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This protein (3HDT) is a putative kinase with possibly cytidylate kinase function utilizing ATP and dCMP as ligands.
Introduction
Online Resources (bioinformatic software)
We used a variety of in silico databases with our protein, 3HDT to find similarities with other amino acid sequences, protein family matches, and structural comparisons to known proteins in the PDB. Below are the recorded results and information from each database. From this information, a hypothesized function was created for 3HDT and potential substrates were selected such as dCMP.
BLASTp
Pfam
DALI
PANZ
Docking
Laboratory Experiments
Coupled kinase assay
Coupled kinase assay diagram (left) with enzymes shown in color and phosphates in yellow. Phosphorylation of dCMP is measured indirectly through the conversion of NADH to NAD+. Background hydrolysis of NADH is measured and subtracted from the conversion rate in the presence of dCMP to produce specific activities (right).
SDS-PAGE
SDS-PAGE results for the purified protein 3HDT. The total weight of this protein is around 25.79 kD. The first lane (left) contains a size standard. The band in the second lane (right) at ~70kD is not the protein of interest (3HDT) but contains a binding metal protein. There is also a faint band around ~26kD, indicating our protein of interest was present.
Results from running an SDS-PAGE with the purified 3HDT
Conclusion/Future Experiments
Our protein was confirmed to be 3HDT using SDS-PAGE and showed activity during coupled kinase assays. While this confirms that 3HDT is a kinase, the true substrate, however, was likely not dCMP. Further research should be done with molecules such as TMP and GMP in the future to narrow down potential nucleotide substrates or elucidate other types of compounds to be considered as ligands for 3HDT.
