IDENTIFICATION OF Ca²⁺ IONS BOUND TO PROTEINS USING AN X-RAY ANOMALOUS DISPERSION TECHNIQUE. K. Hamada¹, Y. Hata², H. Miyatake², T. Fujii², F. Amada^3, K. Fukuyama³, ¹Interdisciplinary Faculty of Science and Engineering, Shimane University, Matsue, 690 Japan, ²Institute for Chemical Research, Kyoto University, Uji, Kyoto, 611 Japan, ³Department of Biology, Osaka University, Toyonaka, Osaka, 560 Japan

Identification of Ca²⁺ ions bound to moderately large proteins by an X-ray anomalous dispersion technique seems to be possible using a high-energy synchrotron source and a sensitive imaging plate. With three kinds of proteins, Serratia protease from Serratia sp E-15 (Hamada, K. et al., 1995, J. Biochem., in press), alkaline protease from Pseudomonas aeruginosa IFO3080 (Miyatake, H. et al., 1995, J. Biochem. 118, 474) and Arthromyces ramosus peroxidase (Kunishima, N. et al., 1995, J. Mol. Biol. 235, 331), the identification of Ca²⁺ ions in their crystals were carried out by the anomalous technique. The X-ray wavelengths used for data collection were 1.283 Å (synchrotron radiation) for Serratia protease, 1.488 Å (synchrotron radiation) for Arthromyces ramosus peroxidase and 1.542 Å (Cu-K[[alpha]]) for alkaline protease. The determination of Ca²⁺ binding sites was performed by Bijvoet difference Fourier maps calculated with coefficients of {F(+)-F(-)} expi([[alpha]]_p-[[pi]]/2). In the Serratia protease, each of five Ca²⁺ ions was located between two neighbors of loops in the [[beta]]-sheet coil formed by the repeated sequence of GGXGXDXBX (B: bulky hydrophobic residue, ideally leucine). In the alkaline protease whose tertiary structure is quite similar to that of Serratia protease, Ca²⁺ ions were identified at seven sites, six of which were shared with the Serratia protease. These Ca²⁺ ions in the both proteins are significantly important in stabilizing the [[beta]]-sheet coil structure. In the peroxidase, the two Ca²⁺ ions were identified at the expected positions. The Ca²⁺ ions contribute to stabilization of the tertiary structure of the peroxidase.