Show simple item record

dc.contributor.authorWatson, Joanna
dc.date.accessioned2017-11-13T10:03:26Z
dc.date.available2017-11-13T10:03:26Z
dc.date.issued2017-11-08
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/268520
dc.description.abstractCdc42 is a member of the Rho family of small GTPases, which, together with its homologues RhoA and Rac1, controls a multitude of cellular functions via the actin cytoskeleton. Cdc42 exerts its effects on the cytoskeleton via effector proteins of the Wiskott-Aldrich Syndrome (WASP) family and the Transducer of Cdc42-dependent Actin assembly (TOCA) family. The WASP family and their activation by Cdc42 have been thoroughly studied in vitro and are well understood. Conversely, understanding of the TOCA family remains limited by a lack of biochemical, biophysical and structural insight. An investigation of the TOCA1-Cdc42 interaction is described here, revealing a relatively low affinity interaction with a dissociation constant in the micromolar range. This is 10-100x weaker than other Rho-effector interactions and suggests that TOCA1 must first be co-localised with Cdc42 to achieve stable binding in vivo. The solution NMR structure of the Cdc42 binding HR1 domain of TOCA1 provides the first structural data on this protein and reveals some interesting structural features that may relate to binding affinity and specificity. A structural model of the Cdc42-HR1 complex provides further insight into differential specificities and affinities of GTPase-effector interactions. NMR and actin polymerisation assays provide insight into the pathway of Cdc42/TOCA1/WASP-dependent actin assembly, suggesting unidirectional displacement of TOCA1 by N-WASP. A comparison of the Cdc42- TOCA1 model with an NMR structure of Cdc42 in complex with the GTPase binding domain of WASP reveals a possible mechanism by which an ‘effector handover’ from TOCA1 to N-WASP could take place. Small GTPases such as Cdc42 are lipid modified and membrane anchored via their C- termini in vivo, so in vitro studies using truncated, unmodified GTPases are limited in their biological interpretation. This project also aimed to develop methods to study full length and membrane-anchored GTPases in vitro. Lipid modified protein was produced, which showed a weak affinity for liposomes, and so structural studies of membrane anchored protein are within reach. Further method development is now required to achieve stable membrane anchoring of lipid modified GTPases for detailed NMR studies.
dc.description.sponsorshipA full PhD studentship was provided by Herchel Smith Studentships, Cambridge UK, for four years. Additional funding was provided by the Cambridge Philosophical Society.
dc.language.isoen
dc.rightsNo Creative Commons licence (All rights reserved)
dc.rightsAll Rights Reserveden
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/en
dc.subjectCdc42
dc.subjectNMR
dc.subjectTOCA1
dc.subjectNWASP
dc.subjectWASL
dc.subjectN-WASP
dc.subjectGTPase
dc.subjectG protein
dc.subjectHR1 domain
dc.subjectFilopodia
dc.subjectEndocytosis
dc.subjectCell signalling
dc.subjectProtein protein interaction
dc.titleStructural and biochemical insight into the interactions of Cdc42 with TOCA1 and N-WASP
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.publisher.departmentBiochemistry
dc.date.updated2017-11-11T17:11:33Z
dc.identifier.doi10.17863/CAM.14730
dc.publisher.collegeCorpus Christi College
dc.type.qualificationtitlePhD in Biochemistry
cam.supervisorMott, Helen
rioxxterms.freetoread.startdate2017-11-11


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record