COASY method
Expression Data
The expression of CoAsy in mouse tissues was determined using SymAtlas (Genomics Institute of Novartis Research Foundation, 2007). SymAtlas was also used to determine expression in mice of the other enzymes in the CoA synthesis pathway, including pantothenate kinase (Pank2), phosphopantothenoylcysteine synthase (6330579B17Rik) and phosphopantothenolycysteine decarboxylase (8430432M10Rik).
Domain Identification
Lalign was used to determine which residues of the full length CoAsy protein were present on chain A (Pearson, 2007).
NCBI Entrez Protein (NCBI, 2002) was used to predict domains of full-length CoAsy, and PFAM was used to predict domains of CoAsy chain A (Sanger Institute, 2005). Results from PFAM for Chain A were compared with those obtained by entering the 2f6rA PDB file into Profunc (Laskoski et. al., 2005).
A DALI (Holm & Sander, 1993) search was performed on the Mus. musculus Coenzyme A Synthase sequence to gather structurally related proteins. Fourteen proteins with a structure relation Z score greater than nine had their PDB entries collected and entered into VMD (Humphrey, Dalke, & Schulten, 1996). Using the STAMP structural alignment tool the molecules were aligned (Russell & Barton, 1992) and the resulting alignment was then coloured by Q per residue (structural Identity) using the MultiSeq (Roberts, Eargle, Wright, & Luthey-Schulten, 2006) tool for VMD. The proteins were then hidden such that only the 2F6R PDB molecule was visible, and the start and end zones of structurally related areas were then labeled. The resulting model can be seen in Figure 3.
Once the structural conservation had been performed the MultiSeq (Roberts, Eargle, Wright, & Luthey-Schulten, 2006) tool was again used to colour the molecules, this time using the sequence identity option. The model was relabeled with highly conserved residues and the results can be seen in Figure 2.
MOTIF identification
MOTIF’s were identified using the PROSITE motif search service (Bairoch, Bucher, & Hofmann, 1997) on the Mus. musculus residue sequence.
Domain Surface Map and Structural Comparison of Surface Maps
Locations of domains on the surface of the Coenzyme A Synthase protein were collected from Mus. musculus sequence entry (NCBI, 2007). The residue ranges for these were highlighted and recoloured on the 2F6R PDB entry using PyMol (Delano, 2007). The results can be seen in Figure 4. PyMol was also used to model the structural surface map comparisons of 2F6R and 1JJV PDB entries with colouring by secondary structure. ATP ligands were placed on the 2F6R PDB entries by aligning 1JJV PDB entry with 2F6R and then hiding all of 1JJV except the attached ATP atoms (Figure 2).
B-Factor Surface Map
The 2F6R PDB entry was modeled using PyMol with colouring set to b-factor. The results can be seen in Figure 2. Electrostatic Surface Potential Map The electrostatic potential map was calculated for the PDB 2F6R entry using APBS (Baker, Sept, Joseph, Holst, & McCammon, 2001) and displayed in PyMol (Delano, 2007). ACO and ATP ligands were added by aligning 1JJV and 2F6R PDB entries and then hiding all but the ligands themselves Figure 3.
Abstract | Introduction | Results | Discussion | Conclusion | Method | References
