Team:Lethbridge/Model

Modelling

Structural Model

To ensure that the fusion of a red fluorescent protein (RFP) to proinsulin peptides or SCI57 would not inhibit appropriate folding of the RFP, or prevent access to the cell-penetrating peptide (CPP) or furin cleavage sites, we performed 3D protein modelling of our construct designs.

Using I-TASSER (Roy, Kucukural, & Zhang, 2010; Yang et al., 2015; Zhang, 2008), we predicted and modeled the secondary and tertiary structure of both the proinsulin and single chain insulin fusion proteins. In both cases, the RFP domain is highly conserved, stable, and predicted to be uninfluenced by the fused elements. The N-terminal 6xHIS tag is structurally accessible and the component residues have solvent accessibility scores in the range of 3-4 (where 0 represents a buried residue and 9 is one that is highly exposed). Though there is less confidence in the folding predictions of the C-terminal proinsulin and single chain insulin domains, the CPP and furin cleavage sites also have solvent accessibility scores in the 3-4 range, and are predicted to have less rigid structure. Based on these predictions, we have no reason to suspect that the functional properties of these fusion proteins will be inhibited by steric hindrance or structural inflexibility.

Figure 1: 3D structural model of 6xHIS-RFP-Proinsulin-CPP. The N-terminal HIS tag is shown in blue and the C-terminal CPP is shown in red. The characteristic beta-barrel scaffold of RFP is clearly evident and appropriately folded.

References

Roy, A., Kucukural, A., Zhang, Y. (2010). I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, 5, 725-738.

Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J., Zhang, Y. (2015). The I-TASSER Suite: Protein structure and function prediction. Nature Methods, 12, 7-8.

Zhang, Y. (2008). I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9, 40.