Computational protocol: Two mutations G335D and Q343R within the amyloidogenic core region of TDP 43 influence its aggregation and inclusion formation

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Protocol publication

[…] NMR data acquirement and processing, and structure calculation were performed as reported previously. The GB1-fused peptides were applied to enhance peptide solubility and obtain high-quality spectra. The 15N/13C-labeled GB1-TDP(311–360) mutant (G335D or Q343R) was dissolved in 20 mM phosphate (pH 6.5), 50 mM NaCl and 8% D2O for NMR data acquirements. All NMR spectra were recorded at 25 °C on a Bruker Avance 600-MHz spectrometer equipped with a TCI CryoProbe (Bruker Biospin). The backbone and side-chain chemical-shift assignments were obtained from the spectra of HNCO, HNHA, HNCACB, CBCA(CO)NH, CC(CO)NH, and HCCH-TOCSY. NOE restraints for structure calculations were obtained from 15N- and 13C-edited NOESY spectra. The NMR data were processed by using NMRPipe and analyzed with SPARKY. The backbone dihedral restraints were derived from TALOS program. The structures were calculated using ARIA2.0 and CNS program, assessed by PROCHECK and displayed by MOLMOL. The structural calculation was performed for 9 cycles and a total of 200 structures were finally obtained. Ten lowest-energy structures were selected and displayed. […]

Pipeline specifications

Software tools Sparky, PROCHECK, MOLMOL
Applications NMR-based proteomics analysis, Protein structure analysis
Diseases TDP-43 Proteinopathies, Frontotemporal Dementia