Team:Western Canada/Dry Lab structures
I-TASSER Modelling
We used I-TASSER to visualize possible structures of our fusion protieins and obtain information about their function. Developed in the Zheng lab at the University of Michigan, I-TASSER is an online server for protein structure prediction; it has been ranked number one in several blinded community-wide experiments designed to evaluate protein structure prediction tools. Using amino acid sequences as input, I-TASSER server searches the Protein Data Bank for protein templates with similar folding. It subsequently generates images of five potential 3-dimensional structures and predicts functional properties of the protein. In total, four variations of enzyme fusion proteins and two variations of csgA fusion proteins were used in the lab. The 3-dimensional structure with the highest confidence score for each our proteins is shown in the table below. In general, the confidence scores associated with the prediction structures were low. Confidence scores are an evaulation of the quality of a predicted structure, and range from [-5, 2] with a higher score indicating higher confidence.
| Protein | Highest Confidence Score | Predicted Structure |
|---|---|---|
| FliC-Laccase-GS-Tag-His (FliC-Laccase) | -1.68 |  |
| CsgA-GS-SpyCatcher-His | -3.13 |  |
| SpyCatcher-GS-CsgA | -2.71 |  |
| FliC-Cutinase-GS-Tag-His | -2.72 |  |
| FliC-Tag-GS-Laccase-His | -2.14 |  |
| FliC-Tag-GS-Cutinase-His | -2.84 |  |
Future Steps
For this project, I-TASSER output was mainly used to visualize the 3-dimensional structures of our fusion proteins. In future work, the predicted structures could be used to determine beneficial modfiications to amino acid sequences by uploading output files to Foldit Standalone. Foldit standalone can be used for the manipulation of protein structures. It would allow us to look at the intramolecular interactions in our predicted protein structures, such as hydrogen bonding, and make modifications to the amino acids involved in these interactions to alter, and possibly improve, the structure of our fusion protein. Furthermore, this software may allow the comparison of the structures of the enzyme-domain of the fusion protein and the native enzyme. Through experimentation, sequence modifications that would make the two structures more similar could be determined. Analysis of this type using Foldit Standalone would be highly complex and extensive enough to be the focus of a separate project entirely. For these reasons, it was not completed as part of this iGEM project, but could be a subject of investigation for future teams should this work be continued.