| As the hub in signaling cascades,Ras proteins are involved in multiple signaling pathways,thereby controlling cellular processes such as cell growth,proliferation,differentiation,etc.Depending on the bound nucleotides,the activity of Ras can be switched on(GTP-bound)or off(GDP-bound),which is regulated by guanine nucleotide exchange factors(GEFs)and GTPase activating proteins(GAPs).The abnormal activation of Ras protein is closely related to human cancer,thus made it an important therapeutic target,which further promotes the investigations of structure and dynamics of active Ras for understanding its signal transduction mechanism and inhibitor development.However,due to the intrinsic GTPase activity of the Ras proteins,non-hydrolyzable or slowly hydrolyzable GTP analogs,such as GppNHp,GTP?S are adopted,which leads to poor knowledge of active Ras,especially the site-specific dynamics of native Ras-GTP directly related to important functions.Furthermore,the specific differences between the conformational dynamics of GTP analogs-bound Ras and natural GTP-bound Ras remain to be elucidated.remains to be elucidated.This thesis aims to characterize the dynamics of native Ras·GTP by solution nuclear magnetic resonance spectroscopy,so as to compare the differences between GTP-bound and GTP analogs-bound Ras proteins,providing a clear direction for RAS-targeted anticancer therapies.In the first part of this thesis,we directly revealed the asymmetry of the Sos-mediated(a kind of GEF)nucleotide exchange process through quantitative analysis of the reaction kinetics for the first time.Then the direction was used to develop an approach for significantly extending the lifetime of Ras·GTP samples,providing the possibility for probing the site-specific dynamics of physiological active Ras.Because the protein sequence and structure of small GTPases are highly homologous,the approach for stabilizing the active state by GEF may serve as an instruction for studying GTP-binding form of other GTPases.In the following part,the above approach,combined with a signal postprocessing method,is applied to site-resolved NMR measurement of the allosteric dynamics of Ras·GTP.Compared to the gloabal dynamics of Ras·GppNHp,the concerted dynamics in Ras·GTP primarily concentrates in the effector lobe,covering the nucleotide-binding site as well as the recently identified allosteric inhibitor-binding sites,supporting the mechanism of conformational selection for Ras-inhibitor recognitions.In addition,We demonstrated that the mutant Ras(T35A)·GTP can faithfully reproduce the spectroscopic characteristics of excited state,providing evidence for its use as a substitute for the structure and dynamics studies of excited state.In the third part of the paper,we used the conformational dynamics data of natural Ras·GTP to test the accuracy of GTPyS,which is favored in recent years,in reproducing the dynamics of natural Ras active state.The hydrolysis rate of GTP yS by the intrinsic GTPase activity of Ras was determined to be 1/24 of that of natural GTP.Furthermore,it has been proved that GTP?S can reproduce the concerted dynamic network of Ras·GTP through NMR relaxation dispersion experiments,revealing the theoretical basis of GTP?S as a good GTP memetic from the perspective of conformational dynamics.In summary,we have developed a Sos-based approach on asymmetric nucleotide exchange catalytic activity,which can significantly extend the lifetime of active Ras,thus making it possible for mutli-dimensional NMR spectroscopic measurements.Plus,we achieved a quantitative analysis of the site-specific details in both native GTP-bound and GTP?S-bound Ras,and revealed the conformational exchange parameters in active Ras and the dynamic network closely related to allosteric communication.In addition,we found that Ras(T35A)·GTP can faithfully represent excited state and GTP?S can perfectly reproduced the natural dynamics network.These findings laid the foundation for the development of allosteric inhibitors targeting active Ras. |
PDF Full Text Request