Maybe you could try a modification that does not require reduction?
Like sulfo-NHS-acetate (will turn primary amines into amides). There
are hundreds of amine-reactive "protein modification" reagents out
there. Most of them listed at piercenet.com.
Also, try doing the reaction at different pH values. Each lysine in
your molecule will have a slightly different pKa, and you can "tune"
which ones get modified that way (to some degree). You may only need to
hit one or two of them to change your crystal form.
-James Holton
MAD Scientist
Nathaniel Clark wrote:
I just tried that protocol, and had essentially all of my protein
crashed out. Any tips on optimizing the methylation reaction to
reduce precipitation? Perhaps reducing the formaldehyde or
dimethylaminoborane, shortening the incubation times, etc.?
Thanks,
Nat
On Wed, Apr 14, 2010 at 6:00 PM, Engin Ozkan <[email protected]> wrote:
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
