Covalent Ras Dimerization on Membrane Surfaces through Photosensitized Oxidation

• Published in: Journal of the American Chemical Society Vol. 138; no. 6; pp. 1800 - 1803
• Main Authors: Chung, Jean K, Lee, Young Kwang, Lam, Hiu Yue Monatrice, Groves, Jay T
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 17.02.2016
• Subjects: Dimerization; Electrophoresis, Polyacrylamide Gel; fluorescence; fluorescence emission spectroscopy; gel electrophoresis; guanosinetriphosphatase; molecular weight; oxidation; Oxidation-Reduction; oxidative stress; oxygen; photosensitivity; Photosensitizing Agents - chemistry; plasma membrane; point mutation; proteins; ras Proteins - chemistry; reactive oxygen species; signal transduction; Spectrometry, Fluorescence; tyrosine
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/jacs.5b12648

Ras, a small GTPase found primarily on the inner leaflet of the plasma membrane, is an important signaling node and an attractive target for anticancer therapies. Lateral organization of Ras on cellular membranes has long been a subject of intense research; in particular, whether it forms dimers on membranes as part of its regulatory function has been a point of great interest. Here we report Ras dimer formation on membranes by Type II photosensitization reactions, in which molecular oxygen mediates the radicalization of proteins under typical fluorescence experimental conditions. The presence of Ras dimers on membranes was detected by diffusion-based fluorescence techniques including fluorescence correlation spectroscopy and single particle tracking, and molecular weights of the stable covalently coupled species were confirmed by gel electrophoresis. Fluorescence spectroscopy implicates interprotein dityrosine as one of the dimerization motifs. The specific surface tyrosine distribution on Ras renders the protein especially sensitive to this reaction, and point mutations affecting surface tyrosines are observed to alter dimerization potential. The photosensitization reactions are reflective of physiological oxidative stress induced by reactive oxygen species, suggesting such processes may occur naturally and influence signaling pathways in cells.