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Study of structural differences among 14-3-3 protein isoforms.
Macáková, Eva ; Gryčová, Lenka (referee) ; Obšil, Tomáš (advisor)
The 14-3-3 proteins are a family of important regulatory proteins, found in all eukaryotes, which are involved in many cellular processes. In this diploma thesis, we studied structure/function relationships of 14-3-3 proteins, in this case it was the influence of the structure of H8-H9 loop on the binding affinity in barley isoform hv 14-3-3A and human isoform 14-3-3ζ. According to former results, hv 14-3-3A binds to a ligand with lowest affinity, which could be caused by present of a glycin in H8-H9 loop, while in other isoforms there is a serin on the same position. We measured the binding affinity in protein hv 14-3-3A WT and its mutant, which contained the serin instead of the glycin in H8-H9 loop. For comparison, we also measured the binding affinity of human isoform 14-3-3ζ containing the serin in H8-H9 loop and its mutant, where the the serin was replaced by the glycin. Proteins were expressed in E. coli cells strain BL21(DE3) and then purified. The dissociation constant for the binding of peptide pRaf-259 labeled with fluorophores FITC and ATTO was measured using both the fluorescence correlated spectroscopy and the steady-state fluorescence intensity. Our results showed that in both isoforms the mutation of H8-H9 loop causes decrease in the binding affinity.
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