Biophysical and structural investigation of ring-shaped DNA binding protein complexes

Kompaniiets, Dmytro (2020) Biophysical and structural investigation of ring-shaped DNA binding protein complexes. PhD thesis, University of Glasgow.

Abstract

Ring-shaped proteins are common for the wide variety of cellular mechanisms. For example: membrane channels involved in cell signaling or energy metabolism (OuYang et al., 2013, Rastogi and Girvin, 1999); protein chaperones and proteasome (Chaudhry et al., 2004, Deville et al., 2017); DNA binding protein complexes (Cannone et al., 2017, Dionne et al., 2003). Their structure/function relationships are extremely important. Despite their similar geometry they have completely different functions, for instance: chaperones are maintaining an environment for the protein folding, whereas DNA binding proteins facilitate DNA unwinding or ligation, or play role of molecular scaffold for other proteins. These proteins are extensively studied, however, there are numerous gaps in knowledge present in this field of protein biology.
Archaea are a domain of unicellular organisms, who live under wide range of conditions including extreme conditions such as high temperature (thermophiles) and high pressure (piezophiles). Certain members of archaea such as Saccharolobus solfataricus and Pyrococcus abyssi are well characterised and are reported to have sets of proteins that are structurally similar to eukaryotic proteins. Moreover, archaea are used as a model system to study DNA replication. PCNA is one of the key players in DNA metabolism. It adopts a ring shape, that enables DNA binding and movement along the DNA strand, as well as accommodation of the variety of interacting partners with their further access the DNA. Proteins, originating from Saccharolobus solfataricus, were used as a model to study the functional assembly of PCNA containing protein complexes during Okazaki fragment maturation. PCNA from extremophilic archaea Pyrococcus abyssi was used as a model to study structure response under extreme conditions.
In this thesis PCNA, as a ring-shaped DNA binding protein, is studied from two sides. The first is a structural comparison of the homomeric PCNA from Pyrococcus abyssi, Saccharomyces cerevisiae, and Homo sapiens, under the effect of extreme conditions such as high temperature and high pressure using CD spectroscopy. This comparison confirms our assumption that proteins from extremophilic organism will display higher structural stability. Interestingly, despite the high sequence and structural similarity, presence or absence of the bulky amino acid residue like Tryptophan might significantly alter protein stability. Moreover, such alterations may be related to the evolution from unicellular prokaryotic organisms to complex multicellular eukaryotic organisms. Higher protein flexibility, which appeared after this amino acid alteration, may be involved in the development of more complex regulation of the molecular mechanisms. The second side is a study of the functional protein assembly of PCNA containing protein complexes during Okazaki fragment maturation using transmission electron microscopy and single particle 3D reconstruction. Improved resolution and level of details for the “Okazakisome” protein complex structure allowed us to gather deeper understanding of the role of PCNA as a molecular “toolbelt” described before (Dionne et al., 2003, Cannone et al., 2015). Also, our approach allowed to get better insights on the dynamic mechanism of the protein/protein interactions within the complex. For example, importance of the DNA Polymerase for the positioning of other client proteins and their movement, as well as conformational changes of the DNA ligase.
NPM1 is a broadly studied protein that forms a ring-shape pentameric assembly. Explicit biochemical characterization of the NPM1 protein and its functions during past years reveals an involvement within the vast variety of mechanisms starting from the genome stability and finishing with apoptosis regulation (Box et al., 2016). Also, NPM1 functions have been reported as a dependent from the protein oligomeric state. However, there are currently no structural information for the full-length protein. Structure of the full-length protein is important in deeper understanding of molecular mechanisms and their regulation. Here, 3D reconstruction of the full-length NPM1 protein pentameric assembly is reported.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: DNA replication, DNA binding, Okazaki fragments maturation, ring shape, PCNA, NPM1, CD, High pressure, Electron microscopy
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Spagnolo, Dr. Laura
• Date of Award: 2020
• Embargo Date: 21 April 2023
• Depositing User: Mr Dmytro Kompaniiets
• Unique ID: glathesis:2020-81293
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 21 May 2020 06:44
• Last Modified: 21 May 2020 06:52
• URI: https://theses.gla.ac.uk/id/eprint/81293