Fibril insulin

A prime example of a Refolding model is that of the insulin protofilament (Jimenez et al., 2002). Insulin is a polypeptide hormone composed of two peptide chains of mainly o -helical secondary structure (Fig. 3A Adams et al., 1969). Its chains (21- and 30-amino acids long) are held together by 3 disulfide bonds, 2 interchain and 1 intrachain (Sanger, 1959). These bonds remain intact in the insulin amyloid fibrils of patients with injection amyloidosis (Dische et al., 1988). Fourier transform infrared (FTIR) and circular dichroic (CD) spectroscopy indicate that a conversion to jS-structure accompanies insulin fibril formation (Bouchard et al., 2000). The fibrils also give a cross-jS diffraction pattern (Burke and Rougvie, 1972). 

Jimenez et al. (2002) proposed a molecular model for the insulin protofilament based on these data and on electron cryomicroscopy (cryo-EM) reconstructions of insulin fibrils. The fibrils show a number of twisted morphologies that seem to be alternative packings of similar protofilaments. The protofilaments have cross sections of 30x40 A. The authors suggest a complete conversion to / -structure and model the amyloid monomer as having four jS-strands (Fig. 3B). Each insulin chain contributes two of these jS-strands, and the chains align in a parallel stack, constrained by the interchain disulfide bonds. One pair of stacked /i-stran ds is curved... 

Burke, M. J., and Rougvie, M. A. (1972). Cross-beta protein structures. I. Insulin fibrils. 

Nielsen, L., S. Frokjaer, J. Brange, V.N. Uversky, and A.L. Fink, Probing the mechanism of insulin fibril formation with insulin mutants. Biochemistry, 2001.40(28) 8397-409. 

Figure 23-3 Infrared absorbance spectra of the amide regions of proteins. (A) Spectra of insulin fibrils illustrating dichroism. Solid line, electric vector parallel to fibril axis broken line, electric vector perpendicular to fibril axis. From Burke and Rougvie.24 Courtesy of Malcolm Rougvie. See also Box 29-E. (B) Fourier transform infrared (FTIR) spectra of two soluble proteins in aqueous solution obtained after subtraction of the background H20 absorption. The spectrum of myoglobin, a predominantly a-helical protein, is shown as a continuous line. That of concanavalin A, a predominantly (3-sheet containing protein, is shown as a broken line. From Haris and Chapman.14 Courtesy of Dennis Chapman.

Figure 11 Atomic Force Microscopy image of aligned PTAA coated insulin fibrils that have been transferred to a glass surface after molecular combing directly onto the surface of a PDMS stamp. The lines have been added to illustrate gaps within the PDMS stamp. The scale bar is 2 mm in length (Herland, Bjdrk et at, 2007). Copyright Wiley-VCH Verlag GmbH Co. KGaA. Reproduced with permission.

Rguie 11.14 Transmission electron micrographs of insulin fibrils formed (a) at 37°C, (b) at 80°C (xlOO 000). 

These aggregates contain nonnative structures, for example, intermolecular P-sheet (50), and can cause irreversible changes to the unfolded species, such as precipitation, aggregation, or fibrillation (18,35,43), where precipitation is the macroscopic equivalent of aggregation (40,42). Aggregation is dependent not only on the protein concentration but also on the stress caused by shaking (49,51). The structure of the aggregated proteins can be more or less well defined, and an example of well-defined structures is the formation of insulin fibrils (52,53). 

Nielsen L, Khurana R, Coats A, et al. Effect of environmental factors on the kinetics of insulin fibril formation elucidation of the molecular mechanism. Biochemistry 2001 40(20) 6036-6046. 

Page 1084 is adapted from crystallographic coordinates deposited with the Protein Data Barrk. PDB ID IPID. Brange, J., Dodson, G. G., Edwards, D. J., Holden, P. H., Whittingham, J. L., A Model of Insulin Fibrils Derived from the X-Ray Crystal Structure of a Monomeric Insulin (Despentapeptide Insulin). To be published. 

Grudzielanek, S., Smirnovas, V., Winter, R. Solvation-assisted pressure tuning of insulin fibrillation from novel aggregation pathways to biotechnological applications, J. Mol. Biol. 356 (2006) 497-509. 

An additional form of insulin, partiaiiy unfoided insuiin, can form a viscous or insoiubie precipitates known as fibriis. Shielding of hydrophobic domains is the principal driving force for the aggregation. Further studies reveaied that when the exposed hydrophobic domain (A2, A3, B11, and B15) interacts with the normally buried aiiphatic residues (A13, B6, B14, and B18) in the hexameric structure, fibrils form (Fig. 32.1) (39). Fibrils also have been studied by electron microscopy, and packing considerations in the crystal lattice explain why fibril formation is accelerated when insulin is in the monomeric state (40). Insulin fibrils do not resuspend on shaking thus, they are pharmaceutically inactive. 

Insulin fibril formation is particularly important with the advent of infusion pumps to deliver insulin. In these devices, insulin is exposed to elevated temperatures, the presence of hydrophobic surfaces, and shear forces, all factors that increase insulin s tendency to aggregate. These problems can be overcome if the insulin is prepared with phosphate buffer or other additives. Another physical stability problem associated with insulin is adsorption to tubing and other surfaces. This normally occurs if the insulin concentration is less than 5 lU/mL (0.03 mM), and it can be prevented by adding albumin to the dosage form if a dilute insulin solution must be used (34). 

Brange J, Andersen L, Laursen ED, et al. Toward understanding insulin fibrillation. J Pharm Sci 1997 86 517-525. 

Burke Ml, Rougvie MA (1972) Cross-protein structures. I. Insulin fibrils. Biochemistry 11 2435-2439 Burley SK, Petsko GA (1985) Aromatic-aromatic interaction a mechanism of protein stmeture stabilization. Science 229 23-28... 

Podesta A, Tiana G, Milan P, Manno M (2006) Early events in insulin fibrillization studied by time-l se atomic force microscopy. Biophys J 90 589-597... 

The results presented in this study provide valuable insights into the protection abilities and mechanism of the used colloid structures, containing cosolvent as a medium for long-term protein stabihty. Moreover, these findings yield valuable information regarding the effect of these colloid structures confinement on insulin fibrillation and/or aggregation. 

FIGURE 13.20 Polythiophene-based probes for amyloid and protein aggregate detection (a) chemical structures of polythiophene derivatives PONT (b) emission spectra of PTAA in free solution (c) emission spectra of PTAA solution complexed with native insulin or amyloid insulin, and (d) kinetics of insulin fibrillation. Reprinted with permission from [190]. Copyright 2005 American Chemical Society. 

The aggregation of protein fibrils in organs is believed to be the cause of several degenerative disorders including Alzheimer s and Parkinson s disease. Amyloid fibrils are structures which, regardless of the identity of the protein, share a common cross-P-sheet core structure. Several Raman spectroscopic studies have focused on insulin amyloid fibrils (14-19). We have recently used DCDR to confirm the previously reported results and perform a more detailed difference spectroscopic analysis to quantify the principal Raman spectral features associated with insulin fibrillation (20). The following results demonstrate that very similar DCDR difference spectral features are observed upon fibrillation of lysozyme. 

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