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A set of 15 cyclic‐hexaalanine and 10 cyclic‐pentaalanine peptides containing one or two backbone N‐aryl amide bonds were synthesized by following a combination of solution‐phase and solid‐phase peptide synthesis. NMR‐based conformation studies of these N‐aryl cyclic‐hexaalanine peptides revealed five distinct template conformations with an antiparallel β‐sheet structure; for N‐aryl cyclic‐pentaalanine peptides three template structures were revealed. All the template structures have distinct peptide‐turn features. The conformations in these N‐aryl peptides were compared to those in the commonly studied N‐methyl peptide analogues. We observed that the N‐aryl peptides exhibit a considerable conformational homogeneity, and their conformations differ significantly from those in N‐methyl analogues. We anticipate that the N‐arylation of backbone amides has the potential for application as a novel tool for conformation and physicochemical modification in peptides. [ABSTRACT FROM AUTHOR]

The dramatic rise in cancer incidence, alongside treatment deficiencies, has elevated cancer to the second-leading cause of death globally. The increasing morbidity and mortality of this disease can be traced back to a number of causes, including treatment-related side effects, drug resistance, inadequate curative treatment and tumor relapse. Recently, anti-cancer bioactive peptides (ACPs) have emerged as a potential therapeutic choice within the pharmaceutical arsenal due to their high penetration, specificity and fewer side effects. In this contribution, we present a general overview of the literature concerning the conformational structures, modes of action and membrane interaction mechanisms of ACPs, as well as provide recent examples of their successful employment as targeting ligands in cancer treatment. The use of ACPs as a diagnostic tool is summarized, and their advantages in these applications are highlighted. This review expounds on the main approaches for peptide synthesis along with their reconstruction and modification needed to enhance their therapeutic effect. Computational approaches that could predict therapeutic efficacy and suggest ACP candidates for experimental studies are discussed. Future research prospects in this rapidly expanding area are also offered. [ABSTRACT FROM AUTHOR]

The neuropeptides orexin A and B regulate various vital functions of the body, such as sleep/wake states, metabolism, and energy homeostasis. A loss of their physiological activity, with reduced ability to recognize their receptors, is suspected to be associated with oxidative stress conditions. These are related to excessive presence of reactive oxygen and nitrogen species, as well as of reactive lipoxidation byproducts. With the aim of evaluating the effects of oxidative stress on the secondary structure of orexin peptides, orexin B was synthesized and characterized by circular dichroism spectroscopy under different conditions. In aqueous solution it presents an unordered conformation, while in a membrane mimetic environment it assumes a helical structure. The effects of oxidative stress were evaluated exposing it to both oxygen and nitrogen radicals as well as to lipoxidation byproducts. The results showed that ROS, but not NRS, induced appreciable conformational changes, and only in the membrane mimetic environment. Lipoxidation byproducts, instead, led to secondary structure modifications much more evident than those induced by the direct action of ROS and RNS, and in both analyzed media. Additionally, MALDI-TOF analyses detected mass variations in the peptide attributable to oxidation of the C-terminal Met residue and deamination of asparagine in the Asn–His sequence. Taken together, all these data seem to confirm the involvement of oxidative processes in dysfunctions of the orexinergic system. [ABSTRACT FROM AUTHOR]

Cyclopurpuracin is a cyclooctapeptide isolated from the methanol extract of Annona purpurea seeds with the following sequence cyclo‐Gly‐Phe‐Ile‐Gly‐Ser‐Pro‐Val‐Pro residue. In this study, a reversed cyclopurpuracin, namely cyclo‐Pro‐Val‐Pro‐Ser‐Gly‐Ile‐Phe‐Gly, was successfully synthesized using a combination of solid and solution phase synthesis methods. The precursor was head‐to‐tail cyclized using 1.25 mM in DCM with 3 eq. HBTU and 1% v/v DIPEA. The final product was obtained (3.8 mg) with an overall yield of 7.4%. The synthetic products were characterized by HR‐ToF‐MS, 1H‐NMR, 13C‐NMR, and 2D NMR, showing different NMR profiles. The presence of the conformation was confirmed by providing additional signals in the 1H NMR and 13C NMR spectrum and the correlation peaks shown by 2D NMR. The presence of the cis conformer in conformer B was proven by the chemical shifts of Ser6 Cβ, Pro7 Cβ, and Pro8 Cβ in conformer B, which were more upfield than the conformer A chemical shifts, and the presence of NOE correlation between Hα/Hα of Val7 and Pro8. The NMR of the reversed cyclopurpuracin revealed the presence of two conformers, conformer A (cyclo‐[trans‐Pro‐Val‐trans‐Pro‐Ser‐Gly‐Ile‐Phe‐Gly]) and conformer B (cyclo‐[cis‐Pro‐Val‐trans‐Pro‐Ser‐Gly‐Ile‐Phe‐Gly]). The presence of dual conformers has not been observed in the NMR spectra of natural cyclopurpuracin. [ABSTRACT FROM AUTHOR]

The N-capping region of an α-helix is a short N-terminal amino acid stretch that contributes to nucleate and stabilize the helical structure. In the VEGF mimetic helical peptide QK, the N-capping region was previously demonstrated to be a key factor of QK helical folding. In this paper, we explored the effect of the chiral inversion of the N-capping sequence on QK folding, performing conformational analysis in solution by circular dichroism and NMR spectroscopy. The effect of such a modification on QK stability in serum and the proliferative effect were also evaluated. [ABSTRACT FROM AUTHOR]

Peptides that increase pro-reparative responses to injury and disease by modulating the functional organization of hyaluronan (HA) with its cell surface binding proteins (e.g., soluble receptor for hyaluronan-mediated motility [RHAMM] and integral membrane CD44) have potential therapeutic value. The binding of RHAMM to HA is an attractive target, since RHAMM is normally absent or expressed at low levels in homeostatic conditions, but its expression is significantly elevated in the extracellular matrix during tissue stress, response-to-injury, and in cancers and inflammation-based diseases. The HA-binding site in RHAMM contains two closely spaced sequences of clustered basic amino acids, in an alpha-helical conformation. In the present communication, we test whether an alpha-helical conformation is required for effective peptide binding to HA, and competitive disruption of HA-RHAMM interaction. The HA-binding RHAMM-competitive peptide P15-1, identified using the unbiased approach of phage display, was examined using circular dichroism spectroscopy and the conformation-predictive AI-based AlphaFold2 algorithm. Unlike the HA-binding site in RHAMM, peptide P15-1 was found to adopt irregular conformations in solution rather than alpha helices. Instead, our structural analysis suggests that the primary determinant of peptide-HA binding is associated with a specific clustering and spacing pattern of basic amino acids, allowing favorable electrostatic interaction with carboxylate groups on HA., Competing Interests: Mary Cowman, Thorsten Kirsc,h and Eva Turley are listed as inventors on US patent 10,449,229 B2. The rest of the authors declare no conflict of interest. The sponsors had no role in the design, execution, interpretation, or writing of the study., (© 2024 The Authors. Proteoglycan Research published by Wiley Periodicals LLC.)

[Display omitted] • Designed and synthesized 10 macrocyclic 14-mer peptides based on Tau PHF peptides. • NMR supported antiparallel β-sheet structure in all peptides, validating the design. • P3 and P8 showed the highest and lowest self-aggregation tendencies, respectively. • In cross-aggregation studies, P3 promoted PHF aggregation, P8 selectively reduced it. • P3 and P8 exhibited pro- and anti-aggregation effects even on Tau macromolecule. Ten macrocyclic peptides, each comprising 14 amino acids, were designed and synthesized based on the Tau aggregation model hexapeptides AcPHF6* and AcPHF6. The design took into account the aggregation tendencies of each residue in AcPHF6* and AcPHF6, their aggregation models, while employing peptide-based structural design principles including N- methylation to promote turns and to block hydrogen bond propagation and elongation of the aggregation chain. NMR analysis supported that all these peptides adopted an antiparallel β-sheet conformation. Self-aggregation studies characterized the aggregation properties of these peptides, identifying two peptides with the highest (P3) and lowest (P8) aggregation tendencies. In cross-aggregation studies with the parent peptides AcPHF6* and AcPHF6, P3 and P8 were found to promote and reduce aggregation, respectively. Furthermore, P3 and P8 demonstrated an enhancement and diminution effect on the aggregation of K18wt, indicating their capacity to modulate aggregation even at the macromolecular level. Thus, the two simple peptides, P3 and P8 selectively exhibit pro- or anti-aggregation effects on PHF peptides and Tau. This study, has thus developed structurally well-defined non-complex peptides, derived from AcPHF6* and AcPHF6, to modulate Tau aggregation as desired, offering applications in Tau model studies and the development of Tau aggregation inhibitors or promoters. [ABSTRACT FROM AUTHOR]

Most cases of Alzheimer's disease (AD) are sporadic, but a small percentage of AD cases, called familial AD (FAD), are associated with mutations in presenilin 1, presenilin 2, or the amyloid precursor protein. Amyloid precursor protein mutations falling within the amyloid-β (Aβ) sequence lead to a wide range of disease phenotypes. There is increasing evidence that distinct amyloid structures distinguished by amyloid conformation-dependent monoclonal antibodies have similarly distinct roles in pathology. It is possible that this phenotypic diversity of FAD associated with mutations within the Aβ sequence is due to differences in the conformations adopted by mutant Aβ peptides, but the effects of FAD mutations on aggregation kinetics and conformational and morphological changes of the Aβ peptide are poorly defined. To gain more insight into this possibility, we therefore investigated the effects of 11 FAD mutations on the aggregation kinetics of Aβ, as well as its ability to form distinct conformations recognized by a panel of amyloid conformation-specific monoclonal antibodies. We found that most FAD mutations increased the rate of aggregation of Aβ. The FAD mutations also led to the adoption of alternative amyloid conformations distinguished by monoclonal antibodies and resulted in the formation of distinct aggregate morphologies as determined by transmission electron microscopy. In addition, several of the mutant peptides displayed a large reduction in thioflavin T fluorescence, despite forming abundant fibrils indicating that thioflavin T is a probe of conformational polymorphisms rather than a reliable indicator of fibrillization. Taken together, these results indicate that FAD mutations falling within the Aβ sequence lead to dramatic changes in aggregation kinetics and influence the ability of Aβ to form immunologically and morphologically distinct amyloid structures.

The dramatic rise in cancer incidence, alongside treatment deficiencies, has elevated cancer to the second-leading cause of death globally. The increasing morbidity and mortality of this disease can be traced back to a number of causes, including treatment-related side effects, drug resistance, inadequate curative treatment and tumor relapse. Recently, anti-cancer bioactive peptides (ACPs) have emerged as a potential therapeutic choice within the pharmaceutical arsenal due to their high penetration, specificity and fewer side effects. In this contribution, we present a general overview of the literature concerning the conformational structures, modes of action and membrane interaction mechanisms of ACPs, as well as provide recent examples of their successful employment as targeting ligands in cancer treatment. The use of ACPs as a diagnostic tool is summarized, and their advantages in these applications are highlighted. This review expounds on the main approaches for peptide synthesis along with their reconstruction and modification needed to enhance their therapeutic effect. Computational approaches that could predict therapeutic efficacy and suggest ACP candidates for experimental studies are discussed. Future research prospects in this rapidly expanding area are also offered.

The neuropeptides orexin A and B regulate various vital functions of the body, such as sleep/wake states, metabolism, and energy homeostasis. A loss of their physiological activity, with reduced ability to recognize their receptors, is suspected to be associated with oxidative stress conditions. These are related to excessive presence of reactive oxygen and nitrogen species, as well as of reactive lipoxidation byproducts. With the aim of evaluating the effects of oxidative stress on the secondary structure of orexin peptides, orexin B was synthesized and characterized by circular dichroism spectroscopy under different conditions. In aqueous solution it presents an unordered conformation, while in a membrane mimetic environment it assumes a helical structure. The effects of oxidative stress were evaluated exposing it to both oxygen and nitrogen radicals as well as to lipoxidation byproducts. The results showed that ROS, but not NRS, induced appreciable conformational changes, and only in the membrane mimetic environment. Lipoxidation byproducts, instead, led to secondary structure modifications much more evident than those induced by the direct action of ROS and RNS, and in both analyzed media. Additionally, MALDI-TOF analyses detected mass variations in the peptide attributable to oxidation of the C-terminal Met residue and deamination of asparagine in the Asn–His sequence. Taken together, all these data seem to confirm the involvement of oxidative processes in dysfunctions of the orexinergic system.

The Ebola delta peptide is an amphipathic, 40-residue peptide encoded by the Ebola virus, referred to as E40. The membrane-permeabilising activity of the E40 delta peptide has been demonstrated in cells and lipid vesicles suggesting the E40 delta peptide likely acts as a viroporin. The lytic activity of the peptide increases in the presence of anionic lipids and a disulphide bond in the C-terminal part of the peptide. Previous in silico work predicts the peptide to show a partially helical structure, but there is no experimental information on the structure of E40. Here, we use circular dichroism spectroscopy to report the secondary structure propensities of the reduced and oxidised forms of the E40 peptide in water, detergent micelles, and lipid vesicles composed of neutral and anionic lipids (POPC and POPG, respectively). Results indicate that the peptide is predominately a random coil in solution, and the disulphide bond has a small but measurable effect on peptide conformation. Secondary structure analysis shows large uncertainties and dependence on the reference data set and, in our system, cannot be used to accurately determine the secondary structure motifs of the peptide in membrane environments. Nevertheless, the spectra can be used to assess the relative changes in secondary structure propensities of the peptide depending on the solvent environment and disulphide bond. In POPC-POPG vesicles, the peptide transitions from a random coil towards a more structured conformation, which is even more pronounced in negatively charged SDS micelles. In vesicles, the effect depends on the peptide-lipid ratio, likely resulting from vesicle surface saturation. Further experiments with zwitterionic POPC vesicles and DPC micelles show that both curvature and negatively charged lipids can induce a change in conformation, with the two effects being cumulative. Electrostatic screening from Na + ions reduced this effect. The oxidised form of the peptide shows a slightly lower propensity for secondary structure and retains a more random coil conformation even in the presence of PG-PC vesicles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The N-capping region of an α-helix is a short N-terminal amino acid stretch that contributes to nucleate and stabilize the helical structure. In the VEGF mimetic helical peptide QK, the N-capping region was previously demonstrated to be a key factor of QK helical folding. In this paper, we explored the effect of the chiral inversion of the N-capping sequence on QK folding, performing conformational analysis in solution by circular dichroism and NMR spectroscopy. The effect of such a modification on QK stability in serum and the proliferative effect were also evaluated.

Amyloidogenic proteins generally form intermolecularly hydrogen-bonded β-sheet aggregates, including parallel, in-register β-sheets (recognized by antiserum OC) or antiparallel β-sheets, β-solenoids, β-barrels, and β-cylindrins (recognized by antiserum A11). Although these groups share many common properties, some amyloid sequences have been reported to form polymorphic structural variants or strains. We investigated the humoral immune response to Aβ42 fibrils and produced 23 OC-type monoclonal antibodies recognizing distinct epitopes differentially associated with polymorphic structural variants. These mOC antibodies define at least 18 different immunological profiles represented in aggregates of amyloid-β (Aβ). All of the antibodies strongly prefer amyloid aggregates over monomer, indicating that they recognize conformational epitopes. Most of the antibodies react with N-terminal linear segments of Aβ, although many recognize a discontinuous epitope consisting of an N-terminal domain and a central domain. Several of the antibodies that recognize linear Aβ segments also react with fibrils formed from unrelated amyloid sequences, indicating that reactivity with linear segments of Aβ does not mean the antibody is sequence-specific. The antibodies display strikingly different patterns of immunoreactivity in Alzheimer disease and transgenic mouse brain and identify spatially and temporally unique amyloid deposits. Our results indicate that the immune response to Aβ42 fibrils is diverse and reflects the structural polymorphisms in fibrillar amyloid structures. These polymorphisms may contribute to differences in toxicity and consequent effects on pathological processes. Thus, a single therapeutic monoclonal antibody may not be able to target all of the pathological aggregates necessary to make an impact on the overall disease process.

Proteins can be targeted to organellar membranes by using a tail anchor (TA), a stretch of hydrophobic amino acids found at the polypeptide carboxyl-terminus. The Fis1 protein (Fis1p), which promotes mitochondrial and peroxisomal division in the yeast Saccharomyces cerevisiae, is targeted to those organelles by its TA. Substantial evidence suggests that Fis1p insertion into the mitochondrial outer membrane can occur without the need for a translocation machinery. However, recent findings raise the possibility that Fis1p insertion into mitochondria might be promoted by a proteinaceous complex. Here, we have performed atomistic and coarse-grained molecular dynamics simulations to analyze the adsorption, conformation, and orientation of the Fis1(TA). Our results support stable insertion at the mitochondrial outer membrane in a monotopic, rather than a bitopic (transmembrane), configuration. Once inserted in the monotopic orientation, unassisted transition to the bitopic orientation is expected to be blocked by the highly charged nature of the TA carboxyl-terminus and by the Fis1p cytosolic domain. Our results are consistent with a model in which Fis1p does not require a translocation machinery for insertion at mitochondria.

Methods to constrain peptides in a bioactive α‐helical conformation for inhibition of protein‐protein interactions represent an ongoing area of investigation in chemical biology. Recently, the first example of a reversible "stapling" methodology was described which exploits native cysteine or homocysteine residues spaced at the i and i + 4 positions in a peptide sequence together with the thiol selective reactivity of dibromomaleimides (a previous study). This manuscript reports on the optimization of the maleimide based constraint, focusing on the kinetics of macrocyclization and the extent to which helicity is promoted with different thiol containing amino acids. The study identified an optimal stapling combination of X1 = L‐Cys and X5 = L‐hCys in the context of the model peptide Ac‐X1AAAX5‐NH2, which should prove useful in implementing the dibromomaleimide stapling strategy in peptidomimetic ligand discovery programmes. [ABSTRACT FROM AUTHOR]

Circular dichroism (CD) spectroscopy is an essential tool for determining the conformation of proteins and peptides in membranes. It can be particularly useful for measuring the free energy of partitioning of peptides into lipid vesicles. The belief is broadly held that such CD measurements can only be made using sonicated small unilamellar vesicles (SUVs) because light scattering associated with extruded large unilamellar vesicles (LUVs) is unacceptably high. We have examined this issue using several experimental approaches in which a chiral object (i.e., peptide or protein) is placed both on the membrane and outside the membrane. We show that accurate CD spectra can be collected in the presence of LUVs. This is important because SUVs, unlike LUVs, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. Our data reveal that undistorted CD spectra of peptides can be measured at wavelengths above 200 nm in the presence of up to 3 mM LUVs and above 215 nm in the presence of up to 7 mM LUVs. We introduce a simple way of characterizing the effect on CD spectra of light scattering and absorption arising from suspensions of vesicles of any diameter. Using melittin as an example, we show that CD spectroscopy can be used to determine the fractional helical content of peptides in LUVs and to measure their free energy of partitioning of into LUVs.

The cross‐strand disulfides (CSDs) found in β‐hairpin antimicrobial peptides (β‐AMPs) show a unique disulfide geometry that is characterized by unusual torsion angles and a short Cα‐Cα distance. While the sequence and disulfide bond connectivity of disulfide‐rich peptides is well studied, much less is known about the disulfide geometry found in CSDs and their role in the stability of β‐AMPs. To address this, we solved the nuclear magnetic resonance (NMR) structure of the β‐AMP gomesin (Gm) at 278, 298, and 310 K, examined the disulfide bond geometry of over 800 disulfide‐rich peptides, and carried out extensive molecular dynamics (MD) simulation of the peptides Gm and protegrin. The NMR data suggests Cα‐Cα distances characteristic for CSDs are independent of temperature. Analysis of disulfide‐rich peptides from the Protein Data Bank revealed that right‐handed and left‐handed rotamers are equally likely in CSDs. The previously reported preference for right‐handed rotamers was likely biased by restricting the analysis to peptides and proteins solved using X‐ray crystallography. Furthermore, data from MD simulations showed that the short Cα‐Cα distance is critical for the stability of these peptides. The unique disulfide geometry of CSDs poses a challenge to biomolecular force fields and to retain the stability of β‐hairpin fold over long simulation times, restraints on the torsion angles might be required. [ABSTRACT FROM AUTHOR]

Depsipeptides and cyclodepsipeptides are analogues of the corresponding peptides in which one or more amide groups are replaced by ester functions. Reports of crystal structures of linear depsipeptides are rare. The crystal structures and conformational analyses of four depsipeptides with an alternating sequence of an α,α‐disubstituted α‐amino acid and an α‐hydroxy acid are reported. The molecules in the linear hexadepsipeptide amide in (S)‐Pms‐Acp‐(S)‐Pms‐Acp‐(S)‐Pms‐Acp‐NMe2 acetonitrile solvate, C47H58N4O9·C2H3N, (3b), as well as in the related linear tetradepsipeptide amide (S)‐Pms‐Aib‐(S)‐Pms‐Aib‐NMe2, C28H37N3O6, (5a), the diastereoisomeric mixture (S,R)‐Pms‐Acp‐(R,S)‐Pms‐Acp‐NMe2/(R,S)‐Pms‐Acp‐(R,S)‐Pms‐Acp‐NMe2 (1:1), C32H41N3O6, (5b), and (R,S)‐Mns‐Acp‐(S,R)‐Mns‐Acp‐NMe2, C30H37N3O6, (5c) (Pms is phenyllactic acid, Acp is 1‐aminocyclopentanecarboxylic acid and Mns is mandelic acid), generally adopt a β‐turn conformation in the solid state, which is stabilized by intramolecular N—H...O hydrogen bonds. Whereas β‐turns of type I (or I′) are formed in the cases of (3b), (5a) and (5b), which contain phenyllactic acid, the torsion angles for (5c), which incorporates mandelic acid, indicate a β‐turn in between type I and type III. Intermolecular N—H...O and O—H...O hydrogen bonds link the molecules of (3a) and (5b) into extended chains, and those of (5a) and (5c) into two‐dimensional networks. [ABSTRACT FROM AUTHOR]

Anticancer peptides (ACPs) could potentially offer many advantages over other cancer therapies. ACPs often target cell membranes, where their surface mechanism is coupled to a conformational change into helical structures. However, details on their binding are still unclear, which would be crucial to reach progress in connecting structural aspects to ACP action and to therapeutic developments. Here we investigated natural helical ACPs, Lasioglossin LL-III, Macropin 1, Temporin-La, FK-16, and LL-37, on model liposomes, and also on extracellular vesicles (EVs), with an outer leaflet composition similar to cancer cells. The combined simulations and experiments identified three distinct binding modes to the membranes. Firstly, a highly helical structure, lying mainly on the membrane surface; secondly, a similar, yet only partially helical structure with disordered regions; and thirdly, a helical monomeric form with a non-inserted perpendicular orientation relative to the membrane surface. The latter allows large swings of the helix while the N-terminal is anchored to the headgroup region. These results indicate that subtle differences in sequence and charge can result in altered binding modes. The first two modes could be part of the well-known carpet model mechanism, whereas the newly identified third mode could be an intermediate state, existing prior to membrane insertion.

The critical role played by solvent environment in maintaining the conformational integrity of peptides and proteins is accepted without question. Numerous experiments have suggested that perturbing the solvent environment of peptides and proteins by the addition of polar organic solvents have important consequences for the conformation of these molecules. However, experimental studies of such perturbations often report different kinds of effects depending on the solvent used and/or the sequence/structure of the molecule under study. In this work we report a simulation based comparative study on the effects of adding two common organic solvents viz. Dimethyl sulfoxide (DMSO) and Acetonitrile (MeCN) on the dynamical conformation of a test peptide Ace-Gly-X-Gly-Nme where X is any amino acid. Our studies identify important differences in peptide solvation by these two solvents, which we attempt to correlate with the kinetic stability of the conformation, as well as the identity of the central 'X' residue in the test peptide. Image 1 • Hydrophobicity influences the preferential hydration of an amino acid. • Non-polar amino acids, tends to be preferentially solvated by Polar Organic Solvents. • Denser water clusters tending to occur in the vicinity of oxygen atoms of the peptide. • State transitions are sensitive to both: conformations and type of Organic Solvents. [ABSTRACT FROM AUTHOR]

Study of amino acid mutation effects on the structure, dynamics and function of peptides/proteins is of considerable prominence. Seminalplasmin (SPLN) is a 47-residue peptide isolated from bovine seminal plasma and has broad spectrum antimicrobial activity, without any hemolytic activity. The 28-40 segment of SPLN with the sequence PKLLETFLSKWIG, designated as SPF, is the most hydrophobic stretch of SPLN with helical structure and primarily responsible for the membrane-perturbing activity of SPLN. An analogue of SPF peptide with K2E and K10E substitutions, designated as SPFE, is lacking helical structure and hence antimicrobial activity. We have synthesized, by solid phase method, and solved the conformation, by NMR spectroscopic method, of two overlapping fragments of SPFE with the sequences PELLETFL (1-8 fragment) and FLSEWIG (7-13 fragment) under hydrophobic environment (DMSO-d6). Results show that the lysine residues at positions 2 and 10 are very crucial for the helical structure and hence the biological activity of SPF peptide. The experimental results are in harmony with the formerly testified molecular dynamic simulation studies. [ABSTRACT FROM AUTHOR]

The work presented below is related to our companion paper in this issue, entitled: Substance P in solution: trans-to-cis configurational changes of penultimate prolines initiate non-enzymatic peptide bond cleavages. Two-dimensional ion mobility spectrometry (IMS-IMS) and mass spectrometry techniques are used to investigate structural transitions for [M+3H]3+ ions of substance P (subP) upon collisional activation (CA) in the gas phase. In this approach, different conformations of ions having a specified mobility are selected after an initial IMS separation, collisionally activated to produce new conformers, and these product structures are separated again using a second IMS region. In this way, it is possible to follow folding and unfolding transitions of different conformations. The analysis shows evidence for five conformations. Unlike other systems, every transition is irreversible. Studies as a function of activation voltage are used to discern pathways of structural changes prior to reaching the energy required for dissociation. Thresholds associated with the onsets of transitions are calibrated to obtain estimates of the energetic barriers between different structures and semi-quantitative potential energy diagrams are presented. Overall, barriers associated with structural transitions of [subP+3H]3+ in the absence of solvent are on the order of ~ 40 kJ mol−1, substantially lower than the ~ 90 kJ mol−1 required for some similar structural transitions in solutions of ethanol. Comparisons of the transition energies in the gas phase with thermochemistry for similar transitions in solution provide clues about why reverse transitions are prohibited. [ABSTRACT FROM AUTHOR]

We report ion mobility spectrometry and mass spectrometry studies of the non-enzymatic step-by-step degradation of substance P (subP), an 11-residue neuropeptide, with the sequence Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-Phe8-Gly9-Leu10-Met11-NH2, in ethanol. At elevated solution temperatures (55 to 75 °C), several reactions are observed, including a protonation event, i.e., [subP+2H]2+ + H+ → [subP+3H]3+, that appears to be regulated by a configurational change and two sequential bond cleavages (the Pro2-Lys3 peptide bond is cleaved to form the smaller nonapeptide Lys3-Met11-NH2 [subP(3–11)], and subsequently, subP(3–11) is cleaved at the Pro4-Gln5 peptide bond to yield the heptapeptide Gln5-Met11-NH2 [subP(5–11)]). Each of the product peptides [subP(3–11) and subP(5–11)] is accompanied by a complementary diketopiperazine (DKP): cyclo-Arg1-Pro2 (cRP) for the first cleavage, and cyclo-Lys3-Pro4 (cKP) for the second. Insight about the mechanism of degradation is obtained by comparing kinetics calculations of trial model mechanisms with experimental data. The best model of our experimental data indicates that the initial cleavage of subP is regulated by a conformational change, likely a trans→cis isomerization of the Arg1-Pro2 peptide bond. The subP(3–11) product has a long lifetime (t1/2 ~ 30 h at 55 °C) and appears to transition through several structural intermediates prior to dissociation, suggesting that subP(3–11) is initially formed with a Lys3-trans-Pro4 peptide bond configuration and that slow trans→cis isomerization regulates the second bond cleavage event as well. From these data and our model mechanisms, we obtain transition state thermochemistry ranging from ΔH‡ = 41 to 85 kJ mol−1 and ΔS‡ = − 43 to – 157 J mol−1 K−1 for each step in the reaction. [ABSTRACT FROM AUTHOR]

There is a need in the field of biological mass spectrometry for structural tools which can report on regional, rather than solely global, structure of gaseous protein ions. Site-specific hydrogen-deuterium (H/D) exchange has shown promise in fulfilling this need, but requires additional method development to prove its utility. In this study, we use H/D exchange and electron capture dissociation (ECD) to probe the gaseous structure of two peptides which are α-helical in solution and which differ by a single point mutation. Global H/D exchange levels, ECD fragmentation profiles, and region specific H/D exchange profiles are compared between wild type (WT) melittin, which adopts a hinged helix conformation in solution, and a mutant P14A melittin which folds into a single helix in solution. High protection from H/D exchange by both peptides is consistent with retention of secondary structure in the gas phase (or refolding into some other compact structure). The P14A mutant melittin exhibits lower ECD fragmentation efficiency than WT melittin, suggesting that it contains more secondary structure in the gas phase, which may indicate that these peptides retain some memory of their solution-phase structures. Examination of the isotopic distributions of fragment ions derived from H/D exchange with subsequent ECD reveals that the C-terminus of these peptides adopts multiple conformations. The results reported here offer insight into the stability of alpha helices in the gas phase, and also highlight the value of combining gas-phase H/D exchange with electron capture dissociation to interrogate gaseous peptide conformation. [ABSTRACT FROM AUTHOR]

Keywords: cyclic peptides; N-arylation; N-modified amino acids; peptide-based drug design; peptide conformation EN cyclic peptides N-arylation N-modified amino acids peptide-based drug design peptide conformation 1 1 1 07/19/23 20230714 NES 230714 I B N b i B -aryl amide bonds for peptide conformation b : In the universe of peptides, the I N i -aryl amide bond, with its large steric and electronic effects and ability to restrain amide bond rotation, is represented as a new star. Cover Feature: Exploration of N-Arylation of Backbone Amides as a Novel Tool for Conformational Modification in Peptides (Chem. Eur. J. 40/2023) Cyclic peptides, N-arylation, N-modified amino acids, peptide-based drug design, peptide conformation. [Extracted from the article]

Protein concentrates were prepared from defatted barley ( Hordeum vulgare L.) flour using alkaline and enzymatic treatments. Milder enzymatic treatments included (i) a bi-enzymatic method involving the use of starch-hydrolyzing enzymes, and (ii) a tri-enzymatic method using the former bi-enzymatic treatment followed by digestion with glucanase. The concentrate obtained through alkaline extraction (AI-BP) was comprised mainly of low molecular weight fractions of proteins. Bi-enzymatic treatment produced a protein concentrate with the highest protein content (49.0%), while those obtained by the tri-enzymatic treatment followed by an isoelectric precipitation step (TEI-BP) gave the highest protein recovery yield (78.3%). In both of the latter concentrates, 35 kDa B-hordeins were the major protein fraction. Divergence in secondary/tertiary structure elements (AI-BP; TEI-BP) was obtained and attributed to the difference in the protein profiles. Further characterization of protein concentrates indicated that they exhibit pseudoplastic behavior. Emulsifying capacity of concentrates was comparable to that of whey protein isolate. [ABSTRACT FROM AUTHOR]

The stabilization of β-sheet secondary structure through peptide backbone modification represents an attractive approach to protein mimicry. Here, we present strategies toward stable β-hairpin folds based on peptide strand N -amination. Novel pyrazolidinone and tetrahydropyridazinone dipeptide constraints were introduced via on-resin Mitsunobu cyclization between α-hydrazino acid residues and a serine or homoserine side chain. Acyclic and cyclic N -amino peptide building blocks were then evaluated for their effect on β-hairpin stability in water using a GB1-derived model system. Our results demonstrate the strong β-sheet stabilizing effect of the peptide N -amino substituent, and provide useful insights into the impact of covalent dipeptide constraint on β-sheet folding. [ABSTRACT FROM AUTHOR]

Vibrational circular dichroism (VCD) has become a standard method for determination of absolute stereochemistry, particularly now that reliable commercial instrumentation has become available. These instruments use a now well-documented Fourier transform infrared-based approach to measure VCD that has virtually displaced initial dispersive infrared-based designs. Nonetheless, many papers have appeared reporting dispersive VCD data, especially for biopolymers. Instrumentation designed with these original methods, particularly after more recent updates optimizing performance in selected spectral regions, has been shown still to have advantages for specific applications. This article presents a mini-review of dispersive VCD instrument designs and includes sample spectra obtained for various biopolymer (particularly peptide) samples. Complementary reviews of Fourier transform-VCD designs are broadly available. [ABSTRACT FROM AUTHOR]

The conventional anticancer therapeutics usually lack cancer specificity, leading to damage of normal tissues that patients find hard to tolerate. Ideally, anticancer therapeutics carrying payloads of drugs equipped with cancer targeting peptides can act like “guided missiles” with the capacity of targeted delivery toward many types of cancers. Peptides are amenable for conjugation to nano drugs for functionalization, thereby improving drug delivery and cellular uptake in cancer-targeting therapies. Peptide drugs are often more difficult to design through molecular docking and in silico analysis than small molecules, because peptide structures are more flexible, possess intricate molecular conformations, and undergo complex interactions. In this review, the development and application of strategies for structure-based design of cancer-targeting peptides against GRP78 are discussed. This Review also covers topics related to peptide pharmacokinetics and targeting delivery, including molecular docking studies, features that provide advantages for in vivo use, and properties that influence the cancer-targeting ability. Some advanced technologies and special peptides that can overcome the pharmacokinetic challenges have also been included. [ABSTRACT FROM AUTHOR]

Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site-specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini-review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra. [ABSTRACT FROM AUTHOR]

The discovery of Intrinsically Disordered Proteins, which contain significant levels of disorder yet perform complex biologically functions, as well as unwanted aggregation, has motivated numerous experimental and theoretical studies aimed at describing residue-level conformational ensembles. Multiple lines of evidence gathered over the last 15 years strongly suggest that amino acids residues display unique and restricted conformational preferences in the unfolded state of peptides and proteins, contrary to one of the basic assumptions of the canonical random coil model. To fully understand residue level order/disorder, however, one has to gain a quantitative, experimentally based picture of conformational distributions and to determine the physical basis underlying residue-level conformational biases. Here, we review the experimental, computational and bioinformatic evidence for conformational preferences of amino acid residues in (mostly short) peptides that can be utilized as suitable model systems for unfolded states of peptides and proteins. In this context particular attention is paid to the alleged high polyproline II preference of alanine. We discuss how these conformational propensities may be modulated by peptide solvent interactions and so called nearest-neighbor interactions. The relevance of conformational propensities for the protein folding problem and the understanding of IDPs is briefly discussed.

The conformational characteristics of protected homo-oligomeric Boc-[β3(R)Val]n-OMe, n = 1, 2, 3, 4, 6, 9, and 12 have been investigated in organic solvents using nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) absorption spectroscopy and circular dichroism (CD) methods. The detailed 1H NMR analysis of Boc-[β3(R)Val]12-OMe reveals that the peptide aggregates extensively in CDCl3, but is disaggregated in 20%, (v/v) dimethyl sulfoxide (DMSO) in CDCl3 and in CD3OH. Limited assignment of the N-terminus NH groups, together with solvent dependence of NH chemical shifts and temperature coefficients provides evidence for 14-helix conformation in the 12-residue peptide. FTIR analysis in CHCl3 establishes that the onset of folding and aggregation, as evidenced by NH stretching bands at 3375 cm−1 (intramolecular) and 3285 cm−1 (intermolecular), begins at the level of the tetrapeptide. The observed CD bands, 214 nm (negative) and 198 nm (positive), support 14-helix formation in the 9 and 12 residue sequences. The folding and aggregation tendencies of homo-oligomeric α-, β-, and γ- residues is compared in the model peptides Boc-[ωVal]n-NHMe, ω = α, β, and γ and n = 1, 2, and 3. Analysis of the FTIR spectra in CHCl3, establish that the tendency to aggregate at the di and tripeptide level follows the order β > α∼γ, while the tendency to fold follows the order γ > β > α. [ABSTRACT FROM AUTHOR]

The human Pin1 WW domain catalyzes the cis- trans isomerization of the proline peptide bond. In this study, the conformation and binding of Cu(II) ions by Pin1 were investigated. It has been found that the affinity of peptide fragments of the human Pin1 WW domain for Cu(II) ions depends on its conformation. In particular, we analyzed three peptides derived from human Pin1: the nonapeptide hPin1(14-22) (with sequence Arg-Met-Ser-Arg-Ser-Ser-Gly-Arg-Val-NH, peptide 1) the undecapeptide hPin1(13-23) (with sequence Lys-Arg-Met-Ser-Arg-Ser-Ser-Gly-Arg-Val-Tyr-NH, peptide 2) and its derivative Ala13Ala23hPin1(13-23) (with sequence Ala-Arg-Met-Ser-Arg-Ser-Ser-Gly-Arg-Val-Ala-NH, peptide 3) to study the role of presence in the sequence of the flanked residues at the N- and C-terminus, i.e., Lys13 and Tyr23. The presence of heat-capacity peaks found by DSC measurements for the systems studied strongly suggests that the conformational equilibria of the peptides studied strongly depend on the temperature. NMR spectroscopy and molecular dynamics simulations were instrumental to verify the conformational preferences of three peptides. The absence of likely or oppositely charged groups at the ends of a short chain fragment destroys chain reversal because the charged groups probably screen the nonpolar core from the solvent. ITC experiment was used to study the interactions with Cu(II) ions. It was found that the most stable complexes with Cu ions are formed with peptide 2, which has the most bent conformation. [ABSTRACT FROM AUTHOR]

Oligomeric aggregates of the amyloid-beta peptide(1-42) (Aβ42) are regarded as a primary cause of cytotoxicity related to membrane damage in Alzheimer's disease. However, a dynamical and structural characterization of pore-forming Aβ42 oligomers at atomic detail has not been feasible. Here, we used Aβ42 oligomer structures previously determined in a membrane-mimicking environment as putative model systems to study the pore formation process in phospholipid bilayers with all-atom molecular dynamics simulations. Multiple Aβ42 oligomer sizes, conformations, and N-terminally truncated isoforms were investigated on the multi-μs time scale. We found that pore formation and ion permeation occur via edge conductivity and exclusively for β-sandwich structures that feature exposed side-by-side β-strand pairs formed by residues 9 to 21 of Aβ42. The extent of pore formation and ion permeation depends on the insertion depth of hydrophilic residues 13 to 16 (HHQK domain) and thus on subtle differences in the overall stability, orientation, and conformation of the aggregates in the membrane. Additionally, we determined that backbone carbonyl and polar side-chain atoms from the edge strands directly contribute to the coordination sphere of the permeating ions. Furthermore, point mutations that alter the number of favorable side-chain contacts correlate with the ability of the Aβ42 oligomer models to facilitate ion permeation in the bilayer center. Our findings suggest that membrane-inserted, layered β-sheet edges are a key structural motif in pore-forming Aβ42 oligomers independent of their size and play a pivotal role in aggregate-induced membrane permeabilization., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The hydrophobic pocket found in the N-heptad repeat (NHR) region of HIV-1 gp41 is a highly conserved epitope that is the target of various HIV-1-neutralizing monoclonal antibodies. Although the high conservation of the pocket makes it an attractive vaccine candidate, it has been challenging to elicit potent anti-NHR antibodies via immunization. Here, we solved a high-resolution structure of the NHR mimetic IQN17, and, consistent with previous ligand-bound gp41 pocket structures, we observed remarkable conformational plasticity of the pocket. The high malleability of this pocket led us to test whether we could improve the immunogenicity of the gp41 pocket by stabilizing its conformation. We show that the addition of five amino acids at the C terminus of IQN17, to generate IQN22, introduces a stabilizing salt bridge at the base of the peptide that rigidifies the pocket. Mice immunized with IQN22 elicited higher avidity antibodies against the gp41 pocket and a more potent, albeit still weak, neutralizing response against HIV-1 compared with IQN17. Stabilized epitope-focused immunogens could serve as the basis for future HIV-1 fusion-inhibiting vaccines., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Ocean acidification is a global challenge that faces marine organisms in the near future with a predicted rapid drop in pH of up to 0.4 units by the end of this century. Effects of the change in ocean carbon chemistry and pH on the development, growth and fitness of marine animals are well documented. Recent evidence also suggests that a range of chemically mediated behaviours and interactions in marine fish and invertebrates will be affected. Marine animals use chemical cues, for example, to detect predators, for settlement, homing and reproduction. But, while effects of high CO2 conditions on these behaviours are described across many species, little is known about the underlying mechanisms, particularly in invertebrates. Here, we investigate the direct influence of future oceanic pH conditions on the structure and function of three peptide signalling molecules with an interdisciplinary combination of methods. NMR spectroscopy and quantum chemical calculations were used to assess the direct molecular influence of pH on the peptide cues, and we tested the functionality of the cues in different pH conditions using behavioural bioassays with shore crabs ( Carcinus maenas) as a model system. We found that peptide signalling cues are susceptible to protonation in future pH conditions, which will alter their overall charge. We also show that structure and electrostatic properties important for receptor binding differ significantly between the peptide forms present today and the protonated signalling peptides likely to be dominating in future oceans. The bioassays suggest an impaired functionality of the signalling peptides at low pH. Physiological changes due to high CO2 conditions were found to play a less significant role in influencing the investigated behaviour. From our results, we conclude that the change of charge, structure and consequently function of signalling molecules presents one possible mechanism to explain altered behaviour under future oceanic pH conditions. [ABSTRACT FROM AUTHOR]