Arrangement of the COOH-terminal and NH2-terminal domains of caldesmon bound to actin.
AuthorsGraceffa P
JournalBiochemistry
PubMed ID9092808
'Smooth muscle caldesmon is a single polypeptide chain with its NH2- and COOH-terminal domains separated by a long alpha-helix. Caldesmon was labeled at either Cys-153 in the NH2 domain or Cys-580 in the COOH domain with a variety of fluorescence probes. Fluorescence intensity, peak position, and polarization of probes on ... More
8-Anilino-1-naphthalenesulfonate is a fluorescent probe of conformational changes in the D-galactose-H+ symport protein of Escherichia coli.
AuthorsWalmsley AR, Martin GE, Henderson PJ
JournalJ Biol Chem
PubMed ID8006005
'The binding of sugars and antibiotics to the overexpressed D-galactose-H+ symport protein (GalP) can be monitored from changes in the fluorescence of 8-anilino-1-naphthalenesulfonate (ANS) equilibrated with inside-out vesicles. Transported sugars, such as D-glucose and D-galactose, cause an enhancement in the ANS fluorescence of up to 13%. Nontransported sugars that have ... More
Analysis of MDR1 P-glycoprotein conformational changes in permeabilized cells using differential immunoreactivity.
AuthorsDruley TE, Stein WD, Roninson IB
JournalBiochemistry
PubMed ID11284687
'The reactivity of the ATP-dependent multidrug transporter P-glycoprotein (Pgp) with the conformation-sensitive monoclonal antibody UIC2 is increased in the presence of Pgp transport substrates, ATP-depleting agents, or mutations that reduce the level of nucleotide binding by Pgp. We have investigated the effects of nucleotides and vinblastine, a Pgp transport substrate, ... More
ATP induces conformational changes of periplasmic loop regions of the maltose ATP-binding cassette transporter.
AuthorsDaus ML, Landmesser H, Schlosser A, Müller P, Herrmann A, Schneider E
JournalJ Biol Chem
PubMed ID16352608
'We have studied cofactor-induced conformational changes of the maltose ATP-binding cassette transporter by employing limited proteolysis in detergent solution. The transport complex consists of one copy each of the transmembrane subunits, MalF and MalG, and of two copies of the nucleotide-binding subunit, MalK. Transport activity further requires the periplasmic maltose-binding ... More
Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli.
AuthorsSahin-Tóth M, le Coutre J, Kharabi D, le Maire G, Lee JC, Kaback HR
JournalBiochemistry
PubMed ID9888822
'Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807]. Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or ... More
Movement of Cys-697 in myosin ATPase associated with ATP hydrolysis.
AuthorsHiratsuka T
JournalJ Biol Chem
PubMed ID1386082
'To detect movement of Cys-697 (SH2) in myosin subfragment-1 (S-1) associated with ATP hydrolysis, SH2 was labeled with the environmentally sensitive fluorescent analog of maleimide, 2-(4''-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). Complex formation of S-1 labeled at Cys-697 with MIANS (MIANS-S-1) with adenyl-5''-yl imidodiphosphate and ADP resulted in a significant decrease in the ... More
Nucleotide-induced conformational changes in P-glycoprotein and in nucleotide binding site mutants monitored by trypsin sensitivity.
AuthorsJulien M, Gros P
JournalBiochemistry
PubMed ID10758006
'Limited trypsin digestion was used to monitor nucleotide-induced conformational changes in wild-type P-glycoprotein (Pgp) as well as in nucleotide binding domain (NBD) Pgp mutants. Purified and reconstituted wild-type or mutant mouse Mdr3 Pgps were preincubated with different hydrolyzable or nonhydrolyzable nucleotides, followed by limited proteolytic cleavage at different trypsin:protein ratios. ... More
Spin-label and fluorescence labeling studies of the thioester bonds in human alpha 2-macroglobulin.
AuthorsZhao BL, Musci G, Sugawara Y, Berliner LJ
JournalBiochemistry
PubMed ID2458760
'Upon cleavage of the reactive thioester bonds (Cys-949-Glx-952) of tetrameric human alpha 2-macroglobulin (alpha 2M) by methylamine, one sulfhydryl group per alpha 2M subunit is exposed. These identical sulfhydryl group sites were labeled with the thiol-specific nitroxide spin-labels (1-oxy-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate and (1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)methyl methanethiosulfonate, a homologous series of maleimide spin-labels, and ... More
The membrane topology of proton-pumping Escherichia coli transhydrogenase determined by cysteine labeling.
AuthorsMeuller J, Rydström J
JournalJ Biol Chem
PubMed ID10383409
'The membrane topology of proton-pumping nicotinamide-nucleotide transhydrogenase from Escherichia coli was determined by site-specific chemical labeling. A His-tagged cysteine-free transhydrogenase was used to introduce unique cysteines in positions corresponding to potential membrane loops. The cysteines were reacted with fluorescent reagents, fluorescein 5-maleimide or 2-[(4''-maleimidyl)anilino]naphthalene-6-sulfonic acid, in both intact cells and ... More
Phosphorylation induces a conformational transition near the lipid-water interface of phospholamban reconstituted with the Ca-ATPase.
AuthorsChen B, Bigelow DJ
JournalBiochemistry
PubMed ID12437353
'We have measured conformational changes of phospholamban (PLB) induced both by its interaction with the SR Ca-ATPase and by phosphorylation of Ser-16 by cAMP-dependent protein kinase (PKA) using an engineered PLB having a single cysteine (Cys-24) derivatized with the fluorophore 2-(4''-maleimidylanilino)naphthalene-6-sulfonic acid (ANSmal). This modified mutant PLB is fully functional ... More
Kinetic partitioning. Poising SecB to favor association with a rapidly folding ligand.
AuthorsDiamond DL, Randall LL
JournalJ Biol Chem
PubMed ID9360972
'Chaperones are a class of proteins that possess the remarkable ability to selectively bind polypeptides that are in a nonnative state. The selectivity of SecB, a molecular chaperone in Escherichia coli, for its ligands can be explained in part by a kinetic partitioning between folding of the polypeptide and association ... More
Cysteine 148 in the lactose permease of Escherichia coli is a component of a substrate binding site. 2. Site-directed fluorescence studies.
AuthorsWu J, Kaback HR
JournalBiochemistry
PubMed ID7918438
'By using site-directed fluorescence spectroscopy, we have carried out structure/function studies on lactose permease purified from Escherichia coli in dodecyl beta, D-maltoside. Initially, permease containing a single native Cys at position 148 (helix V) was studied, since this residue is protected against alkylation by substrates of the permease. In the ... More
Ca2+, caldesmon, and myosin light chain kinase exchange with calmodulin.
AuthorsKasturi R, Vasulka C, Johnson JD
JournalJ Biol Chem
PubMed ID8463316
'Wheat calmodulin (CaM) was labeled at Cys-27 with the sulfhydryl-specific fluorescent probe 2-(4''-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS), to form MIANS.CaM. In the presence of Ca2+, MIANS.CaM undergoes a large fluorescence increase when it binds myosin light chain kinase (MLCK) and caldesmon (CaD), but little fluorescence change when it binds CaM antagonists or ... More
Fluorescence studies of red blood cell membranes from individuals with Huntington's disease.
AuthorsSumbilla C, Lakowicz JR
JournalJ Neurochem
PubMed ID6210762
'Fluorescence spectroscopic methods were used to investigate and compare the properties of erythrocyte membranes from individuals with Huntington''s Disease (HD) and from normal individuals. Erythrocyte ghosts were labeled with four different fluorescent probes: 1,6-diphenylhexatriene (DPH); 6-lauroyl-2-(dimethylamino)-naphthalene (Laurdan); 2-(4-maleimide anilino)-naphthalene-6-sulfonic acid (MIANS) and 5-(iodoacetamidoethyl)aminoaphthalene-1-sulfonic acid (IAEDANS). DPH is sensitive to the ... More
Interaction between residues Glu269 (helix VIII) and His322 (helix X) of the lactose permease of Escherichia coli is essential for substrate binding.
AuthorsHe MM, Kaback HR
JournalBiochemistry
PubMed ID9354639
'Site-directed and Cys-scanning mutagenesis of the lactose permease of Escherichia coli reveals that as few as four residues--Glu269 (helix VIII), Arg302 (helix IV), His322 (helix X), and Glu325 (helix X)--are irreplaceable for coupling substrate and H+ translocation. Interestingly, the four residues are in close physical proximity, Glu269 interacting with His322 ... More
Subunit exchange of lens alpha-crystallin: a fluorescence energy transfer study with the fluorescent labeled alphaA-crystallin mutant W9F as a probe.
AuthorsSun TX, Akhtar NJ, Liang JJ
JournalFEBS Lett
PubMed ID9688580
'A Trp-free alphaA-crystallin mutant (W9F) was prepared by site-directed mutation. This mutant appears to be identical to the wild-type in terms of conformation (secondary and tertiary structures). W9F was labeled with a sulfhydryl-specific fluorescent probe, 2-(4''-maleimidylanilino) naphthalene-6-sulfonate (MIANS), and used in a subunit exchange between alphaA- and alphaA-crystallins as well ... More
Cys(577) is a conformationally mobile residue in the ATP-binding domain of the Na,K-ATPase alpha-subunit.
'2-[4''-Maleimidylanilino]naphthalene 6-sulfonic acid (MIANS) irreversibly inactivates Na,K-ATPase in a time- and concentration-dependent manner. Inactivation is prevented by 3 mM ATP or low K(+) (<1 mM); the protective effect K(+) is reversed at higher concentrations. This biphasic effect was also observed with K(+) congeners. In contrast, Na(+) ions did not protect. ... More
Direct sugar binding to LacY measured by resonance energy transfer.
AuthorsSmirnova IN, Kasho VN, Kaback HR
JournalBiochemistry
PubMed ID17176050
'Trp151 in the lactose permease of Escherichia coli (LacY) is an important component of the sugar-binding site and the only Trp residue out of six that is in close proximity to the galactopyranoside in the structure (1PV7). The short distance between Trp151 and the sugar is favorable for Förster resonance ... More
Structure and stability of gamma-crystallins. II. Differences in microenvironments and spatial arrangements of cysteine residues.
'The gamma-crystallin fractions II, III and IV from calf eye lens were treated with the thiol-specific fluorescent probe 2-(4''-maleimidylanilino)naphthalene-6-sulfonate (MIANS), in order to determine the reactivity of the seven (gamma-II) or six (gamma-III, gamma-IV) cysteine residues. Two classes of reactive cysteines were distinguished by variations in fluorescence intensity with increasing ... More
Nativelike intermediate on the unfolding pathway of pig kidney fructose-1,6-bisphosphatase.
AuthorsReyes AM, Ludwig HC, Yañez AJ, Rodríguez PH, Slebe JC
JournalBiochemistry
PubMed ID12795590
'The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS, MIANS), and size-exclusion chromatography. The unfolding is a multistate process involving ... More
Fluorescence studies on the nucleotide binding domains of the P-glycoprotein multidrug transporter.
AuthorsLiu R, Sharom FJ
JournalBiochemistry
PubMed ID9062112
'One of the major causes of multidrug resistance in human cancers is expression of the P-glycoprotein multidrug transporter, which acts as an efflux pump for a diverse range of natural products, chemotherapeutic drugs, and hydrophobic peptides. In the present study, fluorescence techniques were used to probe the nucleotide binding domains ... More
Change in the protein tertiary structure with non-enzymatic glycosylation of calf alpha-crystallin.
AuthorsLiang JN, Chylack LT
JournalBiochem Biophys Res Commun
PubMed ID6487332
'Non-enzymatic glycosylation of calf alpha-crystallin was induced by incubation with glucose. Glycosylated and non-glycosylated proteins were separated by affinity chromatography on Glyco Gel B boronic acid and were studied by circular dichroism (CD) and fluorescence. CD indicated that the glycosylated protein secondary structure was not altered, but the tertiary structure ... More
Conformational changes at the highly reactive cystein and lysine regions of skeletal muscle myosin induced by formation of transition state analogues.
AuthorsMaruta S, Homma K, Ohki T
JournalJ Biochem (Tokyo)
PubMed ID9722668
'Myosin forms stable ternary complexes with Mg2+-ADP and phosphate analogues of aluminum fluoride (AlF4-), beryllium fluoride (BeFn), and scandium fluoride (ScFn). These complexes are distinct from each other and may mimic different transient states in the ATPase cycle [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. Regions of skeletal ... More
Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains.
AuthorsLiu R, Sharom FJ
JournalBiochemistry
PubMed ID8794769
'P-Glycoprotein is a member of the ABC superfamily of membrane transporters, and functions as an ATP-driven active efflux pump for natural products and chemotherapeutic drugs. Overexpression of P-glycoprotein is a major cause of multidrug resistance in human cancers. Sulfhydryl modification agents are known to inactivate both P-glycoprotein ATPase activity and ... More
Mapping of residues in the NADP(H)-binding site of proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli. A study of structure and function.
AuthorsFjellström O, Axelsson M, Bizouarn T, Hu X, Johansson C, Meuller J, Rydström J
JournalJ Biol Chem
PubMed ID10037725
'Conformational changes in proton pumping transhydrogenases have been suggested to be dependent on binding of NADP(H) and the redox state of this substrate. Based on a detailed amino acid sequence analysis, it is argued that a classical betaalphabetaalphabeta dinucleotide binding fold is responsible for binding NADP(H). A model defining betaA, ... More
Identification of an essential sulfhydryl group in the ouabain binding site of (Na,K)-ATPase.
AuthorsKirley TL, Lane LK, Wallick ET
JournalJ Biol Chem
PubMed ID3007461
'Ellman''s reagent 5,5''-dithiobis-(2-nitrobenzoic acid) inhibits sodium- and potassium-stimulated ATPase, p-nitrophenyl phosphatase activity, and [3H]ouabain binding to lamb kidney (Na,K)-ATPase. The inactivation of [3H]ouabain binding follows pseudo-first order reaction kinetics at pH values less than or equal to 8.2. The inactivation of [3H]ouabain binding, but not of enzymatic activity, can be ... More
P-glycoprotein-mediated colchicine resistance in different cell lines correlates with the effects of colchicine on P-glycoprotein conformation.
AuthorsDruley TE, Stein WD, Ruth A, Roninson IB
JournalBiochemistry
PubMed ID11284688
'The multidrug transporter P-glycoprotein (Pgp) is an ATPase efflux pump for multiple cytotoxic agents, including vinblastine and colchicine. We have found that resistance to vinblastine but not to colchicine in cell lines derived from different types of tissues and expressing the wild-type human Pgp correlates with the Pgp density. Vinblastine ... More
Identification of a region involved in the communication between the NADP(H) binding domain and the membrane domain in proton pumping E. coli transhydrogenase.
AuthorsAlthage M, Bizouarn T, Rydström J
JournalBiochemistry
PubMed ID11502193
'The two hydrophilic domains I and III of Escherichia coli transhydrogenase containing the binding sites for NAD(H) and NADP(H), respectively, are located on the cytosolic side of the membrane, whereas the hydrophobic domain II is composed of 13 transmembrane alpha-helices, and is responsible for proton transport. In the present investigation ... More
Purification and functional characterization of the C-terminal half of the lactose permease of Escherichia coli.
AuthorsWu J, Sun J, Kaback HR
JournalBiochemistry
PubMed ID8611506
'The lactose permease has been expressed in contiguous, non-overlapping polypeptide fragments containing the N-terminal (N6) and C-terminal (C6) transmembrane domains of the protein [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325; Zen, K., et al. (1994) Biochemistry 33, 8198]. When expressed individually, N6 and ... More
Resonance energy transfer measurements between substrate binding sites within the large (Klenow) fragment of Escherichia coli DNA polymerase I.
AuthorsAllen DJ, Benkovic SJ
JournalBiochemistry
PubMed ID2692712
'Resonance energy transfer was used to determine separation distances between fluorescent derivatives of substrates for Klenow fragment and a unique sulfhydryl, cysteine 907, on the enzyme. Fluorescent derivatives of duplex DNA, deoxynucleotide triphosphates (dNTP), and deoxynucleotide monophosphates (dNMP), modified with aminonaphthalenesulfonates (ANS), served as energy-transfer donors to the fluorophore used ... More
Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites.
AuthorsWu J, Frillingos S, Voss J, Kaback HR
JournalProtein Sci
PubMed ID7756985
'By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331-->Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4''-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) enhances ... More
The membrane lipid environment modulates drug interactions with the P-glycoprotein multidrug transporter.
AuthorsRomsicki Y, Sharom FJ
JournalBiochemistry
PubMed ID10346910
'The P-glycoprotein multidrug transporter functions as an ATP-driven efflux pump for a large number of structurally unrelated hydrophobic compounds. Substrates are believed to gain access to the transporter after partitioning into the membrane, rather than from the extracellular aqueous phase. The binding of drug substrates to P-glycoprotein may thus be ... More
Characterization of calponin binding to actin.
AuthorsLu FW, Freedman MV, Chalovich JM
JournalBiochemistry
PubMed ID7547921
'Calponin, a protein isolated from smooth muscle and nonmuscle cells, has previously been shown to inhibit the actin-activated ATPase activity of myosin. Reports of the stoichiometry of binding range from 1 calponin per actin to 1 calponin per 3 actin monomers. We now report a detailed study of the binding ... More
In vitro folding of a membrane protein: effect of denaturation and renaturation on substrate binding by the lactose permease of Escherichia coli.
AuthorsHe MM, Kaback HR
JournalMol Membr Biol
PubMed ID9595550
'Site-directed mutagenesis and site-directed fluorescence spectroscopy demonstrate that Cys148 interacts hydrophobically with the galactosyl moiety of substrates of the lactose permease of Escherichia coli. By taking advantage of the finding that labelling of single-Cys148 permease with the thiol-specific fluorophore 2-(4''-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) is blocked specifically by substrates of the permease, ... More
Involvement of cysteine residues and domain interactions in the reversible unfolding of lipoxygenase-1.
AuthorsSudharshan E, Rao AG
JournalJ Biol Chem
PubMed ID10585402
'Urea-induced unfolding of lipoxygenase-1 (LOX1) at pH 7.0 was followed by enzyme activity, spectroscopic measurements, and limited proteolysis experiments. Complete unfolding of LOX1 in 9 M urea in the presence of thiol reducing or thiol modifying reagents was observed. The aggregation and oxidative reactions prevented the reversible unfolding of the ... More
Interaction of LDS-751 with P-glycoprotein and mapping of the location of the R drug binding site.
AuthorsLugo MR, Sharom FJ
JournalBiochemistry
PubMed ID15641790
'One cause of multidrug resistance is the overexpression of P-glycoprotein, a 170 kDa plasma membrane ABC transporter, which functions as an ATP-driven efflux pump with broad specificity for hydrophobic drugs, peptides, and natural products. The protein appears to interact with its substrates within the membrane environment. Previous reports suggested the ... More
Proximity of reactive cysteine residue and flavin in Escherichia coli pyruvate oxidase as estimated by fluorescence energy transfer.
AuthorsKoland JG, Gennis RB
JournalBiochemistry
PubMed ID6751388
'Pyruvate oxidase of Escherichia coli possesses a reactive cysteine residue believed to be associated with the thiamin pyrophosphate (TPP) binding site. This residue is not reactive in the presence of TPP. Exposure of the enzyme to cysteine-directed fluorescent reagents results in the formation of fluorescent protein conjugates. Although these reagents ... More
Trans-targeting of the phage Mu repressor is promoted by conformational changes that expose its ClpX recognition determinant.
AuthorsMarshall-Batty KR, Nakai H
JournalJ Biol Chem
PubMed ID12424242
'Dominant negative forms of the phage Mu repressor, including the mutant Vir repressors, are not only rapidly degraded by the ClpXP protease but also promote degradation of the unmodified, wild-type repressor. This trans-targeting of the wild-type repressor depends upon a determinant within its C-terminal domain, which is needed for recognition ... More
Reaction of purified (Na,K)-ATPase with the fluorescent sulfhydryl probe 2-(4'-maleimidylanilino)naphthalene 6-sulfonic acid. Characterization and the effects of ligands.
AuthorsGupte SS, Lane LK
JournalJ Biol Chem
PubMed ID226540
Structure, function and regulation of Na-K-ATPase.
AuthorsSchwartz A, Adams RJ, Ball WJ, Collins JH, Gupte SS, Lane LK, Reeves AS, Wallick ET
JournalInt J Biochem
PubMed ID6249660
Spectroscopic investigations of bovine lens crystallins. 2. Fluorescent probes for polar-apolar nature and sulfhydryl group accessibility.
AuthorsAndley UP, Liang JN, Chakrabarti B
JournalBiochemistry
PubMed ID7082651
Myosin structure. Proximity measurements by fluorescence energy transfer.
AuthorsHaugland RP
JournalJ Supramol Struct
PubMed ID127888
Cross-linking of transmembrane helices reveals a rigid-body mechanism in bacteriorhodopsin transport.
AuthorsSimón-Vázquez R, Lazarova T, Perálvarez-Marín A, Bourdelande JL, Padrós E,
JournalAngew Chem Int Ed Engl
PubMed ID19810071
Immobilization of the cytoplasmic ends of helices F and G of bacteriorhodopsin by disulfide crosslinking between engineered cysteines is shown to produce a functionally defective protein. The photocycle of this modified protein is blocked, proton transport is severely decreased, and proton release and uptake kinetics are delayed. The results demonstrate ... More
The sulfhydryl group microenvironment of lactose synthase from bovine milk.
AuthorsWong LJ, Wong SS
JournalInt J Biochem
PubMed ID6088322
Galactosyltransferase from bovine milk was inactivated by a series of sulfhydryl group specific reagents of different structures and sizes. The inactivation rate constants suggest that the thiol is located in a nonpolar microenvironment. The ESR spectrum of a spin labeled galactosyltransferase showed that the sulfhydryl group is in a region ... More
Activation of a dormant ClpX recognition motif of bacteriophage Mu repressor by inducing high local flexibility.
AuthorsMarshall-Batty KR, Nakai H,
JournalJ Biol Chem
PubMed ID18230617
The C-terminal domain (CTD) of bacteriophage Mu immunity repressor (Rep) regulates DNA binding by the N-terminal domain and degradation by ClpXP protease. Five residues at the Rep C terminus (CTD5) can serve as a ClpX recognition motif, but it is dormant unless activated, a state that can be induced by ... More
A FRET-based method to study protein thiol oxidation in histological preparations.
AuthorsMastroberardino PG, Orr AL, Hu X, Na HM, Greenamyre JT,
JournalFree Radic Biol Med
PubMed ID18620047
Cysteine residues in proteins have important biological roles. For example, disulfide bonds are important structural elements; additionally, reversible oxidation of thiols to disulfides functions as a molecular switch and constitutes an early response to oxidative damage. Because organs are heterogeneous structures composed of diverse cell types, there is a compelling ... More
Zinc stabilizes the SecB binding site of SecA.
AuthorsFekkes P, de Wit JG, Boorsma A, Friesen RH, Driessen AJ
JournalBiochemistry
PubMed ID10213615
The molecular chaperone SecB targets preproteins to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA recognizes SecB via its carboxyl-terminal 22 aminoacyl residues, a highly conserved domain that contains 3 cysteines and 1 histidine residue that could potentially be involved in the coordination of a ... More
Spectroscopic study on the effects of nonenzymatic glycation in human alpha-crystallin.
AuthorsLiang JN, Chylack LT
JournalInvest Ophthalmol Vis Sci
PubMed ID3570690
Nonenzymatic glycated lens alpha-crystallin, isolated from human diabetic cataract lenses, does not differ in subunit size and secondary structure from nonglycated alpha-crystallin. The tertiary structure, however, has undergone a significant change as reflected in the changes of near-ultraviolet (UV) circular dichroism (CD) and fluorescence of intrinsic probes (tryptophan, nontryptophan) and ... More
P-glycoprotein shows strong catalytic cooperativity between the two nucleotide sites.
AuthorsSenior AE, Bhagat S
JournalBiochemistry
PubMed ID9454572
P-Glycoprotein (Pgp) (also known as multidrug-resistance protein) contains two nucleotide binding sites, both of which are catalytic ATPase sites. The covalent reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reacts in catalytic sites, and full inactivation of ATPase activity occurs at a reaction stoichiometry of 1 mol of NBD-Cl/mol of Pgp. We show that, at ... More
Engineering the maltose binding protein for reagentless fluorescence sensing.
AuthorsGilardi G, Zhou LQ, Hibbert L, Cass AE
JournalAnal Chem
PubMed ID7802263
This paper describes a mutant of the maltose binding protein (MBP) in which the serine residue at position 337 is replaced by a cysteine residue using site-directed mutagenesis. The mutant MBP has an approximately 2-fold lower affinity for maltose, and the cysteine residue can be modified with 4-[N-(2-(iodoacetoxy)ethyl)-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD) and ... More
Reaction of the ArsA adenosinetriphosphatase with 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid.
AuthorsKsenzenko MY, Kessel DH, Rosen BP
JournalBiochemistry
PubMed ID8241193
The oxyanion-translocating ATPase encoded by the plasmid-borne ars operon catalyzes extrusion of antimonials and arsenicals from cells of Escherichia coli, thus providing resistance to those toxic oxyanions. The purified catalytic subunit of the ATPase, the ArsA protein, exhibits oxyanion-stimulated ATPase activity. The nature of the oxyanion binding site was probed ... More
Direct demonstration that homotetrameric chaperone SecB undergoes a dynamic dimer-tetramer equilibrium.
We have shown here that the cytosolic bacterial chaperone SecB is a structural dimer of dimers that undergoes a dynamic equilibrium between dimer and tetramer in the native state. We demonstrated this equilibrium by mixing two tetrameric species of SecB that can be distinguished by size. We showed that the ... More
Reaction of (Na,K)-ATPase with fluorescent maleimide derivatives. Probes for studying ATP site(s) function.
AuthorsGupte SS, Lane LK
JournalJ Biol Chem
PubMed ID6300109
The fluorescent maleimide derivatives, 2-(4'-maleimidylanilino)naphthalene 6-sulfonic acid (Mal-ANS) and N-(1-pyrene)-maleimide (Mal-pyrene), both alkylate sulfhydryl groups on the alpha subunit of the (Na,K)-ATPase to inhibit (Na,K)-ATPase and p-nitrophenyl phosphatase activities and phosphoenzyme formation. Reaction of the enzyme with Mal-pyrene, but not with Mal-ANS, also inhibits MgPi- and Mg.ATP.Na-supported [3H]ouabain-binding to the ... More
Gbetagamma acts at the C terminus of SNAP-25 to mediate presynaptic inhibition.
Presynaptic inhibition mediated by G protein-coupled receptors may involve a direct interaction between G proteins and the vesicle fusion machinery. The molecular target of this pathway is unknown. We demonstrate that Gbetagamma-mediated presynaptic inhibition in lamprey central synapses occurs downstream from voltage-gated Ca(2+) channels. Using presynaptic microinjections of botulinum toxins ... More
Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis.
P-glycoprotein (P-gp) confers multiple drug resistance on cancer cells by acting as a plasma membrane localized ATP-dependent drug efflux pump. Currently, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug binding sites of P-gp to generate transport of substrate. ... More
Nonequivalence of the nucleotide-binding subunits of an ABC transporter, the histidine permease, and conformational changes in the membrane complex.
AuthorsKreimer DI, Chai KP, Ferro-Luzzi Ames G
JournalBiochemistry
PubMed ID11087367
The membrane-bound complex of the Salmonella typhimurium histidine permease, an ABC transporter (or traffic ATPase), is composed of two membrane proteins, HisQ and HisM, and two identical copies of an ATP-hydrolyzing protein, HisP. We have developed a technique that monitors quantitatively the sulfhydryl modification levels within the intact complex, and ... More
In vitro biotinylation provides quantitative recovery of highly purified active lactose permease in a single step.
AuthorsPouny Y, Weitzman C, Kaback HR
JournalBiochemistry
PubMed ID9843376
Consler et al. [Consler, T. G., Persson, B. L., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6934-6938] described a one-step purification of lactose permease, a hydrophobic membrane transport protein, from Escherichia coli. Permease constructs containing a biotin acceptor domain are biotinylated in vivo, followed by solubilization and avidin ... More
Studies on the spatial arrangement of muscle thin filament proteins using fluorescence energy transfer.
AuthorsLin TI, Dowben RM
JournalJ Biol Chem
PubMed ID6682103
The distance between a pair of fluorophores attached to Cys-36 of beta-tropomyosin and Cys-373 of actin in reconstituted muscle thin filaments was measured by fluorescence energy transfer. Two pairs of donor/acceptor fluorophores, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid/5-iodoacetamidofluorescein and N-(1-pyrene)maleimide/dimethylamino-4-maleimidostilbene, were covalently attached to tropomyosin and actin. The energy transfer efficiencies in various reconstituted ... More
Use of resonance energy transfer to determine the proximity of the guanine nucleotide binding site of transducin relative to a conformationally-sensitive site on the gamma subunit of the cyclic GMP phosphodiesterase.
AuthorsErickson JW, Mittal R, Cerione RA
JournalBiochemistry
PubMed ID7542027
In this work, we have used resonance energy transfer to determine the relative positions of a reactive cysteine residue on the gamma subunit of the retinal cyclic GMP phosphodiesterase (gamma PDE) and a reactive lysine residue on the alpha subunit of transducin (alpha T). The single cysteine residue on gamma ... More
Demonstration of conformational changes associated with activation of the maltose transport complex.
AuthorsMannering DE, Sharma S, Davidson AL
JournalJ Biol Chem
PubMed ID11150310
In Escherichia coli, interaction of a periplasmic maltose-binding protein with a membrane-associated ATP-binding cassette transporter stimulates ATP hydrolysis, resulting in translocation of maltose into the cell. The maltose transporter contains two transmembrane subunits, MalF and MalG, and two copies of a nucleotide-hydrolyzing subunit, MalK. Mutant transport complexes that function in ... More
Spatial proximity of ATP-sensitive tryptophanyl residue(s) and Cys-697 in myosin ATPase.
AuthorsHiratsuka T
JournalJ Biol Chem
PubMed ID1386083
The reactive thiol Cys-697 (SH2) in myosin ATPase was labeled with a fluorescent analog of maleimide, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) (Hiratsuka, T. (1992) J. Biol. Chem. 267, 14941-14948). Although the tryptophan fluorescence of myosin subfragment-1 (S-1) was slightly affected by incorporation of the MIANS fluorophore, the tryptophan fluorescence of the resultant ... More
Movement of a loop in domain 3 of aerolysin is required for channel formation.
AuthorsRossjohn J, Raja SM, Nelson KL, Feil SC, van der Goot FG, Parker MW, Buckley JT
JournalBiochemistry
PubMed ID9425098
Aerolysin is a channel-forming toxin that must oligomerize in order to become insertion-competent. Modeling based on the crystal structure of the proaerolysin dimer and electron microscopic images of the oligomer indicated that a loop in domain 3 must move away from the beta-sheet that forms the main body of the ... More
Small compounds targeted to subunit interfaces arrest maturation in a nonenveloped, icosahedral animal virus.
AuthorsLee KK, Tang J, Taylor D, Bothner B, Johnson JE
JournalJ Virol
PubMed ID15194797
Nudaurelia omega capensis virus (N omega V) capsids were previously characterized in two morphological forms, a T=4, 485-A-diameter round particle with large pores and a tightly sealed 395-A icosahedrally shaped particle with the same quasi-symmetric surface lattice. The large particle converts to the smaller particle when the pH is lowered ... More
A mutation in the lactose permease of Escherichia coli that decreases conformational flexibility and increases protein stability.
AuthorsSmirnova IN, Kaback HR
JournalBiochemistry
PubMed ID12627968
Lactose permease with Cys154 --> Gly (helix V) binds substrate with high affinity but catalyzes little or no transport. The purified, detergent-solubilized mutant protein exhibits much greater thermal stability than the wild type and little tendency to aggregate. Stabilization is also observed in vivo with an unstable mutant that is ... More
Thiol modification as a probe of conformational forms of the F1 ATPase of Escherichia coli and of the structural asymmetry of its beta subunits.
AuthorsStan-Lotter H, Bragg PD
JournalEur J Biochem
PubMed ID2867900
The sulfhydryl groups of soluble and membrane-bound F1 adenosine triphosphatase of Escherichia coli were modified by reaction with the fluorescent thiol reagents 5-iodoacetamidofluorescein, 2-[(4'-iodoacetamido)anilino]naphthalene-6-sulfonic acid 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-d iaz ole and 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid. Whereas gamma and delta subunits were always labeled by these reagents, the beta subunit reacted preferentially in the soluble ... More
ATP-induced opposite changes in the local environments around Cys(697) (SH2) and Cys(707) (SH1) of the myosin motor domain revealed by the prodan fluorescence.
AuthorsHiratsuka T
JournalJ Biol Chem
PubMed ID10506171
To obtain a consistent view of the nucleotide-induced conformational changes around Cys(697) (SH2) and Cys(707) (SH1) in skeletal myosin subfragment-1 (S-1), the two thiols were labeled with the same environmentally sensitive fluorophore, 6-acyl-2-dimethylaminonaphthalene group, using 6-acryloyl-2-dimethylaminonaphthalene (acrylodan, AD) and 6-bromoacetyl-2-dimethylaminonaphthalene (BD), respectively. The resultant fluorescent derivatives, AD-S-1 and BD-S-1, have ... More
Aluminum fluoride activation of bovine transducin induces two distinct conformational changes in the alpha subunit.
AuthorsMittal R, Cerione RA, Erickson JW
JournalBiochemistry
PubMed ID7520280
We have used resonance energy transfer to read out the interactions of the alpha subunit of transducin (alpha T) with the transducin beta gamma subunit complex (beta gamma T) and to compare the rate of aluminum fluoride-induced alpha T activation, as reflected by the enhancement of the alpha T tryptophan ... More
Identification of three oligosaccharide binding sites in ricin.
AuthorsSteeves RM, Denton ME, Barnard FC, Henry A, Lambert JM
JournalBiochemistry
PubMed ID10512623
The galactoside-binding sites of ricin B chain can be blocked by affinity-directed chemical modification using a reactive ligand derived from asialoglycopeptides containing triantennary N-linked oligosaccharides. The terminal galactosyl residue of one branch of the triantennary oligosaccharide is modified to contain a reactive dichlorotriazine moiety. Two separate galactoside-binding sites have been ... More
Characterization of a Mg2+-stabilized state of the (Na+ and K+)--stimulated adenosine triphosphatase using a fluorescent reporter group.
AuthorsForgac MD
JournalJ Biol Chem
PubMed ID6243639
A Mg2+-induced change of the (Na+ and K+)-stimulated adenosine triphosphatase (Na+,K+)-ATPase) from Electrophorus electricus was investigated by kinetics and fluorescence techniques. Binding of Mg2+ to a low affinity site(s) caused inhibition of (Na+,K+)-ATPase activity, an effect which was antagonized by both Na+ and ATP. Mg2+ also caused inhibition of K+-dependent ... More
High-affinity actin-binding nebulin fragments influence the actoS1 complex.
AuthorsRoot DD, Wang K
JournalBiochemistry
PubMed ID11170442
Human nebulin fragments, NA3 and NA4, corresponding to individual superrepeats display high-affinity interactions with individual actin protomers in cosedimentation and solid-phase binding assays. Stoichiometric analysis of nebulin fragment-induced actin polymerization and inhibition of actin-activated S1 ATPase indicate that one superrepeat influences multiple actin molecules along the F-actin filament, consistent with ... More
Spectroscopic examination of the active site of bovine ferrochelatase.
AuthorsDailey HA
JournalBiochemistry
PubMed ID3986176
Spectrofluorometric techniques have been employed to examine the active site of the terminal enzyme of the heme biosynthetic pathway, ferrochelatase (protoheme ferrolyase, EC 4.99.1.1). The fluorescence of both endogenous tryptophan and exogenous 2-(4-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) has been examined. The fluorescence emission of the enzyme's active site bound MIANS is at ... More
Dynamics of lactose permease of Escherichia coli determined by site-directed chemical labeling and fluorescence spectroscopy.
AuthorsWu J, Frillingos S, Kaback HR
JournalBiochemistry
PubMed ID7599118
Mutants with a single Cys residue in place of Phe27, Pro28, Phe29, Phe30, or Pro31 at the periplasmic end of putative transmembrane helix I were used to study the interaction of lactose permease with ligand by site-directed chemical modification or fluorescence spectroscopy. With permease embedded in the native membrane, mutant ... More
Ligand-induced movement of helix X in the lactose permease from Escherichia coli: a fluorescence quenching study.
AuthorsWang Q, Matsushita K, de Foresta B, le Maire M, Kaback HR
JournalBiochemistry
PubMed ID9369484
Five single-Trp mutants were constructed by replacing Val315, Leu318, Val326, Leu329, or Val331 with Trp in transmembrane helix X of a functional lactose permease mutant devoid of Trp residues (Trp-less permease). Taking into account expression levels, each single-Trp permease except for Val331-->Trp exhibits significant activity. The intrinsic fluorescence emission of ... More
Calponin-calmodulin interaction: properties and effects on smooth and skeletal muscle actin binding and actomyosin ATPases.
AuthorsWinder SJ, Walsh MP, Vasulka C, Johnson JD
JournalBiochemistry
PubMed ID8241189
Smooth muscle calponin bound to the biologically active fluorescent calmodulin [2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin] (MIANS.CaM) with a Kd of 80 nM and produced a 3.4-fold fluorescence enhancement. PKC-phosphorylated calponin (1.3 mol of Pi/mol) bound to CaM with approximately 15-fold lower affinity. Calponin inhibited CaM (10 nM) activation of the Ca(2+)-/CaM-activated cyclic nucleotide ... More
Functional significance of a protein conformation change at the cytoplasmic end of helix F during the bacteriorhodopsin photocycle.
AuthorsBrown LS, Váró G, Needleman R, Lanyi JK
JournalBiophys J
PubMed ID8580354
The second half of the photocycle of the light-driven proton pump bacteriorhodopsin includes proton transfers between D96 and the retinal Schiff base (the M to N reaction) and between the cytoplasmic surface and D96 (decay of the N intermediate). The inhibitory effects of decreased water activity and increased hydrostatic pressure ... More
Binding of ligand or monoclonal antibody 4B1 induces discrete structural changes in the lactose permease of Escherichia coli.
By using Cys-scanning mutagenesis with site-directed sulfhydryl modification in situ [Frillingos, S., & Kaback, H. R. (1996) Biochemistry 35, 3950-3956], conformational changes induced by binding of ligand or monoclonal antibody (mAb) 4B1 in the lactose permease of Escherichia coli were studied. Out of 31 single-Cys replacement mutants in helices I, ... More
Structural flexibility modulates the activity of human glutathione transferase P1-1. Role of helix 2 flexibility in the catalytic mechanism.
AuthorsRicci G, Caccuri AM, Lo Bello M, Rosato N, Mei G, Nicotra M, Chiessi E, Mazzetti AP, Federici G
JournalJ Biol Chem
PubMed ID8663072
Presteady-state and steady-state kinetic studies performed on human glutathione transferase P1-1 (EC 2.5.1.18) with 1-chloro-2, 4-dinitrobenzene as co-substrate indicate that the rate-determining step is a physical event that occurs after binding of the two substrates and before the final sigma-complex formation. It may be a structural transition involving the ternary ... More
Time-resolved fluorescence resonance energy transfer shows that the bacterial multidrug ABC half-transporter BmrA functions as a homodimer.
AuthorsDalmas O, Do Cao MA, Lugo MR, Sharom FJ, Di Pietro A, Jault JM
JournalBiochemistry
PubMed ID15766260
Members of the ATP-binding cassette (ABC) transporters share the same basic architecture, with a four-core domain made of two transmembrane plus two nucleotide-binding domains. However, a supramolecular organization has been detected in some ABC transporters, which might be relevant to physiological regulation of substrate transport. Here, the oligomerization status of ... More
Fluorescence of native single-Trp mutants in the lactose permease from Escherichia coli: structural properties and evidence for a substrate-induced conformational change.
AuthorsWeitzman C, Consler TG, Kaback HR
JournalProtein Sci
PubMed ID8563627
Six single-Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87:1638-1642), and fluorescent properties were studied with respect to solvent accessibility, as well ... More
Calcium-promoted resonance energy transfer between fluorescently labeled proteins during aggregation of chromaffin granule membranes.
AuthorsMorris SJ, Südhof TC, Haynes DH
JournalBiochim Biophys Acta
PubMed ID6897615
Proteins of the chromaffin granule membrane were covalently labeled in situ with sulfhydryl-specific fluorophores. Using MIANS (maleimide iodoaminonaphthyl sulfonate) as the donor and fluorescein mercury acetate or fluorescein-5-maleimide as the acceptor. Förster fluorescence resonance energy transfer (FRET) could be employed to measure the degree of inter-membrane and intra-membrane protein-protein contact ... More
Labeling of the beta gamma subunit complex of transducin with an environmentally sensitive cysteine reagent. Use of fluorescence spectroscopy to monitor transducin subunit interactions.
AuthorsPhillips WJ, Cerione RA
JournalJ Biol Chem
PubMed ID2040617
In this study, we have examined the interactions of the beta gamma subunit complex of the retinal GTP-binding protein transducin (beta gamma T) with its alpha subunit (alpha T) using fluorescence spectroscopic approaches. The beta gamma T subunit complex was covalently labeled with 2-(4'-maleimidylanilino)napthalene-6-sulfonic acid (MIANS), an environmentally sensitive fluorescent ... More
Internal movement in myosin subfragment 1 detected by fluorescence resonance energy transfer.
AuthorsXing J, Cheung HC
JournalBiochemistry
PubMed ID7756279
We have determined intersite distances from Cys374 of actin to Cys707 (SH1) and Cys697 (SH2) of myosin subfragment 1 (S1) in actosubfragment 1 (A.S1) by fluorescence resonance energy transfer for rigor complex A.S1 and complexes containing bound ADP and ADP plus orthovanadate (Vi), A.S1.ADP, and A.S1.ADP.Vi. A single energy acceptor ... More
The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity.
AuthorsYe J, Liu LW, Esmon CT, Johnson AE
JournalJ Biol Chem
PubMed ID1317850
The domain of thrombomodulin that binds to the anion-binding exosite of thrombin was identified by comparing the binding of fragments of thrombomodulin to thrombin with that of Hirugen, a 12-residue peptide of hirudin that is known to bind to the anion-binding exosite of thrombin. Three soluble fragments of thrombomodulin, containing ... More
Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.
AuthorsBrune M, Hunter JL, Corrie JE, Webb MR
JournalBiochemistry
PubMed ID8031761
A probe has been developed that can rapidly measure micromolar concentrations of inorganic phosphate (Pi), in particular to follow the release of Pi in real time from enzymes such as phosphatases. Its application is described to investigate the mechanism of actomyosin subfragment 1 ATPase. The probe uses the A197C mutant ... More
Rhodopsin/transducin interactions. I. Characterization of the binding of the transducin-beta gamma subunit complex to rhodopsin using fluorescence spectroscopy.
AuthorsPhillips WJ, Cerione RA
JournalJ Biol Chem
PubMed ID1512242
In this work we have used fluorescence spectroscopic approaches to examine the binding of the beta gamma T subunit complex of transducin to the photoreceptor, rhodopsin. To do this, we have covalently labeled the beta gamma T subunit complex with the environmentally sensitive fluorescent cysteine reagent 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). By ... More
Accessibilities of the sulfhydryl groups of native and photooxidized lens crystallins: a fluorescence lifetime and quenching study.
AuthorsAndley UP, Clark BA
JournalBiochemistry
PubMed ID3349065
Fluorescence lifetime and acrylamide quenching studies on the N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS)-labeled sulfhydryl groups of bovine lens alpha-, beta H-, and gamma-crystallins were carried out to characterize the microenvironment of the sulfhydryls and changes produced by singlet oxygen mediated photooxidation. For the untreated proteins, the lifetimes of the major decay component of ... More
Frequency-domain fluorescence spectroscopy resolves the location of maleimide-directed spectroscopic probes within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum.
AuthorsBigelow DJ, Inesi G
JournalBiochemistry
PubMed ID1825607
We have used fluorescence spectroscopy to characterize three covalently bound spectroscopic maleimide derivatives with respect to their location within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum (SR). These derivatives include (1) 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid, (2) 4-(dimethylamino)azobenzene-4'-maleimide, and (3) fluorescein 5'-maleimide. Biochemical assays demonstrate that modification with any of these ... More
The C-4 hydroxyl group of galactopyranosides is the major determinant for ligand recognition by the lactose permease of Escherichia coli.
AuthorsSahin-Tóth M, Lawrence MC, Nishio T, Kaback HR
JournalBiochemistry
PubMed ID11669639
Binding specificity in lactose permease toward galactopyranosides is governed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while binding affinity can be increased dramatically by nonspecific hydrophobic interactions with the non-galactosyl moiety [Sahin-Tóth, M., Akhoon, K. M., Runner, J., and Kaback, H. R. (2000) Biochemistry 39, 5097-5103]. ... More
Biologically active fluorescent derivatives of spinach calmodulin that report calmodulin target protein binding.
AuthorsMills JS, Walsh MP, Nemcek K, Johnson JD
JournalBiochemistry
PubMed ID3365375
Spinach calmodulin (CaM) has been labeled at cysteine-26 with the sulfhydryl-selective probe 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (MIANS) to produce MIANS-CaM. The interaction of MIANS-CaM with CaM binding proteins was studied by fluorescence enhancement accompanying the protein-protein interactions. MIANS-CaM bound to smooth muscle myosin light-chain kinase with a Kd of 9 nM, causing ... More
Fluorescence studies on calmodulin binding to erythrocyte Ca2(+)-ATPase in different oligomerization states.
AuthorsKosk-Kosicka D, Bzdega T, Johnson JD
JournalBiochemistry
PubMed ID2139581
The fluorescent spinach calmodulin derivative 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin (MIANS-CaM) was used to investigate calmodulin interaction with the purified, detergent-solubilized erythrocyte Ca2(+)-ATPase. Previous studies have shown that the Ca2(+)-ATPase exists in equilibria between monomeric and oligomeric forms. We report here that MIANS-CaM binds to both enzyme forms in a Ca2(+)-dependent manner, with ... More
The substrate-binding site in the lactose permease of Escherichia coli.
AuthorsVenkatesan P, Kaback HR
JournalProc Natl Acad Sci U S A
PubMed ID9707556
Site-directed N-ethylmaleimide labeling was studied with Glu-126 and/or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site. Replacement of either Glu-126 or Arg-144 with Ala markedly decreases Cys-148 reactivity, whereas interchanging the residues, double-Ala replacement, or replacement of Arg-144 with Lys ... More
Monitoring conformational change in the human erythrocyte glucose carrier: use of a fluorescent probe attached to an exofacial carrier sulfhydryl.
AuthorsMay JM, Beechem JM
JournalBiochemistry
PubMed ID8457556
Several fluorescent sulfhydryl reagents were tested as probes for assessing substrate-induced conformational change of the human erythrocyte glucose carrier. Of these, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (Mal-ANS) inhibited 3-O-methylglucose transport most strongly and specifically labeled a previously characterized exofacial sulfhydryl on the glucose carrier. Analysis of equilibrium cytochalasin B binding in cells treated ... More
Proximity of the nucleotide binding domains of the P-glycoprotein multidrug transporter to the membrane surface: a resonance energy transfer study.
AuthorsLiu R, Sharom FJ
JournalBiochemistry
PubMed ID9572868
Very little structural information is available for P-glycoprotein (Pgp), which has been implicated in the multidrug resistance of human tumors because of its ability to act as an ATP-driven efflux pump for hydrophobic compounds. Highly purified Pgp has been labeled on two cysteine residues with the fluorescence probe NBD-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole). ... More
Probing the conformation of the lactose permease of Escherichia coli by in situ site-directed sulfhydryl modification.
AuthorsFrillingos S, Kaback HR
JournalBiochemistry
PubMed ID8672426
By using site-directed chemical labeling of lactose permease, conformational changes induced by ligand binding are observed in the native membrane of Escherichia coli. Membranes containing permease mutants with a single-Cys residue and a biotin-acceptor domain were labeled with radioactive N-ethylmaleimide (NEM) in the presence or absence of beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) ... More
Spectral properties of fluorescent derivatives of the oligomycin sensitivity conferring protein and analysis of their interaction with the F1 and F0 sectors of the mitochondrial ATPase complex.
AuthorsDuszynski J, Dupuis A, Lux B, Vignais PV
JournalBiochemistry
PubMed ID2905894
In order to study the kinetics and the nature of the interactions between the oligomycin sensitivity conferring protein (OSCP) and the F0 and F1 sectors of the mitochondrial ATPase complex, fluorescent derivatives of OSCP, which are fully biologically active, have been prepared by reaction of OSCP with the following fluorescent ... More
Structure and stability of gamma-crystallins: tryptophan, tyrosine, and cysteine accessibility.
AuthorsMandal K, Chakrabarti B
JournalBiochemistry
PubMed ID3166999
The solute perturbation techniques of fluorescence of tryptophan (Trp) and dye-labeled thiol groups of cysteine as well as phosphorescence of tyrosine (Tyr) were utilized to obtain information on the relative solvent exposure and accessibility of these residues in gamma-crystallins. Both acrylamide and iodide quenchers were used to evaluate the quenching ... More
Characterizing the response of calcium signal transducers to generated calcium transients.
AuthorsDavis JP, Tikunova SB, Walsh MP, Johnson JD
JournalBiochemistry
PubMed ID10194340
Cellular Ca2+ transients and Ca2+-binding proteins regulate physiological phenomena as diverse as muscle contraction, neurosecretion, and cell division. When Ca2+ is rapidly mixed with slow Ca2+ chelators, EGTA, or Mg2+/EDTA, artificial Ca2+ transients (ACTs) of varying duration (0.1-50 ms half-widths (hws)) and amplitude can be generated. We have exposed several ... More
FRET analysis indicates that the two ATPase active sites of the P-glycoprotein multidrug transporter are closely associated.
AuthorsQu Q, Sharom FJ
JournalBiochemistry
PubMed ID11170469
Members of the ABC superfamily carry out the transport of various molecules and ions across cellular membranes, powered by ATP hydrolysis. Substantial evidence indicates that the two catalytic sites of the nucleotide binding domains function in a highly cooperative, alternating sites mode, which suggests the possibility that they interact with ... More
Ligand recognition by the lactose permease of Escherichia coli: specificity and affinity are defined by distinct structural elements of galactopyranosides.
AuthorsSahin-Tóth M, Akhoon KM, Runner J, Kaback HR
JournalBiochemistry
PubMed ID10819976
Specificity of substrate recognition in lactose permease is directed toward the galactosyl moiety of lactose. In this study, binding of 31 structural analogues of D-galactose was examined by site-directed N-[(14)C]ethylmaleimide-labeling of the substrate-protectable Cys148 in the binding site. Alkylation of Cys148 is blocked by D-galactose with an apparent affinity of ... More
Equilibrium unfolding of dimeric human prostatic acid phosphatase involves an inactive monomeric intermediate.
AuthorsWójciak P, Mazurkiewicz A, Bakalova A, Kuciel R
JournalInt J Biol Macromol
PubMed ID12719131
Guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular weight, was investigated with activity measurements, size exclusion HPLC, tryptophan fluorescence, 1-anilinonaphtalene-8-sulfonate (ANS) binding and reactivity with 2-(4'-maleimidoanilino)naphthalene-6-sulfonate (MIANS). Equilibrium analysis was performed to shed light on the role of dimerization in the ... More