Conformational states of human H-Ras detected by high-field EPR, ENDOR, and31P NMR spectroscopy

Abstract

Ras is a central constituent of the intracellular signal transduction that switches between its inactive state with GDP bound and its active state with GTP bound. A number of different X-ray structures are available. Different magnetic resonance techniques were used to characterise the conformational states of the protein and are summarised here. 31P NMR spectroscopy was used as probe for the environment of the phosphate groups of the bound nucleotide. It shows that in liquid solution additional conformational states in the GDP as well as in the GTP forms coexist which are not detected by X-ray crystallography. Some of them can also be detected by solid-state NMR in the micro crystalline state. EPR and ENDOR spectroscopy were used to probe the environment of the divalent metal ion (Mg2+ was replaced by Mn2+) bound to the nucleotide in the protein. Here again different states could be observed. Substitution of normal water by 17O-enriched water allowed the determination of the number of water molecules in the first coordination sphere of the metal ion. In liquid solution, they indicate again the existence of different conformational states. At low temperatures in the frozen state ENDOR spectroscopy suggests that only one state exists for the GDP- and GTP-bound form of Ras, respectively.

Surface Change of Ras Enabling Effector Binding Monitored in Real Time at Atomic Resolution

“…This observation is in accordance with ENDOR measurements with GTP. [30] In photolysis spectra of Ras·caged-GTP (Figure 2), the bands of the equilibrated Ras·GTP state point upwards, and absorptions of the Ras· caged-GTP state point downwards. By comparing the spectra of WT and T35S Ras, we see changes in a-, b-, and g-phosphate bands; this indicates a shift to another Ras·GTP state.…”

Solid-state31P NMR investigation on the status of guanine nucleotides in paclitaxel-stabilized microtubules

“…The effects of metal ion binding to the 31 P chemical shifts of nucleotides have been well-documented in the literature, [12,13,20,28,29] which were used for the 31 P resonance assignments in the succeeding texts. The characteristic 31 P signal for GTP is the resonance of betaphosphate (P β ).…”

“…[19] 31 P NMR spectroscopy has been successfully used for detecting the conformations of GTP-binding enzymes in solution [20,21] or in crystals. [12,13] In previous work, we have successfully reported a 31 P cross-polarization magic angle spinning (CPMAS) NMR spectrum obtained from lyophilized microtubules, [22] where the signal-to-noise ratio (SNR = 7.5) was not sufficient enough to provide quantitative information. Here, we report a 31 P CPMAS NMR spectrum of lyophilized, paclitaxel-stabilized microtubules with enhanced resolution (SNR = 25) and show that it is now possible to study the molecular environments in detail for the guanine nucleotides in microtubules.…”

“…[10,11] However, studying the GTP hydrolysis mechanism in microtubules is still challenging, owing in part to the fast dynamics and huge sizes (~10 5 kDa) of microtubules compared with other GTPase proteins. [12,13] Radioactive analogs of GTP were used for analyzing the kinetics of GTP/GDP-P i cap in microtubule. [2,14,15] Distributions of GTP-bound tubulin in microtubules were visualized in vivo by conformation-specific antibodies and immunofluorescence.…”

Pulsed 180-GHz EPR/ENDOR/PELDOR spectroscopy

“…In the past, we have demonstrated that highfield EPR in liquid solution revealed important differences in the number of water ligands between the wild-type and the G12V oncogenic mutant of the protein in both switch states. 27,28 We have recently employed pulsed ENDOR spectroscopy at 94 GHz to investigate these differences in more detail. 29 Here, we present the first representative application of 180-GHz pulsed 1 H-ENDOR in the study of Ras ž Mn 2C ž GDP.…”