Fig. 4-19 This shows the 10th histidine residue in an insulin B chain The different structures shown in (a) and (b) are observed at different pHs by an X-ray diffraction technique. The origin of the different structures is not yet understood.

Fig. 4-20 Hydration and dehydration structure of histidines observed by neutron diffraction techniques (a) The 10th histidine residue. Both the Npi and Ntau nitrogen atoms are hydrated, and the hydrogen atom bound to the Ntau nitrogen atom is dehydrated at pH=10. (b) The 5th histidine residue. Only the Npi nitrogen atom is hydrated.

Insulin is a polypeptide hormone that is critical for the metabolism of glucose. The insulin monomer consists of two chains, a 21-residue A chain and a 30-residue B chain, linked by a pair of disulfide bonds. The structural information of hydrogen and the hydration of insulin is indispensable in understanding the physiological function of insulin. X-ray diffraction techniques have shown that the 10th histidine shows a comformation change while the 5th histidine does not show a change in accordance with pH, but the origin of the different behavior is not yet understood. Neutron diffraction experiments of porcine insulin (crystallized at pH=9) were performed at room temperature using the BIX-3 single-crystal neutron diffractometer installed at the JRR-3 reactor of JAERI. These experiments revealed that hydration and dehydration of histidine residues causes the dependence of the structural change on pH. Both Npi and Ntau nitrogen atoms of the 10th histidine are hydrated at pH=9 (Fig. 4-20 (a)). The hydrogen atom bound to the Ntau nitrogen atom forms a hydrogen bond to Tyr A14, and the structure shown in Fig. 4-19 (a) become stable. The increment in pH causes the dehydration of the Ntau nitrogen atom and the hydrogen bond breaks. However, the hydrogen atom bound to the Npi nitrogen atom remains and maintains the hydrogen bond to a water molecule. A new structure, shown in Fig. 4-19 (b), then becomes stable. On the other hand, in the 5th histidine only the Npi nitrogen atom is hydrated at every pH (Fig. 4-20 (b)). The change in pH does not cause hydration or dehydration, and the 5th histidine does not show any structural change. It is concluded that whether both Npi and Ntau nitrogen atoms in an imidazole ring are hydrated or not is the origin of the structural change and the stabilization of the insulin at different pHs.

Reference M. Maeda et al., Crystallization of a Large Single Crystal of Cubic Insulin for Neutron Protein Crystallography., J. Synchrotron Radiat., 11(1), 41 (2004).

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