Deb McMillen
Institute of Molecular biology
University of Oregon
Eugene OR
On Wed, 12 Aug 1998 Jim.Bloom.B@bayer.com wrote:
> 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
>