Calibration of methanol and ethylene glycol nuclear magnetic resonance thermometers

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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“…Temperature was controlled using the line separation of the methylene and hydroxyl group of external ethylene glycol for calibration (26).…”

Section: Methodsmentioning

confidence: 99%

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

129

187

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“…from the result of the temperature dependency examination. 22 The relationship between the filled gas pressure and the chemical shift of hydrogen gas is shown in Figure 5. The chemical shift of free hydrogen gas at 303 K was ensured to show an upfield shift in accordance with pressure rise.…”

Section: Measurements Of Gaseous Phase Hydrogen (Free Hydrogen Gas)mentioning

confidence: 99%

Evaluation of hydrogen dissolved in rubber materials under high-pressure exposure using nuclear magnetic resonance

Fujiwara

Nishimura

2012

Polym J

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Hydrogen molecules dissolved in rubber due to high-pressure hydrogen gas exposure have been analyzed using 1 H nuclear magnetic resonance (NMR) with solution and solid-state probes. To analyze the characteristics of dissolved hydrogen in rubber materials and to compare them with gaseous phase hydrogen, we measured the NMR spectra of gaseous phase hydrogen and evaluated the chemical shifts and pressure dependency. Measurement using a sealed tube and liquid phase probes enabled the simultaneous analysis of dissolved hydrogen in rubber and gaseous phase hydrogen eliminated from the rubber. Two peaks at 4.3 and 4.8 p.p.m. were observed in the 1 H MAS NMR of the rubber samples after high-pressure hydrogen exposure. Using information from the chemical shift of the free hydrogen gas and the time dependency of hydrogen quantification in the rubber, both of these peaks were confirmed to be attributable to dissolved hydrogen. Their differences in relaxation time confirmed that their mobilities were different. In conclusion, the hydrogen dissolved in acrylonitrile butadiene rubber exists in two different forms with different mobilities. The ratio of those two hydrogens differs and is affected by the exposure pressure and elimination process time.

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“…The NMR spectra were recorded at a calibrated probe temperature of 22 °C using the method described in the literature. 24 Four uranyl/gluconate solutions (I, II, III and IV), containing the same quantity of sodium gluconate but differing amounts of uranyl in D 2 O, were prepared for NMR measurements. A small volume of each uranyl/gluconate solution was transferred into a Teflon liner (Norell, Inc) and placed into a NMR tube (Wilmad).…”

Section: Calorimetrymentioning

confidence: 99%

Complexation of Uranium(VI) by Gluconate in Acidic Solutions: a Thermodynamic Study with Structural Analysis

et al. 2009

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Calibration of methanol and ethylene glycol nuclear magnetic resonance thermometers

Cited by 364 publications

References 5 publications

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

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

Evaluation of hydrogen dissolved in rubber materials under high-pressure exposure using nuclear magnetic resonance

Complexation of Uranium(VI) by Gluconate in Acidic Solutions: a Thermodynamic Study with Structural Analysis

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