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Conformational states of human H‐Ras detected by high‐field EPR, ENDOR, and 31 P NMR spectroscopy
Author(s) -
Spoerner Michael,
Prisner Thomas F.,
Bennati Marina,
Hertel Melanie M.,
Weiden Norbert,
Schweins Thomas,
Kalbitzer Hans Robert
Publication year - 2005
Publication title -
magnetic resonance in chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.483
H-Index - 72
eISSN - 1097-458X
pISSN - 0749-1581
DOI - 10.1002/mrc.1693
Subject(s) - chemistry , electron paramagnetic resonance , crystallography , gtp' , nuclear magnetic resonance spectroscopy , spectroscopy , coordination sphere , nucleotide , molecule , guanosine diphosphate , bound water , conformational change , stereochemistry , nuclear magnetic resonance , crystal structure , guanosine triphosphate , biochemistry , physics , organic chemistry , quantum mechanics , gene , enzyme
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. 31 P 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 (Mg 2+ was replaced by Mn 2+ ) bound to the nucleotide in the protein. Here again different states could be observed. Substitution of normal water by 17 O‐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. Copyright © 2005 John Wiley & Sons, Ltd.