Modifying the protein to crystallize better: Difference between revisions
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There are a number of methods which can be used to modify the crystallization behavior of proteins. | There are a number of methods which can be used to modify the crystallization behavior of proteins. | ||
=== Genetic/Mutagenesis Approach=== | |||
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This method, also called 'surface entropy-reduction mutagenesis', involves mutation of sidechains on the surface of proteins, in order to reduce the entropic cost of forming ordered intermolecular crystal contacts and thus enhance the crystallizability of proteins. | This method, also called 'surface entropy-reduction mutagenesis', involves mutation of sidechains on the surface of proteins, in order to reduce the entropic cost of forming ordered intermolecular crystal contacts and thus enhance the crystallizability of proteins. | ||
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http://nihserver.mbi.ucla.edu/SER/ | http://nihserver.mbi.ucla.edu/SER/ | ||
=== Chemical Modification=== | === Chemical Modification=== | ||
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Several methods are available: | Several methods are available: | ||
====Modification of lysine residues==== | |||
This method, pioneered by the Rayment laboratory, involves the methylation -- under reducing conditions -- of lysine residues. This increases the hydrophobicity of the modified lysine sidechains, reduces the overall solubility of the protein, and -- for some proteins -- promotes the formation of ordered crystal contacts. | This method, pioneered by the Rayment laboratory, involves the methylation -- under reducing conditions -- of lysine residues. This increases the hydrophobicity of the modified lysine sidechains, reduces the overall solubility of the protein, and -- for some proteins -- promotes the formation of ordered crystal contacts. | ||
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Here is a protocol for [[Lysine Methylation]]. | Here is a protocol for [[Lysine Methylation]]. | ||
====Modification of cysteine residues==== | |||
This method involves the carboxymethylation -- under reducing conditions -- of single cysteine residues. This effectively neutralizes the cysteine residues (some of which are chemically reactive), increases the overall solubility of the protein and can help prevent aggregation and denaturation problems. | This method involves the carboxymethylation -- under reducing conditions -- of single cysteine residues. This effectively neutralizes the cysteine residues (some of which are chemically reactive), increases the overall solubility of the protein and can help prevent aggregation and denaturation problems. | ||
Revision as of 16:04, 13 March 2008
Introduction
There are a number of methods which can be used to modify the crystallization behavior of proteins.
Genetic/Mutagenesis Approach
This method, also called 'surface entropy-reduction mutagenesis', involves mutation of sidechains on the surface of proteins, in order to reduce the entropic cost of forming ordered intermolecular crystal contacts and thus enhance the crystallizability of proteins.
Typically, large and charged sidechains -- such as lysine and glutamate -- are mutated to small and uncharged sidechains, mostly alanine. The Derewenda laboratory has championed this approach and the state of the art is described in a recent review (Derewenda ZS, and Vekilov PG [2006]. Acta Crystallogr. D62:116–124)
There is a webserver, called SER (Surface Entropy Reduction Prediction Server), which aims to predict sites that are most suitable for mutation designed to enhance crystallizability:
http://nihserver.mbi.ucla.edu/SER/
Chemical Modification
Another method for enhancing the crystallizability of proteins involves chemical modification of specific sidechains. Several methods are available:
Modification of lysine residues
This method, pioneered by the Rayment laboratory, involves the methylation -- under reducing conditions -- of lysine residues. This increases the hydrophobicity of the modified lysine sidechains, reduces the overall solubility of the protein, and -- for some proteins -- promotes the formation of ordered crystal contacts. A recent study (Walter et al. [2006] "Lysine Methylation as a Routine Rescue Strategy for Protein Crystallization" Structure 14:1617–1622) demonstrated that, out of 10 non-crystallizable proteins, 4 proteins became crystallisable after reductive methylation.
Here is a protocol for Lysine Methylation.
Modification of cysteine residues
This method involves the carboxymethylation -- under reducing conditions -- of single cysteine residues. This effectively neutralizes the cysteine residues (some of which are chemically reactive), increases the overall solubility of the protein and can help prevent aggregation and denaturation problems.
References:
Eiler S et al. (2001) Protein Expr Purif. 22(2):165-73
http://www.ionsource.com/Card/cmc/method.htm -- A protocol for carboxymethylation