Structural and Functional Effects of KRas Monoubiquitination at Lysine 104

K-Ras, a member of the Ras superfamily GTPases, plays a key role in regulating cellular growth, and single point mutations in this protein that promote chronic activation are found in approximately 30% of human cancers.1 Recent findings have suggested that post translational modifications such as monoubiquitination of KRas can alter its intrinsic activity.2 This project invstigated the effects of mUbRas104, the minor site of native in vivo modification, in the context of its impact on protein structure and activity relative to that of WT KRas and mUbRas147. The structural changes of mUbRas104 were characterized using NMR techniques and Rosetta modeling, and the protein’s activity was characterized through various biochemical assays. From the NMR data, chemical shift perturbations, broadening, and changes in secondary structure were observed throughout the protein. Perturbations in the switch regions are consistent with a slightly disturbed binding affinity to downstream effector proteins, but are more localized in switch II and helix 3, contrary to the predominant switch I perturbations found in mUbRas147. These findings suggest that mUbRas104, as a minor site of monoubiquitination, does not cause the same magnitude of functional disturbances as does mUbRas147. From these experiments, a protocol was also developed to irreversibly ligate the ubiquitin molecule, and this protocol can now allow this system to be studied in vivo for future work.Bachelor of Scienc