Two-dimensional IR spectroscopy and segmental 13C labeling reveals the domain structure of human  D-crystallin amyloid fibrils

TitleTwo-dimensional IR spectroscopy and segmental 13C labeling reveals the domain structure of human  D-crystallin amyloid fibrils
Publication TypeJournal Article
Year of Publication2012
AuthorsMoran SD, Woys AM, Buchanan LE, Bixby E., Decatur SM, Zanni MT
Secondary TitleProceedings of the National Academy of Sciences
Volume109
Issue9
Pagination3329-3334
Date Published02/2012
Abstract

The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly 13C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins.

URLhttp://www.ncbi.nlm.nih.gov/pubmed/22328156
DOI10.1073/pnas.1117704109.
Short TitleProceedings of the National Academy of Sciences