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Surface Change of Ras Enabling Effector Binding Monitored in Real Time at Atomic Resolution
Author(s) -
Kötting Carsten,
Kallenbach Angela,
Suveyzdis Yan,
Eichholz Carolin,
Gerwert Klaus
Publication year - 2007
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200600552
Subject(s) - gtp' , effector , gtpase , anti apoptotic ras signalling cascade , signal transduction , chemistry , biophysics , conformational change , microbiology and biotechnology , biochemistry , enzyme , biology , mapk/erk pathway
Ras, the prototype of the Ras superfamily, acts as a molecular switch for cell growth. External growth signals induce a GDP‐to‐GTP exchange. This modifies the Ras surface (Ras on GTP) and enables effector binding, which then activates signal‐transduction pathways. GTP hydrolysis, catalysed by Ras and GAP, returns the signal to “off” (Ras off GDP). Oncogenic mutations in Ras prevent this hydrolysis, and thereby cause uncontrolled cell growth. In the Ras off ‐to‐Ras on transition, the Ras surface is changed by a movement of the switch I loop that controls effector binding. We monitored this surface change at atomic resolution in real time by time‐resolved FTIR (trFTIR) spectroscopy. In the transition from Ras off to Ras on a GTP‐bound intermediate is now identified, in which effector binding is still prevented (Ras off GTP). The loop movement from Ras off GTP to Ras on GTP was directly monitored by the CO vibration of Thr35. The structural change creates a binding site with a rate constant of 5 s −1 at 260 K. A small molecule that shifted the equilibrium from the Ras on GTP state towards the Ras off GTP state would prevent effector binding, even if hydrolysis were blocked by oncogenic mutations. We present a spectroscopic fingerprint of both states that can be used as an assay in drug screening for such small molecules.