Abstract
The interaction between many proteins and hydrophobic functionalized surfaces is known to induce β-sheet and amyloid fibril formation. In particular, insulin has served as a model peptide to understand such fibrillation, but the early stages of insulin misfolding and the influence of the surface have not been followed in detail under the acidic conditions relevant to the synthesis and purification of insulin. Here we compare the adsorption of human insulin on a hydrophobic (-CH3-terminated) silane self-assembled monolayer to a hydrophilic (-NH3(+)-terminated) layer. We monitor the secondary structure of insulin with Fourier transform infrared attenuated total reflection and side-chain orientation with sum frequency spectroscopy. Adsorbed insulin retains a close-to-native secondary structure on both hydrophobic and hydrophilic surfaces for extended periods at room temperature and converts to a β-sheet-rich structure only at elevated temperature. We propose that the known acid stabilization of human insulin and the protection of the aggregation-prone hydrophobic domains on the insulin monomer by adsorption on the hydrophobic surface work together to inhibit fibril formation at room temperature.
Highlights
The formation of insoluble amyloid fibrils is responsible for the deterioration of valuable therapeutic proteins during production, storage, and injection and lies at the origin of several degenerative diseases such as Alzheimer’s and type II diabetes.[1,2] In these areas, insulin has played a pivotal role as model system in understanding fibril formation and inhibition.[3−5] Insulin is a small, globular protein of 51 amino acids arranged in two chains, with a mainly α-helical structure stabilized by three disulfide bonds (Figure 1)
We propose that the known acid stabilization of human insulin and the protection of the aggregation-prone hydrophobic domains on the insulin monomer by adsorption on the hydrophobic surface work together to inhibit fibril formation at room temperature
Insulin has played a pivotal role as model system in understanding fibril formation and inhibition.[3−5] Insulin is a small, globular protein of 51 amino acids arranged in two chains, with a mainly α-helical structure stabilized by three disulfide bonds (Figure 1)
Summary
The formation of insoluble amyloid fibrils is responsible for the deterioration of valuable therapeutic proteins during production, storage, and injection and lies at the origin of several degenerative diseases such as Alzheimer’s and type II diabetes.[1,2] In these areas, insulin has played a pivotal role as model system in understanding fibril formation and inhibition.[3−5] Insulin is a small, globular protein of 51 amino acids arranged in two chains, with a mainly α-helical structure stabilized by three disulfide bonds (Figure 1). Monomers can form dimers to shield a hydrophobic domain on the monomer surface from the solvent. At high concentrations and in the presence of zinc ions, dimers can further self-assemble to hexamers, which protect insulin from fibrillation in vivo. The insulin monomer is the physiologically active species and is the key species in forming amyloids in vitro. In solution at low pH, partially unfolded monomers have been identified as being on the pathway to fibril growth, and their structure has been unravelled.[6,7]
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