| Summary: | Peroxisomes are ubiquitous organelles found in most eukaryotic organisms and are involved in ~-oxidation of fatty acids and the degradation of hydrogen peroxide. They contain no DNA thus all proteins required fo: their functions are identified and imported via a peroxisomal targeting signal (PTS). PTS1, a C-terminal tripeptide approximating the consensus sequence -Ser-Lys-Leu-COO·, is recognized by the tetratricopeptide repeat (TPR) domains of PEX5, a cytosolic receptor that cycles between the cytoplasm and the peroxisome. To gain insight into the thermodynamics of PTS1 binding specificity, a fluorescence- based binding assay that enables the quantification of inhibition constants for PTS1- containing peptide complexes with the full length and TPR region of Arabidopsis thaliana PEX5 was used. A library of PTS1 penta-peptides based on the reference sequence YQSKL and deca-peptides based on the reference sequence, VAKTIRPSRV, were synthesised, tested and found to have a Kj between 160 nM to> 100000 nM with only a small difference observed in affinity between the truncated and full length A. thaliana proteins. Circular dichroism studies showed that the TPR domain of AtPEX5 is 60% alpha helical and that this is the only region of the full length AtPEX5 protein that contains secondary structure, independent of the presence of a PTS1. Binding of the PTS1 by the TPR domain was diminished at pH < 6 however secondary structure was still maintained at pH 6 but lost at pH 5. Finally surface plasmon resonance assays using the immobilised peptides YQSKL, VAKTIRPSRL, VAKTIRQSRL and VAKTIRPRSV showed that the binding response of truncated and full length AtPEX5 to the PTS1 is doubly sigmodial in nature and that full length AtPEX5 has a significantly slower kOffthan the TPR domain alone. Overall the results show that PTS1 sequences which target in vivo may not have affinity for AtPEX5 in vitro, showing a difference between the two techniques. Additionally these results show that the mechanism of binding between AtPEX5 and the PTS1 may not simply be 1:1 but involve the formation of higher order multimeric of complexes of AtPEX5-PTS1.
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