A Sequential Assignment Procedure for Proteins that have Intermediate Line Widths in MAS NMR Spectra: Amyloid Fibrils of Human CA150.WW2

Abstract

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.

Three-Dimensional Structure of CAP-Gly Domain of Mammalian Dynactin Determined by Magic Angle Spinning NMR Spectroscopy: Conformational Plasticity and Interactions with End-Binding Protein EB1

“…This result is not surprising in light of the following: i) as discussed above, the X-ray structures represent complexes of CAP-Gly with EB1 or ZnCLIP, and these complexes are heterotetrameric or heterooctameric species formed upon binding of CAP-Gly to its protein partners; ii) the X-ray data were collected at cryogenic temperatures, in contrast to the MAS NMR spectra acquired at temperatures between −2 °C and 4 °C (arguably, the NMR temperatures are closer to physiological conditions), and iii) our recent work indicates that loop regions of CAP-Gly are flexible on ns-us timescales, as revealed by lower H-C dipolar order parameters compared to the values observed in the β-sheet and α-helical regions of the protein. 28 We also refer the reader to the previous reports on microcrystalline proteins from our group, 14; 31; 32; 33; 34; 35; 36 and from others 37; 38 where similar observations were made for torsion angles in loops vs. rigid secondary structure elements.…”

Methods for structural characterization of prefibrillar intermediates and amyloid fibrils

“…In ss‐NMR the 1 H signals are not observable, thus 15 N and 13 C labeling is necessary, which can be difficult to obtain for larger recombinant proteins, but the labeling has advantages. Dividing the labeling can be utilized in the segmented assignment strategy [57] and labeling can also be exploited to distinguishing between parallel and anti‐parallel arrangement of the β‐strands through single site labeling [58,59]. Distances between single sites of 4.7–5 Å can only be fulfilled in the parallel in‐register β‐strand arrangement, whereas longer distances must be combined with additional constraints to separate off‐register and anti‐parallel arrangements.…”

NMR Studies of Eye Lens Crystallins

“…Mixed samples of 13 C-only and 15 N-only labeled proteins have been used to obtain intermolecular contacts 34,35 via the transferred-echo double-resonance (TEDOR) experiment. 36 A 3-D HNC version of TEDOR has been used in conjunction with sparse 13 C labeling and partial deuteration to generate amide 1 H and 15 N assignments in a demanding system with intermediate linewidths owing to molecular motion 37 The crystallin proteins have served as a test system for the development of new methodology for measuring and utilizing long-range distance constraints, in the form of both residual dipolar coupling (RDC)-based molecular fragment replacement approach 38 -41 and the use of nuclear overhauser effects (NOEs) from distant methyl groups in 13 C methyllabeled samples. 42 Chemical shift perturbation analysis using 1 H-15 N heteronuclear single quantum correlation (HSQC) 43 spectra of a previously assigned protein is an indispensable tool for mapping protein-ligand and protein-protein interactions and has been used extensively to study binding of crystallins to each other and to other molecules in solution.…”