Neutron diffraction study of cubic insulin at two different pDs

Ishikawa, Takuya*; Tanaka, Ichiro*; Chatake, Toshiyuki*; Kurihara, Kazuo; Onishi, Yuki*; Kusaka, Katsuhiro; Tamada, Taro; Kuroki, Ryota; Niimura, Nobuo*

It is known that the protonation status of amino acid side chains can strongly affect the biological function and stability of a protein. It is often hard to estimate it because it depends also on the local environment of the side chain. Neutron diffraction is a powerful tool to observe hydrogen atoms because of its strong interaction with hydrogen. Thus, a neutron study of cubic insulin at pD 6 has been carried out using the BIX-4 diffractometer (JRR-3, JAEA) and the result was compared with the previous neutron study at pD 9 (Maeda, et al. 2004) and by X-ray at pH 6 (Diao, 2003). A large single crystal of cubic insulin was prepared based on the crystallization phase diagram. The crystal diffracted to 2.5 resolution. It was found that only N of His 5 in the B-chain is protonated whereas both N and N of His10 in the B-chain is protonated at pD 6 and the results at pD 9 are also the same (the pK of histidine side chains in the free form is around 6.0). These indicate that neutron diffraction study is useful to determine the protonation status of a protein.