Omplexes with peptide inhibitor, transition state analog, antipain (N-carboxyl-FRVRgl) [26,27], plus the open state was found in the structure of TbOpB in ligand-free type [26]. This allowed a comparative structural evaluation in the open and closed states of protozoan OpB, bacterial PEP and archaeal AAP [26]. A popular mechanism of catalytic activation for all three branches of POP was suggested, which highlighted the value in the interdomain interface and especially of among the interdomain salt bridges (SB1 in TbOpB) within the transition in the enzymes amongst two states [26]. It truly is intriguing that the residues forming this SB1 were not conserved in -proteobacterial OpB [28,29], which includes the well-studied enzymes from E. coli [30], Salmonella enterica [31] and Serratia proteomaculans [32]. This difference strongly suggests there is no direct transfer on the activation mechanism proposed for protozoan OpB for the bacterial enzymes and calls for applications on the structural information obtained for OpB from bacteria to elucidate the mechanisms underlying their catalytic activation. Within this study, we described for the initial time the structures of bacterial OpB from S. proteomaculans (PSP) obtained by X-ray for an enzyme with a modified hinge area (PSPmod) and two of its derivatives. The enzymes were crystallized within the presence of spermine and adopted uncommon intermediate states within the crystal lattices. In the identical time, as outlined by small-angle X-ray scattering (SAXS) wild-type PSP adopts an open state in remedy; spermine causes its transition for the intermediate state, though PSPmod contained molecules inside the open and intermediate states in dynamic equilibrium. The data obtained indicate that the intermediate state, that is seldom located inside the crystal structures of enzymes of the POP family members, could be considerably more popular in vivo. two. Components and Methods 2.1. Mutagenesis Easy single-primer site-directed mutagenesis was performed as described in [33]. Oligonucleotide mutagenesis primer (five -GAG ATG GTG GCG CGC GAG AAC CTG TAT TTC CAA TCG GTG CCT TAT GTC CG-3 ) and check-primer (five -AGA TGG TGG CGC GCG AG-3 ), created for the choice of mutant clones, had been synthetized in (Evrogen, Moscow, Russia). Eighteen cycles of polymerase chain reaction (PCR) had been performed on the templates on the PSP- and PSP-E125A-expressing plasmids [28] applying Tersus Plus PCR kit (Evrogen, Moscow, Russia) in accordance with the manufacturer’s recommendations. The PCR merchandise had been treated with DpnI endonuclease (Thermo Fisher Scientific, MA, USA), which digested the parental DNA template, after which transformed into E. coli Match1 competent cells. The mutant clones had been chosen by PCR performed directly on colonies using Taq DNA polymerase (Evrogen, Moscow, Russia) and check primer with T7 reverse universal primer. Plasmid DNA purified from mutant clones was sequenced to make sure the absence of random mutations related with PCR. The second run of mutagenesis was performed for preparations of PSPmodE75 around the template with the Methyl phenylacetate custom synthesis PSPmod-expressing plasmid. All mutated proteins had been verified by Maldi-TOF mass spectrometry. two.two. Recombinant Proteins Purification and Characterization Proteins had been expressed in E. coli BL21(DE3) (Novagen, Madison, WI, USA) and purified as described in [32]. Protein sizes and purities had been checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie G-250. Protein concentrations had been determined by the Bradford.