Hi again, Thanks for all the suggestions. But no I really have not used either cacodylate-buffers, DTT or BME. Which I am aware can give Cys-aducts. Further I have not treated my protein with protease inhibitors, of which I have heard some can modify cysteins.
I have checked the DNA sequence, since an Arginine side chain fits perfectly in the density but on refinement I get strong positive density on the Arg-beta carbon suggesting a sulfur. Also an Arginine would do a lousy interaction with the lysine in the crystal contact... Maybe it is no big deal, but I want to have control on what happens to my protein and get rid of any sources of heterogeneity. --Björn -----Original Message----- From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Bart Hazes Sent: den 21 februari 2012 18:26 To: [email protected] Subject: Re: [ccp4bb] Modified Cys by TCEP? On 12-02-21 10:11 AM, Björn Kauppi wrote: > Hi all, > > I recently encountered a modified surface cysteine residue in one of my > structures. The protein is expressed in E.coli and my data is to 1.7Å so I > am positive of the number of extra atoms. It really look like one of the > tails (the Propanoic acid) of a TCEP molecule. TCEP was added during > purification at 2 mM. I tried to look in pdb (or google) but I could not find > any reference of that TCEP could do this. The modified Cystein is involved in > a nice crystal contact (salt bridge) to a Lysine, and thereby stabilizing it > further. Looking back at older structures of the same protein, I see the same > modification of this Cys, but not as pronounced since they were done at lower > resolution. > > Does anyone recognize this behavior of TCEP? Or, any other ideas? > > > Björn Kauppi > Structure and Design > Karo Bio AB > > > ______________________________________________________________________ > This e-mail may contain confidential information proprietary to Karo > Bio AB and is meant for the intended addressee(s) only. Any > unauthorized review, use, disclosure or distribution is prohibited. > If you have received this message in error, please advise the sender > and delete the e-mail and any attachments from your files. Thank you! > ______________________________________________________________________ > Did you use cacodylate as buffer. TCEP may have reduced some of the dimethylarsenate, which is really what cacodylate is, to dimethylarsenite which can react with cysteine residues. It works best if the cysteine has a depressed pKa, as it is reactive in the thiolate form, and the proximity to the lysine you mention might do just that. There are several structures with arsenylated cysteine, the one we came across was a poxviral glutaredoxin (2HZE, Bacik & Hazes) Bart ______________________________________________________________________ This e-mail may contain confidential information proprietary to Karo Bio AB and is meant for the intended addressee(s) only. Any unauthorized review, use, disclosure or distribution is prohibited. If you have received this message in error, please advise the sender and delete the e-mail and any attachments from your files. Thank you! ______________________________________________________________________
