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:[email protected]] Im Auftrag von Dipankar Manna Gesendet: Montag, 20. April 2015 21:22 An: [email protected] 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 <[email protected]<mailto:[email protected]>> 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
