A joint project of the Graduate School, Peabody College, and the Jean & Alexander Heard Library

Title page for ETD etd-03232012-133041


Type of Document Dissertation
Author Williams, Christina Kay
Author's Email Address christinakaywilliams@gmail.com
URN etd-03232012-133041
Title Biochemical and structural analysis of SV40 Large T antigen: insights into chaperone mediated inactivation of retinoblastoma tumor suppressor protein
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Walter Chazin, Ph.D Committee Chair
Brandt Eichman Committee Member
Ellen Fanning Committee Member
Richard Armstrong Committee Member
Scott Hiebert Committee Member
Keywords
  • NMR
  • Retinoblastoma protein
  • SV40 T antigen
  • SAXS
Date of Defense 2011-12-21
Availability unrestricted
Abstract
Simian Virus 40 uses its Large T antigen protein to bind and inactivate retinoblastoma tumor suppressor proteins, yielding cell transformation. T antigen is a modular protein with four domains connected by linkers. The N-terminal domain, the J domain, classifies T antigen as a chaperone protein and is necessary for the inactivation of Rb. Physical interaction with Rb is mediated primarily by an LXCXE consensus motif immediately C-terminal to the J domain. An approach integrating nuclear magnetic resonance (NMR) spectroscopy and small angle x-ray scattering (SAXS) was used to study the structural dynamics and interaction of Rb with the LXCXE motif, the J domain and a construct (N260) that extends from the J domain to the adjacent origin binding domain (OBD). NMR and SAXS data revealed substantial flexibility between the two domains in N260. Rb bound to a construct containing the LXCXE motif and the J domain confirmed that weak interactions exist between Rb and the J domain. Analysis of the interaction of Rb with N260 indicated that the OBD is not involved in binding Rb and that the J domain retains dynamic independence from the remainder of T antigen. These results support a T antigen ‘chaperone’ model in which the J domain orientation changes as it acts upon protein complexes to promote cell transformation.

The ubiquitin ligase Cullin 7 (Cul7) has been implicated in chaperone-mediated inactivation of Rb. It is known that Cul7 binds to T antigen via residues within the J domain. However, the Cul7 domain involved in this interaction is unknown. Yeast two hybrid screens in the Ellen Fanning laboratory suggest that the CPH domain of Cul7 bound to T antigen. NMR chemical shift perturbation experiments confirmed the interaction between the J domain of Tag and CPH domain of Cul7. These data identified specific CPH domain residues involved in binding to the Tag J domain, which map to a specific binding surface. Our results set the stage for mutational analysis to establish the functional role of the Tag-Cul7 interaction.

Files
  Filename       Size       Approximate Download Time (Hours:Minutes:Seconds) 
 
 28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access 
  CKWilliams_Dissertation_Final.pdf 3.06 Mb 00:14:11 00:07:17 00:06:23 00:03:11 00:00:16

Browse All Available ETDs by ( Author | Department )

If you have more questions or technical problems, please Contact LITS.