Date of Award

9-10-2019

Publication Type

Master Thesis

Degree Name

M.Sc.

Department

Chemistry and Biochemistry

Keywords

Allosteric site, fluorogenic thiol redox probes, Protein disulfide isomerase, S-nitrosoglutathione reductase

Supervisor

Mutus, B.

Rights

info:eu-repo/semantics/openAccess

Abstract

S-nitrosation is the attachment of a nitric oxide moiety to the thiol side chain of cysteine. S-nitrosoglutathione (GSNO) acts as a bioactive reservoir for NO to maintain an equilibrium in the concentration of NO in the body. Due to this, the study of the enzyme S-nitrosoglutathione reductase has of great interest because of its ability to metabolize GSNO. S-nitrosoglutathione reductase’s activity has been linked to a number of human diseases. Chapter 1 of this thesis presents a proposed allosteric binding domain on GSNOR. Positive cooperativity (sigmoidal deviation) was observed from steady state analysis of GSNOR which indicated an affinity for the binding of GSNO at this site. The presence of such a site was further supported by Molecular docking simulations and HDX-MS which showed that the amino acids Gly321, Lys323, Asn185 and Lys188 interact with molecules bound at this site. Chapter two introduces four reagents that can function as probes or pseudo-substrates for the monitoring of enzymatic activity as well as measuring concentrations of free thiols in vitro and live cells. These reagents are N,N-di(thioamido-fluoresceinyl)-cystine (DTFCys2), N,N-di(thioamido-fluoresceinyl)-homocystine (DTFHCys2), N-amido-O-aminobenzoyl-S-nitrosoglutathione (AOASNOG), and N-thioamido-fluoresceinyl-S-nitroso-glutathione (TFSNOG). They are easy to prepare and purity and can be used in various applications.

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