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Two-dimensional IR spectroscopy and isotope labeling defines the pathway of amyloid formation with residue-specific resolution.

ABSTRACT: There is considerable interest in uncovering the pathway of amyloid formation because the toxic properties of amyloid likely stems from prefibril intermediates and not the fully formed fibrils. Using a recently invented method of collecting 2-dimensional infrared spectra and site-specific isotope labeling, we have measured the development of secondary structures for 6 residues during the aggregation process of the 37-residue polypeptide associated with type 2 diabetes, the human islet amyloid polypeptide (hIAPP). By monitoring the kinetics at 6 different labeled sites, we find that the peptides initially develop well-ordered structure in the region of the chain that is close to the ordered loop of the fibrils, followed by formation of the 2 parallel beta-sheets with the N-terminal beta-sheet likely forming before the C-terminal sheet. This experimental approach provides a detailed view of the aggregation pathway of hIAPP fibril formation as well as a general methodology for studying other amyloid forming proteins without the use of structure-perturbing labels.

SUBMITTER: Shim SH

PROVIDER: S-EPMC2672516 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Two-dimensional IR spectroscopy and isotope labeling defines the pathway of amyloid formation with residue-specific resolution.

Shim Sang-Hee SH Gupta Ruchi R Ling Yun L YL Strasfeld David B DB Raleigh Daniel P DP Zanni Martin T MT

Proceedings of the National Academy of Sciences of the United States of America 20090403 16

There is considerable interest in uncovering the pathway of amyloid formation because the toxic properties of amyloid likely stems from prefibril intermediates and not the fully formed fibrils. Using a recently invented method of collecting 2-dimensional infrared spectra and site-specific isotope labeling, we have measured the development of secondary structures for 6 residues during the aggregation process of the 37-residue polypeptide associated with type 2 diabetes, the human islet amyloid po ...[more]

PMID: 19346479

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Project description:Amyloid formation normally exhibits a lag phase followed by a growth phase, which leads to amyloid fibrils. Characterization of the species populated during the lag phase is experimentally challenging, but is critical since the most toxic entities may be pre-fibrillar species. p-Cyanophenylalanine (F(C[triple bond]N)) fluorescence is used to probe the nature of lag-phase species populated during the formation of amyloid by human islet amyloid polypeptide. The polypeptide contains two phenylalanines at positions 15 and 23 and a single tyrosine located at the C-terminus. Each aromatic residue was separately replaced by F(C[triple bond]N). The substitutions do not perturb amyloid formation relative to wild-type islet amyloid polypeptide as detected using thioflavin T fluorescence and electron microscopy. F(C[triple bond]N) fluorescence is high when the cyano group is hydrogen bonded and low when it is not. It can also be quenched via Förster resonance energy transfer to tyrosine. Fluorescence intensity was monitored in real time and revealed that all three positions remained exposed to solvent during the lag phase but less exposed than unstructured model peptides. The time course of amyloid formation as monitored by thioflavin T fluorescence and F(C[triple bond]N) fluorescence is virtually identical. Fluorescence quenching experiments confirmed that each residue remains exposed during the lag phase. These results place significant constraints on the nature of intermediates that are populated during the lag phase and indicate that significant sequestering of the aromatic side chains does not occur until beta-structure sufficient to bind thioflavin T has developed. Seeding studies and analysis of maximum rates confirm that sequestering of the cyano groups occurs concomitantly with the development of thioflavin T binding capability. Overall, the process of amyloid formation and growth appears to be remarkably homogenous in terms of side-chain ordering. F(C[triple bond]N) also provides information about fibril structure. Fluorescence emission measurements, infrared measurements, and quenching studies indicate that the aromatic residues are differentially exposed in the fibril state with Phe15 being the most exposed. F(C[triple bond]N) is readily accommodated into proteins; thus, the approach should be broadly applicable.

Dataset Information

Two-dimensional IR spectroscopy and isotope labeling defines the pathway of amyloid formation with residue-specific resolution.

Publications

Two-dimensional IR spectroscopy and isotope labeling defines the pathway of amyloid formation with residue-specific resolution.

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