Fig 1.
Unidirectional truncation of UDE gene.
(A) The location of designed starting points along the UDE sequence indicating the predicted disordered segments and the conserved motifs. (B) The truncated UDE gene fragments generated by N-terminal truncation were fused in-frame with the biotin acceptor peptide and out-of-frame with hexahistidine tag, while fragments produced by C-terminal truncation were fused in-frame with hexahistidine tag and out-of-frame with BAP.
Fig 2.
Screening expression level and solubility of UDE truncation libraries.
(A) Size fractionation of UDE fragments generated by unidirectional truncation on agarose gel. In the lanes next to the DNA ladders is the vector with total length UDE gene at higher position while the empty vector is at a lower position. N1–N3 and C1–C3 marked samples show by the exonuclease III truncation generated UDE constructs. (B) Assessment of UDE sublibraries size and diversity by PCR screen. (C) Separation of purified protein fractions on Ni2+-NTA resin from N-terminal (upper panels) and C-terminal (bottom panels) libraries on SDS-PAGE.
Table 1.
The results of analytical gel filtration and the determination of the oligomeric status of each truncated fragments.
Fig 3.
Alignment and scale-up of selected UDE truncated fragments.
(A) The restricted nine UDE truncated fragments from the identified protein clusters that were chosen for scale-up. Arrows show the expression compatible boundaries compared to the previously designated conserved motifs determined by the alignment of UDE homologues sequences. (B) The optimized expression of the nine UDE constructs in E. coli BL21 cells before (-) and after (+) IPTG induction. (C) Purification of recombinant UDE constructs by Ni2+-affinity chromathography. Gel slice images show the supernatant (termed as “Sup” on the figure) of cell lysis and the 300 mM imidazole elution (termed as “Elu” on the figure) fractions for each constructs. Note that the entire purification process can be followed on S1 Fig that shows the whole SDS-PAGE gels, not only the supernatant and imidazole elution samples.
Fig 4.
Secondary structure content determination of UDE and its fragments using VUVCD and SELCON3 program.
(A) Vacuum-ultraviolet circular dichroism (Δε) spectra of the UDE protein and its nine truncated fragments measured over the wavelength region of λ = 170–255 nm. The spectra are sorted into two panels for better visibility and spectra of UDE (red) is shown in both panels for reference (B) Decomposition of the CD spectra of UDE and its selected fragments using six secondary structure components; regular/distorted α-helix (rH/dH), regular/distorted β-strand (rS/dS), turn (T), and disordered structure (D). Upper panel: CD spectrum of UDE as measured and as fitted using the six components [22–24]. Spectra of the components are also plotted with magnitudes proportional to their ratios in the full-length protein. Lower Panel: Difference spectra corresponding to UDE-NA2 and NG3-NA3 together with the fittings based on the spectra of the six basic components.
Fig 5.
Spatial distribution of the secondary structure components along UDE protein.
The location of α-helical segments (A) in the full-length (355 aa) UDE protein and (B) in its nine truncated fragments were determined from the CD spectra and the amino acid sequence using a neural network algorithm [25,26]. The α-helical segments and β-strands are displayed in blue and red, respectively, while both turns and disordered parts appear in yellow. (C) Our final estimate for the secondary structure of UDE obtained as an average of the structure of UDE proposed in panel (A) and the structure of the fragments shown in panel (B) except for CA7. (D) The native structure of the N-terminal end of the full-length UDE was investigated using the evaluation of the CD spectrum of the N- terminal as the difference between the CD spectra of UDE and NA2 according to (Δε1UDE×N1UDE−Δε1NA2×N1NA2)/(N1UDE—N1NA2), where Δε is the molar ellipticity and N is the number of amino acids for UDE and NA2. The same subtraction method was performed with the highly overlapping fragments NG3 and NA3.