Kinetic determination of the GTPase activity of Ras proteins by means of a luminescent terbium complex

Spangler, Christian; Spangler, Corinna M.; Spoerner, Michael; Schäferling, Michael

doi:10.1007/s00216-008-2517-7

Cited by 17 publications

(21 citation statements)

References 35 publications

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“…The same is observed in case of NADP [59], DNA [60], heparin [61], GTP, and GDP [62]. On the other hand, the emission is quenched by phosphate [59], pyrophosphate [63], and lecithin [64].…”

Section: Complexes With Non-chelating Ligandssupporting

confidence: 74%

Luminescent Chemical and Physical Sensors Based on Lanthanide Complexes

Spangler

Schäferling

2010

Lanthanide Luminescence

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Luminescent lanthanide complexes (LLCs) are versatile probes for the determination of a variety of analytes such as metal ions, anions, hydrogen peroxide, ATP, pH, or oxygen. Antenna chromophores have been introduced as ligands for sensitizing lanthanide emission. The ligand system or the lanthanide ion can act as receptors for reversible binding of analytes. Thus, the response of luminescence emission can occur via ligand or metal-centered processes, which will both be discussed. LLCs, particularly Eu 3+ and Tb 3+ complexes, have been incorporated in solid state matrices, e.g., polymer hydrogels or silica to obtain optical sensor layers. A selection of such sensitive materials will be outlined, including sensors for the analytically relevant parameters oxygen, pH, hydrogen peroxide, humidity, and temperature.

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“…The same is observed in case of NADP [59], DNA [60], heparin [61], GTP, and GDP [62]. On the other hand, the emission is quenched by phosphate [59], pyrophosphate [63], and lecithin [64].…”

Section: Complexes With Non-chelating Ligandssupporting

confidence: 74%

Luminescent Chemical and Physical Sensors Based on Lanthanide Complexes

Spangler

Schäferling

2010

Lanthanide Luminescence

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“…One can assume that in the corresponding complex the arginine of Ras at position 32 partly adopts the function of the arginine finger in GAP. This acceleration could be confirmed determining the dissociation of inorganic phosphate as a product of GTP hydrolysis by means of a luminescent terbium complex (42). Finally, the differential intrinsic hydrolysis activity of two triphosphate-bound states might be generalized to explain the very slowly hydrolyzing activity of the Ran.…”

Section: Conformational States Of Ras Bound To Gtp-whenmentioning

confidence: 84%

Conformational States of Human Rat Sarcoma (Ras) Protein Complexed with Its Natural Ligand GTP and Their Role for Effector Interaction and GTP Hydrolysis

Spoerner

Hozsa

Poetzl

et al. 2010

Journal of Biological Chemistry

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187

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The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH 2 p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer 31 P NMR experiments and effector binding studies we show that Ras(wt)⅐Mg 2؉ ⅐GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2. At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1-166) K 12 is 11.3. K 12 of full-length Ras is >20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (k ex ) for Ras(wt)⅐Mg 2؉ ⅐GTP is 7 s ؊1 and thus 18-fold lower compared with that found for the Ras⅐GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/ C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.The guanine nucleotide-binding protein Ras is involved in cellular signal transduction pathways inducing proliferation, differentiation, or apoptosis of cells. It functions as a molecular switch, cycling between an inactive GDP-bound state and an active GTP-bound state. In the active conformation Ras is able to bind different effector proteins such as Raf kinase or RalGDS with nanomolar affinity by interacting with its switch I region. Thus signals can be transmitted resulting in the corresponding cellular response.When bound to guanosine triphosphates Ras exists in two different conformational states, defined as states 1 and 2, which are in chemical equilibrium in solution (1-3). These conformational states are actually only defined for Ras with guanosine triphosphate bound (T), a state that may be different from a state with guanine diphosphate bound (D). Therefore, we will denote the two states in the following as state 1(T) and 2(T) whenever the nucleotide ligand is of concern. The equilibrium between the two states is strongly influenced by the nature of the guanine nucleotide bound to Ras and can be shifted by interaction with effector proteins or regulators such as GTPase activation proteins (GAPs).2 It was found that GTP analogs GppNHp and GppCH 2 p partially shift the equilibrium toward state 1. For the complex of wild-type Ras with physiological GTP itself the existence of the two states could not be...

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“…The amount of OsYchF1 used in the reaction was quantified as described (17). The assays were recorded in 96-well microplates, with each well having a final reaction volume of 100 l. The concentrations of GTP-bound OsYchF1 and ATP-bound OsYchF1 in the assays were 13.4 and 13.0 M, respectively (within the range tested in the method reference, 6 -24 M).…”

Section: Methodsmentioning

confidence: 99%

“…GTPase/ATPase Activity Assays-The GTPase/ATPase activities were examined by monitoring the decrease in lanthanide luminescence of Tb(III)-norfloxacin due to strong quenching by P i during GTP/ATP hydrolysis, following the procedures in a previous report (17). The concentrations of charged GTP and charged ATP were estimated by HPLC.…”

Section: Methodsmentioning

confidence: 99%

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An Ancient P-Loop GTPase in Rice Is Regulated by a Higher Plant-specific Regulatory Protein

Cheung

Xue

Zhou

et al. 2010

Journal of Biological Chemistry

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YchF is a subfamily of the Obg family in the TRAFAC class of P-loop GTPases. The wide distribution of YchF homologues in both eukarya and bacteria suggests that they are descendents of an ancient protein, yet their physiological roles remain unclear. Using the OsYchF1-OsGAP1 pair from rice as the prototype, we provide evidence for the regulation of GTPase/ATPase activities and RNA binding capacity of a plant YchF (OsYchF1) by its regulatory protein (OsGAP1). The effects of OsGAP1 on the subcellular localization/cycling and physiological functions of OsYchF1 are also discussed. The finding that OsYchF1 and OsGAP1 are involved in plant defense response might shed light on the functional roles of YchF homologues in plants. This work suggests that during evolution, an ancestral P-loop GTPase/ ATPase may acquire new regulation and function(s) by the evolution of a lineage-specific regulatory protein.

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Kinetic determination of the GTPase activity of Ras proteins by means of a luminescent terbium complex

Cited by 17 publications

References 35 publications

Luminescent Chemical and Physical Sensors Based on Lanthanide Complexes

Luminescent Chemical and Physical Sensors Based on Lanthanide Complexes

Conformational States of Human Rat Sarcoma (Ras) Protein Complexed with Its Natural Ligand GTP and Their Role for Effector Interaction and GTP Hydrolysis

An Ancient P-Loop GTPase in Rice Is Regulated by a Higher Plant-specific Regulatory Protein

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