Ro52: Structure and interactions of constructs of RING and B-box
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
The ubiquitination process is vital to maintain the protein homeostasis in the cell. With high specificity it regulates degradation of proteins by tagging them with a small protein called ubiquitin. Four proteins are involved to perform the process and in this thesis one of these proteins is studied. This protein is called Ro52 and belongs to the TRIM protein family. It posses E3 ligase activity because of a N-terminal RING-domain and therefore it is responsible for the last step in the ubiquitination process. The structure of Ro52 is not totally solved and the function of the protein’s four domains is not fully understood.
In this thesis three constructs of two domains from Ro52 (RING and B-box) is investigated by circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and auto-ubiquitination assay by Western blot. The goal was to gain deeper insight in structural and functional properties of these domains.
In the end only two constructs were investigated because of time limitations. It was shown by NMR that one construct has similar structure as the wild type but lower stability, possibly due to shorter N-terminal region. Comparison of the results from CD measurements showed that the constructs were well structured but did not reveal any significant differences in secondary structure between the constructs. Functional analysis by Western blot encountered unexpected problems and no results were obtained.
The current thesis provides a basis for further investigation of variant constructs jointly expressing the RING-B-box domains, and shows that even small changes may alter structure and stability in ways that might affect functional properties.
Place, publisher, year, edition, pages
2014. , 64 p.
Structural biology, Ro52, TRIM21, Ubiquitination, Western blot, Protein expression, Protein purification
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:liu:diva-110362ISRN: LiTH-IFM-A-EX--14/2940--SEOAI: oai:DiVA.org:liu-110362DiVA: diva2:744875
Subject / course
2014-06-04, Linköping, 15:00 (English)
Anandapadmanaban, MadhanagopalWallenhammar, Amélie