Iodine is present in plants in inorganic and organic forms and may affect various physiological processes, such as growth, stress response, antioxidant activity, and metabolism. Iodinated metabolites have also been identified in plant tissues, including compounds related to thyroxine (T4) and triiodothyronine (T3); however, their physiological role in plants has not yet been elucidated. The aim of this master's thesis was to identify potential T4-binding proteins in Arabidopsis thaliana in silico and to prepare selected candidates for experimental investigation of their response to T4 and T3.
The candidates were first identified using a bioinformatic approach. Based on predicted protein structures from the AlphaFold database, molecular docking of the T4 ligand was performed using AutoDock Vina. Using two-step filtering based on predicted binding energy and RMSD values, four potential T4-binding proteins were selected: VSP3, ICME, ACX3, and At4g31350. Analysis of the predicted binding sites showed that T4 was predicted to be stabilized in the selected proteins through a combination of hydrophobic and aromatic interactions, as well as polar or electrostatic interactions.
For the selected candidates, various recombinant constructs were prepared from plasmid vectors containing the cDNA of the desired coding sequences, inserted into the pET 32b(+) expression vector, and expressed in the bacterium Escherichia coli. Recombinant proteins VSP3_1 and VSP3_2 were successfully isolated, while ACX3 was obtained in smaller amounts. Isolation of ICME_3 and At4g31350_1 did not allow further analysis due to insufficient quality or quantity. Dynamic light scattering showed that the samples containing VSP3_1 and VSP3_2 were relatively homogeneous, whereas the ACX3 sample contained more aggregates and impurities. Nano differential scanning fluorimetry was used to determine the thermal stability of VSP3_1 and VSP3_2 in the presence of T4; however, changes in melting temperature did not indicate concentration-dependent stabilization or destabilization. Therefore, increased protein stabilization in the presence of T4 could not be reliably confirmed or excluded. Analysis of the interaction of the recombinant proteins ACX3, VSP3_1, and VSP3_2 with fluorescently labelled T3 by native PAGE likewise did not allow a clear conclusion due to low resolution and noise.
The results of the experimental analyses did not allow reliable confirmation of the stabilization of the selected recombinant proteins in the presence of T4 or their interaction with T3. Therefore, the molecular docking predictions cannot be directly linked to actual ligand binding. Further investigation of the potential interactions will require purer protein samples, optimized experimental conditions, and additional biophysical methods.
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