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

Interaction patterns of methoprene-tolerant and germ cell-expressed Drosophila JH receptors suggest significant differences in their functioning

Methoprene-tolerant (Met) and germ cell-expressed (Gce) proteins were shown to be juvenile hormone (JH) receptors of Drosophila melanogaster with partially redundant functions. We raised the question of where the functional differentiation of paralogs comes from. Therefore, we tested Met and Gce interaction patterns with selected partners. In this study, we showed the ability of Gce and its C-terminus (GceC) to interact with 14-3-3 in the absence of JH. In contrast, Met or Met C-terminus (MetC) interactions with 14-3-3 were not observed. We also performed a detailed structural analysis of Met/Gce interactions with the nuclear receptor fushi tarazu factor-1 (Ftz-F1) ligand-binding domain. We showed that GceC comprising an Ftz-F1-binding site and full-length protein interacts with Ftz-F1. In contrast to Gce, only MetC (not full-length Met) can interact with Ftz-F1 in the absence of JH. We propose that the described differences result from the distinct tertiary structure and accessibility of binding sites in the full-length Met/Gce. Moreover, we hypothesize that each interacting partner can force disordered MetC and GceC to change the structure in a partner-specific manner. The observed interactions seem to determine the subcellular localization of Met/Gce by forcing their translocation between the nucleus and the cytoplasm, which may affect the activity of the proteins. The presented differences between Met and Gce can be crucial for their functional differentiation during D. melanogaster development and indicate Gce as a more universal and more active paralog. It is consistent with the theory indicating gce as an ancestor gene

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