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Development and Testing of a Systematic Approach for Computational Enzyme Function Determination

With the success of the Structural Genomics Initiative and other programs, the number of uncharacterized but crystallized proteins is on the rise. To date, there are over 3500 structures in the Protein Data Bank tagged as unknown function. The use of computational tools to speed up the analysis of these structures has been widely adopted, with varying measures of success. ProMOL, a plugin for PyMOL, was developed in a collaboration between RIT and Dowling College. ProMOL functions by searching for conserved active site “motif templates” in a queried protein structure. A combination of ProMOL, NCBI's BLAST algorithm, and University of Helsinki's DALI alignment server were used to attempt to assign functions to 4 previously uncharacterized proteins: PDBIDs 3DS8, 3FLE, 3LP5, and 2L57. The results from the computational analysis were then confirmed in vitro via functional assays. The computational approach successfully assigned the function of 3DS8, 3FLE (both lipases), and 2L57 (a glutaredoxin). It appears that a combination of BLAST alignment, DALI alignment, and ProMOL alignment is sufficient to computationally predict the function of uncharacterized proteins. This approach may be suitable for classroom settings as well, due to the low cost and ease of accessibility of these tools. The research is supported by 2R15GM078077–02 and 3R15GM078077–02S1.