Once thing I don't fully understand: do you have a cleaved density or a cleaved
peptide? If your peptide is sitting in a binding site, and that you can see
only the part inside the pocket, with the rest supposably sitting in a 'more
opened' part of the molecule, or else outside of the molecule, than the cleaved
density could be due to non-specificity of this part of the peptide and high
flexibility / non-recognition. This would obviously have great biological
interest and implications.
Leo
Sent from my iPad (most likely in conference)
On 21 Apr 2015, at 10:25, herman.schreu...@sanofi.com wrote:
Dear Dipankar,
as Bonsor mentioned, 2-3 weeks is something completely different from the few
hours normally used for enzyme assays. A lot may happen during the long
crystallization that does not happen in the assay. Also, your enzyme will
still have the remainder of the catalytic machinery (oxy-anion hole, Hisp-Asp
“proton shuttle”) intact and a water molecule in the cavity left by the
removed sulfur may function as nucleophile.
One question: do you incubate overnight and then set up crystallizations, or
do you grow apo-crystals and incubate fully grown crystals with peptide?
In the first case, I would freeze the crystals after overnight incubation
with fresh peptide.
In the second case, I would incubate only 30 minutes and then freeze the
crystals.
In both cases, I would add as much intact peptide as I can, since it gets
cleaved during incubation.
One other question: do you seen both ends of the peptide, or only half of it.
If you see only half of the peptide, it might be that the full peptide is not
compatible with the crystal packing and only protein molecules with half a
peptide bound may enter the crystal lattice. In this case, you may have to
screen for completely new crystallization conditions in the presence of some
protease inhibitor(s) like PMSF to see if you can get a different crystal
form. You may also want to scan the literature to see if some non-cleavable
substrate-analog (inhibitor) is available.
On the positive side: your structure with the cleaved peptide is also
interesting, since it represents the product complex!
Good luck!
Herman
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
Dipankar Manna
Gesendet: Montag, 20. April 2015 21:22
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Cleaved peptide density!
Dear Bonsor,
Thanks for your suggestions!
It need 2-3 weeks to get the fully grown crystals. And harvest, freeze and
data collection just take usually next 3-5 days. I usually incubated
substrate overnight. Initially I was purifying with the same column as the WT
but in the next batch I used new beads to purify the mutant as you
categorically pointed out. But results are the same, I mean I got the same
cleaved peptide density! I tried soaking with different time frames and with
different peptide concentrations as well but in this case I can't see any
peptide density at all.
Best,
Dipankar
On Mon, Apr 20, 2015 at 9:06 PM, D Bonsor dbon...@ihv.umaryland.edu wrote:
First of all, you don't say how long it took to first set up crystals, for
them to grow, harvest, freeze and collect data on. Secondly how long did
leave the peptide/substrate for your SDS PAGE experiment? If they are of a
different time scale e.g. 6 hours v.s. 30 days, it may be that your enzyme is
not totally dead.
Also how did you purify the alanine mutant? If you purified it on the same
columns/beads as the WT protein you may have a residual amount of active
protein which could cleave your peptide over the course of crystallization.
You may want to use fresh beads, or treat columns with pepsin or sodium
hydroxide.
Not real answers I am afraid, more like suggestions.
--
Dipankar Manna
Research Scholar
Department of Chemistry
University of Oslo
Oslo, Norway
