Tables for
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F, ch. 4.3, pp. 100-101   | 1 | 2 |

Section 4.3.2. Improving solubility

D. R. Daviesa* and A. Burgess Hickmana

aLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0560, USA
Correspondence e-mail:

4.3.2. Improving solubility

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Frequently, a protein is so insoluble that there is only a small probability of direct crystallization. Not only does the limited amount of protein hinder crystallization, but the departure from optimal solubility conditions by the addition of almost any crystallization medium frequently results in rapid precipitation of the protein from solution. When this happens, it is sometimes possible to find surface mutations that enhance solubility. Two strategies have been successfully applied, depending on whether or not the overall topology is known.

An early investigation of the effects of surface mutations (McElroy et al., 1992[link]) involved the crystallization of human thymidylate synthase, where the Escherichia coli enzyme structure was known, but the human enzyme could only be crystallized in an apo form unsuitable for studying inhibitors owing to disorder in the active site. The effect of surface mutations was systematically explored by making 12 mutations in 11 positions, and it was found that some of the mutations dramatically changed the protein solubility. Some of the mutant proteins were easier to crystallize than the wild type, and, furthermore, three crystal forms were obtained that differed from that of the wild type.

A second example of the rational design of surface mutations based on prior knowledge of the structure of a related protein is demonstrated by the studies of the trimethoprim-resistant type S1 hydrofolate reductase (Dale et al., 1994[link]). This protein was rather insoluble and precipitated at concentrations greater than 2 mg ml−1. The authors changed four neutral, amide-containing side chains to carboxylates and examined the expressed proteins for improved solubility. Three of the four mutant proteins were more soluble than the wild-type protein, and a double mutant, [\hbox{Asn48}\rightarrow \hbox{Glu}] and [\hbox{Asn130}\rightarrow \hbox{Asp}], was particularly soluble; this mutant protein crystallized in thick plates, ultimately enabling the structure to be determined.

In the absence of any knowledge of the structure, more heroic procedures are required, as illustrated by the crystallization of the HIV-1 integrase catalytic domain (residues 50–212). This domain had been a focus of intensive crystallization attempts, which were hindered by the low solubility of the protein. The strategy used was to replace all the single hydrophobic residues with lysine and to replace groups of adjacent hydrophobic amino acids with alanines (Jenkins et al., 1995[link]). A simple assay for improved solubility based on the overexpression of the protein was employed, which did not require isolating the purified protein; cell lysis followed by centrifugation and SDS–PAGE analysis were used to determine which mutant proteins were sufficiently soluble to appear in the supernatant. The initial application of this method to 30 mutants resulted in one, [\hbox{Phe185}\rightarrow \hbox{Lys}], which was soluble and which was subsequently crystallized and its structure determined (Dyda et al., 1994[link]). The protein formed a dimer, and the mutated residue was observed at the periphery of the dimer interface where the introduced lysine formed a hydrogen bond with a backbone atom of the second subunit, an interaction not possible for the unmutated protein. The position of the mutation was remote from the active site, and the physiological relevance of the observed dimer interaction was later confirmed by studies on an avian retroviral integrase (Bujacz et al., 1995[link]).

In further mutational work, it was observed that the HIV-1 integrase core-domain mutant suffered from an inability to bind to Mg2+ in the crystal, despite the evidence that Mg2+ or Mn2+ is needed for activity. The original crystallization took place using cacodylate as a buffer and also had dithiothreitol present in the crystallization medium. Under these conditions, cacodylate can react with –SH groups, and there were two cysteines in the structure that were clearly bonded to arsenic atoms. To avoid this problem, attempts were made to crystallize in the absence of cacodylate. These were successful only when a second mutation, designed to improve solubility, was introduced, [\hbox{Trp131}\rightarrow \hbox{Glu}] (Jenkins et al., 1995[link]; Goldgur et al., 1998[link]). The use of this mutant led to crystals that had the desired property of binding to Mg2+ and, in addition, revealed the conformation of a flexible loop that had not been previously defined.


Bujacz, G., Jaskolski, M., Alexandratos, J., Wlodawer, A., Merkel, G., Katz, R. A. & Skalka, A. M. (1995). High resolution structure of the catalytic domain of avian sarcoma virus integrase. J. Mol. Biol. 253, 333–346.
Dale, G. E., Broger, C., Langen, H., D'Arcy, A. & Stüber, 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.
Dyda, F., Hickman, A. B., Jenkins, T. M., Engelman, A., Craigie, R. & Davies, D. R. (1994). Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science, 266, 1981–1986.
Goldgur, Y., Dyda, F., Hickman, A. B., Jenkins, T. M., Craigie, R. & Davies, D. R. (1998). Three new structures of the core domain of HIV-1 integrase: an active site that binds magnesium. Proc. Natl Acad. Sci. USA, 95, 9150–9154.
Jenkins, T. M., Hickman, A. B., Dyda, F., Ghirlando, R., Davies, D. R. & Craigie, R. (1995). Catalytic domain of human immunodeficiency virus type 1 integrase: identification of a soluble mutant by systematic replacement of hydrophobic residues. Proc. Natl Acad. Sci. USA, 92, 6057–6061.
McElroy, H. E., Sisson, G. W., Schoettlin, W. E., Aust, R. M. & Villafranca, J. E. (1992). Studies on engineering crystallizability by mutation of surface residues of human thymidylate synthase. J. Cryst. Growth, 122, 265–272.

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