High-resolution powder X-ray data reveal the T₆hexameric form of bovine insulin

Abstract

A series of bovine insulin samples were obtained as 14 polycrystalline precipitates at room temperature in the pH range 5.0-7.6. High-resolution powder X-ray diffraction data were collected to reveal the T6 hexameric insulin form. Sample homogeneity and reproducibility were verified by additional synchrotron measurements using an area detector. Pawley analyses of the powder patterns displayed pH- and radiation-induced anisotropic lattice modifications. The pronounced anisotropic lattice variations observed for T6 insulin were exploited in a 14-data-set Rietveld refinement to obtain an average crystal structure over the pH range investigated. Only the protein atoms of the known structure with PDB code 2a3g were employed in our starting model. A novel approach for refining protein structures using powder diffraction data is presented. In this approach, each amino acid is represented by a flexible rigid body (FRB). The FRB model requires a significantly smaller number of refinable parameters and restraints than a fully free-atom refinement. A total of 1542 stereochemical restraints were imposed in order to refine the positions of 800 protein atoms, two Zn atoms and 44 water molecules in the asymmetric unit using experimental data in the resolution range 18.2-2.7 Å for all profiles.

In Quest for Improved Drugs against Diabetes: The Added Value of X-ray Powder Diffraction Methods

“…Powder X-ray diffraction data revealed the T 6 hexameric insulin form (space group R3; unit-cell parameters a = 82.5951 (9) Å, c = 33.6089 (3) Å for the sample crystallized at pH 5.0) in agreement with the high-resolution structure of HI, identified earlier by single crystal experiments [69,70].…”

Tyrosine- and tryptophan-coated gold nanoparticles inhibit amyloid aggregation of insulin

“…To further clarify the possible binding partners of a tyrosine residue when it interacts with insulin, we looked at the available crystal structures of insulin dimers (PDB IDs: 2A3G, 2INS, 2ZP6, and 4IDW) (Smith et al 1982(Smith et al , 2005Margiolaki et al 2013). We focused on the interface of insulin-insulin dimers and analyzed the involvement of tyrosine residues in intermolecular interactions.…”

“…Visualizations were performed using DS4. For analysis of crystal structure of bovine insulin multimers, we obtained four multimeric bovine insulin crystal structures from PDB sources (two hetero 12-mers; PDB IDs: 2A3G and 2INS, and two hetero 6-mers; PDB IDs: 2ZP6, 4IDW) (Smith et al 1982(Smith et al , 2005Margiolaki et al 2013). The interface of insulin structures was analyzed to identify the key amino acids involved in multimer formation using DS4.…”

Novel crystalline phase and first-order phase transitions of human insulin complexed with two distinct phenol derivatives

“…Although there is clearly still much scope for improvement of the phasing and structuresolution methods, the molecular-replacement and structurerefinement methods can already be regarded as sufficiently mature for use by adventurous protein crystallographers. The application of powder diffraction to proteins has in fact been proved to be an effective method for high-throughput polymorph identification and crystal screening (Norrman et al, 2006;Collings et al, 2010;Karavassili et al, 2012) as well as to be suitable for structure solution (Margiolaki et al, 2007) and refinement (Margiolaki et al, 2013).…”