Biophysical Investigation of the Function of Enzyme 3DL1

Abstract

Protein function is often tied to structure, so structural comparisons for a protein or enzyme of unknown function may help to elucidate its function. An enzyme of unknown function (PDB ID 3DL1) derived from Klebsiella pneumonia has been studied using various methods to predict and test enzyme function. Crystal structures of 3DL1 with and without zinc bound show that zinc binds at a site with sequence and structural similarity to known metallopeptidases. These crystal structures also show that zinc binding affects the secondary structure of the protein. Computational methods for sequence and structure comparisons were made of 3DL1 to other protein sequences and structures. These show that 3DL1 may be a hydrolase with possible peptidase activity. 3DL1 was expressed in E. coli cells and purified via metal-affinity chromatography. Size exclusion chromatography was used to further purify 3DL1. To study the secondary structure of 3DL1 in the presence and absence of zinc, circular dichroism (CD) spectroscopy was used. The CD data allowed for determination of the stoichiometry of binding of the zinc ion(s) to the protein. It was expected that one zinc ion would bind per protein molecule. Moreover, enzymatic assays were performed using various relevant substrates to monitor the activity of 3DL1 in the presence and absence of zinc. These assays were performed with small molecule substrates and were monitored by UV/visible spectroscopy. Because 3DL1 has some structural similarity to gelatinase A, the activity of 3DL1 with a larger protein substrate was also tested in a gelatin-based assay. The relevance of this project comes from the necessity to identify potential enzymatic action of proteins of unknown function. Through this, some proteins could become useful for treatment of human disease or lead to a greater understanding of their cause.