PAP(248-286) Conformational Changes during the Lag Phase of Amyloid Fibril Formation.

The initial stage of fibril formation of C-terminal region PAP(248-286) of human seminal plasma protein prostatic acid phosphatase was considered. Amyloid fibrils from the peptide PAP(248-286) are termed as a semen-derived enhancer of viral infection (SEVI) found in abundant quantities in semen. The kinetics of the amyloid fibril formation process consists of two characteristic phases (lag phase/nucleation phase and growth phase/elongation phase). The lag phase can be caused by the presence of mature amyloid fibrils (seeds) in protein solution, so-called secondary nucleation. The secondary nucleation includes interaction of protein monomers with the mature fibril surface that leads to protein spatial structural changes for further amyloid fibril formation. In this work, changes of the PAP(248-286) spatial structure were obtained during the secondary nucleation phase. Pulsed-field gradient (PFG) NMR was used to characterize the behavior of monomeric PAP(248-286) in water solution after PAP(248-286) seed addition. The self-diffusion coefficient showed compactization of the peptide monomer due to fibril-monomer interactions. PAP(248-286) spatial structural changes were detected with the help of high-resolution NMR spectroscopy and molecular dynamics (MD) simulation. The folding of PAP(248-286) occurs due to backbone chain bending in the region of H270 and T275 amino acid residues. Obtained folded conformation of PAP(248-286) emerging in the secondary nucleation process is energetically favorable and retains after monomer-amyloid interaction. The structural changes are associated with localization of PAP(248-286) hydrophobic surface regions, which are probably responsible for peptide monomer-amyloid interactions.

Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus.

Ribosome biogenesis is a complex and highly accurate conservative process of ribosomal subunit maturation followed by association. Subunit maturation comprises sequential stages of ribosomal RNA and proteins' folding, modification and binding, with the involvement of numerous RNAses, helicases, GTPases, chaperones, RNA, protein-modifying enzymes, and assembly factors. One such assembly factor involved in bacterial 30S subunit maturation is ribosomal binding factor A (RbfA). In this study, we present the crystal (determined at 2.2 Å resolution) and NMR structures of RbfA as well as the 2.9 Å resolution cryo-EM reconstruction of the 30S-RbfA complex from Staphylococcus aureus (S. aureus). Additionally, we show that the manner of RbfA action on the small ribosomal subunit during its maturation is shared between bacteria and mitochondria. The obtained results clarify the function of RbfA in the 30S maturation process and its role in ribosome functioning in general. Furthermore, given that S. aureus is a serious human pathogen, this study provides an additional prospect to develop antimicrobials targeting bacterial pathogens.

Extent of N-Terminus Folding of Semenogelin 1 Cleavage Product Determines Tendency to Amyloid Formation.

It is known that four peptide fragments of predominant protein in human semen Semenogelin 1 (SEM1) (SEM1(86-107), SEM1(68-107), SEM1(49-107) and SEM1(45-107)) are involved in fertilization and amyloid formation processes. In this work, the structure and dynamic behavior of SEM1(45-107) and SEM1(49-107) peptides and their N-domains were described. According to ThT fluorescence spectroscopy data, it was shown that the amyloid formation of SEM1(45-107) starts immediately after purification, which is not observed for SEM1(49-107). Seeing that the peptide amino acid sequence of SEM1(45-107) differs from SEM1(49-107) only by the presence of four additional amino acid residues in the N domain, these domains of both peptides were obtained via solid-phase synthesis and the difference in their dynamics and structure was investigated. SEM1(45-67) and SEM1(49-67) showed no principal difference in dynamic behavior in water solution. Furthermore, we obtained mostly disordered structures of SEM1(45-67) and SEM1(49-67). However, SEM1(45-67) contains a helix (E58-K60) and helix-like (S49-Q51) fragments. These helical fragments may rearrange into ß-strands during amyloid formation process. Thus, the difference in full-length peptides' (SEM1(45-107) and SEM1(49-107)) amyloid-forming behavior may be explained by the presence of a structured helix at the SEM1(45-107) N-terminus, which contributes to an increased rate of amyloid formation.

Conformational ensemble of amyloid-forming semenogelin 1 peptide SEM1(68-107) by NMR spectroscopy and MD simulations.

SEM1(68-107) is a peptide corresponding to the region of semenogelin 1 protein from 68 to 107 amino acid position. SEM1(68-107) is an abundant component of semen, which participates in HIV infection enhanced by amyloid fibrils forming. To understand the causes influencing amyloid fibril formation, it is necessary to determine the spatial structure of SEM1(68-107). It was shown that the determination of SEM1(68-107) structure is complicated by the non-informative NMR spectra due to the high intramolecular mobility of peptides. The complementary approach based on the geometric restrictions of individual peptide fragments and molecular modeling was used for the determination of the spatial structure of SEM1(68-107). The N- (SEM1(68-85)) and C-terminuses (SEM1(86-107)) of SEM1(68-107) were chosen as two individual peptide fragments. SEM1(68-85) and SEM1(86-107) structures were established with NMR and circular dichroism CD spectroscopies. These regions were used as geometric restraints for the SEM1(68-107) structure modeling. Even though most of the SEM1(68-107) peptide is unstructured, our detailed analysis revealed the following structured elements: N-terminus (70His-84Gln) forms an α-helix, (86Asp-94Thr) and (101Gly-103Ser) regions fold into 310-helixes. The absence of a SEM1(68-107) rigid conformation leads to instability of these secondary structure regions. The calculated SEM1(68-107) structure is in good agreement with experimental values of hydrodynamic radius and dihedral angles obtained by NMR spectroscopy. This testifies the adequacy of a combined approach based on the use of peptide fragment structures for the molecular modeling formation of full-size peptide spatial structure.

Dithiophosphate-Induced Redox Conversions of Reduced and Oxidized Glutathione.

Phosphorus species are potent modulators of physicochemical and bioactive properties of peptide compounds. O,O-diorganyl dithiophoshoric acids (DTP) form bioactive salts with nitrogen-containing biomolecules; however, their potential as a peptide modifier is poorly known. We synthesized amphiphilic ammonium salts of O,O-dimenthyl DTP with glutathione, a vital tripeptide with antioxidant, protective and regulatory functions. DTP moiety imparted radical scavenging activity to oxidized glutathione (GSSG), modulated the activity of reduced glutathione (GSH) and profoundly improved adsorption and electrooxidation of both glutathione salts on graphene oxide modified electrode. According to NMR spectroscopy and GC-MS, the dithiophosphates persisted against immediate dissociation in an aqueous solution accompanied by hydrolysis of DTP moiety into phosphoric acid, menthol and hydrogen sulfide as well as in situ thiol-disulfide conversions in peptide moieties due to the oxidation of GSH and reduction of GSSG. The thiol content available in dissolved GSH dithiophosphate was more stable during air oxidation compared with free GSH. GSH and the dithiophosphates, unlike DTP, caused a thiol-dependent reduction of MTS tetrazolium salt. The results for the first time suggest O,O-dimenthyl DTP as a redox modifier for glutathione, which releases hydrogen sulfide and induces biorelevant redox conversions of thiol/disulfide groups.

Effect of triphenylphosphonium moiety on spatial structure and biointeractions of stereochemical variants of YRFK motif.

Chemical modification of therapeutic peptides is an important approach to improving their physicochemical and pharmacokinetic properties. The triphenylphosphonium (TPP) cation has proved to be a powerful modifier; however, its effects on peptide structure and activity remain uncharacterized. In this study, cytoprotective tetrapeptides based on the YRFK opioid motif with L- or D-Arg residues were linked to (triphenylphosphonio)carboxylic acids with ethylene and pentylene spacers (TPP-3 and TPP-6 groups, respectively). The three-dimensional structure of the oligopeptides was analyzed by NMR spectroscopy, computational methods and circular dichroism (CD). A more compact and bent structure with segregated aromatic groups was revealed for the D-arginine-containing tetrapeptide and its TPP-6 derivative. The TPP moiety caused structure-organizing effect on the tetrapeptides, resulting in transition from random coil to ß-sheet structures, and decreased the peptide backbone flexibility up to ten times. The TPP-3-modified oligopeptide with the lowest RMSD value (ca. 0.05 Å) was characterized by intrapeptide hydrophobic interactions between the TPP and side groups of Tyr and Phe residues accompanied by strong CD induction. The TPP-6-modified oligopeptides showed enhanced ability to form intermolecular associates and disturb liposomal membranes. The relationship between the spatial structure of the oligopeptides and some of their biologically relevant interactions were additionally revealed and are discussed.

The data of heterologous expression protocol for synthesis of 15N, 13C-labeled SEM1(68-107) peptide fragment of homo sapiens semenogelin 1.

The semenogelin 1 protein is secreted in the seminal vesicles. After ejaculation it is split into small peptide fragments using internal proteases. It was shown that the fragments SEM1(45-107), SEM1(49-107), SEM1(68-107) (SEM1(86-107) form amyloid fibrils, which increase the possibility of HIV infection. The article presents a protocol for the synthesis and purification of a 15N, 13C-labeled SEM1(68-107) peptide for further structural studies by high-resolution NMR spectroscopy. The work describes cloning, expression of fusion protein GB1-SEM1(68-107) in E.coli, its purification, removal of GB1 and purification of SEM1(68-107). The purity of SEM1(68-107) samples on each purification steps was evaluated by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) and tricine-SDS-PAGE. The developed protocol allows to obtain SEM1(68-107) peptide for NMR studies (using 3D experiments), instead of costly solid-phase synthesis.

NMR resonance assignment and backbone dynamics of a C-terminal domain homolog of orange carotenoid protein.

Photoprotection in cyanobacteria is mediated by the Orange Carotenoid Protein (OCP), a two-domain photoswitch which has multiple natural homologs of its N- and C-terminal domains. Recently, it was demonstrated that C-terminal domain homologs (CTDHs) of OCP are standalone carotenoproteins participating in multidirectional carotenoid transfer between membranes and proteins. Non-covalent embedment of a ketocarotenoid causes dimerization of the small 16-kDa water-soluble CTDH protein; however, dynamic interactions of CTDH with membranes and other proteins apparently require the monomeric state. Although crystallography recently provided static snapshots of the Anabaena CTDH (AnaCTDH) spatial structure in the apo-form, which predicted mobility of some putative functional segments, no crystallographic information on the holo-form of CTDH is presently available. In order to use NMR techniques to cope with the dynamics of the AnaCTDH protein, it was necessary to obtain 1H, 13C and 15N resonance assignments. AnaCTDH samples enriched with 13C and 15N isotopes were prepared using recombinant protein expression, and NMR resonance assignment was achieved for more than 90% of the residues. The obtained results revealed that the structure of AnaCTDH in solution and in the crystal are largely equivalent. Together with 15N NMR relaxation experiments, our data shed light on the AnaCTDH dynamics and provide the platform for the subsequent analysis of the holo-CTDH structure in solution, for the better understanding of light-triggered protein-protein interactions and the development of antioxidant nanocarriers for biomedical applications in the future.

Backbone and side chain NMR assignments for the ribosome binding factor A (RbfA) from Staphylococcus aureus.

Ribosome binding factor A (RbfA) is a 14.9 kDa adaptive protein of cold shock, which is important for bacterial growth at low temperatures. RbfA can bind to the free 30S ribosomal subunit and interacts with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is important for the efficient processing of 16S rRNA and for the maturation (assembly) of 30S ribosomal subunits. Here we report backbone and side chains 1H, 13C and 15N chemical shift assignments of RbfA from Staphylococcus aureus. Analysis of the backbone chemical shifts by TALOS+ suggests that RbfA contains four α-helixes and three ß-strands with α1-ß1-ß2-α2-α3-ß3-α4 topology. Secondary structure of RbfA have KH-domain fold topology with ßααß subunit which is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, where an Ala residue at position 70 forming an interhelical kink. The solution of the structure of this protein factor and its complex with the ribosome by NMR spectroscopy, X-ray diffraction analysis and cryo-electron microscopy will allow further development of highly selective substances for slowing or completely stopping the translation of the pathogenic bacterium S. aureus, which will interfere with the synthesis and isolation of its pathogenicity factors.

The Role of Metals in the Reaction Catalyzed by Metal-Ion-Independent Bacillary RNase.

Extracellular enzymes of intestinal microbiota are the key agents that affect functional activity of the body as they directly interact with epithelial and immune cells. Several species of the Bacillus genus, like Bacillus pumilus, a common producer of extracellular RNase binase, can populate the intestinal microbiome as a colonizing organism. Without involving metal ions as cofactors, binase depolymerizes RNA by cleaving the 3',5'-phosphodiester bond and generates 2',3'-cyclic guanosine phosphates in the first stage of a catalytic reaction. Maintained in the reaction mixture for more than one hour, such messengers can affect the human intestinal microflora and the human body. In the present study, we found that the rate of 2',3'-cGMP was growing in the presence of transition metals that stabilized the RNA structure. At the same time, transition metal ions only marginally reduced the amount of 2',3'-cGMP, blocking binase recognition sites of guanine at N7 of nucleophilic purine bases.