Abstract
The success of macromolecular crystallization depends on the protein’s ability to form specific, cohesive intermolecular interactions that serve as crystal contacts. In the cases where the protein lacks surface patches conducive to such interactions, crystallization may not occur. However, it is possible to enhance the likelihood of crystallization by engineering such patches through site-directed mutagenesis, targeting specifically residues with high side chain entropy and replacing them with small amino acids (i.e., surface entropy reduction, SER). This method has proven successful in hundreds of crystallographic analyses of proteins otherwise recalcitrant to crystallization. Three representative cases of the application of the SER strategy, assisted by the automated prediction of the mutation sites using the SER prediction (SERp) server are described.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
McElroy HH, Sisson GW, Schottlin WE et al (1992) Studies on engineering crystallizability by mutation of surface residues of human thymidylate synthase. J Cryst Growth 122:265–272
Dale GE, Broger C, Langen H, D’Arcy A, Stuber D (1994) Improving protein solubility through rationally designed amino acid replacements: solubilization of the trimethoprim-resistant type S1 dihydrofolate reductase. Protein Eng 7:933–939
Longenecker KL, Garrard SM, Sheffield PJ, Derewenda ZS (2001) Protein crystallization by rational mutagenesis of surface residues: Lys to Ala mutations promote crystallization of RhoGDI. Acta Crystallogr D 57:679–688
Derewenda ZS (2004) Rational protein crystallization by mutational surface engineering. Structure 12:529–535
Mateja A, Devedjiev Y, Krowarsch D, Longenecker K, Dauter Z, Otlewski J, Derewenda ZS (2002) The impact of Glu → Ala and Glu → Asp mutations on the crystallization properties of RhoGDI: the structure of RhoGDI at 1.3 A resolution. Acta Crystallogr D 58:1983–1991
Derewenda ZS, Vekilov PG (2006) Entropy and surface engineering in protein crystallization. Acta Crystallogr D 62:116–124
Cooper DR, Boczek T, Grelewska K, Pinkowska M, Sikorska M, Zawadzki M, Derewenda Z (2007) Protein crystallization by surface entropy reduction: optimization of the SER strategy. Acta Crystallogr D 63:636–645
Pellicane G, Smith G, Sarkisov L (2008) Molecular dynamics characterization of protein crystal contacts in aqueous solutions. Phys Rev Lett 101:248102
Cieslik M, Derewenda ZS (2009) The role of entropy and polarity in intermolecular contacts in protein crystals. Acta Crystallogr D 65:500–509
Price WN 2nd, Chen Y, Handelman SK, Neely H, Manor P, Karlin R, Nair R, Liu J, Baran M, Everett J, Tong SN, Forouhar F, Swaminathan SS, Acton T, Xiao R, Luft JR, Lauricella A, DeTitta GT, Rost B, Montelione GT, Hunt JF (2009) Understanding the physical properties that control protein crystallization by analysis of large-scale experimental data. Nat Biotechnol 27:51–57
Longenecker KL, Lewis ME, Chikumi H, Gutkind JS, Derewenda ZS (2001) Structure of the RGS-like domain from PDZ-RhoGEF: linking heterotrimeric g protein-coupled signaling to Rho GTPases. Structure 9:559–569
Derewenda U, Mateja A, Devedjiev Y, Routzahn KM, Evdokimov AG, Derewenda ZS, Waugh DS (2004) The structure of Yersinia pestis V-antigen, an essential virulence factor and mediator of immunity against plague. Structure 12:301–306
Janda I, Devedjiev Y, Derewenda U, Dauter Z, Bielnicki J, Cooper DR, Graf PC, Joachimiak A, Jakob U, Derewenda ZS (2004) The crystal structure of the reduced, Zn2+-bound form of the B. subtilis Hsp33 chaperone and its implications for the activation mechanism. Structure 12:1901–1907
Bielnicki J, Devedjiev Y, Derewenda U, Dauter Z, Joachimiak A, Derewenda ZS (2006) B. subtilis ykuD protein at 2.0 A resolution: insights into the structure and function of a novel, ubiquitous family of bacterial enzymes. Proteins 62:144–151
Goldschmidt L, Cooper DR, Derewenda ZS, Eisenberg D (2007) Toward rational protein crystallization: a web server for the design of crystallizable protein variants. Protein Sci 16:1569–1576
Derewenda U, Boczek T, Gorres KL, Yu M, Hung LW, Cooper D, Joachimiak A, Raines RT, Derewenda ZS (2009) Structure and function of Bacillus subtilis YphP, a prokaryotic disulfide isomerase with a CXC catalytic motif. Biochemistry 48:8664–8671
Ubhi D, Kavanagh KL, Monzingo AF, Robertus JD (2011) Structure of Candida albicans methionine synthase determined by employing surface residue mutagenesis. Arch Biochem Biophys 513:19–26
Newman J (2005) Expanding screening space through the use of alternative reservoirs in vapor-diffusion experiments. Acta Crystallogr D 61:490–493
Hsieh FL, Chang TH, Ko TP, Wang AH (2011) Structure and mechanism of an Arabidopsis medium/long-chain-length prenyl pyrophosphate synthase. Plant Physiol 155:1079–1090
Acknowledgment
This work was supported by the National Institutes of Health, grant GM095847.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Goldschmidt, L., Eisenberg, D., Derewenda, Z.S. (2014). Salvage or Recovery of Failed Targets by Mutagenesis to Reduce Surface Entropy. 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_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0354-2_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-0353-5
Online ISBN: 978-1-4939-0354-2
eBook Packages: Springer Protocols