Characteristics of three proposed Malate Dehydrogenases with BASIL.
Start Date
April 2024
Location
2nd floor - Library
Abstract
The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is an association that determines the structures of proteins associated with human diseases in order to understand the function of these proteins. Protein function can be predicted based on sequence similarity with known proteins, but further characterization is needed for publication. From a list of several hundred candidates, we chose enzymes identified as malate dehydrogenase (MDH). We used the in-silico modules of the BASIL curriculum to confirm the proposed function of each enzyme, and each enzyme was further characterized using enzyme kinetics, chemical crosslinking, SDS-PAGE, and gel filtration chromatography. Three MDH enzymes were obtained from SSGCID. Enzyme kinetics assays showed that these enzymes convert oxaloacetate to malate, as expected for MDH. Monomer molecular weight of these enzymes was determined by SDS-PAGE, which showed that the observed MW matched the MW predicted by the protein sequence. Analytical gel filtration and chemical crosslinking were used to determine the quaternary structure of these enzymes. Results from gel filtration suggested that one of these enzymes was a trimer and another was a monomer. Chemical crosslinking showed that the proteins were in their dimeric and tetrameric forms based on data from the gel. Overall, our results confirmed that structurally and functionally these 3 enzymes are MDH enzymes as predicted from the annotations. Our results also show that all 3 enzymes had dimeric and tetrameric forms, consistent with known MDH proteins.
Characteristics of three proposed Malate Dehydrogenases with BASIL.
2nd floor - Library
The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is an association that determines the structures of proteins associated with human diseases in order to understand the function of these proteins. Protein function can be predicted based on sequence similarity with known proteins, but further characterization is needed for publication. From a list of several hundred candidates, we chose enzymes identified as malate dehydrogenase (MDH). We used the in-silico modules of the BASIL curriculum to confirm the proposed function of each enzyme, and each enzyme was further characterized using enzyme kinetics, chemical crosslinking, SDS-PAGE, and gel filtration chromatography. Three MDH enzymes were obtained from SSGCID. Enzyme kinetics assays showed that these enzymes convert oxaloacetate to malate, as expected for MDH. Monomer molecular weight of these enzymes was determined by SDS-PAGE, which showed that the observed MW matched the MW predicted by the protein sequence. Analytical gel filtration and chemical crosslinking were used to determine the quaternary structure of these enzymes. Results from gel filtration suggested that one of these enzymes was a trimer and another was a monomer. Chemical crosslinking showed that the proteins were in their dimeric and tetrameric forms based on data from the gel. Overall, our results confirmed that structurally and functionally these 3 enzymes are MDH enzymes as predicted from the annotations. Our results also show that all 3 enzymes had dimeric and tetrameric forms, consistent with known MDH proteins.
