My guess would be the histidine. Histidine is the next best nucleophile in
both your peptides and it is known to be alkylated by various alkyl halides
under certain conditions. For example in the classic studies of Moore et
al. on ribonuclease, the active site histidines (His-12 and His-119) were
specifically labeled by iodoacetate [cf. Means and Feeney, Chemical
Modification of Proteins, p. 107). Normally histidine is not exceptionally
reactive, but often in cases where His is part of the catalytic site, it
can be super-reactive (another example is the reaction of His-59 with TPCK
in chymotrypsin). I am unaware of a specific case of alkylation of His by
4-VP, but it is not implausible.
Here's a hypothesis. The specific His residue in your protein is in a
(possibly) hydrophobic pocket (or an active site) which happens to bind
4-VP, which then alkylates His . After unfolding the protein, you label
the Cys with IAA. A test of this hypothesis would be the Edman sequencing,
which would show an unusual ("funky"?) residue at position 3 instead of
His. Also, you could try the reverse experiment, alkylation first with
IAA, then unfolding and alkylation with 4-VP. If my hypothesis about a
hydrophobic binding pocket is correct, IAA may not react, because it is not
hydrophobic.
One could make a similar argument for alkylation of Lys, which also is
possible, but I think less likely.
It will be interesting to learn the outcome of this story.
Richard Laursen
--------------------------------------
> The short version of my question...can 4-vinylpyridine or iodoacetic acid
>"modify" any other amino acids than cysteine. At this point I can hear
>the old
>timers go "aha, another young whippersnapper reinventing the
>wheel"...sigh, yep
>its true!
> The long version of my question...we have reason to believe that our protein
>du jour has some free cysteines (12 total in the protein). So we thought that
>it would be fun to figure out which ones are free. So we attempted to be
>"clever"....first we incubated the protein in the presence of 4-VP at 50 C (to
>open it up a little bit and expose any buried cysteines...CD shows it is
>partially unfolded at that temp), dialyze away the 4-VP, add 6M Gd, DTT, 98 C
>for one hour and after it cools add IAA, dialyze into PBS and do a trypsin
>digest...compare with a control that had not received the 4-VP treatment...so
>in theory free cysteines will be labeled with 4-VP and disulfide bonded
>cysteines will be labeled with IAA. Sure enough by comparing the maps we saw
>two new peaks in the 4-VP treated stuff and we got excited. We collected the
>peaks and did Edman sequencing and MALDI-TOF. The Edman was kind of funky but
>we could identify the peptide and it contains one cysteine and the cysteine
>eluted on our HP sequencer where carboxymethyl cysteine should elute
>(note...not PE-CYS like we thought should happen)...the MALDI-TOF gives a nice
>mass that only makes sense if you interpret it as the mass of the peptide plus
>58 (IAA) AND 105 (4-VP). The peptide sequence is SIHDFCLVSK (by the way the
>second peak was SIHDFCLVSKVVGR...same deal with the mass being 163 too high).
>So, it looks to us like the cysteine is IAA modified and another amino acid is
>4-VP modified. Is this possible? Can anyone guess which amino acid is the
>culprit (I wish we had MS/MS capability at this point)? If this hypothesis is
>true what is magical about this part of the protein that this non-Cys amino
>acid is so labeled while it is not somewhere else in the protein (the peptide
>in question is residues 6-15)?
>
>Weather inconsequential: forget things like hurricanes, tornadoes, heat
>waves,
>floods etc. the worse scourge of mankind is the pocket gopher...can you
>imagine having a nice new lawn installed last fall, nice and flat and a deep
>green (out here in CA green is a big deal) and come spring there are all kinds
>of neat mounds of dirt all over the place (so much for the kids croquet
>playing
>area!).
>
> Jim Bloom
> Bayer Corp
> Berkeley, CA
Richard A. Laursen
Department of Chemistry
Boston University
590 Commonwealth Ave.
Boston, MA 02215
Tel (617) 353-2491; FAX (617) 353-6466
email: <laursen@bu.edu>