Thallium counterion distribution in cubic insulin crystals determined from anomalous x-ray diffraction data.

To determine the distribution of monovalent cations around a protein we have measured anomalous scattering diffraction data from Tl-containing cubic insulin crystals at pH 8 and pH 10.5. The differences between Bijvoet reflection pairs within each set of data were used to calculate anomalous scattering difference maps. Both maps show the same six Tl+ sites, which include two well-ordered Tl+ ions previously identified from isomorphous exchange experiments. The other four sites constitute a second class of cations, which, while much more mobile than the protein atoms, are associated with particular ligating groups. Three of the six Tl+ sites are created exclusively by protein main and side chain carbonyl dipoles rather than negatively charged groups. All of the Tl+ ions are positioned so as to interact with both protein atoms and water molecules. The Tl+ occupancies appear to depend in a complex way on interactions with each other and flexibility in the protein structure. The combined occupancies of these cations are slightly less than is required to neutralize the net protein charge of approximately -2e at pH 8 but account for only about half of the approximately -5e protein charge at pH 10.5. Thus, more disordered counterions, not seen in these Bijvoet anomalous scattering difference maps, are more numerous at higher protein net charge.