Weather inconsequential: now the heck with this stuff lets talk about
important things...I quote David Schooley: "At least for the GOPHERS you find
a hardware store that carries gopher bombs- they are mostly sulfur with a
little KNO3 or something to make it burn. You dig up the hole, light the
fuse, and cover it so all the SO2 goes through the burrow. Keep it up for a
few weeks and your problem is gone. Don't do it in front of animal rights
friends!" What I did was call my friendly local extermination company who for
$95 comes out and injects strychnine into the burrows and a week later when one
gopher is dead and his cousin takes over the burrow (apparently moisture in the
soil results in a short half-life for the strychnine)...you call them again and
they repeat the process...this goes on for a month at which point they charge
you another $50...this has gone on for 3 months now, the kids croquet field is
a mess, the extermination company is $195 richer and there are still more
gophers coming!!!!! Are we in the wrong profession?
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