(13)C-detected NMR experiments for automatic resonance assignment of IDPs and multiple-fixing SMFT processing

Abstract

Intrinsically disordered proteins (IDPs) have recently attracted much interest, due to their role in many biological processes, including signaling and regulation mechanisms. High-dimensional (13)C direct-detected NMR experiments have proven exceptionally useful in case of IDPs, providing spectra with superior peak dispersion. Here, two such novel experiments recorded with non-uniform sampling are introduced, these are 5D HabCabCO(CA)NCO and 5D HNCO(CA)NCO. Together with the 4D (HACA)CON(CA)NCO, an extension of the previously published 3D experiments (Pantoja-Uceda and Santoro in J Biomol NMR 59:43-50, 2014. doi: 10.1007/s10858-014-9827-1), they form a set allowing for complete and reliable resonance assignment of difficult IDPs. The processing is performed with sparse multidimensional Fourier transform based on the concept of restricting (fixing) some of spectral dimensions to a priori known resonance frequencies. In our study, a multiple-fixing method was developed, that allows easy access to spectral data. The experiments were tested on a resolution-demanding alpha-synuclein sample. Due to superior peak dispersion in high-dimensional spectrum and availability of the sequential connectivities between four consecutive residues, the overwhelming majority of resonances could be assigned automatically using the TSAR program.

Fig. 1

4D (HACA)CON(CA)NCO technique. a Coherence transfer in a polypeptide chain. b Four ways of fixing the frequencies for SMFT processing, shown using different colors on a coherence transfer scheme: (1i)—green, (1ii)—magenta, (1iii)—red, (1iv)—blue (different fixing symbols are explained in the text). Highlighting the text with an appropriate color shows the fixed frequencies used during SMFT, the frequencies appearing on a cross-section are marked using frames of the same color. c–f 2D cross-sections of the 4D spectrum of alpha-synuclein protein, obtained by SMFT procedure, using (1i) fixing—panel (c), (1ii) fixing (d), (1iii) fixing (e) and (1iv) fixing (f). The cross-sections correspond to the 80K–84G protein fragment. Cross-sections corresponding to the same basis peak are placed one over another

Fig. 2

5D HabCabCO(CA)NCO technique. a Coherence transfer in a polypeptide chain. b Three ways of fixing the frequencies for SMFT processing, shown using different colors on a coherence transfer scheme: (1i)—green, (1ii)—red, (1iii)—magenta (different fixing symbols are explained in the text). Highlighting the text with an appropriate color shows the frequencies fixed during SMFT, the frequencies appearing on a cross-section are marked using frames of the same color. c–e 2D cross-sections of the 5D spectrum of alpha-synuclein protein, obtained by SMFT procedure, using (1i) fixing (c), (1ii) fixing (d), (1iii) fixing (e). The cross-sections correspond to the 80K–84G protein fragment. Cross-sections corresponding to the same basis peak are placed one over another

Fig. 3

5D HNCO(CA)NCO technique. a Coherence transfer in a polypeptide chain. b Three ways of fixing the frequencies for SMFT processing, shown using different colors on a coherence transfer scheme: (1i)—green, (1ii)—red, (1iii)—magenta (different fixing symbols are explained in the text). Highlighting the text with an appropriate color shows the frequencies fixed during SMFT, the frequencies appearing on a cross-section are marked using frames of the same color. c–e 2D cross-sections of the 5D spectrum of alpha-synuclein protein, obtained by SMFT procedure, using (1i) fixing (c), (1ii) fixing (d), (1iii) fixing (e). The cross-sections correspond to the 80K–84G protein fragment. Cross-sections corresponding to the same basis peak are placed one over another

Fig. 4

Schemes of cross-sections corresponding to a single basis peak CO_i–N_i+1–N_i–CO_i−1. For all the techniques, cross-section of each way of fixing is shown. On a right-hand side of each panel, a hypothetical plane containing peaks from all types of fixing is shown (in practice there is no such a cross-section)

Fig. 5

An example of 4D (HACA)CON(CA)NCO (1i) cross-sections overlap: three peaks instead of the expected two appear on both cross-sections (number 13 and 14). The ambiguity can be resolved by comparison with the same cross-sections calculated using (1iv) fixing, where identical peaks are expected