Effect of precipitants and buffers on heavy-atom binding

Carvin, D.; Islam, S. A.; Sternberg, M. J. E.; Blundell, T. L.

doi:10.1107/97809553602060000679

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

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International Tables for Crystallography (2006). Vol. F. ch. 12.1, p. 249 | 1 | 2 |

Section 12.1.3.5. Effect of precipitants and buffers on heavy-atom binding

D. Carvin,^a S. A. Islam,^b M. J. E. Sternberg^b and T. L. Blundell^c ^*

^a Biomolecular Modelling Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Field, London WC2A 3PX, England,^bInstitute of Cancer Research, 44 Lincoln's Inn Fields, London WC2A 3PX, England, and ^cDepartment of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, England
Correspondence e-mail: tom@cryst.bioc.cam.ac.uk

12.1.3.5. Effect of precipitants and buffers on heavy-atom binding

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Components present in the heavy-atom solution can have a profound effect on protein–heavy-atom interactions. The salting in/out agent (precipitant) and buffer are the principal sources of alternative ligands for the heavy-atom reagents, while protons compete with the heavy-atom ion/complex for the reactive amino-acid side chains.

Ammonium sulfate is the most successful precipitant in protein crystallization experiments (Gilliland et al., 1994). However, its continued presence in the mother liquor can cause problems by interfering with protein–heavy-atom interactions. At high hydrogen-ion concentrations, the NH₃ group is protonated (i.e. $[\hbox{NH}_{4}^{+}]$ ), but as the pH rises the proton is lost, typically around pH 6.0–7.0, enabling the group to compete with the protein for the heavy-atom reagent by an S_N2 reaction.

The nucleophilic strength of potential ligands follows the order $[ \hbox{NH}_{3} \gt \hbox{Cl}^{-} \gt \hbox{H}_{2}\hbox{O}.]$ The anionic complex $[\hbox{PtCl}_{4}^{2-}]$ is present in excess ammonia at pH > 7.0 and it will react: $[ \hbox{PtCl}_{4}^{2-} \rightarrow cis\hbox{-PtCl}_{2}(\hbox{NH}_{3})_{2} \rightarrow \hbox{Pt}(\hbox{NH}_{3})_{4}^{2+}.]$

The resultant cationic complex is less susceptible to reaction due to the trans effect of NH₃. Pd, Au, Ag and Hg complexes react in a similar way. Decreasing the pH of the solution reduces the amount of free ammonia available through protonation (Sigler & Blow, 1965). Such a technique may give rise to other problems (e.g. cracked crystal, decreased nucleophilicity of the protein ligands).

Changing the precipitant to sodium/potassium phosphate or magnesium sulfate may alleviate the situation, but it may also present other problems. For instance, $[\hbox{PO}_{4}^{3-}]$ displaces Cl⁻ from $[\hbox{PtCl}_{4}^{2-}]$ , thus increasing the negative charge. Both $[\hbox{PO}_{4}^{3-}]$ and $[\hbox{SO}_{4}^{2-}]$ form insoluble complexes with class A metals (e.g. lanthanide and uranyl cations) (Petsko et al., 1978). Both acetate and citrate form complexes with class A metals, but citrate, a chelating ion, binds more strongly. Tris buffer is probably preferable; it binds many cations, but the complexes formed tend to be relatively unstable.

References

Gilliland, G. L., Tung, M., Blakeslee, D. M. & Ladner, J. E. (1994). Biological Macromolecular Crystallization Database, version 3.0: new features, data and the NASA archive for protein crystal growth data. Acta Cryst. D50, 408–413.Google Scholar

Petsko, G. A., Phillips, D. C., Williams, R. J. P. & Wilson, I. A. (1978). On the protein crystal chemistry of chloroplatinite ions: general principles and interactions with triose phosphate isomerase. J. Mol. Biol. 120, 345–359.Google Scholar

Sigler, P. B. & Blow, D. M. (1965). A means of promoting heavy atom binding in protein crystals. J. Mol. Biol. 14, 640–644.Google Scholar

International Tables for Crystallography (2006). Vol. F. ch. 12.1, p. 249