Fascin Methods

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Predicting Functional Partners

The String 8.0 online database was used to analyze and produce a graph showing fascin-1 interactions with other proteins showing physical and functional interactions.

Cellular Location

Fascin-1 is known to be expressed in many different regions of the body. Using the online Genomic Institute of the Novartis Research Foundation (GNF) SymAtlas v 1.2.4, a graph was constructed showing the levels of fascin-1 in different regions of the body.

Multiple Alignment

NCBI BLAST search was conducted using Fascin 1 sequence as query sequence. From the 112 sequences returned, 100 had FASTA formats and these 100 were run through the multiple-alignment program ClustalX. To help edit the remainding sequences a protDOM search was conducted on Fascin 1, and the results from the search were used. Sequences with excessive amino acids before or after Fascin 1 were deleted, as well as sequences with large gaps, and finally multiple results from the same organism were cleared. A final alignment was conducted on the remainding 32 sequences.

Phylogenetic Tree

Two methods were use to construct the Phylogenetic Tree.

The first used "phlip" software. Using the clustalX alignment, sequences were run through "protdist" to attain a distance matrix calcuation. The resulting data was then constructed into a tree using the neighbor-joining method, where the data was run through the phlip generation program "neighbor". Bootstrapping was conducted using the phlip programs and the original ClustalX alignment and were added the the tree created during neighbor-joining.

The second program utilised MEGA4 software, where the alignment data was formed into a bootstrap neighbor-joining method, with 500 replications.

Structure and domain analysis

Fascin PDB file was retrieved from the RCSB Protein data bank (PDB). 3D visualisations including secondary structure and domain visualisation were generated in PYMOL using this PDB file (1DFC.pdb). 2D secondary structure with domain annotations retrieved from the sequence tab of RCSB PDB entry for Fascin.

Structural allignments

Initial search of structural homologues was done using Dali database search with 1DFC.pdb. Subsequent hits (FGF) were then used in allignments by downloading PDB from RCSB PDB and comparing in PYMOL.

Cleft/Pocket analysis

Pocket analysis was executed using the online softare at Computed Atlas of Surface Topography of proteins (CASTp). 1DFC.pdb was uploaded and pocket analysis used a 1.4 Angstrom probe length. Pocket outputs were analysed manually and relevant pockets taken.

Electrostatic surface model

Using the online PDB2PQR resource in conjunction with the APBS feature in PYMOL an electrostatic surface model for Fascin was generated.

Hydrophobicity Plot

With the help of the online Molecular Toolkit, the hydrophobicity plot for fascin-1 protein was analysed by inserting the protein sequence obtained from NCBI in FASTA format. The hydrophilicity of the protein was then analysed using the online Innovagen website using the peptide property calculator.

Binding sites

3D animation showing binding sites and phosphorylatable Serine residues had individual pictures created by PYMOL. These were then compiled using XnView and UnFREEz conversion and animation programs.

Abstract | Introduction | Methods | Results | Discussion | Conclusion | References
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