High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins

Four novel 5D (HACA(N)CONH, HNCOCACB, (HACA)CON(CA)CONH, (H)NCO(NCA)CONH), and one 6D ((H)NCO(N)CACONH) NMR pulse sequences are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in indirectly detected dimensions. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel pulse sequences were successfully tested using δ subunit (20 kDa) of Bacillus subtilis RNA polymerase that has an 81-amino acid disordered part containing various repetitive sequences

Application of LDA to the resonance list transfer from BMRB Entry 6968 to the experimental spectrum of <i>α</i>-synuclein.

A) A fragment of the 15N-HSQC spectrum with peaks marked with dots. Peaks from the BMRB list are marked with crosses. Labels indicate the corresponding residue name. B) Correct transfer of assignment from BMRB to the experimental peak list (unambiguous assignments shown with solid arrows, more ambiguous ones—with dashed arrows). The experimental and BMRB peaks corresponding to the successors of the residues of the same amino acid type are marked in the same color. C) Hα, Hβ, Cα and Cβ chemical shifts of the residues preceding the residues shown in panels A) and B). For experimental peaks, these chemical shifts were obtained from the 4D HabCab(CO)NH experiment and then analyzed with LDA, enabling assignment transfer. Color coding as on panel B). The full spectrum with both peak-lists is available at 10.5281/zenodo.7032142.</p

Results of LDA of the chemical shifts of <i>α</i>-synuclein: A) subset (i) (H^N, N, CO and C_<i>α</i>); B) subset (ii) (C_<i>α</i>, C_<i>β</i>H_<i>α</i> and H_<i>β</i>); and C) subset (iii) (H^N, N, CO, C_<i>α</i>, C_<i>β</i>, H_<i>α</i> and H_<i>β</i>).

The vertical axis shows the spin system numbers, the horizontal axis the amino acid types (LDA classes). Spin system numbers are colored according to their true amino acid type. Marker sizes indicate the probability, according to LDA, that the spin system in question belongs to a given class.</p

LDA vs. other classification methods.

We analyzed the performance of LDA, Quadratic Discriminant Analysis (QDA), K-Nearest Neighbours (KNN) and Support Vector Machines (SVM) to pick the best classification method for our research. We looked at specific performance parameters such as accuracy, sensitivity, specificity and consistency. (PDF)</p