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Hi all,

I am currently attempting to crystallise a two domain protein which contains several structurally important disulfides. We have a high resolution structure of one domain (1.4 A), 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, 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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Dear Oliver,

I have no experience with crystallography, so I do not know if my suggestion is compatible with it.

Anyway, from a chemical aspect, you could:

a) convert the terminal amino groups of accessible lysines to amides by reacting them with activated esters. In the case of NHS-esters, the coupling can be done at slightly alkaline conditions (borate buffer, pH 8-9). About 5% of DMF or DMSO (final) is required to ensure solubility of the reagents involved. Workup is done by dialysis or gel filtration.

NHS esters may be easily prepared by reacting R-COOH with a soluble carbodiimide (like EDC) and NHS, if not available commercially or you don't want to buy it.

b) another option might be masking accessible amino groups with itaconic anhydride, also at alkaline pH, reversible at pH 3 (eg dialysis against acetic acid).

Both suggestions should not affect SH groups.

HTH

Wolfgang

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