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Team:NYMU-Taipei/modelling-protein-structure-champ-design
From 2011.igem.org
CHAMP Protein Modelling
CHAMP, the computed helical anti-membrane protein, is one of the computational and genetic methods available to engineer antibody-like molecules that target the water-soluble regions of tansmembrane (TM) proteins. (Hang Yin, et al., 2007)
Why We Use the CHAMP Design?
The general purpose of CHAMP design is that though Transmembrane(TM) helices usually play essential roles in biological processes, companion methods to target the TM regions are lacking. The CHAMP design of TM helices that specifically recognize membrane proteins would advance the understanding of sequence-specific recognition in membranes and simultaneously would provide new approaches to modulate protein-protein interactions in membranes. (Hang Yin, et al., 2007)
Now we want to let the CHAMP design play a pivotal role in our project to modulate the protein-protein interactions in membranes. But why is the CHAMP design so important in our iGEM project this year? In this year's [[link here|Optomagnetic design]], we want to use the designed peptide, CHAMP sequence, to inhibit the tight interaction between the two helices of membrane protein Mms13. Then we can successfully use mechanical force to change the conformation of Mms13 to induce the BiFC-based BRET phenomenon. If we do not have the target peptides, CHAMP, the two helices of Mms13 would tightly "stick" together and we can predict the results easily by the fundamental knowledge of physics that the magnetic force applied to the bacteria would not make any change to the conformation of Mms13 protein. If the two helices of Mms13 have tight interaction all the time, the wobbling light we expect in our project derived from BiFC-based BRET would not be excited. As the consequence of lacking the modulation of CHAMP, we would not get our final wobbling fluorescence but the constant light derived from a luciferase reaction; which means that we can only “turn on” neurons with constant light while the wobbling system can excite or inhibit the neurons for both “on and off” functions.
Fig. 1a: Close-up of the predicted tightly packed interface between designed peptides and target protein. The target protein is represented by a red surface with a blue “hot spot”. Fig. 1b: The CHAMP backbone is depicted in ribbon representation with key positions designated for computational design shown in green (Hang Yin, et al., 2007)
Fig. 2: The figure in the left is our initial design by using BRET phenomenon to generate the light. As for the right one, we make a little change using BiFC-based BRET with CHAMP, our designed peptides to make the light wobbling and induce the on-and-off neural control system.
Fig. 6: The critical G-X3-G motifs on helix1 of the Mms13
Selection of a nativelike helical-pair backbone within the chosen structural motif
After the selection of a helical-pair structural motif, we have to select a nativelike helical-pair backbone as the templates for our CHAMP design. First, we follow the instructions to determine the template cluster. (R. F. S. Walters, et al., 2006) A library of 445 helical pairs from 31 proteins in the paper was clustered into groups based on their three-dimensional similarity. Different clusters have specific features and also show varying degrees of homogeneity. We finally choose cluster 2 as our candidates because of the antiparallel helices on Mms13 and the traits which belong to right-handed crossing angle on helix1. (For examples, the helix1 have small residues at the helix–helix interface, and they are also spaced at four-residue intervals.) The pie chart (See Figure 7) shows the fraction of the total number of pairs that fall within a given cluster and Table 1 (See Figure 8) shows some characteristics of the top 14 clusters.
Fig. 11a: The whole 1iwg protein. Figure 11b: The selected template pairs of 1iwg. Figures are graphed using YASARA View (Krieger E., et al., 2011)
Fig. 19: Parts of cross-ranking results.
How to Design the CHAMP Peptides?
We followed the design protocols in the paper- ''Supporting Online Material for Computational Design of Peptides That Target Transmembrane Helices'' (Hang Yin, et al., 2007) and "Understanding Membrane Proteins. How to Design Inhibitors of Transmembrane Protein–Protein Interactions" (J.S. Slusky, et al., 2009). However, because of the limited resources, we made little adjustments by using different programs, such as TMhit (Lo A., et al., 2009), ProtMod (Godzik A., et al., 2011), YASARA (Krieger E., et al., 2011), VMD (Klaus Schulten., et al., 2011), ΔG prediction server v1.0 (Hessa, T., et al., 2007) to finalize our CHAMP design. The study by "Interaction and conformational dynamics of membrane-spanning protein helices'" (D Langosch, et al., 2009) and "Helix-helix interaction patterns in membrane proteins" (D Langosch, et al., 2010) gave us more knowledge about protein-protein interactions in transmembrane domains, and helped us during the CHAMP designing process.
