ABSTRACT
Pancreatic amyloid plaques formed by the pancreatic islet amyloid polypeptide (IAPP) are present in more than 95% of type II diabetes mellitus patients, and their abundance correlates with the severity of the disease. IAPP is currently considered the most amyloidogenic peptide known, but the molecular bases of its aggregation are still incompletely understood. Detailed characterization of the mechanisms of amyloid formation requires large quantities of pure material. Thus, availability of recombinant IAPP in sufficient amounts for such studies constitutes an important step toward elucidation of the mechanisms of amyloidogenicity. Here, we report, for the first time, the successful expression, purification and characterization of the amyloidogenicity and cytotoxicity of recombinant human mature IAPP. This approach is likely to be useful for the production of other amyloidogenic peptides or proteins that are difficult to obtain by chemical synthesis.
Subject(s)
Amyloid/chemistry , Glycine/analogs & derivatives , Amino Acid Sequence , Amyloid/metabolism , Base Sequence , Cells, Cultured , Cloning, Molecular , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Glycine/chemistry , Humans , Islet Amyloid Polypeptide , Microscopy, Electron , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , Peptides/chemistry , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Recombinant Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Time FactorsABSTRACT
Because of their limited size and complexity, de novo designed proteins are particularly useful for the detailed investigation of folding thermodynamics and mechanisms. Here, we describe how subtle changes in the hydrophobic core of a model three-helix bundle protein (GM-0) alter its folding energetics. To explore the folding tolerance of GM-0 toward amino acid sequence variability, two mutant proteins (GM-1 and GM-2) were generated. In the mutants, cavities were created in the hydrophobic core of the protein by either singly (GM-1; L35A variant) or doubly (GM-2; L35A/I39A variant) replacing large hydrophobic side chains by smaller Ala residues. The folding of GM-0 is characterized by two partially folded intermediate states exhibiting characteristics of molten globules, as evidenced by pressure-unfolding and pressure-assisted cold denaturation experiments. In contrast, the folding energetics of both mutants, GM-1 and GM-2, exhibit only one folding intermediate. Our results support the view that simple but biologically important folding motifs such as the three-helix bundle can reveal complex folding plasticity, and they point to the role of hydrophobic packing as a determinant of the overall stability and folding thermodynamic of the helix bundle.