Recombinant expression, purification and structural characterization of Saccharomyces cerevisiae RING finger domain using Nuclear Magnetic Resonance (NMR) spectroscopy
RING finger domain is a domain that is incorporated with other domains to form a large protein known as RBBP6. This is a 250kDa protein that stands for Retinoblastoma binding domain protein 6. It is classified according to different homologues namely RBQ1, PACT, SNAMA, Mpe-1 and P2P-R depending on the organisms in which they are found. P2P-R (a RING finger containing protein) was found to be highly up-regulated in the oesophageal cells compared to normal epithelial cells. This was a pre-indication that P2P-R might serve as an antigen against cancer or be used in the diagnosis of cancer. Saccharomyces cerevisiae RING finger domain is a protein that is represented by the richness of cysteine sequence motif that binds to two zinc atoms. It has a cross-brace topology consisting of 70 amino acids and its sequence is defined as Cys1-Xaa2-Cys2-Xaa9–39-Cys3-Xaa1–3-His4-Xaa2-3-Cys/His5-Xaa2-Cys6-Xaa4–48-Cys7-Xaa2-Cys8 (where Xaa represents any amino acid residue). The RING finger domain is essential in proteins involved in a range of diverse cellular processes such as apoptosis, viral infections, oncogenesis and ubiquitination. The study presented in this dissertation reports on the recombinant expression and partial purification S. cerevisiae RING finger protein, as well as its biophysical and biochemical characterization. Structural predictions using the ProtParam tool on ExPASy provided information regarding the high structural stability due to the abundance of leucine in the protein. The protein has a limited ability for light absorption which is indicative of the absence of two important aromatic amino acids; tryptophan and tyrosine. More so, Swiss-Model showed that the 3D homology model structure is composed of two α-helices contributing to the stability of the structure, two β-sheets that prevents proteolysis and one 310 helix that maintains the helix-coiling transitions. The local quality estimate revealed the structure is rigid and stable. The non-redundant set of the Protein Data Bank analysis showed the structural prediction of the RING finger protein is accurate to a larger extent. Validation of the predicted model by Ramachandran plot showed that 88% of the amino acids residues are in the favoured region meaning that the modelled structure is an acceptable representation of the S. cerevisiae RING finger protein.More so, interacting partners for S. cerevisiae RING finger protein were also ascertained and these protein partners were shown to be involved in cleavage and polyadenylation, however, zinc fingers from YTH1 showed similarity with RING finger domain as promoters of DNA binding. Phylogenetics and sequence alignment showed the ancestral proteins are involved in ubiquitination, which is one of the major pathways of RING finger domains. Lastly, the biochemical characterization using a 1D proton NMR spectroscopy showed that S. cerevisiae RING finger protein is well folded and can be further worked upon. These results provide the baseline information for the structural determination of the S. cerevisiae RING finger domain for future studies.
RING finger domain --RBBP6 --ubiquitination --zinc topology --cancer
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