A sequential assignment procedure for proteins that have intermediate line widths in MAS NMR spectra: amyloid fibrils of human CA150.WW2.
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 beta structure than the native WW-domain fold.
Becker
Johanna
J
Structural Biology, Leipniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
Ferguson
Neil
N
Flinders
Jeremy
J
van Rossum
Barth-Jan
BJ
Fersht
Alan R
AR
Oschkinat
Hartmut
H
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D035602:Transcriptional Elongation Factors
D000682:Amyloid; C108440:TCERG1 protein, human; D015534:Trans-Activators; D035602:Transcriptional Elongation Factors