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
