Peptide Secondary Structure Research Articles

Peptide Retention Time Prediction in Hydrophilic Interaction Liquid Chromatography: Data Collection Methods and Features of Additive and Sequence-Specific Models.

The development of a peptide retention prediction model for hydrophilic interaction liquid chromatography (XBridge Amide column) is described for a collection of ∼40 000 tryptic peptides. Off-line 2D LC-MS/MS analysis (HILIC-RPLC) of S. cerevisiae whole cell lysate has been used to acquire retention information for a HILIC separation. The large size of the optimization data set (more than 2 orders of magnitude compared to previous reports) permits the accurate assignment of hydrophilic retention coefficients of individual amino acids, establishing both the effects of amino acid position relative to peptide termini and the influence of peptide secondary structure in HILIC. The accuracy of a simple additive model with peptide length correction (R2 value of ∼0.96) was found to be much higher compared to an algorithm of similar complexity applied to RPLC (∼0.91). This indicates significantly smaller influence of peptide secondary structure and interactions with counterions in HILIC. Nevertheless, sequence-specific features were found. Helical peptides that tend to retain stronger than predicted in RPLC exhibit negative prediction errors using an additive HILIC model. N-cap helix stabilizing motifs, which increase retention of amphipathic helical peptides in RP, reduce peptide retention in HILIC independently of peptide amphipathicity. Peptides carrying multiple Pro and Gly (residues with lowest helical propensity) retain stronger than predicted. We conclude that involvement of the peptide backbone's carbonyl and amide groups in hydrogen-bond stabilization of helical structures is a major factor, which determines sequence-dependent behavior of peptides in HILIC. The incorporation of observed sequence dependent features into our Sequence-Specific Retention Calculator HILIC model resulted in 0.98 R2 value correlation, significantly exceeding the predictive performance of all RP and HILIC models developed for complex mixtures of tryptic peptides.

Thioether-derived Macrocycle for Peptide Secondary Structure Fixation.

Recently, we developed methods to stabilize peptides into various secondary structures, including α-helix, type III turn and β-hairpin via proper thioether based macrocyclization. These conformationally constrained peptidomimetics confer enhanced biophysical properties and provide a valuable avenue towards clinically-relevant therapeutic molecules. In this personal account, thioether-derived macrocyclization methods developed by our group for stabilization of α-helix, type-III β turn and β-hairpin conformations are discussed.

Antibacterial activity of new peptide from bovine casein hydrolyzed by a serine metalloprotease of Lactococcus lactis subsp lactis BR16

An extracellular serine metalloprotease produced by Lactococcus lactis subsp lactis BR16 isolated from Algerian traditional fermented food was used for hydrolyzing bovine casein in order to identify antibacterial peptide. Antibacterial peptide was separated from peptic hydrolysates by adsorption/desorption then analyzed using reverse phase high performance liquid chromatography (RP-HPLC) and active fraction was analyzed by MALDI-TOF mass spectrometry. A new antibacterial peptide was identified as SSSEESII from the subunit αs2 of casein corresponding of the fragment (24–31). This peptide demonstrated antibacterial activity against two Gram-positive bacteria: Listeria innocua ATCC 33090 and Micrococcus luteus ATCC 4698, and two Gram-negative bacteria: Escherichia coli ATCC 25922 and Salmonella enteritidis ATCC13076. Prediction of peptide secondary structure indicated that this peptide is anionic and must have random coil structure with a small region of hydrophobicity, which confers to the peptide unusual behavior compared to conventional antimicrobial peptides.

Antioxidant Activity Improvement and Evaluation of Structure Changes of SHECN Treated by Pulsed Electric Field (PEF) Technology

Abstract The purpose of the study was to evaluate the relationship between antioxidant activity improvement and structure changes of peptide (Ser-His-Glu-Cys-Asn (SHECN) isolated from soybean treated by pulsed electric field (PEF). The two-factor-at-a–time (TFAT) was performed to investigate interaction of electric field intensity (5, 10, 15 and 20 kV/cm) and pulse frequency (1,800 and 2,400 Hz) on antioxidant activity improvement and structure changes. Compared with untreated peptide, DPPH radical inhibition of SHECN was significantly increased to 95.54 ± 0.16 % at optimal conditions (electric field intensity 15 kV/cm, pulse frequency 1,800 Hz and a retention time of 2 h). Results showed that the primary structure of SHECN had not been changed based on the nuclear magnetic resonance analysis. However, the secondary structure of peptide, especially α-helix can be changed. These results suggested that mechanism of antioxidant activity improvement is related to secondary structure changes.

Cyclopropane-Based Peptidomimetics Mimicking Wide-Ranging Secondary Structures of Peptides: Conformational Analysis and Their Use in Rational Ligand Optimization.

Detailed conformational analyses of our previously reported cyclopropane-based peptidomimetics and conformational analysis-driven ligand optimization are described. Computational calculations and X-ray crystallography showed that the characteristic features of cyclopropane function effectively to constrain the molecular conformation in a three-dimensionally diverse manner. Subsequent principal component analysis revealed that the diversity covers the broad chemical space filled by peptide secondary structures in terms of both main-chain and side-chain conformations. Based on these analyses, a lead stereoisomer targeting melanocortin receptors was identified, and its potency and subtype selectivity were improved by further derivatization. The presented strategy is effective not only for designing non-peptidic ligands from a peptide ligand but also for the rational optimization of these ligands based on the plausible target-binding conformation without requiring the three- dimensional structural information of the target and its peptide ligands.

Predicting Electrophoretic Mobility of Tryptic Peptides for High-Throughput CZE-MS Analysis

A multiparametric sequence-specific model for predicting peptide electrophoretic mobility has been developed using large-scale bottom-up proteomic CE-MS data (5% (∼0.8M) acetic acid as background electrolyte). Peptide charge (Z) and size (molecular mass, M) are the two major factors determining electrophoretic mobility, in complete agreement with previous studies. The extended size of the data set (>4000 peptides) permits access to many sequence-specific factors that impact peptide mobility. The presence of acidic residues Asp and Glu near the peptide N-terminus is by far the most prominent among them. The induction effect of the side chain of N-terminal Asp reduces the basicity of the N-terminal amino group and, as hence, its charge, by ∼0.27 units, lowering mobility. The correlation of the model (R2 ∼ 0.995) indicates that the peptide separation process in CZE is relatively simple and can be predicted to a much higher precision than current RP-HPLC models. Similar to RP-HPLC prediction studies, we anticipate future developments that introduce peptide migration standards, collect larger data sets for modeling through the alignment of multiple CZE-MS acquisitions, and study of the behavior of peptides carrying post-translational modifications. The increased size of data sets will also permit investigation of the fine-scale effects of peptide secondary structure on peptide mobility. We observed that peptides with higher helical propensity tend to have higher than predicted electrophoretic mobility; the incorporation of these features into CZE migration models will require significantly larger data sets.

Low pH-triggering changes in peptide secondary structures

We developed a novel methodology using cyclic α,α-disubstituted α-amino acids (dAAs) with an acetal-side chain to control peptide secondary structures. The introduction of cyclic dAAs into peptides contributed to the stabilization of peptide secondary structures as a helix, while an acidic treatment of peptides resulted in a marked conformational change.

Self-assembly of peptide amphiphiles for drug delivery: the role of peptide primary and secondary structures.

Peptide amphiphiles (PAs), functionalized with alkyl chains, are capable of self-assembling into various nanostructures. Recently, PAs have been considered as ideal drug carriers due to their good biocompatibility, specific biological functions, and hypotoxicity to normal cells and tissues. Meanwhile, the nanocarriers formed by PAs are able to achieve controlled drug release and enhanced cell uptake in response to the stimulus of the physiological environment or specific biological factors in the location of the lesion. However, the underlying detailed drug delivery mechanism, especially from the aspect of primary and secondary structures of PAs, has not been systematically summarized or discussed. Focusing on the relationship between the primary and secondary structures of PAs and stimuli-responsive drug delivery applications, this review highlights the recent advances, challenges, and opportunities of PA-based functional drug nanocarriers, and their potential pharmaceutical applications are discussed.

Small Molecule-Assisted PET: Approaches to Imaging of Conformational Diseases of the Brain

PET (positron emission tomography) in vivo imaging of cerebral conformational diseases is essentially based on non-peptide small molecule ligands used to detect early alterations in peptide secondary structures and subsequent accumulation of aberrant oligomers and protein deposits involved in progressive neurodegeneration, cognitive and movement disorders. In this article, an overview is given about tracers currently available and lead structures of potential PET probes for detection of β-amyloid (Aβ), tau protein, α-synuclein, constitutive (PrPc) and infectious isoforms (PrPsc) of prions (proteinaceous infectious particles) as imaging targets. Whereas the styrylpyridine derivative florbetapir, approved for clinical applications, the stilbene derivative florbetaben and the benzoxazole derivative BF227 show high affinity binding to Aβ, preclinical investigations promise improved pharmacokinetics for benzoimidazothiazoles, aryloxazoles and benzofuran derivatives. Tau protein imaging based clinically, presently, on the pyridine-pyridoindole T807 has got new incentives following identification of a series of pyrrolopyridine quinolines and pharmacokinetic improvements of fluoropropoxy quinolines including for instance THK-5351. The pyridine isoquinoline MK6240 is involved now in clinical trials. Most forward-looking efforts apply to small molecule ligands of α-synuclein, which are expected to permit a breakthrough in differential diagnostics of Parkinson-related dementia and Lewy body diseases. However, at the moment the proposed lead structures are in affinity and blood brain barrier delivery properties below the possibilities of Aβ and tau protein ligands. This is the case also for potential tracers of prion proteins.

Simultaneous Analysis of Secondary Structure and Light Scattering from Circular Dichroism Titrations: Application to Vectofusin-1.

Circular Dichroism data are often decomposed into their constituent spectra to quantify the secondary structure of peptides or proteins but the estimation of the secondary structure content fails when light scattering leads to spectral distortion. If peptide-induced liposome self-association occurs, subtracting control curves cannot correct for this. We show that if the cause of the light scattering is independent from the peptide structural changes, the CD spectra can be corrected using principal component analysis (PCA). The light scattering itself is analysed and found to be in good agreement with backscattering experiments. This method therefore allows to simultaneously follow structural changes related to peptide-liposome binding as well as peptide induced liposome self-association. We apply this method to study the structural changes and liposome binding of vectofusin-1, a transduction enhancing peptide used in lentivirus based gene therapy. Vectofusin-1 binds to POPC/POPS liposomes, causing a reversal of the negative liposome charge at high peptide concentrations. When the peptide charges exactly neutralise the lipid charges on both leaflets reversible liposome self-association occurs. These results are in good agreement with biological observations and provide further insight into the conditions required for efficent transduction enhancement.

Synthesis of a biological active β-hairpin peptide by addition of two structural motifs

The idea of privileged scaffolds – that there seem to be more bioactive compounds found around some structures than others – is well established for small drug molecules, but has little significance for standalone peptide secondary structures whose adaptable shapes escape the definition of a 3D motif in the absence of a protein scaffold. Here, we joined two independent biological functions in a single highly restricted peptide to support the hypothesis that the β-hairpin shape is the common basis of two otherwise unrelated biological recognition processes. To achieve this, the hydrophobic cluster HWX4LV from the decapeptide cyclic hairpin model peptide C1-C10cyclo-CHWEGNKLVC was included in the bicyclic peptide 2. The designed β-hairpin peptide C4-C17, C8-C13bicyclo-KHQCHWECTZGRCRLVCGRSGS (2, Z=citrulline), serves, on the one hand, as a specific epitope for rheumatoid autoantibodies and, on the other hand, shows a not negligible antibiotic effect against the bacterial strain E. coli AS19.

Insights into the aggregation mechanism of Aβ(25–40)

The hydrophobic fragment of the Alzheimer's related β-amyloid (Aβ) peptide, Aβ(25–40), aggregates and forms insoluble amyloid fibrils at a rate similar to the full-length peptide. In order to gain insight into the fibrillization of Aβ(25–40) and the ability of the flavonoid myricetin to inhibit its aggregation, the isoleucine at position 32 (I32A) and the glycine at position 37 (G37A) in the full-length peptide were replaced with alanine. Thioflavin T assays indicate that substitution of isoleucine for alanine significantly reduces the rate and extent of fibrillization compared to the Aβ(25–40) and G37A peptides. Although all three peptides are fully disordered initially, circular dichroism studies suggest the structure of the I32A and G37A peptides are different from the parent peptide Aβ(25–40). Introduction of myricetin to the peptide samples results in modest structural changes for the Aβ(25–40) and G37A peptides but not the I32A peptide. Aβ(25–40) oligomers were predominantly tetramers, whereas I32A and G37A oligomers were a mixture of trimers and dimers. After 48h of incubation at 37°C, the amount of tetramers and trimers in solution dropped for the Aβ(25–40) and G37A peptides but remained similar for the I32A peptide. Incubation of Aβ(25–40) with myricetin increased the relative proportion of trimers to tetramers. Ultraviolet resonance Raman studies suggests that the I32A peptide may be more hydrated than the Aβ(25–40) and G37A peptides. Taken together, these data indicate the structural changes observed for the Aβ(25–40) and G37A peptides upon introduction of myricetin are localized around residue 32 and could arise from hydrophobic interactions between the peptide and the flavonoid or interference with the self-association of the peptide in this region. Substitution of isoleucine at position 32 with alanine had little effect on the peptide's secondary structure but dramatically decreased the propensity of the peptide fibrillize.

Are AMBER Force Fields and Implicit Solvation Models Additive? A Folding Study with a Balanced Peptide Test Set.

Implicit solvation models have long been sought as routes to significantly increase the speed and capabilities of biomolecular simulations. However, it has not always been clear that force fields developed independently of solvation models can together accurately predict secondary structure and folding, and whether the separate influences of the solvation and force field models can be described as independent and additive (versus synergistic). Here, we test two implicit solvation models with several recently developed protein force fields, within the AMBER simulation package. We create a representative set of five helical and five hairpin peptides, 11-20 amino acid residues in length, and calculate folded structures using replica exchange molecular dynamics simulations for all force field/solvent/peptide combinations, each with two instances using distinct starting configurations. In general, we find that no force field/solvent combination successfully folds all peptides and that the hairpin peptides are more difficult to capture. That being said, the older ff96/igb5* combination does a reasonable job in folding multiple secondary structures, while ff14SB/igb5* and ff14ipq/igb8 work well for helical and hairpin motifs, respectively. All combinations give rise to similar numbers of salt bridges, except for solvent models paired with ff14ipq, which slightly enhances them. Interestingly, we are unable statistically to decouple the effects of force field, solvent model, and peptide secondary structure on performance, such that particular combinations can have specific effects. These results suggest that future efforts might benefit from codevelopment of implicit models with force fields or from the use of emerging coarse-graining strategies that extract solvation effects in a bottom-up manner.

In vitro antioxidant activities of the novel pentapeptides Ser-His-Glu-Cys-Asn and Leu-Pro-Phe-Ala-Met and the relationship between activity and peptide secondary structure.

Using high-performance liquid chromatography/tandem mass spectrometry, two novel antioxidant pentapeptides [Ser-His-Glu-Cys-Asn (SHECN) and Leu-Pro-Phe-Ala-Met (LPFAM)] were identified from 1-3-kDa soybean protein hydrolysates (SPH). The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used to evaluate cytotoxicity in HepG2 cells. Antioxidant activity was measured using in vitro assays, including the cellular antioxidant activity assay (CAA), 2,2-diphenyl-1-picrylhydrazyl or 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) inhibition, and oxygen radical absorbance capacity (ORAC) assays. Finally, the secondary structure was determined using circular dichroism (CD). The results revealed that two novel peptides were nontoxic and possessed antioxidant activity. SHECN had significantly higher antioxidant activity than LPFAM (P < 0.05). The CAA value of SHECN was 776.22 µmol QE 100 g-1 . SHECN also showed significant DPPH inhibition (70.18 ± 4.06%) and ABTS inhibition (88.16 ± 0.76%). It had normalized ORAC values of 0.3000 ± 0.0070 µmol GE mg-1 and 0.0900 ± 0.0020 µmol TE mg-1 , respectively. The results of the CD analysis demonstrated that, compared to LPFAM, which had much lower antioxidant activity, SHECN had a high β-sheet content and reduced α-helix content. The results indicated that SHECN possessed high antioxidant activity. A higher β-sheet content and lower content levels of α-helix appear to be correlated with antioxidant activity. © 2016 Society of Chemical Industry.

Solution State Structure of P1, the Mimetic Peptide Derived from IgM Antigen Apo B-100 by NMR

Apolipoprotein B-100 (Apo-B100) is a major component of low density lipoprotein (LDL). Apo B-100 protein has 4,536 amino acid sequence and these amino acids are classified into peptide groups A to G with subsequent 20 amino acids (P1-P302). The peptide groups were act as immunoglobulin (Ig) antigens which oxidized via malondialdehyde (MDA). The mimetic peptide P1 (EEEMLENVSLVCPKDAT RFK) out of D-group peptides carrying the highest value of IgG antigens were selected for structural studies that may provide antigen specificity. Circular Dichroism (CD) spectra were measured for peptide secondary structure in the range of 190-250 nm. Experimental results show that P1 exhibit partial of <TEX>${\beta}-sheet$</TEX> and random coil structure. Homonuclear (COSY, TOCSY, NOESY) 2D-NMR experiments were carried out for NMR signal assignments and structure determination for P1. On the basis of these completely assigned NMR spectra and distance data, distance geometry (DG) and Molecular dynamics (MD) were carried out to determine the structures of P1. The proposed structure was selected by comparisons between experimental NOE spectra and back calculated 2D NOE results from determined structure showing acceptable agreement. The total Root-Mean-Square-Deviation (RMSD) value of P1 obtained upon superposition of all atoms was in the range <TEX>$0.33{\AA}$</TEX>. The solution state P1 has mixed structure of <TEX>${\beta}-sheet$</TEX> (Glu[1] to Cys[12]) and random coil (Pro[13] to Lys[20]). These NMR results are well consistent with secondary structure from experimental results of circular dichroism. Structural studies based on NMR may contribute to the studies of atherosclerosis and observed conformational characteristics of apo B-100 in LDL using monoclonal antibodies.

Constraining an Irregular Peptide Secondary Structure through Ring-Closing Alkyne Metathesis.

Macrocyclization can be used to constrain peptides in their bioactive conformations, thereby supporting target affinity and bioactivity. In particular, for the targeting of challenging protein–protein interactions, macrocyclic peptides have proven to be very useful. Available approaches focus on the stabilization of α‐helices, which limits their general applicability. Here we report for the first time on the use of ring‐closing alkyne metathesis for the stabilization of an irregular peptide secondary structure. A small library of alkyne‐crosslinked peptides provided a number of derivatives with improved target affinity relative to the linear parent peptide. In addition, we report the crystal structure of the highest‐affinity derivative in a complex with its protein target 14‐3‐3ζ. It can be expected that the alkyne‐based macrocyclization of irregular binding epitopes should give rise to new scaffolds suitable for targeting of currently intractable proteins.

Alzheimer Disease Amyloid Beta Peptides: AFM and Voltammetric Characterization

Alzheimer’s disease (AD) is the most widespread form of dementia and was recently estimated to affect 44.4 million people worldwide. The main clinical manifestations of AD are loss of memory, cognitive skills, speech, and normative behaviour; however, accumulation of fibrillary amyloid beta (Aβ) peptides in plaques can occur and be observed decades before the AD symptoms. Native human amyloid beta (Aβ) peptides exist as short isoforms, soluble monomers, varying from 39 to 43 amino acids. In AD, dysregulation and overproduction of Aβ peptides occurs, leading to a cascade mechanism in which the peptides undergo morphological changes, from soluble, monomeric random coil or α-helix conformations, into aggregated β-sheet structures, a fibrillization process that involves numerous intermediate structures. While at the beginning of ’90 it was considered that the neurodegeneration in AD is caused by the deposition of Aβ peptide plaques (large Aβ aggregates) in the brain tissue, nowadays, what appeared to be primarily responsible for neurodegeneration are these transient heterogeneous small soluble oligomers and protofibrils, which are difficult to be investigated due to the fast aggregation rate of Aβ peptide. The time dependent structural modifications undergone by the synthetic human Aβ isoform peptides containing 40 and 42 amino acids, Aβ1-40 and Aβ1-42 peptides, i.e.the intermediary structures which appear during fibrilization, were studied by atomic force microscopy (AFM) at highly oriented pyrolytic graphite and differential pulse (DP) voltammetry at a glassy carbon electrode. The Aβ peptide solutions were incubated at room temperature, and in free chloride media, to limit the effect of temperature and salt concentration on the aggregation rate,. In order to fully understand how the electron transfer reaction was influenced by both the specific amino acid side-chain position and the fibrilization process, the Aβ1-40 and Aβ1-42 peptides aggregation was compared with the aggregation of four Aβ peptides control sequences that: (i) do not aggregate (inverse Aβ40-1 and Aβ42-1 and rat Aβ1-40Rat peptides), and (ii) lack specific electroactive amino acids (mutants Aβ1-40Phe10 and Aβ1-40Nle35peptides). The fibrilization process was investigated by AFM, via changes in the adsorption morphology, from initially random coiled structures, corresponding to the Aβ peptide monomers in random coil or in α-helix conformations, to aggregates, protofibrils and two types of fibrils, corresponding to the Aβ peptide in a β-sheet configuration. The hydrophobic carbon surface induced rapid changes in the Aβ peptides conformation, and differences between the adsorbed fibrils, formed at the carbon surface or in solution, were detected. The Aβ peptides fibrilization was electrochemically detected via the decrease of the Aβ peptide electroactive amino acids oxidation peak currents, in the DP voltammograms, that occurred in a time dependent manner. Structurally, the Aβ peptide sequences contained five electroactive amino acid residues, one tyrosine (Tyr10), three histidines (His6, His13 and His14) and one methionine (Met35). DP voltammograms showed that, according to their primary structure, the Aβ peptides undergo oxidation in one or two steps, the first electron transfer reaction corresponding to the Tyr10 residue oxidation and the second to His6, His13, His14 and Met35 residues. The highest contribution to the second oxidation peak current was from the His13 residue, followed by His14 and His6 residues, while the Met35residue presented the lowest oxidation peak current. The Aβ peptides electron transfer reaction depended on the sequence hydrophobicity, on the position of the electroactive amino acid residues in the sequence (the residues close to N-termini giving the highest oxidation peak currents), being also strongly influenced by the Aβ peptide secondary structure. The AFM and voltammetric results were consistent with the in vitro fibrilization mechanism, observed for different Aβ peptide sequences. The Aβ peptide monomers followed a nucleation process, leading to the formation of small nuclei. Once the nucleation process started, Aβ free monomers in solution were added to the nuclei and their dimensions grown forming various aggregate types, such as small oligomers and long fibrils with different morphologies. This polymerization reaction followed first-order kinetics, and is thermodynamically favored by the hydrophobic regions of Aβ peptides, as well as their β-sheet secondary structure. As fibrilization proceeded, the electroactive residues became more protected, and the transfer of electrons became more difficult.

New Insight in Copper-Ion Binding to Human Islet Amyloid: The Contribution of Metal-Complex Speciation To Reveal the Polypeptide Toxicity.

Type-2 diabetes (T2D) is considered to be a potential threat on a global level. Recently, T2D has been listed as a misfolding disease, such as Alzheimer's and Parkinson's diseases. Human islet amyloid polypeptide (hIAPP) is a molecule cosecreted in pancreatic β cells and represents the main constituent of an aggregated amyloid found in individuals affected by T2D. The trace-element serum level is significantly influenced during the development of diabetes. In particular, the dys-homeostasis of Cu(2+) ions may adversely affect the course of the disease. Conflicting results have been reported on the protective role played by complex species formed by Cu(2+) ions with hIAPP or its peptide fragments in vitro. The histidine (His) residue at position 18 represents the main binding site for the metal ion, but contrasting results have been reported on other residues involved in metal-ion coordination, in particular those toward the N or C terminus. Sequences that encompass regions 17-29 and 14-22 were used to discriminate between the two models of the hIAPP coordination mode. Due to poor solubility in water, poly(ethylene glycol) (PEG) derivatives were synthesized. A peptide fragment that encompasses the 17-29 region of rat amylin (rIAPP) in which the arginine residue at position 18 was substituted by a histidine residue was also obtained to assess that the PEG moiety does not alter the peptide secondary structure. The complex species formed by Cu(2+) ions with Ac-PEG-hIAPP(17-29)-NH2 , Ac-rIAPP(17-29)R18H-NH2 , and Ac-PEG-hIAPP(14-22)-NH2 were studied by using potentiometric titrations coupled with spectroscopic methods (UV/Vis, circular dichroism, and EPR). The combined thermodynamic and spectroscopic approach allowed us to demonstrate that hIAPP is able to bind Cu(2+) ions starting from the His18 imidazole nitrogen atom toward the N-terminus domain. The stability constants of copper(II) complexes with Ac-PEG-hIAPP(14-22)-NH2 were used to simulate the different experimental conditions under which aggregate formation and oxidative stress of hIAPP has been reported. Speciation unveils: 1) the protective role played by increased amounts of Cu(2+) ions on the hIAPP fibrillary aggregation, 2) the effect of adventitious trace amounts of Cu(2+) ions present in phosphate-buffered saline (PBS), and 3) a reducing fluorogenic probe on H2 O2 production attributed to the polypeptide alone.

Orientation Determination of a Hybrid Peptide Immobilized on CVD-Based Reactive Polymer Surfaces

Antimicrobial peptides (AMPs), due to their unique structure/function relationship, have great opportunities to be developed into novel diagnostic and therapeutic agents for a variety of pathogens and illnesses. Often such peptides are administered using powder or suspension, limiting their reusability or recyclability. Immobilization of these antimicrobial peptides on biotic/abiotic surfaces may circumvent such disadvantages, but such immobilization is likely to not only change the peptide secondary structures and their orientations but also ultimately affect functionality. In order to better understand surface-bound structures of AMPs on abiotic surfaces, cecropin A (1–8)–melittin (1–18) hybrid peptides were chemically immobilized on polymer surfaces prepared by chemical vapor deposition (CVD) polymerization. Measurements by sum frequency generation (SFG) vibrational spectroscopy and circular dichroism were used to characterize the peptides immobilized on the CVD-based polymer in situ. In addition, coarse-grained molecular dynamics (MD) simulations were used to understand the orientation of these peptides on the molecular level. Simulation results were highly consistent with experimental data. Results indicated that, unlike other linear peptides immobilized on similar abiotic surfaces, this hybrid peptide immobilized on CVD-based polymer surfaces exhibited two bending points. Such conclusions help further understand the role surface immobilization for such unique molecules.

Conformational study of melectin and antapin antimicrobial peptides in model membrane environments

Antimicrobial peptides have long been considered as promising compounds against drug-resistant pathogens. In this work, we studied the secondary structure of antimicrobial peptides melectin and antapin using electronic (ECD) and vibrational circular dichroism (VCD) spectroscopies that are sensitive to peptide secondary structures. The results from quantitative ECD spectral evaluation by Dichroweb and CDNN program and from the qualitative evaluation of the VCD spectra were compared. The antimicrobial activity of the selected peptides depends on their ability to adopt an amphipathic α-helical conformation on the surface of the bacterial membrane. Hence, solutions of different zwitterionic and negatively charged liposomes and micelles were used to mimic the eukaryotic and bacterial biological membranes. The results show a significant content of α-helical conformation in the solutions of negatively charged liposomes mimicking the bacterial membrane, thus correlating with the antimicrobial activity of the studied peptides. On the other hand in the solutions of zwitterionic liposomes used as models of the eukaryotic membranes, the fraction of α-helical conformation was lower, which corresponds with their moderate hemolytic activity.