The effect of pH on beta(2) adrenoceptor function. Evidence for protonation-dependent activation.
AuthorsGhanouni P, Schambye H, Seifert R, Lee TW, Rasmussen SG, Gether U, Kobilka BK
JournalJ Biol Chem
PubMed ID10652295
'The transition of rhodopsin from the inactive to the active state is associated with proton uptake at Glu(134) (1), and recent mutagenesis studies suggest that protonation of the homologous amino acid in the alpha(1B) adrenergic receptor (Asp(142)) may be involved in its mechanism of activation (2). To further explore the ... More
The kinetics of phagosome maturation as a function of phagosome/lysosome fusion and acquisition of hydrolytic activity.
AuthorsYates RM, Hermetter A, Russell DG
JournalTraffic
PubMed ID15813751
'Professional phagocytes function at the hinge of innate and acquired immune responses by internalizing particulate material that is digested and sampled within the phagosome of the cell. Despite intense interest, assays to measure phagosome maturation remain insensitive and few in number. In this current study, we describe three novel assays ... More
Probing the unfolding pathway of alpha1-antitrypsin.
AuthorsJames EL, Whisstock JC, Gore MG, Bottomley SP
JournalJ Biol Chem
PubMed ID10092631
'Protein misfolding plays a role in the pathogenesis of many diseases. alpha1-Antitrypsin misfolding leads to the accumulation of long chain polymers within the hepatocyte, reducing its plasma concentration and predisposing the patient to emphysema and liver disease. In order to understand the misfolding process, it is necessary to examine the ... More
A fluorescent probe study of plasminogen activator inhibitor-1. Evidence for reactive center loop insertion and its role in the inhibitory mechanism.
AuthorsShore JD, Day DE, Francis-Chmura AM, Verhamme I, Kvassman J, Lawrence DA, Ginsburg D
JournalJ Biol Chem
PubMed ID7890653
'A mutant recombinant plasminogen activator inhibitor 1 (PAI-1) was created (Ser-338-->Cys) in which cysteine was placed at the P9 position of the reactive center loop. Labeling this mutant with N,N''-dimethyl-N-(acetyl)-N''-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) provided a molecule with a fluorescent probe at that position. The NBD-labeled mutant was almost as reactive ... More
Activation of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) involves Rubisco activase Trp16.
Authorsvan de Loo FJ, Salvucci ME
JournalBiochemistry
PubMed ID8679566
'The role of the N-terminal region of tobacco Rubisco activase in ATP hydrolysis and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation was examined by construction of mutant proteins. Deletion of the first 50 amino acids of Rubisco activase almost completely eliminated the ability to activate Rubisco, without changing the ATP-hydrolyzing and self-associating properties ... More
A molecular switch of chemokine receptor selectivity. Chemical modification of the interleukin-8 Leu25 --> Cys mutant.
AuthorsLusti-Narasimhan M, Chollet A, Power CA, Allet B, Proudfoot AE, Wells TN
JournalJ Biol Chem
PubMed ID8621714
'Interleukin-8 (IL-8), a member of the CXC chemokine family, is a key activator of neutrophils. We have previously shown that two novel CC chemokine-like properties, namely monocyte chemoattraction and binding to CC CKR-1, are introduced into IL-8 by mutating Leu25 to the conserved tyrosine present in CC chemokines. To further ... More
Evidence for a pre-latent form of the serpin plasminogen activator inhibitor-1 with a detached beta-strand 1C.
AuthorsDupont DM, Blouse GE, Hansen M, Mathiasen L, Kjelgaard S, Jensen JK, Christensen A, Gils A, Declerck PJ, Andreasen PA, Wind T
JournalJ Biol Chem
PubMed ID17018527
'Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in beta-sheet A and detachment of beta-strand 1C (s1C) at the C-terminal hinge of the RCL. This is ... More
'Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share ... More
Lifetime fluorescence method for determining membrane topology of proteins.
AuthorsPosokhov YO, Ladokhin AS
JournalAnal Biochem
PubMed ID16298322
'Recently, we introduced a sensitive method for determining the bilayer topology (cis- or trans-leaflet location) of single-site cysteine-linked 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorescent labels on membrane proteins. It uses a novel quencher, LysoUB, composed of a single acyl chain attached to a UniBlue chromophore. In its original version, the method relied on ... More
Two lipid-packing sensor motifs contribute to the sensitivity of ArfGAP1 to membrane curvature.
AuthorsMesmin B, Drin G, Levi S, Rawet M, Cassel D, Bigay J, Antonny B
JournalBiochemistry
PubMed ID17253781
'ArfGAP1 (Arf GTPase activating protein 1) controls the cycling of the COPI coat on Golgi membranes by catalyzing GTP hydrolysis in the small G protein Arf1. ArfGAP1 contains a central motif named ALPS (ArfGAP1 lipid-packing sensor) that adsorbs preferentially onto highly curved membranes. This motif allows coupling of the rate ... More
Inducible expression of the cell surface heparan sulfate proteoglycan syndecan-2 (fibroglycan) on human activated macrophages can regulate fibroblast growth factor action.
AuthorsClasper S, Vekemans S, Fiore M, Plebanski M, Wordsworth P, David G, Jackson DG
JournalJ Biol Chem
PubMed ID10446183
'Monocyte/macrophages play important roles in regulating tissue growth and angiogenesis through the controlled release of heparin-binding growth factors such as fibroblast growth factor (FGF), vascular endothelial growth factor, and heparin binding epidermal growth factor. The action of these potent growth mediators is known to be regulated by adsorption to heparan ... More
Outer membrane phospholipase A is dimeric in phospholipid bilayers: a cross-linking and fluorescence resonance energy transfer study.
AuthorsUbarretxena-Belandia I, Hozeman L, van der Brink-van der Laan E, Pap EH, Egmond MR, Verheij HM, Dekker N
JournalBiochemistry
PubMed ID10353852
'In the cell, the activity of outer membrane phospholipase A (OMPLA) is strictly regulated to prevent uncontrolled breakdown of the membrane lipids. Previously, it has been shown that the enzymatic activity is modulated by reversible dimerization. The current studies were carried out to define the oligomeric state of OMPLA in ... More
Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.
AuthorsGether U, Lin S, Ghanouni P, Ballesteros JA, Weinstein H, Kobilka BK
JournalEMBO J
PubMed ID9362488
'Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that ... More
Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility.
AuthorsGether U, Ballesteros JA, Seifert R, Sanders-Bush E, Weinstein H, Kobilka BK
JournalJ Biol Chem
PubMed ID9006889
'Mutations in several domains can lead to agonist-independent, constitutive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the beta2 adrenergic receptor (CAM) is characterized ... More
Phosphorylation induces conformational changes in the leukocyte NADPH oxidase subunit p47(phox).
AuthorsPark HS, Kim IS, Park JW
JournalBiochem Biophys Res Commun
PubMed ID10334912
'The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the reduction of oxygen to at the expense of NADPH. The enzyme is dormant in resting neutrophils but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(phox) ... More
Insights into the action of the superfamily of cholesterol-dependent cytolysins from studies of intermedilysin.
'The cholesterol-dependent cytolysins (CDCs), a superfamily of pore-forming toxins, are characterized by a conserved undecapeptide motif that is believed to be critical for membrane recognition by means of cholesterol. Intermedilysin (ILY), an unusual member of the CDCs, exhibits specificity for human cells and contains nonconservative substitutions in the motif. We ... More
Import of a mitochondrial presequence into protein-free phospholipid vesicles.
AuthorsMaduke M, Roise D
JournalScience
PubMed ID8385804
'A synthetic mitochondrial presequence has been shown to translocate across pure phospholipid bilayers. The presequence was fluorescently labeled so that its association with membranes could be monitored spectroscopically. In the presence of large unilamellar vesicles, the presequence showed time- and potential-dependent protection from reaction with added trypsin and dithionite. The ... More
Accelerated conversion of human plasminogen activator inhibitor-1 to its latent form by antibody binding.
AuthorsVerhamme I, Kvassman JO, Day D, Debrock S, Vleugels N, Declerck PJ, Shore JD
JournalJ Biol Chem
PubMed ID10364183
'The serpin plasminogen activator inhibitor-1 (PAI-1) slowly converts to an inactive latent form by inserting a major part of its reactive center loop (RCL) into its beta-sheet A. A murine monoclonal antibody (MA-33B8), raised against the human plasminogen activator (tPA).PAI-1 complex, rapidly inactivates PAI-1. Results presented here indicate that MA-33B8 ... More
The elongation of yeast prion fibers involves separable steps of association and conversion.
AuthorsScheibel T, Bloom J, Lindquist SL
JournalProc Natl Acad Sci U S A
PubMed ID14983002
'A self-perpetuating change in the conformation of the translation termination factor Sup35p is the basis for the prion [PSI+], a protein-based genetic element of Saccharomyces cerevisiae. In a process closely allied to in vivo conversion, the purified soluble, prion-determining region of Sup35p (NM) converts to amyloid fibers by means of ... More
Fluorescent peptide probes for high-throughput measurement of protein phosphatases.
AuthorsNoble JE, Ganju P, Cass AE
JournalAnal Chem
PubMed ID12720338
'A homogeneous microplate assay for the serine/threonine protein phosphatases PP1 and PP2A, employing fluorescent-labeled phosphopeptides, has been developed. Phosphopeptides derived from a phosphoacceptor site in myelin basic protein were designed with a cysteine adjacent to the phosphoresidue, allowing site-selective labeling with dyes. The fluorescence emission from the environmentally sensitive fluorophore ... More
Antichymotrypsin interaction with chymotrypsin. Reactions following encounter complex formation.
AuthorsNair SA, Cooperman BS
JournalJ Biol Chem
PubMed ID9651334
'Serpins, serine proteinase inhibitors, form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases, that only slowly release I*, in which the P1-P1'' linkage is cleaved. Recently we presented evidence that the serpin antichymotrypsin (ACT, I) reacts with the serine proteinase chymotrypsin (Chtr, E) to form an E*I* complex ... More
Viroporin-mediated membrane permeabilization. Pore formation by nonstructural poliovirus 2B protein.
AuthorsAgirre A, Barco A, Carrasco L, Nieva JL
JournalJ Biol Chem
PubMed ID12183456
'Enterovirus nonstructural 2B protein is involved in cell membrane permeabilization during late viral infection. Here we analyze the pore forming activity of poliovirus 2B and several of its variants. Solubilization of 2B protein was achieved by generating a fusion protein comprised of poliovirus 2B attached to a maltose-binding protein (MBP) ... More
Purification and fluorescent labeling of the human serotonin transporter.
AuthorsRasmussen SG, Gether U
JournalBiochemistry
PubMed ID15736959
'To establish a purification procedure for the human serotonin transporter (hSERT) we expressed in Sf9 insect cells an epitope-tagged version of the transporter containing a FLAG epitope at the N-terminus and a polyhistidine tail at the C-terminus (FLAG-hSERT-12H). For purification, the transporter was solubilized in digitonin followed by nickel affinity ... More
Fluorescent labeling of the leukocyte NADPH oxidase subunit p47(phox): evidence for amphiphile-induced conformational changes.
AuthorsPark HS, Park JW
JournalArch Biochem Biophys
PubMed ID9851827
'The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O-2 from oxygen using NADPH as the electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47(phox) and p67(phox) ... More
Time-resolved polarized fluorescence spectroscopy studies of plasminogen activator inhibitor type 1: conformational changes of the reactive center upon interactions with target proteases, vitronectin and heparin.
AuthorsFa M, Karolin J, Aleshkov S, Strandberg L, Johansson LB, Ny T
JournalBiochemistry
PubMed ID7577977
'Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1''), in the reactive center loop, ... More
Use of fluorescence spectroscopy to study conformational changes in the beta 2-adrenoceptor.
AuthorsKobilka BK, Gether U
JournalMethods Enzymol
PubMed ID11665566
Potential for interactions between the carboxy- and amino-termini of Rubisco activase subunits.
AuthorsSalvucci ME
JournalFEBS Lett
PubMed ID14988023
The subunit interactions of Rubisco activase were investigated using mutants containing an introduced Cys near the N- and/or C-terminus. Chemical cross-linking of the C-terminal and double insertion mutant produced subunit dimers and dimers plus high ordered oligomers, respectively. Fluorescence measurements with N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine showed that the environment around the introduced Cys ... More
Engineering a polarity-sensitive biosensor for time-lapse imaging of apoptotic processes and degeneration.
AuthorsKim YE, Chen J, Chan JR, Langen R,
JournalNat Methods
PubMed ID19966809
Apoptosis is of central importance to many areas of biological research, but there is a lack of methods that permit continuous monitoring of apoptosis or cell viability in a nontoxic and noninvasive manner. Here we report the development of a tool applicable to live-cell imaging that facilitates the visualization of ... More
Structural insights into the membrane-anchoring mechanism of a cholesterol-dependent cytolysin.
AuthorsRamachandran R, Heuck AP, Tweten RK, Johnson AE
JournalNat Struct Biol
PubMed ID12368903
Perfringolysin O (PFO), a cytolytic toxin secreted by pathogenic Clostridium perfringens, forms large pores in cholesterol-containing membranes. Domain 4 (D4) of the protein interacts first with the membrane and is responsible for cholesterol recognition. By using several independent fluorescence techniques, we have determined the topography of D4 in the membrane-inserted ... More
Specific protein-membrane contacts are required for prepore and pore assembly by a cholesterol-dependent cytolysin.
AuthorsSoltani CE, Hotze EM, Johnson AE, Tweten RK
JournalJ Biol Chem
PubMed ID17412689
Three short hydrophobic loops and a conserved undecapeptide at the tip of domain 4 (D4) of the cholesterol-dependent cytolysins (CDCs) mediate the binding of the CDC monomers to cholesterol-rich cell membranes. But intermedilysin (ILY), from Streptococcus intermedius, does not bind to cholesterol-rich membranes unless they contain the human protein CD59. ... More
The major myosin-binding site of caldesmon resides near its N-terminal extreme.
AuthorsLi Y, Zhuang S, Guo H, Mabuchi K, Lu RC, Wang CA
JournalJ Biol Chem
PubMed ID10753900
The primary myosin-binding site of caldesmon was thought to be in the N-terminal region of the molecule, but the exact nature of the caldesmon-myosin interaction has not been well characterized. A caldesmon fragment that encompasses residues 1-240 (N240) was found to bind full-length smooth muscle myosin on the basis of ... More
Conformational changes of Escherichia coli RNA polymerase sigma70 factor induced by binding to the core enzyme.
AuthorsCallaci S, Heyduk E, Heyduk T
JournalJ Biol Chem
PubMed ID9830052
Mutants of RNA polymerase sigma70 subunit from Escherichia coli with unique cysteine residues engineered into conserved region 1 (autoinhibition domain of sigma70), region 2.4 (-10 DNA element binding domain), region 4.2 (-35 DNA element binding domain), and a nonconserved region between regions 1 and 2 were prepared. The chemical reactivity ... More
A newly designed microspectrofluorometer for kinetic studies on protein crystals in combination with x-ray diffraction.
AuthorsKlink BU, Goody RS, Scheidig AJ
JournalBiophys J
PubMed ID16698776
We present a new design for a fluorescence microspectrophotometer for use in kinetic crystallography in combination with x-ray diffraction experiments. The FLUMIX device (Fluorescence spectroscopy to monitor intermediates in x-ray crystallography) is built for 0 degrees fluorescence detection, which has several advantages in comparison to a conventional fluorometer with 90 ... More
Structure of a serpin-enzyme complex probed by cysteine substitutions and fluorescence spectroscopy.
The x-ray crystal structure of the serpin-proteinase complex is yet to be determined. In this study we have investigated the conformational changes that take place within antitrypsin during complex formation with catalytically inactive (thrombin(S195A)) and active thrombin. Three variants of antitrypsin Pittsburgh (an effective thrombin inhibitor), each containing a unique ... More
The mechanism of pore assembly for a cholesterol-dependent cytolysin: formation of a large prepore complex precedes the insertion of the transmembrane beta-hairpins.
AuthorsShepard LA, Shatursky O, Johnson AE, Tweten RK
JournalBiochemistry
PubMed ID10956018
Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of membrane-penetrating toxins. The CDCs form large homooligomers (estimated to be comprised of up to 50 CDC monomers) that are responsible for generating a large pore in cholesterol-containing membranes of eukaryotic cells. The assembly of the PFO cytolytic ... More
Intracellular Ca(2+) regulates the cellular iron uptake in K562 cells.
AuthorsCi W, Li W, Ke Y, Qian ZM, Shen X
JournalCell Calcium
PubMed ID12618146
Fluorescence quenching was used to study the kinetics of the transferrin receptor (TfR)-mediated iron uptake in the calcein-loaded K562 cells. It was found that elevation of intracellular free Ca(2+) ([Ca(2+)](i)) by thapsigargin (TG) speeds up the initial rate of iron uptake and increases the overall capacity of the cells in ... More
Manipulating the amyloid-beta aggregation pathway with chemical chaperones.
AuthorsYang DS, Yip CM, Huang TH, Chakrabartty A, Fraser PE
JournalJ Biol Chem
PubMed ID10551864
Amyloid-beta (Abeta) assembly into fibrillar structures is a defining characteristic of Alzheimer's disease that is initiated by a conformational transition from random coil to beta-sheet and a nucleation-dependent aggregation process. We have investigated the role of organic osmolytes as chemical chaperones in the amyloid pathway using glycerol to mimic the ... More
The use of fluorescent probes to characterize conformational changes in the interaction between vitronectin and plasminogen activator inhibitor-1.
AuthorsGibson A, Baburaj K, Day DE, Verhamme I, Shore JD, Peterson CB
JournalJ Biol Chem
PubMed ID9030577
Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue-type plasminogen activator and urokinase, is known to convert readily to a latent form by insertion of the reactive center loop into a central beta-sheet. Interaction with vitronectin stabilizes PAI-1 and decreases the rate of conversion to the latent form, but conformational ... More
Calmodulin binds to caldesmon in an antiparallel manner.
AuthorsWang E, Zhuang S, Kordowska J, Grabarek Z, Wang CL
JournalBiochemistry
PubMed ID9398229
Two of the five tryptophan residues (W659 and W692) in chicken gizzard smooth muscle caldesmon (CaD) are located within the calmodulin (CaM) binding sites in the C-terminal region of the molecule. When these Trp residues are replaced with Gly in either recombinant fragments or synthetic peptides of CaD, the affinity ... More
Determining the membrane topology of proteins: insertion pathway of a transmembrane helix of annexin 12.
AuthorsLadokhin AS, Isas JM, Haigler HT, White SH
JournalBiochemistry
PubMed ID12427023
We describe a sensitive method for determining the bilayer topology of single-site cysteine-linked NBD fluorescent labels on membrane proteins. Based upon a method developed for peptides [W. C. Wimley and S. H. White (2000) Biochemistry 39, 161-170], it utilizes a novel fluorescence quencher, lysoUB, comprised of a single acyl chain ... More
Structural similarity of the covalent complexes formed between the serpin plasminogen activator inhibitor-1 and the arginine-specific proteinases trypsin, LMW u-PA, HMW u-PA, and t-PA: use of site-specific fluorescent probes of local environment.
AuthorsBackovic M, Stratikos E, Lawrence DA, Gettins PG
JournalProtein Sci
PubMed ID11967374
We have used two fluorescent probes, NBD and dansyl, attached site-specifically to the serpin plasminogen activator inhibitor-1 (PAI-1) to address the question of whether a common mechanism of proteinase translocation and full insertion of the reactive center loop is used by PAI-1 when it forms covalent SDS-stable complexes with four ... More
Room temperature trapping of rhodopsin photointermediates.
AuthorsSikora S, Little AS, Dewey TG
JournalBiochemistry
PubMed ID8161500
By suspending bovine rhodopsin in trehalose-water glass films, it is possible to trap photostates in the light-activation process. Because of the unusually high vitrification temperature of trehalose-water mixtures, this trapping can be accomplished at room temperature. This allows for a facile investigation of the spectroscopic properties of rhodopsin's photointermediates. Depending ... More
The F-G loop region of cytochrome P450scc (CYP11A1) interacts with the phospholipid membrane.
AuthorsHeadlam MJ, Wilce MC, Tuckey RC
JournalBiochim Biophys Acta
PubMed ID14637024
Cytochrome P450scc (CYP11A1) is a protein attached to the inner surface of the inner mitochondrial membrane that uses cholesterol from the membrane phase as its substrate for the first step in steroid hormone synthesis. We investigated the mechanism by which CYP11A1 interacts with the membrane. Hydrophobicity profiles of CYP11A1 and ... More
Role of the catalytic serine in the interactions of serine proteinases with protein inhibitors of the serpin family. Contribution of a covalent interaction to the binding energy of serpin-proteinase complexes.
AuthorsOlson ST, Bock PE, Kvassman J, Shore JD, Lawrence DA, Ginsburg D, Björk I
JournalJ Biol Chem
PubMed ID8530403
The contribution of a covalent bond to the stability of complexes of serine proteinases with inhibitors of the serpin family was evaluated by comparing the affinities of beta-trypsin and the catalytic serine-modified derivative, beta-anhydrotrypsin, for several serpin and non-serpin (Kunitz) inhibitors. Kinetic analyses showed that anhydrotrypsin had little or no ... More
Interaction between ion channel-inactivating peptides and anionic phospholipid vesicles as model targets.
Studies of rapid (N-type) inactivation induced by different synthetic inactivating peptides in several voltage-dependent cation channels have concluded that the channel inactivation "entrance" (or "receptor" site for the inactivating peptide) consists of a hydrophobic vestibule within the internal mouth of the channel, separated from the cytoplasm by a region with ... More
Resolution of Michaelis complex, acylation, and conformational change steps in the reactions of the serpin, plasminogen activator inhibitor-1, with tissue plasminogen activator and trypsin.
AuthorsOlson ST, Swanson R, Day D, Verhamme I, Kvassman J, Shore JD
JournalBiochemistry
PubMed ID11570875
Michaelis complex, acylation, and conformational change steps were resolved in the reactions of the serpin, plasminogen activator inhibitor-1 (PAI-1), with tissue plasminogen activator (tPA) and trypsin by comparing the reactions of active and Ser 195-inactivated enzymes with site-specific fluorescent-labeled PAI-1 derivatives that report these events. Anhydrotrypsin or S195A tPA-induced fluorescence ... More
A proposed common structure of substrates bound to mitochondrial processing peptidase.
AuthorsKojima K, Kitada S, Ogishima T, Ito A
JournalJ Biol Chem
PubMed ID11031253
Mitochondrial processing peptidase (MPP), a metalloendopeptidase consisting of alpha- and beta-subunits, specifically cleaves off the N-terminal presequence of the mitochondrial protein precursor. Structural information of the substrate bound to MPP was obtained using fluorescence resonance energy transfer (FRET) measurement. A series of the peptide substrates, which have distal arginine residues ... More
Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies.
AuthorsSokal I, Li N, Klug CS, Filipek S, Hubbell WL, Baehr W, Palczewski K
JournalJ Biol Chem
PubMed ID11524415
Guanylyl cyclase-activating proteins are EF-hand Ca(2+)-binding proteins that belong to the calmodulin superfamily. They are involved in the regulation of photoreceptor membrane-associated guanylyl cyclases that produce cGMP, a second messenger of vertebrate vision. Here, we investigated changes in GCAP1 structure using mutagenesis, chemical modifications, and spectroscopic methods. Two Cys residues ... More
Inactivation of NADP(+)-dependent isocitrate dehydrogenase by nitric oxide.
AuthorsYang ES, Richter C, Chun JS, Huh TL, Kang SS, Park JW
JournalFree Radic Biol Med
PubMed ID12361803
Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. NO donors such as S-nitrosothiols, diethylamine NONOate, spermine NONOate, and 3-morpholinosydnomine N-ethylcarbamide (SIN-1)/superoxide dismutase inactivated ICDH in ... More
Agonist-induced conformational changes at the cytoplasmic side of transmembrane segment 6 in the beta 2 adrenergic receptor mapped by site-selective fluorescent labeling.
The environmentally sensitive, sulfhydryl-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine (IANBD) was used as a molecular reporter of agonist-induced conformational changes in the beta(2) adrenergic receptor, a prototype hormone-activated G protein-coupled receptor. In the background of a mutant beta(2) adrenergic receptor, with a minimal number of endogenous cysteine residues, new cysteines were ... More
Heparan sulfate proteoglycan isoforms of the CD44 hyaluronan receptor induced in human inflammatory macrophages can function as paracrine regulators of fibroblast growth factor action.
AuthorsJones M, Tussey L, Athanasou N, Jackson DG
JournalJ Biol Chem
PubMed ID10713114
The CD44 glycoprotein is expressed in multiple isoforms on a variety of cell types where it functions as a receptor for hyaluronan-mediated motility. Recently, interest has centered on CD44 heparan sulfate proteoglycan (HSPG) isoforms because of their potential to sequester heparin-binding growth factors and chemokines. Expression of these isoforms on ... More
Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an alpha-helical to beta-sheet transition identified by fluorescence spectroscopy.
AuthorsShepard LA, Heuck AP, Hamman BD, Rossjohn J, Parker MW, Ryan KR, Johnson AE, Tweten RK
JournalBiochemistry
PubMed ID9772185
Clostridium perfringens perfringolysin O (PFO or theta-toxin) is a cytolytic toxin that binds to cholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varying size in the bilayer. In this study, a membrane-spanning domain has been identified in PFO by a combination of fluorescence spectroscopic methods using the ... More
Fluorescent labeling of purified beta 2 adrenergic receptor. Evidence for ligand-specific conformational changes.
AuthorsGether U, Lin S, Kobilka BK
JournalJ Biol Chem
PubMed ID7499324
The purpose of the present study was to develop an approach to directly monitor structural changes in a G protein-coupled receptor in response to drug binding. Purified human beta 2 adrenergic receptor was covalently labeled with the cysteine-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)ethylenediamine (IANBD). IANBD is characterized by a fluorescence which ... More