Phase changes in T3R3f human insulin: temperature or pressure induced?

The structure of T3R3f hexameric human insulin has been determined at 100 K from two different crystals at 1.2 and 1.3 Angstrom resolution and refined to residuals of 0.169 and 0.176, respectively. Owing to a phase change, th e c axis is double its room-temperature value and the asymmetric unit conta ins two independent TRf insulin dimers. Compared with the orientation in th e room-temperature structure, one dimer undergoes a rotation about the c ax is of -5 degrees, while the second is rotated +4 degrees. A superposition o f the backbone atoms of the two independent dimers shows that the C-alpha a toms of five residues within the R-f-state monomers are displaced by more t han 1.0 Angstrom; smaller displacements are observed for the T-state monome rs. Four zinc ions lie on the crystallographic threefold axis and each form s bonds to three symmetry-related HisB10 N-epsilon2 atoms from the T- and R -f-state trimers. While three of the zinc ions are tetrahedrally coordinate d with a chloride ion completing the coordination sphere, mixed tetrahedral /octahedral coordination is observed for one of the T-state zinc ions. The three symmetry-related 'phenolic binding sites' in one hexamer contain wate r molecules and a glycerol molecule, but the same sites in the second hexam er are occupied by a zinc ion coordinated to an alternate conformation of H isB10, a symmetry-related HisB5 and two chloride ions. Two additional and p artially occupied zinc ion sites are observed at the interface between the two independent dimers. One zinc ion is coordinated by a T-state HisB5 of o ne dimer, an R-state HisB5 of the second dimer and two water molecules; the second zinc ion is coordinated by an alternate side-chain conformation of the T-state HisB5 and three water molecules. The carboxyl group of one GluB 13 side chain, which exists in two discrete conformations, appears to be pr otonated, because short contacts exist to a second carboxyl group or to a c arbonyl O atom.