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
