Browsing by Subject "Guanosine Triphosphate"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Recombinant αβ-Tubulin and a Simple Computational Model Shed Light on the Molecular Mechanisms of Microtubule Dynamics(2015-02-06) Piedra, Felipe-Andrés; Yu, Hongtao; Ranganathan, Rama; Ross, Elliott M.; Rice, Luke M.Microtubules (MTs) are essential to all eukaryotic organisms. They help segregate chromosomes and organize the cytoplasm. MTs are hollow barrels of the protein αβ-tubulin that exhibit a non-equilibrium behavior called dynamic instability: the stochastic switching of single polymers from a state of gradual growth to one of rapid disassembly. Dynamic instability underlies the MT cytoskeleton's rapid reorganizability and enables its diversity of functions. MTs can be reconstituted from purified αβ-tubulin and have been studied in vitro for over 40 years. Over this time, huge strides have been made in the development of an understanding of dynamic instability. Nevertheless, the mechanistic basis of important phenomena like GTP-dependent assembly and GTP hydrolysis-induced conformational change and catastrophe (the switch from growing to shrinking) remain controversial or unexplained. In Chapter 2, I discuss a study in which we used a computational model to investigate the consequences of a new way of thinking about the effect of nucleotide-state on αβ-tubulin and MT assembly. Our results suggest that GDP exposure on the MT plus-end can frustrate elongation and lead to catastrophe. We therefore predicted that GDP to GTP exchange on the MT plus-end might reduce the frequency of catastrophe. We tested our prediction by analyzing the effects of a mutant αβ-tubulin and a GTP analog designed to increase the rate of terminal nucleotide exchange on MT dynamics in vitro. Our experimental results support the results from our model. Thus, we believe that GDP exposure on the MT plus-end increases the likelihood of catastrophe, and can be countered by GDP to GTP exchange. In Chapter 3, I discuss a comparison of yeast and porcine MT dynamics in vitro. My measurements reveal striking differences between yeast and mammalian MT dynamics, and provide new constraints for models of MT dynamics. I conclude my thesis in Chapter 4 with my view of what my work means, what remains to be done and what paths my work has opened for further exploration.Item Structural Bases of Plexin Signal Transduction(2015-07-14) Pascoe, Heath Garrick; Chen, Zhe; Zhang, Xuewu; Jiang, Qiu-Xing; Chook, YuhMinPlexins are a family of transmembrane receptors for the Semaphorin family of repulsive axon guidance molecules. Plexin mediated signal transduction is critical for a variety of cellular processes including regulation of adhesion and actin organization. Aberrant plexin signaling is associated with numerous pathologies. Despite plexin's essential cellular functions, a detailed understanding of the structural basis underlying plexin singling remains elusive. Here, the structural bases for two aspects of plexin signaling are revealed. Plexins relay signals in part by accelerating the GTP hydrolysis reaction catalyzed by the small GTPase Rap. This discovery appears at odds with the known structural features of plexin GAP domains. Plexin GAPs are structurally related to RasGAPs and possess RasGAP catalytic machinery. Conversely, plexins are structurally unrelated to canonical RapGAPs and don't possess their associated catalytic machinery. Here, the structural basis underlying the non-canonical RapGAP activity of plexins is revealed. Plexins induce a unique configuration of Rap's Switch II loop to utilize a non-canonical catalytic residue. Plexins also regulate RhoA activity. The B family plexins recruit two RhoGEFs (PDZ-RhoGEF and Leukemia Associated RhoGEF) by specifically binding to the PDZ domains of these GEFs. Conversely, these PDZ domains bind promiscuously to a variety of ligands. The structural basis by which plexins specifically recognize these two PDZ domains is revealed here. B family plexins interact with the PDZ domain of PDZ-RhoGEF using a secondary interface outside the canonical PDZ binding site. This secondary interface contributes to tight binding and is important for RhoA activation following plexin activation. Secondary interfaces may be a general mechanism utilized by modular protein-protein interaction domains to achieve specificity. Structural and biochemical characterization of plexins can be difficult. Here, two methods for studying plexins are described. These methods were critical to the success of the above studies. First, a method used to generate fusion proteins in-vitro for use in crystallography is described. This method allowed for the successful crystallization of a plexin/Rap complex. Second, a biochemical assay is described for easily measuring Plexin GAP activity in-vitro. This method circumvents the difficulties of many alternative approaches to measuring GAP activity.