Abstract
The growth of diffraction-quality single crystals is of primary importance in protein X-ray crystallography. Chemical modification of proteins can alter their surface properties and crystallization behavior. The Midwest Center for Structural Genomics (MCSG) has previously reported how reductive methylation of lysine residues in proteins can improve crystallization of unique proteins that initially failed to produce diffraction-quality crystals. Recently, this approach has been expanded to include ethylation and isopropylation in the MCSG protein crystallization pipeline. Applying standard methods, 180 unique proteins were alkylated and screened using standard crystallization procedures. Crystal structures of 12 new proteins were determined, including the first ethylated and the first isopropylated protein structures. In a few cases, the structures of native and methylated or ethylated states were obtained and the impact of reductive alkylation of lysine residues was assessed. Reductive methylation tends to be more efficient and produces the most alkylated protein structures. Structures of methylated proteins typically have higher resolution limits. A number of well-ordered alkylated lysine residues have been identified, which make both intermolecular and intramolecular contacts. The previous report is updated and complemented with the following new data; a description of a detailed alkylation protocol with results, structural features, and roles of alkylated lysine residues in protein crystals. These contribute to improved crystallization properties of some proteins.
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References
Ferré-D’Amaré AR, Burley SK (1994) Use of dynamic light scattering to assess crystallizability of macromolecules and macromolecular assemblies. Structure 2:357–359
Dong A, Xu X, Edwards AM et al (2007) In situ proteolysis for protein crystallization and structure determination. Nat Methods 4:1019–1021
Derewenda ZS (2004) Rational protein crystallization by mutational surface engineering. Structure 12:529–535
Kim Y, Quartey P, Li H et al (2008) Large-scale evaluation of protein reductive methylation for improving protein crystallization. Nat Methods 5:853–854
D’Arcy A, Stihle M, Kostrewa D et al (1999) Crystal engineering: a case study using the 24 kDa fragment of the DNA gyrase B subunit from Escherichia coli. Acta Crystallogr D Biol Crystallogr 55:1623–1625
Rypniewski WR, Holden HM, Rayment I (1993) Structural consequences of reductive methylation of lysine residues in hen egg white lysozyme: an X-ray analysis at 1.8-A resolution. Biochemistry 32:9851–9858
Means GE, Feeney RE (1990) Chemical modifications of proteins: history and applications. Bioconjug Chem 1:2–12
Rayment I (1997) Reductive alkylation of lysine residues to alter crystallization properties of proteins. Methods Enzymol 276:171–179
Rayment I, Rypniewski WR, Schmidt-Base K et al (1993) Three-dimensional structure of myosin subfragment-1: a molecular motor. Science 261:50–58
Derewenda ZS, Vekilov PG (2006) Entropy and surface engineering in protein crystallization. Acta Crystallogr D Biol Crystallogr 62:116–124
Sledz P, Zheng H, Murzyn K et al (2010) New surface contacts formed upon reductive lysine methylation: improving the probability of protein crystallization. Protein Sci 19:1395–1404
Walter TS, Meier C, Assenberg R et al (2006) Lysine methylation as a routine rescue strategy for protein crystallization. Structure 14:1617–1622
Schubot FD, Waugh DS (2004) 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 Crystallogr D Biol Crystallogr 60:1981–1986
Shaw N, Cheng C, Tempel W et al (2007) (NZ)CH…O contacts assist crystallization of a ParB-like nuclease. BMC Struct Biol 7:46
Kobayashi M, Kubota M, 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
Fan Y, Joachimiak A (2010) Enhanced crystal packing due to solvent reorganization through reductive methylation of lysine residues in oxidoreductase from Streptococcus pneumoniae. J Struct Funct Genomics 11:101–111
Means GE (1977) Reductive alkylation of amino groups. Methods Enzymol 47:469–478
Zhang M, Thulin E, Vogel HJ (1994) Reductive methylation and pKa determination of the lysine side chains in calbindin D9k. J Protein Chem 13:527–535
Zhang M, Vogel HJ (1993) Determination of the side chain pKa values of the lysine residues in calmodulin. J Biol Chem 268:22420–22428
Kim Y, Dementieva I, Zhou M et al (2004) Automation of protein purification for structural genomics. J Struct Funct Genomics 5:111–118
Means GE, Feeney RE (1995) Reductive alkylation of proteins. Anal Biochem 224:1–16
Anashkina A, Kuznetso E, Esipova N et al (2007) Comprehensive statistical analysis of residues interaction specificity at protein–protein interfaces. Proteins 67:1060–1077
Glaser F, Steinberg DM, Vakser IA et al (2001) Residue frequencies and pairing preferences at protein–protein interfaces. Proteins 43:89–102
Juers DH, Matthews BW (2001) Reversible lattice repacking illustrates the temperature dependence of macromolecular interactions. J Mol Biol 311:851–862
Magalhaes A, Maigret B, Hoflack J et al (1994) Contribution of unusual arginine–arginine short-range interactions to stabilization and recognition in proteins. J Protein Chem 13:195–215
Wang J, Dauter M, Alkire R et al (2007) Triclinic lysozyme at 0.65 Å resolution. Acta Crystallogr D Biol Crystallogr 63:1254–1268
Acknowledgments
We wish to thank all members of the Structural Biology Center and Midwest Center for Structural Genomics at Argonne National Laboratory for their help in conducting these experiments. This work was supported by National Institutes of Health Grant number GM GM094585, Contract numbers HHSN272200700058C and HHSN272201200026C and by the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.
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Tan, K. et al. (2014). Salvage of Failed Protein Targets by Reductive Alkylation. In: Anderson, W.F. (eds) Structural Genomics and Drug Discovery. Methods in Molecular Biology, vol 1140. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-0354-2_15
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DOI: https://doi.org/10.1007/978-1-4939-0354-2_15
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