Hi Brad,
For DosR, a stable disulfide bond was reported even in presence of 150 mM DTT - was reduced only after Thermal denaturation. see J Mol Biol. 2008 Nov 21;383(4):822-36 Best Clemens Von: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] Im Auftrag von Brad Bennett Gesendet: Tuesday, August 11, 2009 12:06 AM An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Disulfide bond "survival" in the presence of DTT? Hi all- Got a perplexing thiol chemistry/phasing issue. To prevent non-native disulfide bonds from forming between free Cys but to preserve native disulfides, I have heard of using a very low (say < 0.5 mM) concentration of DTT. I've recently come across a paper where the assignment of 3 disulfides in a protein structure was carried out by calculating a 4A resolution anomalous difference map (detecting the sulfur anomalous signal with 1.7A x-rays). However, at every step in the protein prep and in the crystallization solution, there exists mM concentrations of DTT. Every protein is different, so the microenvironment and the protection of a disulfide will be different for every one you come across. But I am surprised that a disulfide can "withstand" that much DTT. I guess the proof is in the pudding, which would be the anomalous peaks observed in the maps. What is the upper limit on DTT (or B-ME or TCEP) concentration that people have used and still maintained native disulfides? Thanks- Brad
