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A Sequential Assignment Procedure for Proteins that have Intermediate Line Widths in MAS NMR Spectra: Amyloid Fibrils of Human CA150.WW2
Author(s) -
Becker Johanna,
Ferguson Neil,
Flinders Jeremy,
van Rossum BarthJan,
Fersht Alan R.,
Oschkinat Hartmut
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700706
Subject(s) - fibril , chemistry , crystallography , nuclear magnetic resonance spectroscopy , chemical shift , nuclear magnetic resonance , biophysics , stereochemistry , biochemistry , biology , physics
The second WW domain (WW2) of CA150, a human transcriptional activator, forms amyloid fibrils in vitro under physiological conditions. Based on experimental constraints from MAS NMR spectroscopy experiments, alanine scanning and electron microscopy, a structural model of CA150.WW2 amyloid fibrils was calculated earlier. Here, the assignment strategy is presented and suggested as a general approach for proteins that show intermediate line width. The 13 C, 13 C correlation experiments were recorded on fully or partially 13 C‐labelled fibrils. The earlier 13 C assignment (26 residues) was extended to 34 of the 40 residues by direct 13 C‐excitation experiments by using a deuterated sample that showed strongly improved line width. A 3D HNC‐TEDOR (transferred‐echo double‐resonance) experiment with deuterated CA150.WW2 fibrils yielded 14 amide nitrogen and proton resonance assignments. The obtained chemical shifts were compared with the chemical shifts determined with the natively folded WW domain. TALOS (Torsion angle likelihood obtained from shift and sequence similarity) predictions confirmed that, under physiological conditions, the fibrillar form of CA150.WW2 adopts a significantly different β structure than the native WW‐domain fold.