THALLIUM COUNTERION DISTRIBUTION IN CUBIC INSULIN CRYSTALS DETERMINEDFROM ANOMALOUS X-RAY-DIFFRACTION DATA

To determine the distribution of monovalent cations around a protein w e have measured anomalous scattering diffraction data from Tl-containi ng cubic insulin crystals at pH 8 and pH 10.5. The differences between Bijvoet reflection pairs within each set of data were used to calcula te anomalous scattering difference maps. Both maps show the same six T l+ sites, which include two well-ordered Tl+ ions previously identifie d from isomorphous exchange experiments. The other four sites constitu te a second class of cations, which, while much more mobile than the p rotein atoms, are associated with particular ligating groups. Three of the six Tl+ sites are created exclusively by protein main and side ch ain 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 wa y on interactions with each other and flexibility in the protein struc ture. 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 prot ein 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.