Team:Lethbridge/Modeling
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- | ==<font color="white">The Method | + | ==<font color="white">The Method== |
To model the xylE structure, the sequence for xylE from <i>Pseudomonas putida</i> (NCBI accession number NP_542866) was aligned with the primary sequence from the crystal structure of xylE from the same organism (pdb: 1MPY; several differences in amino acid sequence were observed) using CLUSTALW (Higgins <i>et al.</i>, 1996). Based on this sequence alignment, a homology model was generated using the alignment mode in SWISSMODEL (Guex <i>et al.</i>, 1997; Kiefer <i>et al.</i>, 2009; Arnold <i>et al.</i>, 2006). To model the placement of an N-terminal arginine tag, the tag was manually added to the N-terminus of the model. Energy minimization was carried out in SWISS-PDB viewer in vacuo utilizing a GROMOS96 energy minimization (Guex <i>et al.</i>, 1997). The resulting pdb file was visualized and manipulated using PYMOL, images were taken using the same software (DeLano, 2006). | To model the xylE structure, the sequence for xylE from <i>Pseudomonas putida</i> (NCBI accession number NP_542866) was aligned with the primary sequence from the crystal structure of xylE from the same organism (pdb: 1MPY; several differences in amino acid sequence were observed) using CLUSTALW (Higgins <i>et al.</i>, 1996). Based on this sequence alignment, a homology model was generated using the alignment mode in SWISSMODEL (Guex <i>et al.</i>, 1997; Kiefer <i>et al.</i>, 2009; Arnold <i>et al.</i>, 2006). To model the placement of an N-terminal arginine tag, the tag was manually added to the N-terminus of the model. Energy minimization was carried out in SWISS-PDB viewer in vacuo utilizing a GROMOS96 energy minimization (Guex <i>et al.</i>, 1997). The resulting pdb file was visualized and manipulated using PYMOL, images were taken using the same software (DeLano, 2006). | ||
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+ | ==<font color="white">Results and Limitations== | ||
+ | Homology modeling of xylE shows that while the C arginine tag cannot interfere with the active site, the N terminal tag is actually a concern. As shown in figure 2, the arginine tag is located perilously close to one side of the active site and has a real potential for at least partially blocking substrate access. | ||
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+ | However, these results are from only using one force field and by holding the structure of the rest of the protein static. The use of simulated annealing methods or even molecular dynamics would greatly increase our ability to predict the effect of these tags. | ||
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===<font color="white">References=== | ===<font color="white">References=== | ||
Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006) The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling., Bioinformatics 22, 195-201. | Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006) The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling., Bioinformatics 22, 195-201. |