Structural Genomics (SG) efforts have given rise to the need for better methods to predict protein function. Protein structures are often correlated with their function, but this relationship is complex. Proteins with the same function may have different structural folds. Proteins within a superfamily can have different biochemical functions although the structures are highly similar. Members of the Crotonase Superfamily have the common feature of recognizing an acyl-CoA substrate and stabilizing an enolate anion intermediate. However, this superfamily includes diverse biochemical functions, including hydrolase, isomerase, and dehalogenase activities. This project focuses on the functional characterization of proteins within the Crotonase Superfamily through computational and experimental methods. The computational methods, THEMATICS, POOL, and SALSA, are used to analyze the local structures at predicted active sites and are employed to predict the functions of the SG proteins of unknown function within the superfamily. Functional assignment is based on the predicted sets of catalytically active amino acid residues for the proteins of known function within the superfamily. Predicted sets of active residues for the SG proteins are compared with the local structural signatures obtained from the previously characterized proteins. Biochemical assays serve to validate the computational predictions. Thirty-four SG proteins were analyzed and 13 aligned with known members of the Crotonase Superfamily. Twenty-one SG proteins in the Crotonase Superfamily did not align with the consensus signatures of the known functional subfamilies. Several of the proteins were expressed recombinantly and purified to be used for functional assays to validate predictions. Accurate functional annotation by computational methods facilitates practical application of SG data.

Predicting the function of structural genomics proteins of unknown function in the crotonase superfamily

249th National Meeting (2015)

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