Methods & Materials

=Structural analysis

Structural analysis of our protein was carried out by submitting the PDB file of our protein 2gnx to a number of online databases and analysing their output for usage in our project. These databases include -

a) Dali - A database which predicts the closest structural matches to the submitted protein. This database was very extensively used for the purposes of our structural analysis.

b) CE - A database using which comparisons can be drawn as to sequence similarity of 2 submitted proteins, as well as to the 3-D structures of both proteins. We used this database in combination with the Dali database, by comparing our protein with structurally related proteins as per the Dali results.

c)Q-siteFinder - A website that predicts the ligand binding sites on the protein that has been submitted into the database.

A software named RasMol was used in the structural analysis of our protein as well. We used this program to compare the 3-D structure of our protein with the closest structually related proteins generated from the Dali search in order to draw conclusions as to which residues of our protein would be involved in ligand binding.

Functional Analysis

Functional analysis was undertaken in two phases – the first relying on the protein sequence comparisons, and the second relying on structural analysis.

Deriving Function from Sequence Data

This phase involved submitting the BC048403 FASTA sequence to several predictive databases including:

• STRING – a database providing the ability to predict functional associations between proteins.

• Locate – a database containing data describing the membrane organization and sub-cellular location of proteins.

• CDART – a tool that displays the functional domains that make up the protein and other proteins with similar domain architectures.

The default search variables were used in most cases. In the case of the protein BC048403, these tools returned no results and thus there was no further analysis concerning them.

It is important to note that solely depending on data from sequence analysis is not reliable due to the way in which the sequencing databases work. These databases work by comparing similarities to homologs, however genes that are homologous can in fact be paralogs and have divergent functions. Thus sequence analysis should form part of your research but not be the only foundation of your results.

Deriving Function from Structural Data

The sequence was submitted to the following tools which can be used to determine functional information from structural data:

• Profunc – a tool used to analyse the 3D structure of a protein to help identify its likely biochemical function

• Proknow – a tool used to achieve the same ends as Profunc but with a slightly different process

• Interpro – a tool used to predict protein domains

• Pfam – identifies protein families

These tools provided limited or no results and so a different approach was investigated.

A search was conducted using Symatlas, a gene atlas of mouse and human expression patterns across diverse tissue sets. This tool also has the functionality to find correlated micro-array expression data allowing the discovery of proteins with similar expression data.

Once structural analysis had progressed and some basic data was known concerning the structure of the protein, this information was used in furthering the functional analysis. Specifically, research was performed on discovering a link between the results from the micro-array analysis, and the presence of structural domains identified through structural analysis. more coming...

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