Proinsulin: Crystallization and Preliminary X-Ray Diffraction Studies

Abstract

Abstract. Bovine proinsulin has been crystallized under a variety of conditions at both neutral and acid pH. Microtechniques were employed with sample weights of about 200 jig and volumes of 5-20 1AL The crystalline preparations all differ from each other morphologically.X-ray photographs of one form, tetragonal bipyramids grown at pH 3 with added ammonium sulphate solution, established the space group P41212 (or its enlantiomorph P43212). The cell dimensions are a = 50.8 i 0.2 A, c = 148.0 + 0.4 A. The asymmetric unit in this form is a dimer of proinsulin which is also the dominant species in solution at this pH.Introduction. Following the studies of Steiner arid his co-w%-orkersl2 who established that insulin is synthesized via a single chain precursor protein, proinsulin has been isolated and characterized from bovine,3-6 porcine,' cod,8 anglerfish,9 and human10"1 tissues. The connecting peptide segment of proinsulin which links the carboxyl-terminal group of the insulin B chain to the amino-terminal group of the insulin A chain is cleaved in vivo by proteolysis. The lengths and sequences of the C-peptide vary markedly between species. In bovine proinsulin, where the connecting peptide is 30 amino acid residues long," the proinsulin molecule with a single 81 residue chain has a molecular weight of 868.5.

Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells.

“…Thus, we propose that proinsulin enters IGs in the soluble phase in pancreatic B-cells. These data are entirely consistent with earlier biophysical studies indicating that randomly coiled C-peptides interfere with close-packing of soluble proinsulin hexamers (Steiner, 1973;Weiss et al, 1990), thereby rendering proinsulin incapable of forming large, insoluble complexes in an aqueous environment above pH 3 (Fullerton et al, 1970;Grant et al, 1972). While we do not preclude the possibility that some proteins in some cell types might undergo complex Figure 9.…”

Conservation of chain reversal regions in proteins

“…Only one helix is predicted for bovine at C6-12, whereas helices at C1-12 and C21-27 are predicted for human C-peptide. The x-ray crystallographic studies of proinsulin is still at a preliminary stage (Fullerton et al, 1970). Although earlier CD studies showed that the C-peptide region of proinsulin to be essentially devoid of secondary structure (Frank and Veros, 1968), recent laser Raman spectroscopy studies (Yu et al, 1972) revealed a considerable fraction of a-helical structure.…”

Molecular dynamics and circular dichroism studies of human and rat C-peptides

“…Crystallographic data in low resolution showed that the tertiary structure of the insulin moiety of bovine proinsulin is very similar to the crystal structure of the insulin molecule and that bovine C-peptide would exhibit two short segments of a-helix (alpha helix) [17,19]. Circular dichroism (CD) spectra of bovine proinsulin described the occurrence of six residues in a-helix and nine residues in b (beta) structure in the C-peptide moiety [17].…”

“…Initial information on C-peptide structure came from studies of bovine proinsulin in solution [17,18] and in crystal form, by X-ray diffraction [19]. Crystallographic data in low resolution showed that the tertiary structure of the insulin moiety of bovine proinsulin is very similar to the crystal structure of the insulin molecule and that bovine C-peptide would exhibit two short segments of a-helix (alpha helix) [17,19].…”