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Re: [ccp4bb] Lysine methylation for proteins containing disulfide bonds? |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: Lysine methylation for proteins containing disulfide bonds? From: Engin Ozkan eozkan {- at -} STANFORD {- dot -} EDU Date: 2010-04-14 I was surprised to get a few messages asking for our protocol for reductive methylation of proteins for crystallization. We employ almost exactly the protocol published by Walter et al, Structure, 2006. This is a "Ways and Means" article that made us realize how easy it was to do this regularly on failing projects, and it has a section titled "The Protocol" for those wanting to do it. A couple of things to add: We use methanol-free formaldehyde. Also, remember that your protein has to be in an amine-free buffer; Tris is no good during the reaction, but it can be used to quench the reaction. Engin On 4/13/10 9:51 PM, Engin Özkan wrote: > Dear Oliver, > > In our lab, reductive methylation using dimethylaminoborane is > regularly performed, and nearly everything we work on have native > disulfides. Among five or six reactions I've performed on molecules > with disulfides, I have not had a case where solubility or stability > was affected. In one case, however, methylation broke up a protein > complex (the interface is lysine heavy). I did also hear from lab > members a few cases where there was protein precipitating during > methylation, but that seems to be the exception rather than the norm. > > Engin > > On 4/13/10 7:55 PM, Oliver Clarke wrote: >> Hi all, >> >> I'm currently trying to crystallise a two domain protein which >> contains several structurally important disulfides. We have a high >> resolution structure of one domain (~1.4 A resolution), which reveals >> quite a few solvent-exposed lysines, some of which are involved in >> crystal-contacts. >> >> The two-domain construct also crystallises, but the only crystals >> obtained after extensive optimisation are stacks of thin-plates that >> show poor diffraction (multiple lattices, streaky spots) to around 3 A. >> >> I would like to attempt modification of the lysine residues by >> reductive methylation or cyclic pentylation (in the hope of improving >> morphology and/or diffraction), but I am worried that the reducing >> conditions required for the reaction (due to the presence of the >> dimethylaminoborane complex) will disrupt the native disulfides and >> result in protein aggregation or denaturation. Does anyone have any >> experience with reductive methylation of disulfided proteins, or know >> of any references describing the same? >> >> Thanks in advance, >> >> Oliver Clarke. >> >> ______________________________________________________________________ >> The information in this email is confidential and intended solely for >> the addressee. >> You must not disclose, forward, print or use it without the >> permission of the sender. >> ______________________________________________________________________ > > -- Engin Özkan Post-doctoral Scholar Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 ph: (650)-498-7111 CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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