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Hi Yadong,
Indeed most of the newly synthesized proteins loose their initiator
methionine while the protein synthesis is still underway! Initiator
methionine is removed by an enzyme called methionine aminopeptidase
(MetAP). The only condition is the second residue should be small and
uncharged (Gly, Ala, Ser).
Just to introduce to these MetAPs, please look at these references.
A. Addlagatta et al, 2005, Biochemistry, 44, 14741-14749.
A. Addlagatta et al, 2005, Biochemistry, 44, 7166-7174.
Anthony
________________________________
Anthony Addlagatta, PhD
HHMI Research Associate
Institute of Molecular Biology
University of Oregon
Eugene, OR-97403
Phone: (541) 346-5867
Fax: (541)346-5270
Web: http://darkwing.uoregon.edu/~anthony
On Sep 26, 2006, at 8:47 AM, Yadong Yu wrote:
Hi,
This is a ccp4 irrelevant problem but it may be interesting to this
group.
Recently I sent 2 freshly purified protein samples for MALDI mass
spectra to determine their molecular weights. The results of these
2 protein both were ~135 Da lower than their theoretical molecular
weights. It is certainly out of the range of normal error as my
proteins have MW less than 20 kDa. The accuracy of these MALDI
experiments is reliable, as evidenced by the exact 2-fold ratios
between the single-charge value and double-charge value. The
expression vector of these 2 proteins were sequenced. They were
expressed in Rosetta(DE3), an E.coli strain, and purified in native
conditions. Taken account of the size of this difference, it would
not be cleavage of some small group, e.g. amino group of Gln/Asn,
from certain residues, the more possible reason would be
deprivation of amino acid at either end of the protein. In this
secenario, the initial methionine may be lost because its peptide
fragment (-H2O), of 131 Da in size, matches the difference very
well.
Anyone experienced such problem? Is the head of protein already cut
during expression, or just lost in MALDI experiments?
Yadong Yu
LCMN/NICHD/NIH
Bldg 35, Room 3B 1006
35 Lincoln Drive
Bethesda MD 20892
(301)496-9347
Fax (301)496-2396
[EMAIL PROTECTED]
