[Protein-analysis] Re: deglycosylated protein amino acid sequence help!!

Dr Engelbert Buxbaum via proteins%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Thu May 20 10:26:49 EST 2010

In article <mailman.656.1274118593.25217.proteins from net.bio.net>, 
h.e.pagett from newcastle.ac.uk says...
> Hello, 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 
thesame as the one used in this paper (An a2-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!!
> Thanks, Helen

There is another group, bionet.glycosci, where you may ask about the 
sweet things ;-).

Now about the protein part of your questions:

When you treat your protein with ßME, you indeed split disulphide bonds. 
That you get 3 bands in SDS-PAGE means, that your protein is a hetero-
oligomer with 3 subunits of different molecular mass (not: weight!). 
There may be just 3 subunits, or the 3 subunits form a protomer, of 
which several come together for the functional protein (if you want to 
read up on this, the textbook example is hemoglobin, a diprotomer. Each 
protomer consists of an alpha- and a beta-subunit). Assuming a 
monoprotomer, the molecular mass would be around 262 kDa. You should be 
able to check the mass of the complete protein by doing the SDS-PAGE in 
the absence of ßME (just leave it out of the sample buffer, then proceed 
as usual). Obviously, you need a lower percentage gel to resolve such 
big proteins.

None of your bands corresponds to AY423545, which has a protein 
molecular mass of 170 kDa (check the uniprot link in the genebank 
entry), to which the sugar chains would have to be added. Unless the 
molecular mass differences are caused solely by differences in 
glycosylation, the 3 bands correspond to 3 different protein sequences. 
These may however be produced by proteolytic splitting of AY423545, the 
difference between the 262 kDa found and the 170 expected might be the 
contribution of the sugar chain to the electrophoretic mobility (which, 
however, is not the molecular mass of the bound sugar!). You could blot 
the bands onto a PVDF membrane and give it to a protein science core 
facility for N-terminal sequencing. That gives you the first 30 or so 
amino acids of each protein, which you can use for a BLAST search.  

In order to locate the disulphide bonds, you take the native protein and 
label all free Cys with iodoacetamide. Those Cys, which take part in 
disulphide bonds are not modified at this step. Then you split the 
disulphides with TCEP and label the now free disulphide-forming Cys with 
a fluorescing or radioactive SH-reagent, e.g. IAEDANS. The protein is 
then fragmented by protease digestion, the peptides isolated 
(chromatography and/or electrophoresis) and the fluorescent ones 
sequenced. Again, you locate the position by BLAST search. The next step 
would be to find out which of those Cys reacts with which other, but you 
can come back here once you got that far ;-).

The other method of course would be to crystallize your protein and 
determine the structure by X-ray diffraction. There you would actually 
see the disulphide bonds. This is how most of the data in PDB were 
obtained (some also by NMR, but your proteins are too big for that). To 
view PDB files, use DeepView (http://spdbv.vital-it.ch/).

Either way, obtaining such structural information on a protein is not a 
task done between now and lunch, and deserves more than a paragraph in 
your thesis!

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