Structural and functional studies of recombinant human MsrB3A

Paul, Adityo (2017) Structural and functional studies of recombinant human MsrB3A. MSc(R) thesis, University of Glasgow.

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In humans, MsrB3 occur in two different isoforms, which is a distinct characteristic found in no other organism. MsrB3A is the MsrB3 isoform which is localised within the endoplasmic reticulum of the human cell, while, the MsrB3B isoform is found in the mitochondria along with MsrB2. The primary role of MsrB3A is to repair the oxidised methionine residues in nascent polypeptides and the unfolded protein chains found in the endoplasmic reticulum, which become oxidised by ROS species produced within the endoplasmic reticulum. However, the exact mode of action of MsrB3A and the stereo-specificity of MsrB3A is not entirely clear.
Hence, this thesis aims - (i) to determine the structure of MsrB3A, and (ii) to find out the functionality and stereospecificity of MsrB3A protein. The mature gene sequence for MsrB3A protein lacking the sequence for the N-terminal signal peptide was successfully cloned into recombinant E. coli cells and subsequently over-expressed to generate the recombinant MsrB3A protein for further studies.
From a sequence alignment, it was predicted that the 126th position cysteine residue in the amino acid sequence of MsrB3A was an active site cysteine and responsible for the function of the protein, as it was a conserved residue. Based on this assumption, the 126th position cysteine was substituted with alanine residue using site directed mutagenesis to generate a mutant protein which could be further used in determination of protein activity.
With respect to determination of MsrB3A structure, we used three approaches- (i) CD Spectroscopy for determining the secondary structural elements of MsrB3A, (ii) NMR spectroscopy and (iii) protein crystallisation to determine the tertiary structure of MsrB3A protein.
The results obtained from CD spectroscopy and NMR spectroscopy proved that the recombinant MsrB3A protein had a folded structure. It was also noted that the results which were obtained by CD spectroscopy were consistent with previous studies performed by other groups with respect to MsrB proteins. Though certain crystallisation hits were obtained, good quality crystals required for determining the crystal structure by X-ray spectroscopy were not achieved, hence, the crystal trials were not successful and requires further work.
To address the functionality of MsrB3A, two different approaches were used- (i) a gel shift assay and (ii) a HPLC assay. The gel shift assay involved the change in mobility of SurA protein in its oxidised and reduced states which formed the basis of determining the protein functionality, when analysed on SDS-PAGE gel. Hence, when SurA was oxidised by treatment with H2O2, the mobility of SurA decreased with respect to its native form when analysed on SDS-PAGE gel. Hence, when the oxidised form was repaired by treatment with MsrB3A protein in presence of DTT, there was a slight shift in the mobility of oxidised SurA towards its native form, but could not gain complete mobility, suggesting an incomplete repair and indicating that MsrB3A is stereo-specific, but cannot say which isoform of methionine sulfoxide it reduces. Similar results were also achieved by HPLC assay in which approximately half of the dabsylated methionine sulfoxide concentration was reduced to dabsylated methionine, which supports and suggests that MsrB3A is stereo-specific, but further experiments using individually dabsylated methionine-R-sulfoxide and methionine-S-sulfoxide is required as substrate to have accurate determination of stereo-specificity. Another significant observation which was made by the HPLC assay was the non-functionality of the C126A MsrB3A mutant protein. When the dabsylated methionine sulfoxide was treated with the C126A MsrB3A mutant no reduction was observed upon separation using the HPLC. This indicated that the cysteine residue being substituted was an active site cysteine which is responsible for the protein function.
Therefore, to conclude, we could gain a stable recombinant protein product which could be actively used for study and experimentation.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Keywords: Methionine, methionine sulfoxide, methionine sulfoxide reductase (Msrs).
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Supervisor's Name: Bulleid, Professor Neil and Smith, Dr. Brian
Date of Award: 2017
Depositing User: Mr. Adityo Paul
Unique ID: glathesis:2017-8546
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 01 Nov 2017 09:45
Last Modified: 27 Nov 2017 13:25

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