Reductive alkylation of lysine residues to change protein properties (pI, solubility and hydropathy) which may promote crystallization via improved crystal packing.

Reductive alkylation of proteins has been successfully applied to obtain a significant number of high-quality crystals from proteins previously unable to be crystallized. Alkylating the e amino group of lysines alters the hydropathy, solubility and pI of the protein which may promote crystallization by altering sample-sample, sample-solvent and crystal packing interactions.

Reductive alkylation does not change the intrinsic charge on a protein but may change the isoelectric point (pI) slightly. The N-terminal amino group on the backbone will also be reductively alkylated. In general, alkylated proteins retain their original biochemical function. This protocol is designed with the goal of generating a high degree of modification with few side reactions, resulting in a homogeneous population of protein.

Reductive Alkylation Kit

1. Large-scale evaluation of protein reductive methylation for improving protein crystallization. Kim et al. Nature Methods, Volume 5, Number 10, page 853-854, October 2008.

2. Lysine methylation as a routine rescue strategy for protein crystallization. Walter et al. Structure 14, 1617-1622, November 2006.

3. A pivotal role for reductive methylation in the de novo crystallization of a ternary complex composed of Yersinia pestis virulence factors YopN, SycN and YscB. Florian D. Schubot and David S.Waugh. Acta Cryst. (2004). D60, 1981-1986.

4. Reductive alkylation of lysine residues to alter crystallization properties of proteins. Ivan Rayment. Methods in Enzymology, Volume 276, 171-179, (1997).

5. Crystallization and improvement of crystal quality for X-ray diffraction of maltooligosyl trehalose synthase by reductive methylation of lysine residues. Matsuura et al. Acta Cryst. (1999). D55, 931-933.

6. Structural consequences of reductive methylation of lysine residues in hen egg white lysozyme: An X-ray analysis at 1.8 angstrom resolution. Rypniewski, W.R., Holden, H.M., and Rayment, I. (1993). Biochemistry 32, 9851-9858.

7. Reductive alkylation of amino groups in proteins. Means, G. E. & Feeney, R. E. (1968). Biochemistry, 7, 2192-2201.

8. Double chromodomains cooperate to recognize the methylated histone H3 tail. Flanagan et al. Nature, 438, 1181-1185 (2005).

9. Reductive Isopropylation of Amino Groups in Lysine Containing Peptides. Brown and Greenberg. Analytical Letters, Volume 17, Issue 12 1984 , pages 1429-1445.

10. Selectivity in the Modification of the a-Amino Groups of Hemoglobin on Reductive Alkylation with Aliphatic Carbonyl Compounds. Acharya et al. Journal of Biological Chemistry, Vol. 260, No. 10, Issue of May 25, pp. 6039-6046, 1985.

11. Accessibility and mobility of lysine residues in beta-lactoglobulin. Brown et al. Biochemistry, 1988, 5601-5610.

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