Document Type
Article
Language
eng
Publication Date
9-2015
Publisher
Elsevier (Cell Press)
Source Publication
Biophysical Journal
Source ISSN
0006-3495
Abstract
Ras GTPase is a molecular switch controlling a number of cellular pathways including growth, proliferation, differentiation, and apoptosis. Recent reports indicated that Ras undergoes dimerization at the membrane surface through protein-protein interactions. If firmly established this property of Ras would require profound reassessment of a large amount of published data and modification of the Ras signaling paradigm. One proposed mechanism of dimerization involves formation of salt bridges between the two GTPase domains (G domains) leading to formation of a compact dimer as observed in Ras crystal structures. In this work, we interrogated the intrinsic ability of Ras to self-associate in solution by creating conditions of high local concentration through irreversibly tethering the two G domains together at their unstructured C-terminal tails. We evaluated possible self-association in this inverted tandem conjugate via analysis of the time-domain fluorescence anisotropy and NMR chemical shift perturbations. We did not observe the increased rotational correlation time expected for the G domain dimer. Variation of the ionic strength (to modulate stability of the salt bridges) did not affect the rotational correlation time in the tandem further supporting independent rotational diffusion of two G domains. In a parallel line of experiments to detect and map weak self-association of the G domains, we analyzed NMR chemical shifts perturbations at a number of sites near the crystallographic dimer interface. The nearly complete lack of chemical shift perturbations in the tandem construct supported a simple model with the independent G domains repelled from each other by their overall negative charge. These results lead us to the conclusion that self-association of the G domains cannot be responsible for homodimerization of Ras reported in the literature.
Recommended Citation
Kovrigina, Elizaveta A.; Galiakhmetov, Azamat R.; and Kovriguine, Evgueni, "The Ras G Domain Lacks the Intrinsic Propensity to Form Dimers" (2015). Chemistry Faculty Research and Publications. 420.
https://epublications.marquette.edu/chem_fac/420
Comments
Accepted version. Biophysical Journal, Vol. 109, No. 5 (September 2015): 1000-1008. DOI. © 2015 Biophysical Society. Published by Elsevier Inc. Used with permission.