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Updated: Apr 7, 2026

Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Published on: October 8, 2015
Locking GTPases covalently in their functional states
David Wiegandt1, Sophie Vieweg1, Frank Hofmann1
1Department of Physical Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Abstract:
GTPases act as key regulators of many cellular processes by switching between active (GTP-bound) and inactive (GDP-bound) states. In many cases, understanding their mode of action has been aided by artificially stabilizing one of these states either by designing mutant proteins or by complexation with non-hydrolysable GTP analogues. Because of inherent disadvantages in these approaches, we have developed acryl-bearing GTP and GDP derivatives that can be covalently linked with strategically placed cysteines within the GTPase of interest. Binding studies with GTPase-interacting proteins and X-ray crystallography analysis demonstrate that the molecular properties of the covalent GTPase-acryl-nucleotide adducts are a faithful reflection of those of the corresponding native states and are advantageously permanently locked in a defined nucleotide (that is active or inactive) state. In a first application, in vivo experiments using covalently locked Rab5 variants provide new insights into the mechanism of correct intracellular localization of Rab proteins.
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