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.
>>
>> ______________________________________________________________________
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>
>
--
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
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