Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Gabriel Conte Cortez Martins - Miami Dade College
Co-Author(s): Edwin Ginés-Candelaria, Miami Dade College, Miami, FL
The DNA replication initiator protein, DnaA, from the Gram-positive, obligate anaerobic bacterium Clostridium taeniosporum binds to the DnaA boxes located at origin of replication, oriC, promoting strand opening and recruiting DnaB Helicase and other DNA polymerase Holoenzyme proteins to the replication fork, which starts DNA replication. In this study, we investigated the production of a three-dimensional structure from the primary amino acid sequence of DnaA, the replication initiator protein and thus predicted the three-dimensional structure of DnaA using bioinformatics tools. We extracted the coding gene sequence and translated it using Bioinformatics tool Geneious 10.2.2. We also used online bioinformatics servers PSIPRED and ANGLOR to determine secondary structures and dihedral angles, respectively, to guide protein folding. The graphical user interface for the biomolecular modeling suit Rosetta Commons, Foldit Standalone, was used to fold the protein and generate its tridimensional structure. We confirmed our model utilizing a Ramachandran Plot generated by web server RAMPAGE, along with a quality assessment evaluation from the online server ModFold. Finally, we further supported our domain structure model by comparing it to homologous DnaA proteins obtained from bacteria in the protein databank. We observed that DnaA contains 456 amino acids, and secondary structure prediction, obtained via PSIPRED, indicates that the protein contains six β-sheets and twenty ⍺-helices. We identified a 98-amino, winged-helix that corresponds to the DnaA-box binding domain, and locates to the middle portion of the amino acid sequence. The latter contains four ⍺-helices and two anti-parallel β-sheets. The three-dimensional structure prediction for DnaA obtained from Foldit Standalone possessed the lowest energy, which is consistent with predictions of native protein structure. These results will allow us to better understand DnaA’s function and activity, and provide us with new insights about the protein as well. Furthermore, a better understanding of DnaA will allow us to better understand the DNA replication process in Clostridium taeniosporum, consistent with all structure-function relationships, which permeate Life and originate at the chemical level. We will investigate additional bioinformatics tools and algorithms that will assess the quality of the predicted model and will allow refinements of such based on distance profiles of consecutive backbone alpha carbon atoms. References: Geneious version 10.2.2 (http://www.geneious.com, Kearse et al., 2012) Jones DT. (1999) Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292: 195-202 Kleffner, Robert, et al. Foldit Standalone: a video game-derived protein structure manipulation interface using Rosetta. Bioinformatics 2017 btx283.Gabriel Conte Cortez Martins - ERN Abstract F.docx
Funder Acknowledgement(s): This project would not have been possible without the support of Department of Education STEM SPACE grant P03C1160161 and principles of research developed by the National Science Foundation Advanced Technology Education Program NSF ATE DUE 0802508.
Faculty Advisor: Edwin Ginés-Candelaria, firstname.lastname@example.org
Role: I was responsible for generating the three-dimensional structure prediction for the DNA replication initiator protein, DnaA, analyzing its structural data, and assessing the model's quality.