[Protein-analysis] deglycosylated protein amino acid sequence help!!
(by h.e.pagett from newcastle.ac.uk)
Mon May 17 12:37:48 EST 2010
Im not sure if this is the best forum to place this but Im hoping that someone might be able to help!!
Im a marine biologist by training but have veered into the dark art of proteomics/glycobiology for my PhD...Im feeling a little out of my depth due to a complete lack of knowledge of basic principles!
So, basically I have a glycoprotein that I have purified which is the same as the one used in this paper (An α2-macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus amphitrite. Dreanno, Matsumura, Dohmae, Takio, Hirota, Kirby and Clare. PNAS 2006 vol. 103 no. 39 14396-14401) so have the amino acid sequence as found in that paper (GenBank accession number AY423545). I have been concentrating on the sugars side of the glycoprotein but am curious about the protein structure too.
I know that it is possible to guess possible glycosylation sites on the protein from the amino acid sequence (there are 7) but is it possible to find out other things?:
1) when treated with mercaptoethanol the native protein denatures into 3 subunits (98, 88 and 76kDa) on SDS-PAGE gel. This means they are disulphide bonds (correct me if I'm wrong?!). Can the amino acid sequence tell me where these subunits split (ie where the disulphide bonds are?)...this would mean I can find out which of the possible glycosylation sites is on which subunit.
2) any other structural information that can be found from the sequence?
Ive been doing some TEM work on the deglycosylated protein too to get some more info about its structural characteristics. I have found another protein to compare it to on the RCSB website, which is TEP1r (accession numer AF291654). They are ~26% similar. In regard to this I would like to know:
1) Where the 3 disulphide bonds are in this sequence (I think its the last 6 Cys's of the sequence from the RCBS PDB sequence details as it shows green lines between them...how has this been worked out?)
2) Where is the thioester bond found in the sequence? And can I display it in a PyMOL type image?
If I can combine all of this info into one neat paragraph for my thesis discussion it would be fantastic!!
If anyone could be able to help (even if its to tell me I cant find out this information from the amino acid sequence!) I would be very very very grateful!!
Miss Helen E Pagett
School of Marine Science and Technology
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Newcastle upon Tyne
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