Methods and Materials

Following the evolution of the protein

The protein sequence was blasted in the NCBI website. The blasted sequence was scanned using a non-redundand database to obtain the most significant sequences. 57 sequences with significant E-values were recovered. 55 of these sequences were used to perform a multiple alignment sequence by using the program ClustalX. When this was completed, a distance matrix calculation was performed on the sequences. The program Phylip-3.63 was used. The calculations from the matrix distance calculation were transformed by using a program in Phylip-3.63, Neighbor.exe. Afterwards, the bootstrap values for the sequences were calculated. This was done by running the alignment produced from the Neighbor program through Seqboot. 100 bootstrap samples were produced. The program Protdist.exe from Phylip-3.63 was used with the settings M for multiple, D for multiple data sets, and 100 for the replicates. The file created was run through Neighbor.exe. The same settings needed for Protist.exe were used. The treefile that was created was run through the program Consenses.exe. The resulting file was opened using the program Treefile. The phylogenetic tree created had the bootstrap values displayed on the branch nodes.

Identifying the functionof the protein.

Using the Blast results FASTA sequences for 2i2O and closely related sequences including 1hu3 (Danio Renio and Mouse) were found and inserted into the following databases for analysis.

• ProKnow
• ProFunc
• Locate
• String
• PDB
• DALI

Determining the protein structure

A protein search on PDB, NCBI Entrez, Pfam and InterPro databases was performed as the initial step in identifying the structure of the given protein. The given PDB code 2i2o was used in the searches. Next, proteins with similar structures were identified using the DALI server. A CE comparison was performed among the identified structures.

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