Re: [ccp4bb] Lysine methylation for proteins containing disulfide bonds?

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: To model or not to model...
From: Bernhard Rupp br {- at -} RUPPWEB {- dot -} ORG
Date: 2010-04-14

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Subject: Re: Lysine methylation for proteins containing disulfide bonds?
From: Engin_Özkan eozkan {- at -} STANFORD {- dot -} EDU
Date: 2010-04-14

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.
>
> ______________________________________________________________________
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--
Engin Özkan
Post-doctoral Scholar
Laboratory of K. Christopher Garcia
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 navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: To model or not to model...
From: Bernhard Rupp br {- at -} RUPPWEB {- dot -} ORG
Date: 2010-04-14