Thallium Counterion Distribution in Cubic Insulin Crystals Determined from Anomalous X-ray Diffraction Data

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 91; no. 4; pp. 1224 - 1228
• Main Authors: Badger, John, Li, Youli, Caspar, D. L. D.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 15-02-1994; National Acad Sciences; National Academy of Sciences
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Atomic interactions; Atoms; Biological and medical sciences; Biology; Cations; Cations - chemistry; Cattle; Crystal structure; Crystalline structure; Crystals; Cubic crystals; Fundamental and applied biological sciences. Psychology; Insulin; Insulin - chemistry; Ions; Models, Molecular; Molecular biophysics; Molecules; Physics; Protein hormones. Growth factors. Cytokines; Proteins; Reproducibility of Results; Scattering, Radiation; Structure in molecular biology; Thallium - chemistry; X-Ray Diffraction; X-Rays; Protein hormone; Vertebrata; Mammalia; Bovine; Thallium ion; Counter ion; Artiodactyla; Localization; Insulin; Ungulata; Structural analysis; Crystalline structure
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.4.1224

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.