Functional Characterization of 3CBW Using Bioinformatics and Wet-Lab Experiments
Start Date
August 2024
End Date
August 2024
Location
ALT 206
Abstract
BASIL is a course-based undergraduate research experience that includes using a computer and various websites and services to assign functions to proteins. The first four modules of BASIL were used to investigate the protein, 3CBW. First, SPRITE was used to find active sites of known enzymes that have a similar structure to the side chains on 3CBW. Next, BLAST was used to perform a sequence-based search for 3CBW to identify known proteins with similar sequence. Third, InterPro was used to compare the sequence of 3CBW to find proteins with similar protein families. Finally, DALI and Foldseek were used to determine possible functions of 3CBW by comparing the structure to other enzymes and determine how similar structures must be to have the same function. The results of these tests led to the hypothesis that 3CBW is likely a beta-mannosidase. Cells containing a plasmid that includes the 3CBW protein were grown and then the protein was purified. Several malate dehydrogenase enzymes were evaluated kinetically to confirm their function and to learn how to perform a kinetic analysis of an enzyme. Kinetic analysis of 3CBW was performed with both alpha- and beta-mannose substrates to evaluate the hypothesis that 3CBW is a beta-mannosidase enzyme.
Functional Characterization of 3CBW Using Bioinformatics and Wet-Lab Experiments
ALT 206
BASIL is a course-based undergraduate research experience that includes using a computer and various websites and services to assign functions to proteins. The first four modules of BASIL were used to investigate the protein, 3CBW. First, SPRITE was used to find active sites of known enzymes that have a similar structure to the side chains on 3CBW. Next, BLAST was used to perform a sequence-based search for 3CBW to identify known proteins with similar sequence. Third, InterPro was used to compare the sequence of 3CBW to find proteins with similar protein families. Finally, DALI and Foldseek were used to determine possible functions of 3CBW by comparing the structure to other enzymes and determine how similar structures must be to have the same function. The results of these tests led to the hypothesis that 3CBW is likely a beta-mannosidase. Cells containing a plasmid that includes the 3CBW protein were grown and then the protein was purified. Several malate dehydrogenase enzymes were evaluated kinetically to confirm their function and to learn how to perform a kinetic analysis of an enzyme. Kinetic analysis of 3CBW was performed with both alpha- and beta-mannose substrates to evaluate the hypothesis that 3CBW is a beta-mannosidase enzyme.
