4,4-Difluoroproline as a Unique 19F NMR Probe of Proline Conformation.

Despite the importance of proline conformational equilibria (trans versus cis amide and exo versus endo ring pucker) on protein structure and function, there is a lack of convenient ways to probe proline conformation. 4,4-Difluoroproline (Dfp) was identified to be a sensitive 19F NMR-based probe of proline conformational biases and cis-trans isomerism. Within model compounds and disordered peptides, the diastereotopic fluorines of Dfp exhibit similar chemical shifts (ΔδFF = 0-3 ppm) when a trans X-Dfp amide bond is present. In contrast, the diastereotopic fluorines exhibit a large (ΔδFF = 5-12 ppm) difference in chemical shift in a cis X-Dfp prolyl amide bond. DFT calculations, X-ray crystallography, and solid-state NMR spectroscopy indicated that ΔδFF directly reports on the relative preference of one proline ring pucker over the other: a fluorine which is pseudo-axial (i.e., the pro-4R-F in an exo ring pucker, or the pro-4S-F in an endo ring pucker) is downfield, while a fluorine which is pseudo-equatorial (i.e., pro-4S-F when exo, or pro-4R-F when endo) is upfield. Thus, when a proline is disordered (a mixture of exo and endo ring puckers, as at trans-Pro in peptides in water), it exhibits a small Δδ. In contrast, when the Pro is ordered (i.e., when one ring pucker is strongly preferred, as in cis-Pro amide bonds, where the endo ring pucker is strongly favored), a large Δδ is observed. Dfp can be used to identify inherent induced order in peptides and to quantify proline cis-trans isomerism. Using Dfp, we discovered that the stable polyproline II helix (PPII) formed in the denatured state (8 M urea) exhibits essentially equal populations of the exo and endo proline ring puckers. In addition, the data with Dfp suggested the specific stabilization of PPII by water over other polar solvents. These data strongly support the importance of carbonyl solvation and n → π* interactions for the stabilization of PPII. Dfp was also employed to quantify proline cis-trans isomerism as a function of phosphorylation and the R406W mutation in peptides derived from the intrinsically disordered protein tau. Dfp is minimally sterically disruptive and can be incorporated in expressed proteins, suggesting its broad application in understanding proline cis-trans isomerization, protein folding, and local order in intrinsically disordered proteins.

Effects of Proline on Internal Friction in Simulated Folding Dynamics of Several Alanine-Based α-Helical Peptides.

We have studied in silico the effect of proline, a model cosolvent, on local and global friction coefficients in (un)folding of several typical alanine-based α-helical peptides. Local friction is related to dwell times of a single, ensemble-averaged hydrogen bond (HB) within each peptide. Global friction is related to energy dissipated in a series of configurational changes of each peptide experienced by increasing the number of HBs during folding. Both of these approaches are important in relation to future atomic force microscopic-based measurements of internal friction via force-clamp single-molecule force spectroscopy. Molecular dynamics (MD) simulations for six peptides, namely, ALA5, ALA8, ALA15, ALA21, (AAQAA)3, and H2N-GN(AAQAA)2G-COONH2, have been conducted at 2 and 5 M proline solutions in water. Using previously obtained MD data for these peptides in pure water as well as upgraded theoretical models, we obtained variations of local and global internal friction coefficients as a function of solution viscosity. The results showed the substantial role of proline in stabilizing the folded state and slowing the overall folding dynamics. Consequently, larger friction coefficients were obtained at larger viscosities. The local and global internal friction, i.e., respective, friction coefficients approximated to zero viscosity, was also obtained. The evolution of friction coefficients with viscosity was weakly dependent on the number of concurrent folding pathways but was rather dominated by a stabilizing effect of proline on the folded states. Obtained values of local and global internal friction showed qualitatively similar results and a clear dependency on the structure of the studied peptide.

Misacylation of tRNA with Ser-Pro Dipeptide for In Vitro Transcription-Translation.

Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins. To that end, we synthesized the 3,5-dinitrobenzyl (DNB)-activated esters of a native Ser-Pro dipeptide and its cis- and trans-locked alkene analogs. Murakami et al. created the DNB flexizyme (dFx), a ribozyme that acylates tRNA with DNB esters of amino acids to permit unnatural amino acids to be incorporated into proteins. A tRNA from yeast that recognizes the amber stop codon, along with the dFx flexizyme, were generated by in vitro transcription with T7 RNA polymerase. dFx was used to successfully catalyze the chemical misacylation of truncated amber tRNA with the Ser-Pro-DNB activated dipeptide. This method allows the introduction of non-native Ser-Pro dipeptide mimics into full-length proteins by in vitro transcription-translation. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3,5-dinitrobenzyl activated esters of Ser-Pro Basic Protocol 2: Preparation of truncated amber tRNA Basic Protocol 3: Acylation of amber-tRNA by the dFx flexizyme Basic Protocol 4: PAGE electrophoresis of tRNASerPro.

4-Thiaproline accelerates the slow folding phase of proteins containing cis prolines in the native state by two orders of magnitude.

The cis/trans isomerization of peptidyl-prolyl peptide bonds is often the bottleneck of the refolding reaction for proteins containing cis proline residues in the native state. Proline (Pro) analogues, especially C4-substituted fluoroprolines, have been widely used in protein engineering to enhance the thermodynamic stability of peptides and proteins and to investigate folding kinetics. 4-thiaproline (Thp) has been shown to bias the ring pucker of Pro, to increase the cis population percentage of model peptides in comparison to Pro, and to diminish the activation energy barrier for the cis/trans isomerization reaction. Despite its intriguing properties, Thp has been seldom incorporated into proteins. Moreover, the impact of Thp on the folding kinetics of globular proteins has never been reported. In this study, we show that upon incorporation of Thp at cisPro76 into the thioredoxin variant Trx1P the half-life of the refolding reaction decreased from ~2 h to ~35 s. A dramatic acceleration of the refolding rate could be observed also for the protein pseudo wild-type barstar upon replacement of cisPro48 with Thp. Quantum chemical calculations suggested that the replacement of the Cγ H2 group by a sulfur atom in the pyrrolidine ring, might lower the barrier for cis/trans rotation due to a weakened peptide bond. The protein variants retained their thermodynamic stability upon incorporation of Thp, while the catalytic and enzymatic activities of the modified Trx1P remained unchanged. Our results show that the Pro isostere Thp might accelerate the rate of the slow refolding reaction for proteins containing cis proline residues in the native state, independent from the local structural environment.

Phase Separation and Fibrillization of Human Annexin A7 Are Mediated by Its Proline-Rich Domain.

Human annexin A7, a calcium- and phospholipid-binding protein, governs calcium homeostasis, plasma membrane repair, apoptosis, and tumor progression. A7 contains an N-terminal proline-rich domain (PRD; 180 residues, ∼24% prolines) that determines its functional specificity. Using microscopy and dye-binding assays, we show that recombinant A7 and its isolated PRD spontaneously phase separate into spherical condensates, which subsequently transform into ß-sheet-rich fibrils. We demonstrate that fibrillization of A7-PRD proceeds via primary nucleation and fibril-catalyzed secondary nucleation processes, as determined by chemical kinetics, providing a mechanistic basis for its amyloid assembly. This study confirms and highlights a subclass of eukaryotic PRDs prone to forming aggregates with important physiological and pathological implications.

Assignment of the disordered, proline-rich N-terminal domain of the tumour suppressor p53 protein using 1HN and 1Hα-detected NMR measurements.

Protein p53 is mostly known for playing a key role in tumour suppression, and mutations in the p53 gene are amongst the most frequent genomic events accompanying oncogenic transformation. Continuous research is conducted to target disordered proteins/protein regions for cancer therapy, for which atomic level information is also necessary. The disordered N-terminal part of p53 contains the transactivation and the proline-rich domains-which besides being abundant in proline residues-contains repetitive Pro-Ala motifs. NMR assignment of such repetitive, proline-rich regions is challenging due to the lack of amide protons in the 1HN-detected approaches, as well as due to the small chemical shift dispersion. In the present study we perform the full assignment of the p531-100 region by applying a combination of 1HN- and 1Hα-detected NMR experiments. We also show the increased information content when using real-time homo- and heteronuclear decoupled acquisition schemes. On the other hand, we highlight the presence of minor proline species, and using Pro-selective experiments we determine the corresponding cis or trans conformation. Secondary chemical shifts for (Cα-Cß) atoms indicate the disordered nature of this region, with expected helical tendency for the TAD1 region. As the role of the proline-rich domain is yet not well understood our results can contribute to further successful investigations.

Effects of structural changes on antibacterial activity and cytotoxicity due to proline substitutions in chimeric peptide HnMc.

Owing to the rapidly increasing emergence of multidrug-resistant pathogens, antimicrobial peptides (AMPs) are being explored as next-generation antibiotics. However, AMPs present in nature are highly toxic and exhibit low antibacterial activity. Simple modifications, such as amino acid substitution, can enhance antimicrobial activity and cell selectivity. Herein, we show that HnMc-W, substituted by the Phe1Trp analog of HnMc, a chimeric peptide, resulted in membranolytic antibacterial action and enhanced salt tolerance, whereas HnMc-WP1 with one Ser9Pro substitution resulted in a proline-kink helical structure that increased salt-tolerant antibacterial effects and reduced cytotoxicity. In addition, the HnMc-WP2 peptide, designed with a PXXP motif, had a flexible central hinge in its α-helical structure due to the introduction of two Pro and two Gln (X positions, by deletion of two Gln at positions 16 and 17) residues instead of Ser at position. HnMc-WP2 exhibited excellent antibacterial effects without cytotoxicity in vitro. Moreover, its potent antibacterial activity was demonstrated in a drug-resistant Pseudomonas aeruginosa-infected mouse model in vivo. Our findings provide valuable information for the design of peptides with high antibacterial activity and cell selectivity.

Efficient and accurate calculation of proline cis/trans isomerization free energies from Hamiltonian replica exchange molecular dynamics simulations.

Proline cis/trans isomerization plays an important role in many biological processes but occurs on time scales not accessible to brute-force molecular dynamics (MD) simulations. We have designed a new Hamiltonian replica exchange scheme, ω-bias potential replica exchange molecular dynamics (ωBP-REMD), to efficiently and accurately calculate proline cis/trans isomerization free energies. ωBP-REMD is applied to various proline-containing tripeptides and a biologically important proline residue in the N2-domain of the gene-3-protein of phage fd in the wildtype and mutant variants of the protein. Excellent cis/trans transition rates are obtained. Reweighting of the sampled probability distribution along the peptide bond dihedral angle allows construction of the corresponding free-energy profile and calculation of the cis/trans isomerization free energy with high statistical precision. Very good agreement with experimental data is obtained. ωBP-REMD outperforms standard umbrella sampling in terms of convergence and agreement with experiment and strongly reduces perturbation of the local structure near the proline residue.

Proline cis/trans Isomerization in Intrinsically Disordered Proteins and Peptides.

BACKGROUND: Intrinsically disordered proteins and protein regions (IDPs/IDRs) are important in diverse biological processes. Lacking a stable secondary structure, they display an ensemble of conformations. One factor contributing to this conformational heterogeneity is the proline cis/trans isomerization. The knowledge and value of a given cis/trans proline ratio are paramount, as the different conformational states can be responsible for different biological functions. Nuclear Magnetic Resonance (NMR) spectroscopy is the only method to characterize the two co-existing isomers on an atomic level, and only a few works report on these data. METHODS: After collecting the available experimental literature findings, we conducted a statistical analysis regarding the influence of the neighboring amino acid types (i ± 4 regions) on forming a cis-Pro isomer. Based on this, several regularities were formulated. NMR spectroscopy was then used to define the cis-Pro content on model peptides and desired point mutations. RESULTS: Analysis of NMR spectra prove the dependence of the cis-Pro content on the type of the neighboring amino acid-with special attention on aromatic and positively charged sidechains. CONCLUSIONS: Our results may benefit the design of protein regions with a given cis-Pro content, and contribute to a better understanding of the roles and functions of IDPs.

Gas Phase Fragmentation Behavior of Proline in Macrocyclic b7 Ions.

The fragmentation characteristics of b7 ions produced from proline-containing heptapeptides have been studied in detail. The study has utilized the following C-terminally amidated model peptides: PA6, APA5, A2PA4, A3PA3, A4PA2, A5PA, A6P, PYAGFLV, PAGFLVY, PGFLVYA, PFLVYAG, PLVYAGF, PVYAGFL, YPAGFLV, YAPGFLV, YAGPFLV, YAGFPLV, YAGFLPV, YAGFLVP, PYAFLVG, PVLFYAG, A2PXA3, and A2XPA3 (where X = C, D, F, G, L, V, and Y, respectively). The results have shown that b7 ions undergo head-to-tail cyclization and form a macrocyclic structure. Under the collision-induced dissociation (CID) condition, it generates nondirect sequence ions regardless of the position of the proline and the neighboring amino acid residues. This study highlights the unusual and unique fragmentation behavior of proline-containing heptapeptides. Following the head-to-tail cyclization, the ring opens up and places the proline residue in the N-terminal position while forming a regular oxazolone form of b2 ions for all peptide series. Then, the fragmentation reaction pathway is followed by the elimination of proline with its C-terminal neighbor residue as an oxazolone (e.g., PXoxa) for all proline-containing peptide series.

Isonitrile-Proline - A Versatile Handle for the Chemoselective Derivatization of Collagen Peptides.

Functional groups that allow for chemoselective and bioorthogonal derivatization are valuable tools for the labelling of peptides and proteins. The isonitrile is such a group but synthetic methods for its incorporation into peptides by solid-phase peptide synthesis are not known. Here, we introduce (4S)- and (4R)-isonitrileproline (Inp) as building blocks for solid-phase peptide synthesis. Conformational studies of (4S)- and (4R)-Inp and thermal stability analysis of Inp-containing collagen triple helices revealed that the isonitrile group exerts a stereoelectronic gauche effect. We showcase the value of Inp for bioorthogonal labelling by derivatization of Inp-containing collagen model peptides (CMPs). Dual labelling with a pair of bioorthogonal reactions of a CMP containing Inp and azidoproline residues further highlights the versatility of the new isonitrile-containing amino acids.

ß-Turn editing in Gramicidin S: Activity impact on replacing proline α-carbon with stereodynamic nitrogen.

Gramicidin S, natural antimicrobial peptide is used commercially in medicinal lozenges for sore throat and Gram-negative and Gram-positive bacterial infections. However, its clinical potential is limited to topical applications because of its high red blood cells (RBC) cytotoxicity. Given the importance of developing potential antibiotics and inspired by the cyclic structure and druggable features of Gramicidin S, we edited proline α-carbon with stereodynamic nitrogen to examine the direct impact on biological activity and cytotoxicity with respect to prolyl counterpart. Natural Gramicidin S (12), proline-edited peptides 13-16 and wild-type d-Phe-d-Pro ß-turn mimetics (17 and 18) were synthesized using solid phase peptide synthesis and investigated their activity against clinically relevant bacterial pathogens. Interestingly, mono-proline edited analogous peptide 13 showed moderate improvement in antimicrobial activity against E. coli ATCC 25922 and K.pneumoniae BAA 1705 as compared to Gramicidin S. Furthermore, proline edited peptide 13 exhibited equipotent antimicrobial effect against MDR S. aureus and Enterococcus spp. Analysis of cytotoxicity against VERO cells and RBC, reveals that proline edited peptides showed two-fivefold lesser cytotoxicity than the counterpart Gramicidin S. Our study suggests that introducing single azPro/Pro mutation in Gramicidin S marginally improved the activity and lessens the cytotoxicity as compared with the parent peptide.

Collagen-Derived Dipeptides and Amino Acids Have Immunomodulatory Effects in M1-Differentiated RAW264.7 Cells and PBMC.

A number of food components, such as polyphenols and phytonutrients, have immunomodulatory effects. Collagen has various bioactivities, such as antioxidative effects, the promotion of wound healing, and relieving symptoms of bone/joint disease. Collagen is digested into dipeptides and amino acids in the gastrointestinal tract and subsequently absorbed. However, the difference in immunomodulatory effects between collagen-derived dipeptides and amino acids is unknown. To investigate such differences, we incubated M1 macrophages or peripheral blood mononuclear cells (PBMC) with collagen-derived dipeptides (hydroxyproline-glycine (Hyp-Gly) and proline-hydroxyproline (Pro-Hyp)) and amino acids (proline (Pro), hydroxyproline (Hyp), and glycine (Gly)). We first investigated the dose dependency of Hyp-Gly on cytokine secretion. Hyp-Gly modulates cytokine secretion from M1 macrophages at 100 µM, but not at 10 µM and 1 µM. We then compared immunomodulatory effects between dipeptides and mixtures of amino acids on M1 macrophages and PBMC. There was, however, no difference in cytokine secretion between dipeptides and their respective amino acids. We conclude that collagen-derived dipeptides and amino acids have immunomodulatory effects on M1-differentiated RAW264.7 cells and PBMC and that there is no difference in the immunomodulatory effects between dipeptides and amino acids.

The Peptidyl-Prolyl cis-trans isomerase, Pin1, associates with Protein Kinase C θ via a critical Phospho-Thr-Pro motif in the V3 regulatory domain.

Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCθ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCθ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCθ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCθ, indicating the specificity of the Pin1-PKCθ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCθ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCθ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCθ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase.

Early-Stage Oligomerization of Prion-like Polypeptides Reveals the Molecular Mechanism of Amyloid-Disrupting Capacity by Proline Residues.

Proline cis/trans isomerization governs protein local conformational changes via its local mechanical rigidity. The amyloid-disrupting capacity of proline is widely acknowledged; however, the molecular mechanism is still not clear. To understand how proline residues in polypeptide chains influence amyloid propensity, we study several truncated sequences of the TDP-43 C-terminal region (287-322) and their triple proline variants (308PPP310). We use coarse-grained molecular simulation to study the time evolution of the process of aggregation in the early stages in an effective high-concentration condition (∼25 mM). This ensures the long time scales for protein association at laboratory concentrations. We use several experimentally determined structure templates as initial structures of monomer conformations. We carry out oligomer size analysis and cluster analysis, along with several structural measures, to characterize the size distributions of oligomers and their morphological/structural properties. We show that average oligomer size is not a good indicator of amyloid propensity. Structural order and/or morphological properties are better alternatives. We show that proline variants can efficiently maintain the formation of large "ordered" oligomers of shorter truncated sequences, i.e., 307-322. This "order" maintenance is weakened when using longer truncated sequences (i.e., 287-322), leading to the formation of "disordered" oligomers. From an energy trade-off perspective, if the entropic effect is weak (short sequence length), the shape-complementarity of proline variants effectively guides the oligomerization process to form "ordered" oligomer intermediates. This leads to a distinct aggregation pathway that promotes amyloid formation (on-pathway). Strong entropic effects (long sequence length), however, would cause the formation of "disordered" oligomers. This in turn will suppress amyloid formation (off-pathway). The proline shape-complementary effects provide a guided morphological restraint to facilitate the pathways of amyloid formation. Our study supports the importance of structure-based kinetic heterogeneity of prion-like sequence fragments in driving different aggregation pathways. This work sheds light on the role of morphological and structural order of early-stage oligomeric species in regulating amyloid-disrupting capacity by prolines.

Expression and kinetic characterization of PYCR3.

PYCRs are proline biosynthetic enzymes that catalyze the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline in humans. PYCRs - especially PYCR1 - are upregulated in many types of cancers and have been implicated in the altered metabolism of cancer cells. Of the three isoforms of PYCR, PYCR3 remains the least studied due in part to the lack of a robust recombinant expression. Herein, we describe a procedure for the expression of soluble SUMO-PYCR3 in Escherichia coli, purification of the fusion protein, and removal of the SUMO tag. PYCR3 is active with either NADPH or NADH as the coenzyme. Bi-substrate kinetic measurements obtained by varying the concentrations of both L-P5C and NADH, along with product inhibition data for l-proline, suggest a random ordered bi bi mechanism. A panel of 19 proline analogs was screened for inhibition, and the kinetics of competitive inhibition (with L-P5C) were measured for five of the compounds screened, including N-formyl-l-proline, a validated inhibitor of PYCR1. N-formyl-l-proline was found to be ten times more selective for PYCR1 over PYCR3. The SUMO-PYCR3 expression system should be useful for testing the isoform specificity of PYCR1 inhibitors.

Predicting Collagen Triple Helix Stability through Additive Effects of Terminal Residues and Caps.

Collagen model peptides (CMPs) consisting of proline-(2S,4R)-hydroxyproline-glycine (POG) repeats have provided a breadth of knowledge of the triple helical structure of collagen, the most abundant protein in mammals. Predictive tools for triple helix stability have, however, lagged behind since the effect of CMPs with different frames ([POG]n , [OGP]n , or [GPO]n ) and capped or uncapped termini have so far been underestimated. Here, we elucidated the impact of the frame, terminal functional group and its charge on the stability of collagen triple helices. Combined experimental and theoretical studies with frame-shifted, capped and uncapped CMPs revealed that electrostatic interactions, strand preorganization, interstrand H-bonding, and steric repulsion at the termini contribute to triple helix stability. We show that these individual contributions are additive and allow for the prediction of the melting temperatures of CMP trimers.

Influence of Proline Chirality on Neighbouring Azaproline Residue Stereodynamic Nitrogen Preorganization.

We report herein the first systematic crystal structural investigation of azaproline incorporated in homo- and heterochiral diprolyl peptides. The X-ray crystallography data of peptides 1-5 illustrates that stereodynamic nitrogen in azaproline adopted the stereochemistry of neighbouring proline residue without depending on its position in the peptide sequence. Natural bond orbital analysis of crystal structures indicates OazPro -C'Pro of peptides 4 and 5 participating in n→π* interaction with stabilization energy about 1.21-1.33 kcal/mol. Density functional theory calculations suggested that the endo-proline ring puckering favoured over exo-conformation by 6.72-7.64 kcal/mol. NBO and DFT data reveals that the n→π* interactions and proline ring puckering stabilize azaproline chirality with the neighbouring proline stereochemistry. The CD, solvent titration, variable-temperature and 2D NMR experimental results further supported the crystal structures conformation.

Probing Peptidylprolyl Bond cis/trans Status Using Distal 19 F NMR Reporters.

A method for measuring peptidylprolyl bond cis-trans conformational status in peptide models is described, using 4-fluorophenylalanine (4FPhe) as a distal reporter for 19 F NMR. The %cis-Pro population was measured for peptides of the general structure Ac-X-Pro-Z-Ala-Ala-4FPhe (X and Z are proteinogenic amino acids) at pH 7.4, and provided conformational populations consistent with literature values obtained by more complex methods. This approach was applied to probe the prolyl bond status in pentapeptide models of the intrinsically disordered C-terminal region of α-synuclein, which mirrored the preferences in the Ac-X-Pro-Z-Ala-4FPhe models. Advantageously, the 19 F reporter group does not need to be adjacent to or attached to proline to provide quantifiable signals and distal 4-fluorophenylalanines can be placed so as not to influence prolyl bond conformation. Finally, we demonstrated that the prolyl bond status is not significantly affected by pH when there are ionisable amino acid residues at the carboxyl side of proline, which makes 19 F NMR an invaluable tool with which to study proline isomerism at a range of pHs and in different solvents and buffers.

First report on the synthesis and structural studies of trans-Phakellistatin 18: a rotamer of marine natural product phakellistatin 18.

Phakellistatin peptides from marine organisms are the sources of proline-rich cyclic peptides with reported significant antitumor activities. Phakellistatin 18 (1), reported from marine sponge Phakellia fusca, contains three proline-peptide linkages in cis form. We attempted the total synthesis of natural product 1 through solution-phase macrocyclization approach, as a result, the synthetic cyclic peptide 2 was obtained as a rotamer of natural product having all three proline residues in trans-conformation. Here, we describe the synthesis, structural, and cytotoxicity studies of trans-Phakellistatin 18 (2), and its analog [Ala1,3,6]-Phakellistatin 18 (3). Detailed NMR studies were carried out to characterize the synthesized peptides, and anti-cancer screening was performed by using MTT assay. The synthetic trans-Phakellistatin 18 (2) (IC50=67.5 ± 2.938 µM) showed comparable cytotoxicity against HepG2 cancer cell line with standard drug doxorubicin (IC50=63.88 ± 6.48 µM). Here, the first synthetic and structural studies on trans-Phakellistatin 18 (2), and its anticancer screening against HepG2 cell line was reported.