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Analysis of the left-handed β-helical fold toward understanding of amyloid fibril structure


The left-handed parallel β-helix (LβH) is a structurally repetitive, highly regular, symmetrical fold, formed by coiling of elongated β-sheets into helical `rungs'. This canonical fold has recently received interest as a possible solution to the fibril structure of amyloid. However, sequence and structural requirements, and stability and folding of the LβH are not fully understood. To analyze this protein fold and investigate the possibility of its involvement in amyloid formations, combined approaches using computational and experimental methods were applied. (1) Sequence characteristics of the LβH were determined by analyzing known structures. A genome-wide sequence analysis was undertaken to determine the prevalence of this unique protein fold across the genomes. Molecular dynamics (MD) simulations were used to demonstrate the stabilizing effect of successive rungs and the hydrophobic core of the LβH. (2) An in-vivo folding assay using a known LβH protein was developed to investigate the residue tolerance and effect of mutations at structurally critical regions of the β-helical structure. Tyrosine fluorescence spectroscopy was also used to study the thermodynamic stability of the LβH protein. It showed that the β-helix could tolerate non-hydrophobic residues at interior positions, and proline residues in the β-helical domain were shown to be important, but not critical, in folding of the β-helix. A recombinant protein consisting of the LβH with a prion fragment was designed, constructed and tested. The results suggest that the amyloid prone PrP fragment may fold into a β-helix. (3) Modeling of possible monomeric subunits of the insulin amyloid fibril was conducted using β-solenoid folds, namely the β-helix and β-roll. While both the β-helix and β-roll models agreed with currently available biophysical data from misfolded insulin, MD simulations showed that the β-roll model was relatively more stable. Insulin monomeric subunit models were incorporated into plausible models of insulin fibrils. Simulated X-ray fiber diffraction patterns of these models demonstrated that the model fibers constructed from a β-solenoid subunit provided the reasonable fit to available experimental diffraction patterns.

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