The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins.
AuthorsZhang B, Zhang Y, Wang Z, Zheng Y
JournalJ Biol Chem
PubMed ID10843989
'The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which ... More
Kinetics of Cdc42 membrane extraction by Rho-GDI monitored by real-time fluorescence resonance energy transfer.
AuthorsNomanbhoy TK, Erickson JW, Cerione RA
JournalBiochemistry
PubMed ID10026253
'The mechanisms underlying the ability of the Rho-GDP dissociation inhibitor (RhoGDI) to elicit the release of Rho-related GTP-binding proteins from membranes is currently unknown. In this report, we have set out to address this issue by using fluorescence resonance energy transfer approaches to examine the functional interactions of the RhoGDI ... More
Biochemical analysis of SopE from Salmonella typhimurium, a highly efficient guanosine nucleotide exchange factor for RhoGTPases.
'RhoGTPases are key regulators of eukaryotic cell physiology. The bacterial enteropathogen Salmonella typhimurium modulates host cell physiology by translocating specific toxins into the cytoplasm of host cells that induce responses such as apoptotic cell death in macrophages, the production of proinflammatory cytokines, the rearrangement of the host cell actin cytoskeleton ... More
Alanine scan mutagenesis of the switch I domain of the Caulobacter crescentus CgtA protein reveals critical amino acids required for in vivo function.
AuthorsLin B, Skidmore JM, Bhatt A, Pfeffer SM, Pawloski L, Maddock JR
JournalMol Microbiol
PubMed ID11251813
'The Caulobacter crescentus CgtA protein is a member of the Obg/GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP and displays rapid exchange rate constants for either nucleotide, indicating that the guanine nucleotide-binding and exchange properties of CgtA are different from those ... More
Different structural and kinetic requirements for the interaction of Ran with the Ran-binding domains from RanBP2 and importin-beta.
'The cytoplasmic disassembly of Ran.GTP.importin and Ran.GTP.exportin. cargo complexes is an essential step in the corresponding nuclear import and export cycles. It has previously been shown that such disassembly can be mediated by RanBP1 in the presence of RanGAP. The nuclear pore complex protein RanBP2 (Nup358) contains four Ran-binding domains ... More
Molecular role for the Rab binding platform of guanine nucleotide dissociation inhibitor in endoplasmic reticulum to Golgi transport.
AuthorsWu SK, Luan P, Matteson J, Zeng K, Nishimura N, Balch WE
JournalJ Biol Chem
PubMed ID9756941
'Guanine nucleotide dissociation inhibitor (GDI) regulates the recycling of Rab GTPases involved in vesicle targeting and fusion. We have analyzed the requirement for conserved amino acid residues in the binding of Rab1A and the function of GDI in transport of cargo between the endoplasmic reticulum (ER) and the Golgi apparatus. ... More
Targeting of Rac1 to the phagocyte membrane is sufficient for the induction of NADPH oxidase assembly.
AuthorsGorzalczany Y, Sigal N, Itan M, Lotan O, Pick E
JournalJ Biol Chem
PubMed ID11007780
'The superoxide (O(2))-generating NADPH oxidase complex of phagocytes consists of a membrane-associated flavocytochrome (cytochrome b(559)) and four cytosolic proteins, p47(phox), p67(phox), p40(phox), and the small GTPase Rac (Rac1 or -2). NADPH oxidase activation (O(2) production) is elicited as the consequence of assembly of some or all cytosolic components with cytochrome ... More
Characterization of the ternary complex between Rab7, REP-1 and Rab geranylgeranyl transferase.
AuthorsAlexandrov K, Simon I, Yurchenko V, Iakovenko A, Rostkova E, Scheidig AJ, Goody RS
JournalEur J Biochem
PubMed ID10491170
'Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein ... More
S-glutathiolation by peroxynitrite of p21ras at cysteine-118 mediates its direct activation and downstream signaling in endothelial cells.
AuthorsClavreul N, Adachi T, Pimental DR, Ido Y, Schöneich C, Cohen RA
JournalFASEB J
PubMed ID16415107
'The highly reactive species, peroxynitrite, is produced in endothelial cells in pathological states in which the production of superoxide anion and NO is increased. Here, we show that peroxynitrite added exogenously or generated endogenously in response to exposure to an NO donor or oxidized low-density lipoproteins (oxLDL) increases p21ras activity ... More
Kinetics of interaction of Rab5 and Rab7 with nucleotides and magnesium ions.
AuthorsSimon I, Zerial M, Goody RS
JournalJ Biol Chem
PubMed ID8702787
'We describe here the kinetics of the interaction of GTP and GDP with the small GTP-binding proteins Rab5 and Rab7. It was possible to make use of the intrinsic fluorescence of these proteins, since Rab5 contains two and Rab7 three tryptophan residues, respectively. With both enzymes, there is a significant ... More
The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding.
AuthorsJault JM, Fieulaine S, Nessler S, Gonzalo P, Di Pietro A, Deutscher J, Galinier A
JournalJ Biol Chem
PubMed ID10636874
'Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on ... More
RanBP10 is a cytoplasmic guanine nucleotide exchange factor that modulates noncentrosomal microtubules.
AuthorsSchulze H, Dose M, Korpal M, Meyer I, Italiano JE, Shivdasani RA,
JournalJ Biol Chem
PubMed ID18347012
'Microtubule spindle assembly in mitosis is stimulated by Ran.GTP, which is generated along condensed chromosomes by the guanine nucleotide exchange factor (GEF) RCC1. This relationship suggests that similar activities might modulate other microtubule structures. Interphase microtubules usually extend from the centrosome, although noncentrosomal microtubules function in some differentiated cells, including ... More
Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide exchange factor Cdc25Mm.
AuthorsLenzen C, Cool RH, Prinz H, Kuhlmann J, Wittinghofer A
JournalBiochemistry
PubMed ID9585556
'Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. We present fluorescence measurements to define the kinetic constants that characterize the interactions between Ras, GEF, and nucleotides, ... More
Vav2 is an activator of Cdc42, Rac1, and RhoA.
AuthorsAbe K, Rossman KL, Liu B, Ritola KD, Chiang D, Campbell SL, Burridge K, Der CJ
JournalJ Biol Chem
PubMed ID10744696
'Vav and Vav2 are members of the Dbl family of proteins that act as guanine nucleotide exchange factors (GEFs) for Rho family proteins. Whereas Vav expression is restricted to cells of hematopoietic origin, Vav2 is widely expressed. Although Vav and Vav2 share highly related structural similarities and high sequence identity ... More
The kinetic mechanism of the GAP-activated GTPase of p21 ras.
AuthorsMoore KJ, Lowe PN, Eccleston JF
JournalPhilos Trans R Soc Lond B Biol Sci
PubMed ID1351296
'Guanine nucleotides modified by acetylation of the ribose moiety with the small fluorophore N-methylanthranilic acid(mant) have been shown to bind to p21 ras with similar equilibrium and kinetic rate constants as the parent nucleotides. Hydrolysis of p21.mantGTP to p21.mantGDP results in a 10% decrease in fluorescence intensity occurring at the ... More
Glucosylation and ADP ribosylation of rho proteins: effects on nucleotide binding, GTPase activity, and effector coupling.
AuthorsSehr P, Joseph G, Genth H, Just I, Pick E, Aktories K
JournalBiochemistry
PubMed ID9548761
'We studied the effects of glucosylation of RhoA, Rac1, and Cdc42 at threonine-35 and -37 by Clostridium difficile toxin B on nucleotide binding, GTPase activity, and effector coupling and compared these results with the ADP ribosylation of RhoA at asparagine-41 catalyzed by Clostridium botulinum C3 transferase. Whereas glucosylation and ADP ... More
Nucleotide binding by the erythrocyte transglutaminase/Gh protein, probed with fluorescent analogs of GTP and GDP.
AuthorsMurthy SN, Lorand L
JournalProc Natl Acad Sci U S A
PubMed ID10869438
'GTP is known to be a potent inhibitor of the protein crosslinking activity of transglutaminase (TG), probably the most abundant G protein in the human red cell. Nucleotide binding to TG was examined by fluorescence spectroscopy and anisotropy in mixtures of TG with methylanthraniloyl analogs of GTP and GDP. A ... More
Fluorescence methods for monitoring interactions of Rab proteins with nucleotides, Rab escort protein, and geranylgeranyltransferase.
AuthorsAlexandrov K, Scheidig AJ, Goody RS
JournalMethods Enzymol
PubMed ID11210530
Fluorescence approaches to the study of the p21ras GTPase mechanism.
AuthorsEccleston JF, Moore KJ, Brownbridge GG, Webb MR, Lowe PN
JournalBiochem Soc Trans
PubMed ID1889625
The use of ribose-modified guanine nucleotides and tryptophan mutants of p21ras, neither of which have significant effect on the kinetic mechanism of the p21ras GTPase and the GAP-activated p21ras GTPase, will now allow a detailed kinetic study of how GAP and other regulatory proteins interact with p21ras. This will lead ... More
Stimulation of the GTPase activity of translation elongation factor G by ribosomal protein L7/12.
AuthorsSavelsbergh A, Mohr D, Wilden B, Wintermeyer W, Rodnina MV
JournalJ Biol Chem
PubMed ID10625623
Elongation factors (EFs) Tu and G are GTPases that have important functions in protein synthesis. The low intrinsic GTPase activity of both factors is strongly stimulated on the ribosome by unknown mechanisms. Here we report that isolated ribosomal protein L7/12 strongly stimulates GTP hydrolysis by EF-G, but not by EF-Tu, ... More
Mechanism of Rab geranylgeranylation: formation of the catalytic ternary complex.
AuthorsAnant JS, Desnoyers L, Machius M, Demeler B, Hansen JC, Westover KD, Deisenhofer J, Seabra MC
JournalBiochemistry
PubMed ID9730828
Rab proteins are geranylgeranylated on one or two C-terminal cysteines by Rab geranylgeranyl transferase (RabGGTase). The reaction is dependent on a Rab-binding protein, termed Rab escort protein (REP). Here, we studied the role of REP in the geranylgeranylation reaction. We first characterized the interaction between REP and ungeranylgeranylated Rab using ... More
Transient kinetic studies on the interaction of Ras and the Ras-binding domain of c-Raf-1 reveal rapid equilibration of the complex.
AuthorsSydor JR, Engelhard M, Wittinghofer A, Goody RS, Herrmann C
JournalBiochemistry
PubMed ID9760267
Transient kinetic methods have been used to analyze the interaction between the Ras-binding domain (RBD) of c-Raf-1 and a complex of H-Ras and a GTP analogue. The results obtained show that the binding is a two-step process, with an initial rapid equilibrium step being followed by an isomerization reaction occurring ... More
Substrate and product structural requirements for binding of nucleotides to H-ras p21: the mechanism of discrimination between guanosine and adenosine nucleotides.
AuthorsRensland H, John J, Linke R, Simon I, Schlichting I, Wittinghofer A, Goody RS
JournalBiochemistry
PubMed ID7819254
The interaction of the protein product of the H-ras oncogene with a series of nucleoside di- and triphosphates has been examined to investigate the tolerance of the active site to departures from the GTP or GDP structures. Nucleotides which bind relatively strongly could be used as competitors of GDP in ... More
Interaction of guanine nucleotides with the signal recognition particle from Escherichia coli.
AuthorsJagath JR, Rodnina MV, Lentzen G, Wintermeyer W
JournalBiochemistry
PubMed ID9799502
The bacterial signal recognition particle (SRP) is an RNA-protein complex. In Escherichia coli, the particle consists of a 114 nt RNA, a 4.5S RNA, and a 48 kDa GTP-binding protein, Ffh. GDP-GTP exchange on, and GTP hydrolysis by, Ffh are thought to regulate SRP function in membrane targeting of translating ... More
Interferon-induced MxA protein. GTP binding and GTP hydrolysis properties.
AuthorsRichter MF, Schwemmle M, Herrmann C, Wittinghofer A, Staeheli P
JournalJ Biol Chem
PubMed ID7539429
MxA is a GTPase encoded by an interferon-activated human gene which inhibits the multiplication of several RNA viruses. Recombinant histidine-tagged MxA protein (His-MxA) was expressed in Escherichia coli and purified to near homogeneity. Gel filtration showed that it formed high molecular weight oligomers. Purified His-MxA exhibited specific GTP hydrolysis rates ... More
Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1.
AuthorsKlebe C, Bischoff FR, Ponstingl H, Wittinghofer A
JournalBiochemistry
PubMed ID7819259
The guanine nucleotide dissociation and GTPase reactions of Ran, a Ras-related nuclear protein, have been investigated using different fluorescence techniques to determine how these reactions are stimulated by the guanine nucleotide exchange factor RCC1 and the other regulatory protein, RanGAP1 (GTPase-activating protein). The intrinsic GTPase of Ran is one-tenth of ... More
Mg2+ and a key lysine modulate exchange activity of eukaryotic translation elongation factor 1B alpha.
To sustain efficient translation, eukaryotic elongation factor B alpha (eEF1B alpha) functions as the guanine nucleotide exchange factor for eEF1A. Stopped-flow kinetics using 2'-(or 3')-O-N-methylanthraniloyl (mant)-GDP showed spontaneous release of nucleotide from eEF1A is extremely slow and accelerated 700-fold by eEF1B alpha. The eEF1B alpha-stimulated reaction was inhibited by Mg2+ ... More
Individual rate constants for the interaction of Ras proteins with GTPase-activating proteins determined by fluorescence spectroscopy.
AuthorsAhmadian MR, Hoffmann U, Goody RS, Wittinghofer A
JournalBiochemistry
PubMed ID9109662
Individual rate constants for the interaction of H-, K-, and N-Ras with GAP-334 and NF1-333 were determined using fluorescent derivatives of guanine nucleotides at the active site of the Ras proteins. Stopped-flow experiments with NF1-333 show a fast concentration-dependent initial phase corresponding to the binding reaction followed by a slower ... More
Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome.
AuthorsWilden B, Savelsbergh A, Rodnina MV, Wintermeyer W
JournalProc Natl Acad Sci U S A
PubMed ID16940356
The translocation of tRNA and mRNA through the ribosome is promoted by elongation factor G (EF-G), a GTPase that hydrolyzes GTP during the reaction. Recently, it was reported that, in contrast to previous observations, the affinity of EF-G was much weaker for GTP than for GDP and that ribosome-catalyzed GDP-GTP ... More
Elucidation of binding determinants and functional consequences of Ras/Raf-cysteine-rich domain interactions.
AuthorsWilliams JG, Drugan JK, Yi GS, Clark GJ, Der CJ, Campbell SL
JournalJ Biol Chem
PubMed ID10777480
Raf-1 is a critical downstream target of Ras and contains two distinct domains that bind Ras. The first Ras-binding site (RBS1) in Raf-1 has been shown to be essential for Ras-mediated translocation of Raf-1 to the plasma membrane, whereas the second site, in the Raf-1 cysteine-rich domain (Raf-CRD), has been ... More
The interaction between rac1 and its guanine nucleotide dissociation inhibitor (GDI), monitored by a single fluorescent coumarin attached to GDI.
The interaction of rac with guanine nucleotide dissociation inhibitor protein (rhoGDI) is described, using GDI fluorescently labeled on its single cysteine with N-[2-(1-maleimidyl)ethyl]-7-diethylaminocoumarin-3-carboxamide (MDCC). The labeled GDI shows a 70% decrease in fluorescence emission on binding geranylgeranylated rac1.GDP and has an affinity for rac1 within a factor of 2 of ... More
Fluorescent guanine nucleotide analogs and G protein activation.
AuthorsRemmers AE, Posner R, Neubig RR
JournalJ Biol Chem
PubMed ID8188654
The N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Both MANT-GTP gamma S (mGTP gamma S) and MANT-GTP (mGTP) displayed a magnesium-dependent increase in fluorescence upon binding to bovine brain G(o). A much greater increase in MANT-guanine nucleotide fluorescence was observed with ... More
SopE and SopE2 from Salmonella typhimurium activate different sets of RhoGTPases of the host cell.
AuthorsFriebel A, Ilchmann H, Aepfelbacher M, Ehrbar K, Machleidt W, Hardt WD
JournalJ Biol Chem
PubMed ID11440999
The bacterial enteropathogen Salmonella typhimurium employs a specialized type III secretion system to inject toxins into host cells, which trigger signaling cascades leading to cell death in macrophages, secretion of pro-inflammatory cytokines, or rearrangements of the host cell cytoskeleton and thus to bacterial invasion. Two of the injected toxins, SopE ... More
Mechanism of nucleotide release from Rho by the GDP dissociation stimulator protein.
AuthorsHutchinson JP, Eccleston JF
JournalBiochemistry
PubMed ID10985780
Guanine nucleotide dissociation stimulator (GDS) promotes the release of tightly bound GDP from various Ras superfamily proteins, including RhoA, Rac1, K-Ras, Rap1A, and Rap1B. It displays no significant sequence homology to other known exchange factors for small G-proteins. Studies are reported here of the mechanism of GDS-mediated nucleotide release from ... More
Structural mapping of catalytic site with respect to alpha-subunit and noncatalytic site in yeast mitochondrial F1-ATPase using fluorescence resonance energy transfer.
AuthorsDivita G, Goody RS, Gautheron DC, Di Pietro A
JournalJ Biol Chem
PubMed ID8514756
The intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1 is a very sensitive probe to differentiate nucleotide binding to catalytic and noncatalytic sites (Divita, G., Di Pietro, A., Roux, B., and Gautheron, D. C. (1992) Biochemistry 31, 5791-5798), the catalytic site saturation producing quenching of Trp-257 fluorescence (Divita, G., Jault, ... More
Characterization of p190RhoGEF, a RhoA-specific guanine nucleotide exchange factor that interacts with microtubules.
Rho family GTPases control numerous cellular processes including cytoskeletal reorganization and transcriptional activation. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) which stimulate the exchange of bound GDP for GTP. We recently isolated a putative GEF, termed p190RhoGEF that binds to RhoA and, when overexpressed in neuronal cells, ... More
Hydrolysis of GTP by p21NRAS, the NRAS protooncogene product, is accompanied by a conformational change in the wild-type protein: use of a single fluorescent probe at the catalytic site.
AuthorsNeal SE, Eccleston JF, Webb MR
JournalProc Natl Acad Sci U S A
PubMed ID2185475
2'(3')-O-(N-Methyl)anthraniloylguanosine 5'-triphosphate (mantGTP) is a fluorescent analogue of GTP that has similar properties to the physiological substrate in terms of its binding constant and the kinetics of its interactions with p21NRAS, the NRAS protooncogene product. There is a 3-fold increase in fluorescence intensity when mantGTP binds to p21NRAS. The rate ... More
Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy.
AuthorsLeonard DA, Evans T, Hart M, Cerione RA, Manor D
JournalBiochemistry
PubMed ID7918454
We have developed several high-resolution assays for the nucleotide state of a rho-subfamily low molecular weight GTP-binding protein, Cdc42Hs. The first involves the use of the fluorescent N-methylanthraniloyl derivative of GDP (mant-GDP). As has been shown for the ras protein, mant-dGDP fluorescence is significantly enhanced (approximately 20%) upon binding to ... More
Dynamic and equilibrium studies on the interaction of Ran with its effector, RanBP1.
AuthorsKuhlmann J, Macara I, Wittinghofer A
JournalBiochemistry
PubMed ID9315840
Ran, a small nuclear GTP-binding protein, is one of the most abundant Ras-related proteins in eucaryotic cells. Ran is essential for nucleo-cytoplasmatic transport and is primarily localized in the nucleus and at the nuclear pore complex. Here, we characterize the kinetics and equilibrium of the interaction between Ran and RanBP1 ... More
Neosynthesis and activation of Rho by Escherichia coli cytotoxic necrotizing factor (CNF1) reverse cytopathic effects of ADP-ribosylated Rho.
Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA ... More
cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension.
AuthorsChristensen AE, Selheim F, de Rooij J, Dremier S, Schwede F, Dao KK, Martinez A, Maenhaut C, Bos JL, Genieser HG, Døskeland SO
JournalJ Biol Chem
PubMed ID12819211
Little is known about the relative role of cAMP-dependent protein kinase (cAPK) and guanine exchange factor directly activated by cAMP (Epac) as mediators of cAMP action. We tested cAMP analogs for ability to selectively activate Epac1 or cAPK and discriminate between the binding sites of Epac and of cAPKI and ... More
Interactions of Escherichia coli primary replicative helicase DnaB protein with nucleotide cofactors.
AuthorsJezewska MJ, Kim US, Bujalowski W
JournalBiophys J
PubMed ID8889182
Interactions between the Escherichia coli primary replicative helicase DnaB protein and nucleotide cofactors have been studied using several fluorescent nucleotide analogs and unmodified nucleotides. The thermodynamically rigorous fluorescent titration technique has been used to obtain true binding isotherms, independently of the assumptions of any relationships between the observed quenching of ... More
Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy.
AuthorsNomanbhoy TK, Cerione R
JournalJ Biol Chem
PubMed ID8626553
The GDP-dissociation-inhibitor (GDI) for Rho-like GTP-binding proteins is capable of three different biochemical activities. These are the inhibition of GDP dissociation, the inhibition of GTP hydrolysis, and the stimulation of the release of GTP-binding proteins from membranes. In order to better understand how GDI interactions with Rho-like proteins mediate these ... More
Kinetics of the interaction of translation factor SelB from Escherichia coli with guanosine nucleotides and selenocysteine insertion sequence RNA.
AuthorsThanbichler M, Bock A, Goody RS
JournalJ Biol Chem
PubMed ID10781605
The kinetics of the interaction of GTP and GDP with SelB, the specific translation factor for the incorporation of selenocysteine into proteins, have been investigated using the stopped-flow method. Useful signals were obtained using intrinsic (i.e. tryptophan) fluorescence, the fluorescence of methylanthraniloyl derivatives of nucleotides, or fluorescence resonance energy transfer ... More
Partial G protein activation by fluorescent guanine nucleotide analogs. Evidence for a triphosphate-bound but inactive state.
AuthorsRemmers AE, Neubig RR
JournalJ Biol Chem
PubMed ID8617747
N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Previously, we showed that MANT fluorescence intensity when bound to G protein was related to the degree of G protein activation where MANT-guanosine-5'-O-(3-thiotriphosphate) (mGTP gammaS) had the highest fluorescence followed by mGTP and mGDP, ... More
The kinetic mechanism of Ran--nucleotide exchange catalyzed by RCC1.
AuthorsKlebe C, Prinz H, Wittinghofer A, Goody RS
JournalBiochemistry
PubMed ID7548002
The interaction of Ran, a Ras-related nuclear GTP-binding protein, with its guanine nucleotide exchange factor RCC1 has been studied by equilibrium and transient kinetic measurements using fluorescent nucleotides. The four-step mechanism of catalyzed nucleotide exchange involves the formation of ternary complexes consisting of Ran, RCC1, and GXP as well as ... More
Prodan fluorescence reflects differences in nucleotide-induced conformational states in the myosin head and allows continuous visualization of the ATPase reactions.
AuthorsHiratsuka T
JournalBiochemistry
PubMed ID9585528
The noncovalent fluorescent probe 6-propionyl-2-(dimethylamino)naphthalene (prodan) binds stoichiometrically to myosin subfragment-1 (S-1) without affecting the ATPase and actin-binding properties of S-1. Neither ATP nor actin interferes with the prodan binding. Free prodan exhibits a green emission peak at 520 nm. However, the prodan bound to S-1 and the S-1.ADP complex ... More
RABIF/MSS4 is a Rab-stabilizing holdase chaperone required for GLUT4 exocytosis.
Authors
JournalProc Natl Acad Sci U S A
PubMed ID28894007
NRBF2 is a RAB7 effector required for autophagosome maturation and mediates the association of APP-CTFs with active form of RAB7 for degradation.
Authors
JournalAutophagy
PubMed ID32543313
Enhancing autophagy maturation with CCZ1-MON1A complex alleviates neuropathology and memory defects in Alzheimer disease models.