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Six- and seven-dimensional experiments by combination of sparse random sampling and projection spectroscopy dedicated for backbone resonance assignment of intrinsically disordered proteins.

Żerko S, Koźmiński W - J. Biomol. NMR (2015)

Bottom Line: The resulted data sets could be processed as five-dimensional data using existing software.The novel experiments were successfully tested using 1 mM sample of α-synuclein on 600 and 800 MHz NMR spectrometers equipped with standard room temperature probes.The experiments allowed backbone assignment from a 1-day acquisition.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, 02089, Warsaw, Poland.

ABSTRACT
Two novel six- and seven-dimensional NMR experiments are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in four indirectly detected dimensions and synchronous sampling in the additional dimensions using projection spectroscopy principle. The resulted data sets could be processed as five-dimensional data using existing software. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel experiments were successfully tested using 1 mM sample of α-synuclein on 600 and 800 MHz NMR spectrometers equipped with standard room temperature probes. The experiments allowed backbone assignment from a 1-day acquisition.

No MeSH data available.


Eight 2D cross-sections from 7D HNCO(N)CACONH experiment (++++ , ++−, +−+− and +− + correspond to the signs in the co-addition of obtained raw data sets) corresponding to the HNCO peak positions of G36CO-V37N and G73CO-V74N signals showing CA i−1COi−1(NiHiN) sequential peaks. While coevolution of HN leads to a more robust connectivity adding of CA frequency further differentiate signal positions due to the difference in CA chemical shifts of V37 and V74. Note that y-axis at the cross-sections from the 7D HNCO(N)CACONH spectrum is labelled with 15N chemical shift scale, however, the peak frequencies in this dimension are given by the Eq. (4)
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Fig7: Eight 2D cross-sections from 7D HNCO(N)CACONH experiment (++++ , ++−, +−+− and +− + correspond to the signs in the co-addition of obtained raw data sets) corresponding to the HNCO peak positions of G36CO-V37N and G73CO-V74N signals showing CA i−1COi−1(NiHiN) sequential peaks. While coevolution of HN leads to a more robust connectivity adding of CA frequency further differentiate signal positions due to the difference in CA chemical shifts of V37 and V74. Note that y-axis at the cross-sections from the 7D HNCO(N)CACONH spectrum is labelled with 15N chemical shift scale, however, the peak frequencies in this dimension are given by the Eq. (4)

Mentions: Performing sequential assignment of α-synuclein due to its disordered nature can be regarded as a challenging task. Despite α-synuclein’s moderate size (140 a.a.), full potential of the “HNCO-HNCO” strategy can be seen, for example, in the case of G36CO-V37N and G73CO-V74N signals which are hardly distinguishable using COi−1Ni connectivity (see Fig. 5). Application of 6D experiment quickly resolves such ambiguity without using any additional experiment as the HN chemical shifts of aforementioned residues vary by almost 0.2 ppm (see Fig. 6). Moreover, peak positions in the resulting spectra are further differentiated when seventh dimension (CAi−1) is introduced (see Fig. 7). In addition to a better signal dispersion CA chemical shift provide partial information about residue type.Fig. 5


Six- and seven-dimensional experiments by combination of sparse random sampling and projection spectroscopy dedicated for backbone resonance assignment of intrinsically disordered proteins.

Żerko S, Koźmiński W - J. Biomol. NMR (2015)

Eight 2D cross-sections from 7D HNCO(N)CACONH experiment (++++ , ++−, +−+− and +− + correspond to the signs in the co-addition of obtained raw data sets) corresponding to the HNCO peak positions of G36CO-V37N and G73CO-V74N signals showing CA i−1COi−1(NiHiN) sequential peaks. While coevolution of HN leads to a more robust connectivity adding of CA frequency further differentiate signal positions due to the difference in CA chemical shifts of V37 and V74. Note that y-axis at the cross-sections from the 7D HNCO(N)CACONH spectrum is labelled with 15N chemical shift scale, however, the peak frequencies in this dimension are given by the Eq. (4)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4642589&req=5

Fig7: Eight 2D cross-sections from 7D HNCO(N)CACONH experiment (++++ , ++−, +−+− and +− + correspond to the signs in the co-addition of obtained raw data sets) corresponding to the HNCO peak positions of G36CO-V37N and G73CO-V74N signals showing CA i−1COi−1(NiHiN) sequential peaks. While coevolution of HN leads to a more robust connectivity adding of CA frequency further differentiate signal positions due to the difference in CA chemical shifts of V37 and V74. Note that y-axis at the cross-sections from the 7D HNCO(N)CACONH spectrum is labelled with 15N chemical shift scale, however, the peak frequencies in this dimension are given by the Eq. (4)
Mentions: Performing sequential assignment of α-synuclein due to its disordered nature can be regarded as a challenging task. Despite α-synuclein’s moderate size (140 a.a.), full potential of the “HNCO-HNCO” strategy can be seen, for example, in the case of G36CO-V37N and G73CO-V74N signals which are hardly distinguishable using COi−1Ni connectivity (see Fig. 5). Application of 6D experiment quickly resolves such ambiguity without using any additional experiment as the HN chemical shifts of aforementioned residues vary by almost 0.2 ppm (see Fig. 6). Moreover, peak positions in the resulting spectra are further differentiated when seventh dimension (CAi−1) is introduced (see Fig. 7). In addition to a better signal dispersion CA chemical shift provide partial information about residue type.Fig. 5

Bottom Line: The resulted data sets could be processed as five-dimensional data using existing software.The novel experiments were successfully tested using 1 mM sample of α-synuclein on 600 and 800 MHz NMR spectrometers equipped with standard room temperature probes.The experiments allowed backbone assignment from a 1-day acquisition.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Żwirki i Wigury 101, 02089, Warsaw, Poland.

ABSTRACT
Two novel six- and seven-dimensional NMR experiments are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in four indirectly detected dimensions and synchronous sampling in the additional dimensions using projection spectroscopy principle. The resulted data sets could be processed as five-dimensional data using existing software. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel experiments were successfully tested using 1 mM sample of α-synuclein on 600 and 800 MHz NMR spectrometers equipped with standard room temperature probes. The experiments allowed backbone assignment from a 1-day acquisition.

No MeSH data available.