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

International Tables for Crystallography (2012). Vol. F, ch. 4.3, p. 134   | 1 | 2 |

Section 4.3.8. Stabilization of protein targets

Z. S. Derewendaa*

aDepartment of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908–0736, USA
Correspondence e-mail: zsd4n@virginia.edu

4.3.8. Stabilization of protein targets

| top | pdf |

There is currently no clear consensus regarding a possible correlation between the thermostability of a protein and its propensity to form crystals. It is often assumed on the basis of somewhat anecdotal evidence that thermostable proteins are more readily crystallizable and therefore if a specific target protein is recalcitrant to crystallization then a homologue from a thermophilic organism should instead be used. In some cases, low thermostability may correlate with the presence of unstructured loops or termini and consequently the construct-optimization strategies, as described above, are likely to yield a more crystallizable variant with a concomitantly increased stability. For example, a study of MAPKAP kinase 2 showed that truncated variants with increased thermal stability also showed higher crystallization propensity (Malawski et al., 2006[link]). However, it is uncertain whether the relationship is causal or serendipitous. A recent analysis of large-scale data from a structural genomics project showed that when partly or fully unfolded proteins and hyperstable proteins (with melting temperatures Tm of greater than 90 °C) are excluded from comparisons, thermostability per se does not correlate with propensity for crystallization (Price et al., 2009[link]). Consequently, it appears that in a general case of a well behaving protein, attempts to increase thermostability by site-directed mutagenesis may not necessarily yield variants with enhanced crystallization properties, although when the prospective crystallization target is inherently unstable, engineering more stable variants may be helpful. In fact, this strategy has been successfully used for membrane proteins, which are often unstable in detergent environments. The first structure of a recombinant G-protein-coupled receptor (GPCR), i.e. bovine rhodopsin in complex with 11-cis retinal, was obtained using a thermostable variant with an engineered disulfide bond (Standfuss et al., 2007[link]). In the more recent case of the turkey adrenergic β2 receptor, 318 variants were screened and six mutations were identified that increased thermostability. A variant containing all six mutations had an apparent Tm that was 21 °C higher than that of the native protein in dodecyl­maltoside (DDM), was more stable in short-chain detergents and was successfully crystallized (Warne et al., 2008[link], 2009[link]). The effort required for such vast screening is substantial, but it appears that within protein families (such as GPCRs) the pattern of mutations enhancing thermostability is preserved, thus making it possible to transfer the mutations from one family member to another (Serrano-Vega & Tate, 2009[link]).

Finally, it should be noted that protein thermostability is strongly dependent on solution parameters such as the ionic strength. In the case of ribonuclease SA, the melting temp­erature (Tm) increased from ∼40 to ∼60 °C on transfer from pure 50 mM diglycine buffer to 0.9 M ammonium sulfate (Trevino et al., 2007[link]). Similarly, binding small molecules, either physiological or non­physiological ligands, typically promote stability (Matulis et al., 2005[link]). High-throughput screening methods have been developed to aid in screening for conditions and ligands that enhance stability (Vedadi et al., 2006[link]; Mezzasalma et al., 2007[link]) and in a general case this strategy appears to hold better promise than attempts to engineer higher stability through mutagenesis.

References

Malawski, G. A., Hillig, R. C., Monteclaro, F., Eberspaecher, U., Schmitz, A. A., Crusius, K., Huber, M., Egner, U., Donner, P. & Muller-Tiemann, B. (2006). Identifying protein construct variants with increased crystallization propensity – a case study. Protein Sci. 15, 2718–2728.
Matulis, D., Kranz, J. K., Salemme, F. R. & Todd, M. J. (2005). Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry, 44, 5258–5266.
Mezzasalma, T. M., Kranz, J. K., Chan, W., Struble, G. T., Schalk-Hihi, C., Deckman, I. C., Springer, B. A. & Todd, M. J. (2007). Enhancing recombinant protein quality and yield by protein stability profiling. J. Biomol. Screen. 12, 418–428.
Price, W. N. II et al. (2009). Understanding the physical properties that control protein crystallization by analysis of large-scale experimental data. Nat. Biotechnol. 27, 51–57.
Serrano-Vega, M. J. & Tate, C. G. (2009). Transferability of thermostabilizing mutations between beta-adrenergic receptors. Mol. Membr. Biol. 26, 385–396.
Standfuss, J., Xie, G., Edwards, P. C., Burghammer, M., Oprian, D. D. & Schertler, G. F. (2007). Crystal structure of a thermally stable rhodopsin mutant. J. Mol. Biol. 372, 1179–1188.
Trevino, S. R., Scholtz, J. M. & Pace, C. N. (2007). Amino acid contribution to protein solubility: Asp, Glu, and Ser contribute more favorably than the other hydrophilic amino acids in RNase Sa. J. Mol. Biol. 366, 449–460.
Vedadi, M. et al. (2006). Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc. Natl Acad. Sci. USA, 103, 15835–15840.
Warne, T., Serrano-Vega, M. J., Baker, J. G., Moukhametzianov, R., Edwards, P. C., Henderson, R., Leslie, A. G., Tate, C. G. & Schertler, G. F. (2008). Structure of a beta1-adrenergic G-protein-coupled receptor. Nature (London), 454, 486–491.
Warne, T., Serrano-Vega, M. J., Tate, C. G. & Schertler, G. F. (2009). Development and crystallization of a minimal thermostabilized G protein-coupled receptor. Protein Expr. Purif. 65, 204–213.








































to end of page
to top of page