Mutagenising phosphorylation sites

[David Micklem]

Dear All,

I wonder if someone out there could give me some advice about mutagenising
Serine residues suspected of being phosphorylation sites.

As far as I can gather, it is common to change the Serine to an Alanine to
mimic the always-unphosphorylated state, and to an Aspartate to mimic
phospho-serine.

Is this correct?  Does anyone know why this is done rather than Serine->
Cysteine, which should be both unphosphorylatable and (presumably - I'm
not a great chemist) more chemically similar to Serine? (This is an
intracellular protein, so formation of disulfide bonds shouldn't be a
problem(?))

Similarly, a naive look at the structures of phosphoserine, aspartate and
glutamate suggest to me that glutamate is more like phosphoserine than
aspartate is. Am I mistaken?

Any guidance on why Ala and Asp should be used instead of Cys and Glu (or vice
versa) would be much appreciated.

Thanks for any help!

David

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D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
Fax: [+44] (0)1223 334089               
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