PDBsum entry 1guj

Hormone

PDB id: 1guj

Contents

Protein chains

21 a.a.

30 a.a. *

Ligands

SO4 ×2

Waters ×127

* Residue conservation analysis

PDB id:

1guj

Name:

Hormone

Title:

Insulin at ph 2: structural analysis of the conditions promoting insulin fibre formation.

Structure:

Insulin. Chain: a, c. Engineered: yes. Insulin. Chain: b, d. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: saccharomyces cerevisiae. Expression_system_taxid: 4932. Expression_system_taxid: 4932

Biol. unit:

Hetero-Dimer (from PDB file)

Resolution:

1.62Å R-factor: 0.172 R-free: 0.207

Authors:

J.L.Whittingham,D.J.Scott,K.Chance,A.Wilson,J.Finch,J.Brange, G.G.Dodson

Key ref:

J.L.Whittingham et al. (2002). Insulin at pH 2: structural analysis of the conditions promoting insulin fibre formation. J Mol Biol, 318, 479-490. PubMed id: 12051853 DOI: 10.1016/S0022-2836(02)00021-9

Date:

28-Jan-02 Release date: 08-Mar-02

Protein chains

P01308  (INS_HUMAN) -  Insulin from Homo sapiens

Seq: 110 a.a.

Struc: 21 a.a.

Protein chains

P01308  (INS_HUMAN) -  Insulin from Homo sapiens

Seq: 110 a.a.

Struc: 30 a.a.

DOI no: 10.1016/S0022-2836(02)00021-9

J Mol Biol 318:479-490 (2002)

PubMed id: 12051853

Insulin at pH 2: structural analysis of the conditions promoting insulin fibre formation.

J.L.Whittingham, D.J.Scott, K.Chance, A.Wilson, J.Finch, J.Brange, G.Guy Dodson.

ABSTRACT

When insulin solutions are subjected to acid, heat and agitation, the normal pattern of insulin assembly (dimers-->tetramers-->hexamers) is disrupted; the molecule undergoes conformational changes allowing it to follow an alternative aggregation pathway (via a monomeric species) leading to the formation of insoluble amyloid fibres. To investigate the effect of acid pH on the conformation and aggregation state of the protein, the crystal structure of human insulin at pH 2.1 has been determined to 1.6 A resolution. The structure reveals that the native fold is maintained at low pH, and that the molecule is still capable of forming dimers similar to those found in hexameric insulin structures at higher pH. Sulphate ions are incorporated into the molecule and the crystal lattice where they neutralise positive charges on the protein, stabilising its structure and facilitating crystallisation. The sulphate interactions are associated with local deformations in the protein, which may indicate that the structure is more plastic at low pH. Transmission electron microscopy analysis of insulin fibres reveals that the appearance of the fibres is greatly influenced by the type of acid employed. Sulphuric acid produces distinctive highly bunched, truncated fibres, suggesting that the sulphate ions have a sophisticated role to play in fibre formation, rather as they do in the crystal structure. Analytical ultracentrifugation studies show that in the absence of heating, insulin is predominantly dimeric in mineral acids, whereas in acetic acid the equilibrium is shifted towards the monomer. Hence, the effect of acid on the aggregation state of insulin is also complex. These results suggest that acid conditions increase the susceptibility of the molecule to conformational change and dissociation, and enhance the rate of fibrillation by providing a charged environment in which the attractive forces between the protein molecules is increased.

Selected figure(s)

Figure 2.
Figure 2. Negative staining transmission electron micrographs of human insulin fibres prepared under a variety of conditions. Unless otherwise stated, all solutions contained 5 mg/ml human insulin. The conditions used were: (a) 0.004 M H[2]SO[4] (pH 2.1), 70 °C; (b) 0.01 M HCl (pH 2.0), 70 °C; (c) H[3]PO[4] (pH 2.0), 90 °C; (d) 0.5 M citric acid (pH 1.9), 90 °C; (e) 20 mg/ml bovine insulin in 8.3 M acetic acid (pH 1.6), 37 °C; (f) 20% acetic acid (pH 2.0) and 0.004 M Na[2]SO[4], 90 °C.

Figure 3.
Figure 3. Stereo view illustrations showing comparisons of the pH 2 insulin dimer (green) with the dimer of (a) the T[6] insulin hexamer (blue),[20.] and (b) the B9 Ser->Glu mutant insulin dimer (red). [9.] Only C^a atoms and the sulphate ions (ball and stick representation in the low pH structure) are shown. The dimers were overlapped using an alignment on residues B9-B19 and D9-D19, which constitute the B chain a-helices in each dimer. This Figure was made using MOLSCRIPT.[36.]

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 318, 479-490) copyright 2002.

Figures were selected by an automated process.