Designed peptides with homochiral and heterochiral diproline templates as conformational constraints

Diproline segments have been advanced as templates for nucleation of folded structure in designed peptides. The conformational space available to homochiral and heterochiral diproline segments has been probed by crystallographic and NMR studies on model peptides containing L-Pro-L-Pro and D-Pro-L-Pro units. Four distinct classes of model peptides have been investigated: a) isolated D-Pro-L-Pro segments which form type II β-turn; b) D-Pro-L-Pro-L-Xxx sequences which form type II′-I (βII′-I′, consecutive β-turns) turns; c) D-Pro-L-Pro-D-Xxx sequences; d) L-Pro-L-Pro-L-Xxx sequences. A total of 17 peptide crystal structures containing diproline segments are reported. Peptides of the type Piv-D-Pro-L-Pro-L-Xxx-NHMe are conformationally homogeneous, adopting consecutive β-turn conformations. Peptides in the series Piv-D-Pro-L-Pro-D-Xxx-NHMe and Piv-L-Pro-L-Pro-L-Xxx-NHMe, display a heterogeneity of structures in crystals. A type VIa β-turn conformation is characterized in Piv-L-Pro-L-Pro-L-Phe-OMe (18), while an example of a 5→1 hydrogen bonded α-turn is observed in crystals of Piv-D-Pro-L-Pro-D-Ala-NHMe (11). An analysis of pyrrolidine conformations suggests a preferred proline puckering geometry is favored only in the case of heterochiral diproline segments. Solution NMR studies, reveal a strong conformational influence of the C-terminal Xxx residues on the structures of diproline segments. In L-Pro-L-Pro-L-Xxx sequences, the Xxx residues strongly determine the population of Pro-Pro cis conformers, with an overwhelming population of the trans form in L-Xxx = L-Ala (19)

A proline insertion-deletion in the spike glycoprotein fusion peptide of mouse hepatitis virus strongly alters neuropathology.

Fusion peptides (FPs) in spike proteins are key players mediating early events in cell-to-cell fusion, vital for intercellular viral spread. A proline residue located at the central FP region has often been suggested to have a distinctive role in this fusion event. The spike glycoprotein from strain RSA59 (PP) of mouse hepatitis virus (MHV) contains two central, consecutive prolines in the FP. Here, we report that deletion of one of these proline residues, resulting in RSA59 (P), significantly affected neural cell syncytia formation and viral titers postinfection in vitro. Transcranial inoculation of C57Bl/6 mice with RSA59 (PP) or RSA59 (P) yielded similar degrees of necrotizing hepatitis and meningitis, but only RSA59 (PP) produced widespread encephalitis that extended deeply into the brain parenchyma. By day 6 postinfection, both virus variants were mostly cleared from the brain. Interestingly, inoculation with the RSA59 (P)- carrying MHV significantly reduced demyelination at the chronic stage. We also found that the presence of two consecutive prolines in FP promotes a more ordered, compact, and rigid structure in the spike protein. These effects on FP structure were due to proline\u27s unique stereochemical properties intrinsic to its secondary amino acid structure, revealed by molecular dynamics andNMRexperiments.Wetherefore propose that the differences in the severity of encephalitis and demyelination between RSA59 (PP) and RSA59 (P) arise from the presence or absence, respectively, of the two consecutive prolines in FP. Our studies define a structural determinant of MHV entry in the brain parenchyma important for altered neuropathogenesis. © 2019 Singh et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc

Design of a peptide hairpin containing a central three-residue loop

The construction of a designed β-hairpin structure, containing a central three-residue loop has been successfully achieved in the synthetic nonapeptide Boc-Leu-Phe-Val-DPro-LPro-DAla-Leu-Phe-Val-OMe (2). The design is based on expanding the two-residue loop established in the peptide β-hairpin Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe (1). Characterization of the registered β-hairpins in peptides 1 and 2 is based on the observation of key nuclear Overhauser effects (NOEs) in CDCl3 and CD3OH. Solvent titration and temperature dependence of NH chemical shifts establish the identity of NH groups involved in interstrand hydrogen bonding. In peptide 2, the antiparallel registry is maintained, with the formation of a DPro-LPro-DAla loop, stabilized by a 5→1 hydrogen bond between Val3 CO and Leu7 NH groups (C13, β-turn) and a 3→1 hydrogen bond between dPro4 CO and DAla6 NH groups (C7, γ-turn). NMR derived structures suggest that in peptide 2, DAla(6) adopts an αL conformation. In peptide 1, the DPro-LPro segment adopts a type II' β-turn. Replacement of DAla (6) in peptide 2 by lAla in peptide 3 yields a β-hairpin conformation, with a central DPro-LPro two-residue loop. Strand slippage at the C-terminus results in altered registry of the antiparallel strands

NMR analysis of aromatic interactions in designed peptide β-hairpins

Designed octapeptide β-hairpins containing a central DPro-Gly segment have been used as a scaffold to place the aromatic residues Tyr and Trp at various positions on the antiparallel β-strands. Using a set of five peptide hairpins, aromatic interactions have been probed across antiparallel β-sheets, in the non-hydrogen bonding position (Ac-L-Y-V-DP-G-L-Y/W-V-OMe: peptides 1 and 2), diagonally across the strands (Boc-Y/W-L-V-DP-G-W-L-V-OMe: peptides 3 and 6), and along the strands at positions i and i + 2 (Boc-L-L-V-DP-G-Y-L-W-OMe: peptide 4). Two peptides served as controls (Boc-L-L-V-DP-G-Y-W-V-OMe: peptide 5; Boc-L-Y-V-DP-G-L-L-V-OMe: peptide 7) for aromatic interactions. All studies have been carried out using solution NMR methods in CDCl3 + 10% DMSO-d6 and have been additionally examined in CD3OH for peptides 1 and 2. Inter-ring proton-proton nuclear Overhauser effects (NOEs) and upfield shifted aromatic proton resonances have provided firm evidence for specific aromatic interactions. Calculated NMR structures for peptides 1 and 2, containing aromatic pairs at facing non-hydrogen bonded positions, revealed that T-shaped arrangements of the interacting pairs of rings are favored, with ring current effects leading to extremely upfield chemical shifts and temperature dependences for specific aromatic protons. Anomalous far-UV CD spectra appeared to be a characteristic feature in peptides where the two aromatic residues are spatially proximal. The observation of the close approach of aromatic rings in organic solvents suggests that interactions of an electrostatic nature may be favored. This situation may be compared to the case of aqueous solutions, where clustering of aromatic residues is driven by solvophobic (hydrophobic) forces

Design of a Peptide Hairpin Containing a Central Three-Residue Loop

The construction of a designed $\beta$-hairpin structure, containing a central three-residue loop has been successfully achieved in the synthetic nonapeptide $Boc-Leu-Phe-Val-^DPro-^LPro-^DAla-Leu-Phe-Val-OMe$ (2). The design is based on expanding the two-residue loop established in the peptide $\beta$-hairpin $Boc-Leu-Phe-Val-^DPro-^LPro-Leu-Phe-Val-OMe$ (1). Characterization of the registered $\beta$-hairpins in peptides 1 and 2 is based on the observation of key nuclear Overhauser effects (NOEs) in $CDCl_3$ and $CD_3OH$. Solvent titration and temperature dependence of NH chemical shifts establish the identity of NH groups involved in interstrand hydrogen bonding. In peptide 2, the antiparallel registry is maintained, with the formation of a $^DPro-^LPro-^DAla$ loop, stabilized by a $5\rightarrow1$ hydrogen bond between Val3 CO and Leu7 NH groups $(C_{13}, \alpha-turn)$ and a $3\rightarrow1$ hydrogen bond between $^DPro4$ CO and $^DAla6$ NH groups $(C_7, \gamma-turn)$. NMR derived structures suggest that in peptide 2, $^DAla(6)$ adopts an $\alpha_L$ conformation. In peptide 1, the $^DPro-^LPro$ segment adopts a type II' $\beta$-turn. Replacement of $^DAla (6)$ in peptide 2 by $^LAla$ in peptide 3 yields a $\beta$-hairpin conformation, with a central $^DPro-^LPro$ two-residue loop. Strand slippage at the C-terminus results in altered registry of the antiparallel strands

The rα structure, chemical shift anisotropy, and the abnormal orientation of methyldichlorophosphine from the NMR spectrum

NMR studies of methyldichlorophosphine have been undertaken in the nematic phase of mixed liquid crystals of opposite diamagnetic anisotropies. The rα structure is derived. The proton chemical-shift anisotropy has been determined from the studies without the use of a reference compound and without a change of experimental conditions. It is shown that the molecule orients in the liquid crystal with positive diamagnetic anisotropy in such a way that the C3 symmetry axis of the CH3P moiety is preferentially aligned perpendicular to the direction of the magnetic field, unlike other similar systems. This is interpreted in terms of the formation of a weak solvent-solute molecular complex. The heteronuclear indirect spin-spin coupling constants are determined. The sign of the two-bond JPH is found to be positive

Synthesis of 2-deoxy cyclic and linear oligosaccharides by oligomerization of monomers

Cyclic and linear oligosaccharides constituted with 2-deoxy sugar units are synthesized by an oligomerization reaction involving activated thioglycoside monomers, consisting of a 2-deoxy sugar unit. The oligomerization promoter plays an important role in the formation of either the cyclic- or the linear oligosaccharides. Encapsulation abilities of a 2-deoxy cyclic hexamer with p-nitrophenot, by a H-1 NMR method, showed complexation of the guest molecule with the host molecule

Tuning the β-turn segment in designed peptide β-hairpins: Construction of a stable type I′ β-turn nucleus and hairpin-helix transition promoting segments Ч

Designed octapeptides Boc-Leu-Val-Val-Aib-DXxx-Leu-Val-Val-OMe (DXxx = DAla, 3a;DVal, 3c and DPro, 5a) and Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (3b) have been investigated to construct models of a stable type I′ β-turn nucleated hairpin and to generate systems for investigating helix-hairpin conformational transitions. Peptide 5a, which contains a central Aib-DPro segment, is shown to adopt a stable type I′ β-turn nucleated hairpin structure, stabilized by four cross-strand hydrogen bonds. The stability of the structure in diverse solvents is established by the observation of all diagnostic NOEs expected in a β-hairpin conformation. Replacement of DPro5 by DAla/DVal (3a-c) results in sequences that form β-hairpins in hydrogen bonding solvents like CD3OH and DMSO-d6. However, in CDCl3 evidence for population of helical conformations is obtained. Peptide 6b (Boc-Leu-Phe-Val-Aib-Aib-Leu-Phe-Val-OMe), which contains a centrally positioned Aib-Aib segment, provides a clear example of a system, which exhibits a helical conformation in CDCl3 and a significant population of both helices and hairpins in CD3OH and DMSO-d6. The coexistence of multiple conformations is established by the simultaneous observation of diagnostic NOEs. Control over stereochemistry of the central β-turn permits generation of models for robust -hairpins and also for the construction of systems that may be used to probe helix-hairpin conformational transitions

Stabilizing effect of electrostatic vs. aromatic interactions in diproline nucleated peptide beta-hairpins

The contribution of Tyr-His vs. Cys-His interacting pairs to the scaffold stability of (D)Pro-(L)Pro nucleated peptide beta-hairpins has been examined. We present direct evidence for the superiority of the Cys-His pairs, mediated by sulphur-imidazole interactions, as added stabilizing agents of the beta-hairpin scaffold

C12-Helix development in ()n sequences - spectroscopic characterization of Boc-Aib-4(R)Val]-OMe oligomers

The solution conformations of the -hybrid oligopeptides Boc-Aib-4(R)Val]n-OMe (n = 1-8) in organic solvents have been probed by NMR, IR, and CD spectroscopic methods. In the solid state, this peptide series favors C12-helical conformations, which are backbone-expanded analogues of 310 helices in -peptide sequences. NMR studies of the six- (n = 3) and 16-residue (n = 8) peptides reveal that only two NH protons attached the N-terminus residues Aib(1) and 4(R)Val(2) are solvent-exposed. Sequential NiH-Ni+1H NOEs characteristic of local helical conformations are also observed at the residues. IR studies establish that chain extension leads to a large enhancement in the intensities of the hydrogen-bonded NH stretching bands (3343-3280 cm-1), which suggest elongation of intramolecularly hydrogen-bonded structures. The development of C12-helical structures upon lengthening of the sequence is supported by the NMR and IR observations. The CD spectra of the ()n peptides reveal a negative maximum at ca. 206 nm and a positive maximum at ca. 192 nm, spectral feature that are distinct from those of 310 helices in -peptides