Site-directed mutagenesis of p97/VCP to gain insight on the role of ATP binding to D1 domain affecting adaptor protein selectivity.

p97/VCP is a homohexameric AAA-ATPase associated with diverse cellular activities such as DNA repair and endoplamic reticulum-associated degradation (ERAD). The diversity of its functions lies within the interaction with different adaptor proteins such as p47 and Ufd1/Npl4 (UN) heterodimer. By bindi...

Full description

Saved in:
Bibliographic Details
Main Author: Rizal Ismail.
Other Authors: Susana Geifman Shochat
Format: Final Year Project
Language:English
Published: 2013
Subjects:
Online Access:http://hdl.handle.net/10356/52920
Tags: Add Tag
No Tags, Be the first to tag this record!
Institution: Nanyang Technological University
Language: English
id sg-ntu-dr.10356-52920
record_format dspace
spelling sg-ntu-dr.10356-529202023-02-28T18:01:27Z Site-directed mutagenesis of p97/VCP to gain insight on the role of ATP binding to D1 domain affecting adaptor protein selectivity. Rizal Ismail. Susana Geifman Shochat School of Biological Sciences DRNTU::Science p97/VCP is a homohexameric AAA-ATPase associated with diverse cellular activities such as DNA repair and endoplamic reticulum-associated degradation (ERAD). The diversity of its functions lies within the interaction with different adaptor proteins such as p47 and Ufd1/Npl4 (UN) heterodimer. By binding to the major adaptor protein binding site at the N-terminal domain of p97/VCP, adaptor proteins direct p97/VCP to different sites and different activities in the cell, utilizing the energy generated by ATP hydrolysis in the D2 domains of p97/VCP. This process should be regulated. Competition binding assays by Chia et al., 2012, showed that ATP binding to D1 was able to increase p97/VCP affinity to UN compared to p47. In this research, we aimed to gain further insight on Chia et al. findings by mutating critical ATP binding residues, K251A, R359A and H384A in D1 and D478A, K524A and K658A in D2, and comparing the binding affinities of UN to wild type and mutant p97/VCP by SPR. Due to time limitations, we could focus only on one of the mutants, K251A. However, the mutant protein did not generate hexamers for us to proceed with binding assays. Future hexamerization and binding studies will be done with the mutants. Bachelor of Science in Biological Sciences 2013-05-29T03:15:28Z 2013-05-29T03:15:28Z 2013 2013 Final Year Project (FYP) http://hdl.handle.net/10356/52920 en Nanyang Technological University 38 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Science
spellingShingle DRNTU::Science
Rizal Ismail.
Site-directed mutagenesis of p97/VCP to gain insight on the role of ATP binding to D1 domain affecting adaptor protein selectivity.
description p97/VCP is a homohexameric AAA-ATPase associated with diverse cellular activities such as DNA repair and endoplamic reticulum-associated degradation (ERAD). The diversity of its functions lies within the interaction with different adaptor proteins such as p47 and Ufd1/Npl4 (UN) heterodimer. By binding to the major adaptor protein binding site at the N-terminal domain of p97/VCP, adaptor proteins direct p97/VCP to different sites and different activities in the cell, utilizing the energy generated by ATP hydrolysis in the D2 domains of p97/VCP. This process should be regulated. Competition binding assays by Chia et al., 2012, showed that ATP binding to D1 was able to increase p97/VCP affinity to UN compared to p47. In this research, we aimed to gain further insight on Chia et al. findings by mutating critical ATP binding residues, K251A, R359A and H384A in D1 and D478A, K524A and K658A in D2, and comparing the binding affinities of UN to wild type and mutant p97/VCP by SPR. Due to time limitations, we could focus only on one of the mutants, K251A. However, the mutant protein did not generate hexamers for us to proceed with binding assays. Future hexamerization and binding studies will be done with the mutants.
author2 Susana Geifman Shochat
author_facet Susana Geifman Shochat
Rizal Ismail.
format Final Year Project
author Rizal Ismail.
author_sort Rizal Ismail.
title Site-directed mutagenesis of p97/VCP to gain insight on the role of ATP binding to D1 domain affecting adaptor protein selectivity.
title_short Site-directed mutagenesis of p97/VCP to gain insight on the role of ATP binding to D1 domain affecting adaptor protein selectivity.
title_full Site-directed mutagenesis of p97/VCP to gain insight on the role of ATP binding to D1 domain affecting adaptor protein selectivity.
title_fullStr Site-directed mutagenesis of p97/VCP to gain insight on the role of ATP binding to D1 domain affecting adaptor protein selectivity.
title_full_unstemmed Site-directed mutagenesis of p97/VCP to gain insight on the role of ATP binding to D1 domain affecting adaptor protein selectivity.
title_sort site-directed mutagenesis of p97/vcp to gain insight on the role of atp binding to d1 domain affecting adaptor protein selectivity.
publishDate 2013
url http://hdl.handle.net/10356/52920
_version_ 1759854764527452160