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

PROTEOMICS -- SST2 & YDR078C

THE FUN NEVER ENDS

Click here to view the protein information I have discovered on:

SST2

YDR078C


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:

Domain

Start

End

Bits

Evalue

Alignment

DEP

279

358

94.40

2.3e-24

Align

RGS

420

689

195.70

7.1e-55

Align

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:

E-value

Sequence

Region

Superfamily

Alignment

Genome

4.8e-23

YourInputSequence

420-698

Regulator of G-protein signalling, RGS


E-value

Sequence

Region

Superfamily

Alignment

Genome

4.6e+00

YourInputSequence

78-632

DNA/RNA polymerases


Please send feed back to

gough@supfam.org

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.

Interaction
Information

PIR

SWISSPROT

Number
of references
or techniques

Interactions
(text)

Interactions
(graphical)
blue-static
purple-java

Protein names

A25906:S55974

GBA1_YEAST:SST2_YEAST

1

GTP-binding regulatory protein G alpha chain

 

SST2 protein

S64195:S55974

MPT5_YEAST:SST2_YEAST

2

HTR1 protein

 

SST2 protein

6. The next step was to find the protein on several interaction maps (PDFs.)

NB_Figure1: Based on the green branch from SST2 we can determine that SST2 is part of the cell structure. Black, however, implies an unknown cellular role or localization.

Degradation: SST2 is labelled orange in this tree, indicating that it is involved in mating-response. This has already been determined in projects 2 and 3.

Membrane: SST2 is invovled in membrane fusion according to this interaction map. Because SST2 is involved in pheromone response, which is initialized on the cell membrane, it is not surprising that it would be clustered here. However, it is unclear if SST2 is actually located on the membrane because most research related here does not indicate it.

Aging: Here SST2 is involved in protein translocation. Protein translocation is an unspecific description of protein-protein interaction in which one protein is translocated, or, simply, moved.

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 SST2 based on the amino acid sequence. The data can be found here. The amount of data generated by this program is truly amazing. One could spend weeks or months dissecting the various aspects presented here. It is interesting to note that the largest composition of the protein, according to this program, is the signal transduction inhibitor domain(s.) Because of the role of SST2 in pheromone response, discussed in project 2, this is confirmational and exciting.

 



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:

E-value

Sequence

Region

Superfamily

Alignment

Genome

Sorry No Significant Hits.


E-value

Sequence

Region

Superfamily

Alignment

Genome

3.7e-02

YourInputSequence

101-149

Nucleotidylyl transferase

3.2e+00

YourInputSequence

66-104

Copper amine oxidase, domain 3 (catalytic)


Please send feed back to

gough@supfam.org

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.

Interaction
Information

PIR

SWISSPROT

Number
of references
or techniques

Interactions
(text)

Interactions
(graphical)
blue-static
purple-java

Protein names

HJBYDH:S48765

SRS2_YEAST:YD78_YEAST

1

helicase HPR5

 

probable membrane protein YDR078c

S46822:S48765

YHA6_YEAST:YD78_YEAST

3

hypothetical protein YHL006c

 

probable membrane protein YDR078c

S47544:S48765

not in SW:YD78_YEAST

2

probable membrane protein YLR376c

 

probable membrane protein YDR078c

S48765:S48382

YD78_YEAST:YIP2_YEAST

2

probable membrane protein YDR078c

 

hypothetical protein YIL152w

OKBYS1:S48765

CKS1_YEAST:YD78_YEAST

1

cell division control protein CKS1

 

probable membrane protein YDR078c

 

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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