Re applied for the processing of SAXS profiles. The radius of gyration (Rg) and total forward scattering at zero angle (I(0)) were calculated making use of the Guinier approximation. Pair-distance distribution function (PDDF) and maximum particle dimension (Dmax) had been computed employing GNOM [44]. Globularity and flexibility with the proteins were analyzed by using dimensionless Kratky volume-of-correlation (Vc)-based plot [45]. Low-resolution bead (dummy atom) model reconstructions of protein shape from SAXS information had been generated in DAMMIN [46] utilizing slow regime. The visualization in the bead model, derived 2-Cyanopyrimidine custom synthesis density maps along with the subsequent fitting of full-atomic models have been performed working with UCSF Chimera [47]. Theoretical scattering curves for the crystal structure (pdb id: 7ob1) and for the homology models of open and closed forms were calculated and compared with experimental curves utilizing FOXS [48] and CRYSOL [49]. 3. Outcomes and Discussion 3.1. Comparative Analysis of Physicochemical Attributes and Enzymatic Activity of PSPmod The peptidases on the POP family, including OpB, contain two domains—propeller and catalytic domains–linked through two hinge peptides (Supplementary Figure S2). The first peptide follows the N-terminal part of the catalytic domain (N-terminal loop) and hyperlinks it with the -propeller domain. The second follows the -propeller and hyperlinks it for the C-terminal part of the catalytic domain–/-hydrolase tertiary fold. Earlier, we showed that the chymotrypsinolysis of PSP in native conditions results in the appearance of a 66 kDa enzyme with a truncated N-terminal loop, which retains activity towards lowmolecular weight substrates and is also active towards a high-molecular weight substrate, azocasein [50]. This item of limited proteolysis was not stable and suffered from further degradation. The production of truncated PSP, in which the N-terminal loop was deleted by site-directed mutagenesis, in E. coli resulted in the expression of insoluble proteins with undetectable catalytic activity soon after refolding (information not shown). Ultimately, the tobacco etch virus (TEV) protease digestion internet site (ENLYFQS) was introduced by site-directed mutagenesis alternatively of amino acids 71-77 (IPQQEHS), forming the very first hinge peptide (Supplementary Figure S2). The recombinant protein using a modified hinge region (PSPmod) was expressed in E. coli and purified to homogeneity. The TEV recognition internet site was confirmed by MaldiTOF mass spectrometry but was not susceptible to TEV-protease hydrolysis. This low accessibility for TEV-protease was supposedly caused by powerful interactions of the peptide with neighboring protein regions. To make sure that PSPmod doesn’t possess important distortions within the oligomeric state or secondary structure composition when compared with wild-type PSP, the proteins have been evaluated by size-exclusion chromatography (SEC) and circular dichroism spectroscopy (CD), respectively, when their thermal stabilities were measured by differential scanning calorimetry (DSC) (Figure 1A ). Based on SEC, both proteins have been eluted as monomers with molecular weights of 80 kDa (calculated MW is 76 kDa) (Figure 1A). This corresponds for the previously obtained data that discovered that monomers are functional forms of -proteobacterial OpB [302]. In contrast, protozoan OpB, which includes TbOpB and LmOpB, have been verified active dimers in resolution [26,27]. Analogously, the CD spectra of PSP and PSPmod were close to each other, excluding minor differences (Figure 1B).Biology 2021, ten,7 of.