Fig 1.
Segmental isotopic labeling via Sortase-Mediated Ligation (SML).
Strategy for segmental isotopic labeling of villin 4 utilizing SML between the disordered linker and folded headpiece. LPXTG represents the requisite sortase recognition site.
Fig 2.
Schematic representation of villin 4.
The N-terminal core fragment and C-terminal headpiece domain (HP63) are joined by a mostly disordered linker (positions 721–911). The linker has N-terminal basic (pI 11.5, shown in blue) and C-terminal acidic (pI 4.1, shown in orange) regions. A predicted 11-residue PEST motif (shown in black) separates the basic and acidic regions of the linker.
Fig 3.
Effect of linker structure on SML using villin 4 IDR substrates.
(A) Model SML reaction for comparing ligation efficiency with IDR substrates containing different spacers. (B) Time course demonstrating improved ligation efficiency when spacers were included between the IDR and LPXTG motif. Reaction progress was estimated using LC-ESI-MS. (C) ESI-MS spectrum of SML reaction mixture for the substrate containing a single glycine (G) spacer following a 4 h incubation at room temperature (calculated MWs: unmodified substrate = 14062 Da, ligation product = 13296 Da, * = MeCN adducts from LC-ESI-MS mobile phase).
Fig 4.
Segmental isotopic labeling of truncated villin 4.
(A) SML strategy for producing segmentally labeled derivatives containing an IDR segment (35-residue acidic fragment) and the headpiece domain of villin 4. (B) ESI-MS spectrum of purified FH8-IDR-HP63 with selective incorporation of 15N in the HP63 segment (calculated MW assuming 100% 15N incorporation in HP63 = 20797 Da, * = MeCN adducts from LC-ESI-MS mobile phase). (C) 15N-HSQC (500 MHz, 25°C) of uniformly labeled FH8-IDR-HP63. Based on the sequence of FH8-IDR-HP63, 176 resonances originating from non-proline residues are expected and 173 were observed. (D) 15N-HSQC (500 MHz, 25°C) of segmentally labeled FH8-IDR-HP63 in which 15N labeling is restricted to HP63. Based on the sequence of the labeled segment, 61 resonances originating from non-proline residues are expected and 58 were observed.
Fig 5.
15N-HSQC NMR spectra (25°C) of segmentally labeled FH8-IDR-HP63 and IDR-HP63.
(A) Spectrum recorded at 600 MHz (1H frequency) with 256 indirect increments using a FH8-IDR-HP63 sample with the FH8-IDR portion segmentally labeled with 13C/15N isotopes. All observed resonances for the labeled FH8-IDR segment are represented by open red contours, and signals that have been segmentally assigned to the IDR linker portion are overlaid with solid red ovals. Residue-specific NMR assignments for 36 residues in the linker region (27 from the villin 4 IDR and 9 residues from the flanking TEV and sortase ligation sites) and four FH8 domain peaks (false positives from the segmental assignment) are indicated with residue numbers. The numbers for the TEV site (7 residues) are shown in grey, FH8 domain (4 residues) are shown in blue, and two residues corresponding to the SML ligation site (G114, L115) are shown in green. Seven segmentally assigned peaks for which no residue specific assignments were obtained are marked with “+”. Residue numbers shown correspond to the sequence of FH8-IDR-HP63 (see S2 Fig). To relate the assigned IDR residues to the corresponding positions in wild type villin 4, a value of 798 should be added. (B) Spectrum recorded at 500 MHz (1H frequency) with 196 indirect increments using an IDR-HP63 sample with the IDR segmentally labeled with 15N isotopes. All observed resonances for the labeled IDR segment are represented by solid red signals. Residue-specific NMR assignments for 25 residues in the linker region were unambiguously transferred from panel A (out of 27 listed on panel A). Three new resonances were observed (marked with “x”) following removal of FH8. Two of these are presumed to correspond to residues E79 and E80, which are adjacent to the newly formed N-terminal residue G78. The two residues corresponding to the SML ligation site (G114, L115) are shown in green. As in the case of FH8-IDR-HP63 (panel A), additional peaks (6 total) for which no residue specific assignments were obtained are marked with “+”.
Fig 6.
Temperature sensitivity in the 35-residue acidic segment of the villin 4 IDR.
Representative regions of 15N-HSQC NMR spectra (500 MHz, 1H frequency) for IDR-HP63 with the IDR segmentally labeled with 15N isotopes recorded at (A) 45°C (black) / 15°C (red) and (B) 45°C (black) / 25°C (red). Spectra recorded at 15°C and 45°C were re-referenced to the 1H / H2O chemical shift value (4.78 ppm) of the spectrum recorded at 25°C according to a standard approach [63]. The number of scans for the three spectra were 64 (15°C), 128 (25°C), and 192 (45°C), with 196 indirect increments (15N dimension) in all three cases. (C) Ratios of 15N-HSQC backbone amide signal intensity for assigned IDR residues at 45°C and 15°C, and (D) a corresponding data set for signal intensity ratios at 45°C and 25°C. In both panels C and D, data points for residues 81–99 are colored in light grey, and dark grey for residues 100–115. Light grey and dark grey arrows on panels A and B point to select signals representing each of these two respective groups. Data points for residues 87 (from the light grey group) and 106 (dark grey group) are excluded from this analysis due to their resonance overlap. Values shown above the brackets represent the average intensity ratio (± standard deviation) for the residues indicated.