This web page was produced as an assignment for an undergraduate course at Davidson College.

SST2

1. I began the search at the Sanger Institute, where you can searh "Pfam", otherwise known as the Protein families database of alignemnts and HMMs.

By entering the amino acid sequence of SST2, the program can determine sequence similarity to known protein domains whose function and structure might be known. The results agree with known information on SST2. The DEP domain has an unknown function, but is involved in G-protein signalling, and possibly in GTPase activity. The RGS domain is a regulator of G-protein signalling. SST2's function in response to pheromone signalling, specifically with GTPase activation makes it clear that these structural results are in line with its function. The results are below:

Here is the actual (text) alignment of SST2 with DEP and RGS.

2. The next stop on the road to discovering the structure of the SST2 protein was PSIPRED, the protein structure prediction center.

A. The MEMSAT results predict transmembrane topology, based on the prediction and location of helices. They are hard to interpret manually, but are useful to continue to extrapolate structure with 3D modelling programs.

B. The PSIPRED results predict secondary structure: helix, strand and coil. The secondary structure results can help to build a 3D model or predict functional and conserved domains.

C. The GenTHREADER results examine specific domains and assign protein fold hypothesis. The hypothesis are then ranked based on degree of certainty from certain (CERT) to guess (GUESS.) As noted on the home page, GenThreader is unique in that it examines evolutionary relationships in order to more accurately predict folds and eliminate random matches.

3. At SCOP, a site for the Structural Classification of Proteins, I searched the superfamily sequence database.

The results are similar to the Pfam results. They show that SST2 has a an RGS domain as discussed above. However, the superfamily results also show alignment with DNA/RNA polymerarse domains, which is somewhat surprising and a novel discovery. Although, it is important to note, as the website indicates, that the sequence is only aligned against a model. It is possible that the alignment is random or incomplete.

HMM library:

Gough et al. 2001. "Assignment of Homology to Genome Sequences using a Library of Hidden Markov Models that Represent all Proteins of Known Structure." J. Mol. Biol., 313(4), 903-919.

4. The Structural Genomics Initiative allows you to search either by amino acid sequence of accession number. By entering the accession number, a wealth of information in chart format. Click here to view the chart.

5. At the DIP database, protein-protein interactions can be assessed.

The live chart below shows the two interactions found for SST2. Click on the colored dots to view interactions of SST2 with several proteins in several formats.

YDR078C

For the un-annotated gene, I will follow the same search pattern as above for organization purposes.

1. Began at the Sanger Institute, and searched Pfam using the amino acid sequence of YDR078C: No results were found for the unknown gene.

2. The next stop on the road to discovering the structure of the SST2 protein was PSIPRED, the protein structure prediction center.

A. The MEMSAT results created an error and therefore are unavailable.

B. The PSIPRED results*

C. The GenTHREADER results*

*Results are somewhat indechiperable, but are important in further predictions.

3. Went to SCOP again, a site for the Structural Classification of Proteins, and searched the superfamily sequence database. No significant results were found as shown below.

HMM library:

Sorry No Significant Hits.

Gough et al. 2001. "Assignment of Homology to Genome Sequences using a Library of Hidden Markov Models that Represent all Proteins of Known Structure." J. Mol. Biol., 313(4), 903-919.

4. The Structural Genomics Initiative allows you to search either by amino acid sequence of accession number. By entering the accession number, a wealth of information in chart format. Click here to view the chart

5. At the DIP database, protein-protein interactions can be assessed.

The live image below can display various hypothetical interactions of YDR078C with other proteins. Many of these interactions are hypothetical, but the degree of possibilities is exciting and a good place to start for further investigating the function of YDR078C, as I shall do in the experimental protocol.

6. Finding YDR078C on the interaction maps.

NB_Figure1: The branches off of YDR078C are black and indicate that the cellular role and localization are unknown. The box is pale yellow and is not described in the legend in our class textbook.

Degradation: In this figure, YDR078C is involved in RNA-processing/modification. The evidence below corroborates with this function and begins to direct the narrow the focus of research that might investigate the function YDR078C.

Membrane: YDR078C is implicated in RNA turnover. The exact definition of "RNA turnover" is unclear, but one can assume it is somehow involved in either translation or transcription because RNA is the cornerstone of these two cellular functions.

Aging: YDR078C is also involved in protein translocation, as is SST2 (see above description of "protein translocation.")

7. The final step used a 3D modelling program, from the Biomolecular Modelling Laboratory.

The data generated by the 3d-PSSM server identified individual structures for YDR078C based on the amino acid sequence. The data can be found here. They are pretty astounding. It appears that each model could be live, although I was not able to interact with them. The various hypothetical structures represent a powerful modelling tool.

Email the author at: amhartman@davidson.edu

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