University of Limerick
Browse

Towards X-ray structural analysis of Thermus thermophilus sulphite reductase: purification characterisation and crystallisation

Download (2.34 MB)
thesis
posted on 2022-10-10, 11:50 authored by Michael D O'Donnell
The process of sulfur remediation is an ancient process credited with the origins of life, through the interconversion of sulfur molecule. The sulfite reductases play an essential role, allowing an organism to reduce sulfite, building a range of sulfur based molecules such as the amino acids and vitamins. The sulfite reductases can be divided into different groups. This depends on the number of subunits and whether the sulfite reductase operates via an assimilatory or a dissimilatory mode. While there have been a number of structures solved and a mechanism for this family proposed, the overall understanding of the function of this class of enzymes is still scarce. Here, the sulfite reductase of Thermus thermophilus was studied, with the protein previously cloned BL21* E. coli cells. Initial work revolved around increasing the yield, through the optimization of the lysis and purification protocol leading to homogenous population of the sample required for structural studies. Research was also performed on the optimisation of the crystallisation conditions for the protein. Crystals obtained have been used for the structure determination by X-ray crystallography. The crystal structure has been solved to 2.38 Å resolution. Over the course of the study the yield was increased to 24 mg/L (a 400% increase). The conversion of the dimeric species to the monomer was also achieved. The isolation of the monomer produced much higher resolution crystals than the mixed population samples with the best crystals being acquired from using 30 mg/mL of sulfite reductase with 12.5 mM sodium sulfite as the additive.

Funding

Using the Cloud to Streamline the Development of Mobile Phone Apps

Innovate UK

Find out more...

History

Degree

  • Master (Research)

First supervisor

Soulimane, Tewfik

Note

peer-reviewed

Other Funding information

IRC

Language

English

Department or School

  • Chemical Sciences

Usage metrics

    University of Limerick Theses

    Categories

    No categories selected

    Exports

    RefWorks
    BibTeX
    Ref. manager
    Endnote
    DataCite
    NLM
    DC