Procedure for reductive methylation of protein to improve crystallizability
Lab/Group: Z.-J. Liu Lab
Related Journal & Article Information
Journal: Nature Structural & Molecular Biology
Introduction
Post genomics era has seen a number of new challenges emerge while moving towards the goal of covering the protein fold space such that eventually a protein could be modeled from its primary sequence using known protein structures. While expression is still a major challenge for a number of medically important proteins, a soluble expression may not always guarantee a 3D structure. Many soluble proteins purified to homogeneity, resist crystallization because of the intrinsic flexible nature of the surface residues. Lysine and glutamic acid residues found on the surface of the proteins, have long side chains that are mobile. These side chains need to be localized in space in order to promote homogenous inter molecular interactions necessary for formation of a crystal lattice. Addition of methyl groups to the side chain amine of lysines1-9 has been shown to promote crystallization presumably by immobilizing these side chains. The protocol outlined here describes the process of reductively methylating a protein for improving its crystallizability.
Materials
Reagents
1. Dimethylamine borane complex (DMAB) (Sigma, cat. no. – 15584)
2. 36 % solution of formaldehyde (Sigma, cat. no. – F8775)
Stock Solutions
1M DMAB Prepare 1ml of 1M stock solution of DMAB by dissolving 59mg of DMAB in 1ml of deionized water taken in a 1.5ml eppendorf tube. Cover the eppendorf tube with aluminum foil to prevent exposure of solution to light.
1M Formaldehyde Prepare 1ml of 1M stock solution of formaldehyde by dissolving 84µl of formaldehyde (36 % solution) in 1ml of deionized water taken in a 1.5ml eppendorf tube.
Equipment
1. Gel shaker
2. Acta PurifierTM (Amersham Biosciences, USA)
Procedure
1. Pipette out 1ml of 10mg ml-1 protein sample (critical in PBS and pure and homogeneous) each in two 1.5ml eppendorf tubes.
2. Cover the tubes with aluminum foil. Label one tube as “control”. Do not add any chemicals to this tube. Label “methylated” on the other tube.
3. Add 20μl of 1M DMAB solution followed by 40μL of 1M formaldehyde solution to the “methylated” tube.
4. Shake both the tubes (“control” and “methylated”) at 4 °C in dark on a gel shaker maintained at 100 rpm for 2 h.
5. Repeat steps 3 and 4 twice.
6. Add 10μL of DMAB and incubate for 12 h at 4 °C in the dark while shaking.
7. Run size exclusion chromatography on “control” and “methylated” protein using 20mM Tris, 150mM NaCl, pH8.0 for buffer exchange.
8. Concentrate “control” and “methylated” protein to ~ 10-15 mg ml-1
9. Analyze proteins using SDS PAGE and mass spectroscopy.
10. Screen control and methylated protein for crystallization under identical conditions.
Troubleshooting
Step 9: If there is no increase in molecular weight prepare fresh stock of reagents and repeat the procedure.
Critical Steps
Step 1: Protein should be pure, homogenous and in a buffer without primary amines. Phosphate buffered saline could be a good choice for this step.
Step 6: Size exclusion chromatography using Tris-based buffer helps quench and remove excess chemicals.
Anticipated Results
The above procedure results in dimethylation of free amine groups. An increase in molecular weight by 28 daltons for each free amine group (lysines and the N-terminal amine) is expected. Usually a qualitative shift in protein band on SDS PAGE and size exclusion elution profiles is observed for the methylated protein when compared to the control. Mass spectroscopy is used to quantitatively confirm the success of the procedure.
The methylated protein is expected to behave different than control during crystallization. Therefore, when methylation is used to improve crystal quality, the methylated protein may not crystallize under the same conditions where the control protein crystallized previously. In such cases, the methylated protein needs to be screened again for crystallization.
References
1. Liu, Z.-J. et. al.: The high-throughput protein-to-structure pipeline at SECSG. Acta Crystallogr. D 61, 679-84 (2005).
2. Rayment, I. Reductive alkylation of lysine residues to alter crystallization properties of proteins. Methods in Enzymology 276, 171-179 (1997).
3. Geoghegan, K., Cabacungan, J., Dixon, H. & Feeney, R. Alternative reducing agents for reductive methylation of amino groups in proteins. Int. J. Pept. Protein Res. 17, 345-352 (1981).
4. Gerken, T., Jentoft, J., Jentoft, N. & Dearborn, D. Intramolecular interactions of amino groups in 13C reductively methylated hen egg-white lysozyme. J. Biol. Chem. 257, 2894-2900 (1982).
5. Means, G. & Feeney, R. Reductive alkylation of amino groups in proteins. Biochemistry 7, 2192-2201 (1968).
6. Rypniewski, W., Holden, H. & Rayment, I. Structural consequences of reductive methylation of lysine residues in hen egg white lysozyme: An X-ray analysis at 1.8Ǻ resolution. Biochemistry 32, 9851-9858 (1993).
7. Schubot, F. & Waugh, D. 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. Acta Cryst. D 60, 1981-1986 (2004).
8. Kobayashi, M., Kubota, M. & Matsuura, Y. Crystallization and improvement of crystal quality for X-ray diffraction of maltooligosyl trehalose synthase by reductive methylation of lysine residues. Acta Cryst. D 55, 931-933 (1999).
9. Walter, T., Meier, C., Assenberg, R., Au, K.-F., Ren, J., Verma, A., Nettleship, J., Owens, R., Stuart, D. & Grimes J. Lysine methylation as a routine rescue strategy for protein crystallization. Structure 14, 1617-1622 (2006)
Acknowledgements
This work was funded by the 863 (Grant 2006AA02A316) and 973 (Grant 2006CB910901) Project of the Ministry of Science and Technology of China，the National Natural Science Foundation of China (30670427) and the Institute of Biophysics, CAS, China.
Keywords
methylation of lysine