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Reductive methylation

References


Rayment, I. Reductive alkylation of lysine residues to alter crystallization properties of proteins. Meth Enzymol 276, 171–179 (1997)

"Reductive alkylation of lysine residues involves the initial formation of a Schiff base between the Ɛ-amino group of a lysine residue and either a ketone or aldehyde that is then reduced to a secondary or tertiary amine [...]
[...] In principle a wide variety of alkyl moieties can be added to an amino group by reductive alkylation. In practice the majority of cases employing this chemical modification have focused on adding methyl groups by the use of formaldehyde. This has occurred primarily because of the greater reactivity of formaldehyde than other ketones or aldehydes, and because this modification has the mildest effect on the biochemical properties of the protein"

Walter, T.S. et al. Lysine methylation as a routine rescue strategy for protein crystallization. Structure 14, 1617–1622 (2006)

"Originally used as a method of isotope labeling (Means and Feeney, 1968; Rice and Means, 1971), this has proven value for protein crystalli-zation, allowing, in certain instances, the formation of diffraction-quality crystals for proteins that had previously proved refractory to crystallization" (REFs)

REFs.

Kobayashi, M., Kubota, M., and Matsuura, Y. (1999). Crystallization and improvement of crystal quality for X-ray diffraction of maltooligosyl trehalose synthase by reductive methylation of lysine residues. Acta Crystallogr. D Biol. Crystallogr. 55, 931–933.

Kurinov, I.V., Mao, C., Irvin, J.D., and Uckun, F.M. (2000). X-ray crystallographic analysis of pokeweed antiviral protein-II after reductive methylation of lysine residues. Biochem. Biophys. Res. Commun. 275, 549–552.

Saxena, A.K., Singh, K., Su, H.P., Klein, M.M., Stowers, A.W., Saul, A.J., Long, C.A., and Garboczi, D.N. (2006). The essential mosquito-stage P25 and P28 proteins from Plasmodium form tile-like triangular prisms. Nat. Struct. Mol. Biol. 13, 90–91.


Protocol


I. Protein preparation
This protocol requires approximately 10-20 mg of purified protein at concentration 5-10mg/ml
Purified protein should be stored in lysis buffer:
50 mM HEPES pH 8.0, 500 mM NaCl, 5% glycerol and 10 mM beta-mercaptoethanol.

II. Reagent Preparation (prepare fresh in the day of experiment)
1M dimethylamine-borane complex (ABC) in water (FW 58.92)
1M formaldehyde in water
(Dilute 12M stock (FLUKA) with water)
50mg/ml glycine
1M dithiothreitol (DTT)

III. Reductive Methylation: Day 1
Add 20 μl of 1M ABC per 1 ml of protein solution
Add 40 μl of 1M formaldehyde per 1ml of protein solution immediately and mix the solution gently
Incubate solution at 4 C degrees for 2 hours
Repeat steps a, b and c
At the end of the 2nd incubation period, add an 10ul of ABC per ml of protein (initial volume)
Incubate at 4C degrees overnight (12-14 hrs)

IV. Reductive Methylation: Day 2
Add 1 mL of 50 mg/ml glycine and 1M DTT (final concentration 5mM) to quench the reaction
Incubate on ice for 2 hours
Dialyze protein overnight against a large volume of crystallization buffer: 20 mM HEPES pH 8.0, 200 mM NaCl, 2 mM DTT 
Concentrate protein to desired concentration
Screen modified protein for crystallization conditions