular Sequence Data; Mutation; Recombinant Proteins; SEC Translocation Channels; Stilbenes; Sulfonic Acids

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, and five conserved glycine- and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[(14)C]ethylmaleimide ([(14)C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.

Topics: Amino Acid Sequence; Antiporters; Bacterial Proteins; Binding Sites; Biological Transport; DNA Transposable Elements; Ethylmaleimide; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Sequence Homology, Amino Acid; Stilbenes; Sulfonic Acids; Tetracycline

We cloned a gene encoding a 17-kDa protein from a cDNA library of the plant Sedum lineare and found that its deduced amino acid sequence showed similarities to those of Escherichia coli bacterioferritin co-migratory protein (Bcp) and its homologues, which comprise a discrete group associated with the peroxiredoxin (Prx) family. Studies of the recombinant 17-kDa protein produced in E. coli cells revealed that it actually had a thioredoxin-dependent peroxidase activity, the hallmark of the Prx family. PrxQ, as we now designate the 17-kDa protein, had two cysteine residues (Cys-44 and Cys-49) well conserved among proteins of the Bcp group. These two cysteines were demonstrated to be essential for the thioredoxin-dependent peroxidase activity by analysis of mutant proteins, suggesting that these residues are involved in the formation of an intramolecular disulphide bond as an intermediate in the reaction cycle. Expression of PrxQ suppressed the hypersensitivity of an E. coli bcp mutant to peroxides, indicating that it might exert an antioxidant activity in vivo.

Topics: Amino Acid Sequence; Antioxidants; Bacterial Proteins; Cloning, Molecular; Conserved Sequence; Cysteine; Dimerization; Disulfides; Escherichia coli; Genetic Complementation Test; Magnoliopsida; Molecular Sequence Data; Molecular Weight; Mutation; Peroxidases; Peroxides; Peroxiredoxins; Sequence Alignment; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stilbenes; Sulfonic Acids; Thioredoxins

Escherichia coli DsbB has four essential cysteine residues, among which Cys41 and Cys44 form a CXXC redox active site motif and the Cys104-Cys130 disulfide bond oxidizes the active site cysteines of DsbA, the disulfide bond formation factor in the periplasm. Functional respiratory chain is required for the cell to keep DsbA oxidized. In this study, we characterized the roles of essential cysteines of DsbB in the coupling with the respiratory chain. Cys104 was found to form the inactive complex with DsbA under respiration-defective conditions. While DsbB, under normal aerobic conditions, is in the oxidized state, having two intramolecular disulfide bonds, oxidation of Cys104 and Cys130 requires the presence of Cys41-Cys44. Remarkably, the Cys41-Cys44 disulfide bond is refractory to reduction by a high concentration of dithiothreitol, unless the membrane is solubilized with a detergent. This reductant resistance requires both the respiratory function and oxygen, since Cys41-Cys44 became sensitive to the reducing agent when membrane was prepared from quinone- or heme-depleted cells or when a membrane sample was deaerated. Thus, the Cys41-Val-Leu-Cys44 motif of DsbB is kept both strongly oxidized and strongly oxidizing when DsbB is integrated into the membrane with the normal set of respiratory components.

Topics: Bacterial Proteins; Binding Sites; Cysteine; Disulfides; Dithiothreitol; Electron Transport; Escherichia coli; Membrane Proteins; Mutation; Oxidation-Reduction; Oxygen; Protein Disulfide-Isomerases; Stilbenes; Sulfonic Acids

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 (a) and plasminogen kringle 4, significantly attenuate the fluorescence intensity of the FLP. Other proteins, such as bovine serum albumin, did not affect the quantum yield of FLP fluorescence. When human lipoprotein (a) is bound to a lysine-Sepharose affinity column, FLP was found to effectively elute the protein, indicating that the peptide can compete with lysine for the kringle-binding site on lipoprotein (a). The data suggest that FLP binds specifically to kringles through the lysine residues on the peptide, and that binding significantly affects the fluorescence from the labeled peptide. These properties of FLP make it a potentially useful tool for studying the relative affinity of different kringles for lysine binding, which is thought to be an important mechanism for kringle-target protein interactions.

Topics: Amino Acid Sequence; Fluorescent Dyes; Humans; Lipoprotein(a); Molecular Sequence Data; Oligopeptides; Peptide Fragments; Plasminogen; Protein Binding; Stilbenes; Sulfonic Acids