A neutron crystallographic analysis of a cubic porcine insulin at pD 6.6

The pKa values of ionizable amino acid side chains are tabulated in standard textbooks. However, whether a certain amino acid side chain in a protein is charged or not cannot be estimated from standard pH values measured from protein solutions. Protonation and deprotonation of various ionizable amino acid residues were observed by a neutron diffraction experiment and discussed on the basis of the charged states estimated by the pKa values of the amino acid residues. The neutron diffraction study has been carried out at 2.7 A resolution on cubic porcine insulin at pD 6.6 using the BIX-4 single crystal diffractometer at the JRR-3 reactor of the Japan Atomic Energy Agency. For the present work, a large single crystal of 2.7 mm3 (=2.0 x 1.7 x 0.8 mm) was obtained by dialysis. The structure refinement was carried out using the program CNS. The resulting Rcryst is 21.6% and the Rfree is 29.1% at a resolution of 2.7 A. In the case of HisB5, both Nπ and Nτ of an imidazole ring are protonated at pD 6.6, but at pD 9 only Nπ is protonated. In contrast, for HisB10, both Nπ and Nτ are protonated at pD 6.6 as well as at pD 9. The ionization states of several amino acids in porcine insulin have been obtained at pD 6.6 and they are compared with those at pD 9 obtained by neutron diffraction as well as those at pH 6.50 and 6.98 obtained by X-ray diffraction. In this manuscript, the difference between these forms will be discussed