Peptide Model System Research Articles

Peptide Probes Reveal a Hydrophobic Steric Ratchet in the Anthrax Toxin Protective Antigen Translocase

Anthrax toxin is a tripartite virulence factor produced by Bacillus anthracis during infection. Under acidic endosomal pH conditions, the toxin's protective antigen (PA) component forms a transmembrane channel in host cells. The PA channel then translocates its two enzyme components, lethal factor and edema factor, into the host cytosol under the proton motive force. Protein translocation under a proton motive force is catalyzed by a series of nonspecific polypeptide binding sites, called clamps. A 10-residue guest/host peptide model system, KKKKKXXSXX, was used to functionally probe polypeptide-clamp interactions within wild-type PA channels. The guest residues were Thr, Ala, Leu, Phe, Tyr, and Trp. In steady-state translocation experiments, the channel blocked most tightly with peptides that had increasing amounts of nonpolar surface area. Cooperative peptide binding was observed in the Trp-containing peptide sequence but not the other tested sequences. Trp substitutions into a flexible, uncharged linker between the lethal factor amino-terminal domain and diphtheria toxin A chain expedited translocation. Therefore, peptide-clamp sites in translocase channels can sense large steric features (like tryptophan) in peptides, and while these steric interactions may make a peptide translocate poorly, in the context of folded domains, they can make the protein translocate more rapidly presumably via a hydrophobic steric ratchet mechanism.

Testing the role of charge and structure on the stability of peptide-porphyrin complexes.

This study aims to extend a structural and biophysical understanding of a coiled-coil based peptide model system that serves as a scaffold for the anionic porphyrin, TPPS4. This is part of an ongoing biomaterials effort to create photoelectronically active mesoscale fibrils for surface deposition and characterization of conductivity properties. The goals are two-fold: (1) to explore optimal basic side-chain moieties for tight binding to TPPS4 and (2) to test the binding of various metalated TPPS4 derivatives to our peptide model system. The latter goal is to control the electronic and redox properties of the fibrillar biomaterials. A soluble version of the peptide biomaterial was used in order to probe binding and to extract thermodynamically rigorous equilibrium binding constants. UV-visible spectroscopy and circular dichroism spectropolarimtery are used to measure the effects of binding on the Soret band of the porphyrin and the helical signal of the peptide, respectively. For the first study, it was found that lysine, ornithine, and arginine are equally robust at engaging TPPS4 with low micromolar binding affinity. In the case of the metalated porphyrins, submicromolar binding affinity was observed for Cu(II), Ni(II), and Pd(II). The ability of these metalated porphyrins to bind with high affinity is dependent largely on structural perturbations of the porphyrin molecule, rather than on induced electronic effects.

Effect of helical flanking sequences on the morphology of polyglutamine-containing fibrils.

A peptide model system has been developed to study the effects of helical flanking sequences on polyglutamine aggregation. In a companion manuscript, the kinetics of aggregation are described, comparing the influence of a well-defined heterotetrameric coiled coil to that of the helix-rich structure found in Htt(NT), a 17-residue flanking sequence found in the huntingtin protein, on polyglutamine aggregation. Here, the morphological characterization of the resultant fibrils that form for a set of peptides is reported, only one of which, KKQ25KK, has been previously studied. A careful analysis of TEM and AFM images of KKQ25KK confirms that it forms bundled fibrils of varying length and reveals, unexpectedly, that they are composed of fully extended cross-β-strands. Second, it is shown that helical flanking sequences do not disrupt the assembly of a core cross-β-sheet structure, but such flanking sequences can influence higher order processes, such as inhibiting the bundling of the fibrils.

A fibril-like assembly of oligomers of a peptide derived from β-amyloid.

A macrocyclic β-sheet peptide containing two nonapeptide segments based on Aβ15–23 (QKLVFFAED) forms fibril-like assemblies of oligomers in the solid state. The X-ray crystallographic structure of macrocyclic β-sheet peptide 3 was determined at 1.75 Å resolution. The macrocycle forms hydrogen-bonded dimers, which further assemble along the fibril axis in a fashion resembling a herringbone pattern. The extended β-sheet comprising the dimers is laminated against a second layer of dimers through hydrophobic interactions to form a fibril-like assembly that runs the length of the crystal lattice. The second layer is offset by one monomer subunit, so that the fibril-like assembly is composed of partially overlapping dimers, rather than discrete tetramers. In aqueous solution, macrocyclic β-sheet 3 and homologues 4 and 5 form discrete tetramers, rather than extended fibril-like assemblies. The fibril-like assemblies of oligomers formed in the solid state by macrocyclic β-sheet 3 represent a new mode of supramolecular assembly not previously observed for the amyloidogenic central region of Aβ. The structures observed at atomic resolution for this peptide model system may offer insights into the structures of oligomers and oligomer assemblies formed by full-length Aβ and may provide a window into the propagation and replication of amyloid oligomers.

Mechanistic insights into Pin1 peptidyl-prolyl cis-trans isomerization from umbrella sampling simulations.

The peptidyl-proyl isomerase Pin1 plays a key role in the regulation of phospho(p)-Ser/Thr-Pro proteins, acting as a molecular timer of the cell cycle. After recognition of these motifs, Pin1 catalyzes the rapid cis-trans isomerization of proline amide bonds of substrates, contributing to maintain the equilibrium between the two conformations. Although a great interest has arisen on this enzyme, its catalytic mechanism has long been debated. Here, the cis-trans isomerization of a model peptide system was investigated by means of umbrella sampling simulations in the Pin1-bound and unbound states. We obtained free energy barriers consistent with experimental data, and identified several enzymatic features directly linked to the acceleration of the prolyl bond isomerization. In particular, an enhanced autocatalysis, the stabilization of perturbed ground state conformations, and the substrate binding in a procatalytic conformation were found as main contributions to explain the lowering of the isomerization free energy barrier.

Amyloid fibril nucleation: effect of amino acid hydrophobicity.

We consider the nucleation of amyloid fibrils when the process occurs by direct polymerization of fully extended peptides (i.e., β-strands) into fibrils composed of successively layered β-sheets with alternating weak and strong hydrophobic surfaces. We extend our recently developed nucleation model (Kashchiev, D.; Cabriolu, R.; Auer, S. J. Am. Chem. Soc. 2013, 135, 1531-1539) to derive general expressions for the work to form such fibrils, the fibril solubility, the nucleation work, the equilibrium concentration of nuclei, and the fibril nucleation rate as explicit functions of the supersaturation of the protein solution. Analysis of these expressions illustrates the effect of increased asymmetry between the weak and strong hydrophobic β-sheet surfaces on the thermodynamics and kinetics of the polymerization process. In particular, the application of our theoretical framework to a simple model peptide system shows that lowering the hydrophobicity of one β-sheet surface can hamper protein fibrillation because the threshold concentration below which the fibril nucleation is practically arrested, and above which the process occurs vigorously--because then each monomer in the solution acts as a fibril nucleus--is shifted to higher concentrations. This effect is entirely due to the effect of asymmetry of the two hydrophobic β-sheet surfaces on the fibril solubility. In addition, with increasing asymmetry, the nucleation rate of one fibril polymorph becomes increasingly dominant, illustrating that there is a morphological selection between the two possible polymorphs.

Selectivity in fragmentation of N-methylacetamide after resonant K-shell excitation.

The fragmentation pattern of the peptide model system, N-methylacetamide, is investigated using ion time-of-flight (TOF) spectroscopy after resonant K-shell excitation. Corresponding near-edge X-ray absorption fine structure (NEXAFS) spectra recorded at high resolution at the C1s, N1s and O1s edges are presented. Analysis of the ion TOF data reveals a multitude of fragmentation channels and dissociation pathways. Comparison between the excitation of six different resonances in the vicinity of the C1s, N1s and O1s edges suggests evidence for site-selective bond breaking. In particular the breaking of the peptide bond and the N-C(α) bond show a clear correlation with resonant excitation at the N1s edge. Also, stronger tendencies towards site-selective bond breaking are found for the generation of single ions compared with ion pairs. Analysis of angular distributions of ions from breakage of the peptide bond yields a fragmentation time of <400 fs.

Biological activities and physicochemical properties of Maillard reaction products in sugar–bovine casein peptide model systems

The purpose of this study was to evaluate the biological activities and physicochemical properties of Maillard reaction products (MRPs), derived from aqueous reducing sugar (ribose, galactose and lactose) and bovine casein peptide (BCP) model systems. The fluorescence intensity of ribose-BCP MRPs reached the maximum value within 1h, while it took 3h for galactose-BCP MRPs. Size exclusion chromatography of all the MRPs indicated molecular rearrangements and production of new smaller molecules, as a function of the heating time. The consumption of ribose and amino groups was the highest in the ribose-BCP MRPs. BCP lost its known angiotensin-I-converting enzyme (ACE) inhibitory activity by the Maillard reaction with reducing sugars. Ribose–BCP MRPs had the lowest ACE inhibitory activity, but they showed the highest 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity and ferrous reducing power among all the MRPs. Galactose-BCP MRPs inhibited, slightly the growth of Caco-2 cells, while ribose-BCPand lactose-BCP MRPs had no cytotoxicity.

Unusual Folding Propensity of an Unsubstituted β,γ‐Hybrid Model Peptide: Importance of the CH⋅⋅⋅O Intramolecular Hydrogen Bond

The single-crystal X-ray diffraction analysis of a β,γ-hybrid model peptide Boc-β-Ala-γ-Abu-NH2 revealed the existence of four crystallographically independent molecules (A, B, C and D conformers) in the asymmetric unit. The analysis revealed that unusual β-turn-like folded structures predominate, wherein the conformational space of non-proteinogenic β-Ala and γ-Abu residues are restricted to gauche-gauche-skew and skew-gauche-trans-skew orientations, respectively. Interestingly, the U-shaped conformers are seemingly stabilised by an effective unconventional C-H⋅⋅⋅O intramolecular hydrogen bond, encompassing a non-covalent 14-membered ring-motif. Taking into account the signs of torsion angles, these conformers could be grouped into two distinct categories, A/B and C/D, establishing the incidence of non-superimposable stereogeometrical features across a non-chiral one-component peptide model system, that is, "mirror-image-like" relationships. The natural occurrence of β-Ala and γ-Abu entities in various pharmacologically important molecules, coupled with their biocompatibilities, highlight how the non-functionalised β,γ-hybrid segment may offer unique advantages for introducing and/or manipulating a wide spectrum of biologically relevant hydrogen bonded secondary structural mimics in short synthetic peptides.

Adaptive Aggregation of Peptide Model Systems

Jet-cooled infrared spectra of acetylated glycine, alanine, and dialanine esters and their dimers are reported in the amide A and amide I-III regions. They serve as particularly simple peptide aggregation models and are found to prefer a single backbone conformation in the dimer that is different from the most stable monomer backbone conformation. In the case of alanine, evidence for topology-changing chirality discrimination upon dimer formation is found. The jet spectroscopic results are compared to gas phase spectra and quantum chemical calculations. They provide reliable benchmarks for the evaluation of the latter in the field of peptide interactions.

Synthetic Peptides Model Instability of Cardiac Myosin Subfragment-2

Hypertrophic cardiomyopathy (HCM), a heart-related abnormality, is the most prevalent cause of sudden death in young athletes at sporting events. A cluster of cardiomyopathy mutations are localized in β-cardiac myosin at the N-terminal region of subfragment-2. Using resonance energy transfer probes, a synthetic peptide model system was developed to study stability of the coiled coil (S2 fragment) structure by determining monomer-dimer equilibrium of the peptide. Fluorescence resonance energy transfer and MacroModel software suite were used to obtain distance measurements along with measurement of coiled coil formation. The model peptide was used to characterize the effects of disease-causing-mutations and examine potential candidate drugs (polyamines) to counteract effects of mutations causing HCM. Distance measurements between donor and acceptor probes obtained by computational simulation and fluorescence resonance energy transfer (FRET) were consistent. Measurements also agreed with simulations of unlabeled wildtype, indicating coiled coil structural stability of the peptide. Interaction of the site-specific antibody with the peptide strongly inhibited dimerization and destabilized coiled coil structure of the peptide. Presence of negatively charged glutamate residues in the region of subfragment-2 strongly suggested a potential interaction site for positively charged polyamines. Binding of certain polyamines, such as poly-L-Lysine 11 residues and poly-D-Lysine 17 residues, demonstrated the ability to enhance dimerization and improve stability of the coiled coil structure, while some other polyamines were shown to have insignificant impact on the structure. In an attempt to characterize the effect of HCM-causing-mutations, peptides containing E924K mutation and lethal mutation E930 deletion were synthesized. Fluorescence resonance probes were conjugated to the mutant peptides to determine coiled coil formation. Results obtained from both dynamic simulations and resonance energy transfer experiments indicated that these mutations strongly inhibit dimerization, and thus, destabilize coiled coil structure of the peptide. Further experiments were conducted using heterodimers containing a chain of wildtype and a chain of mutant peptide. Both E924K & Edel930 mutations destabilized coiled coil formation and prevented dimerization. This peptide model system would provide a promising tool for drug development targeting HCM-causing-mutations along the S2 region of myosin.

Exploring the Interactions between a Coiled-Coil Peptide Model System and Metallated Porphyrins towards the Design of Photoelectronically Active Biomaterials

Porphyrin-peptide structures hold great potential for design of environmentally responsive nanowires, and porphyrin metallation may modulate these structures' electronic properties. To better understand these effects, the binding of metallated meso-tetrakis(4-sulfonatophenyl)porphine (TPPS4) to the peptide Cp3K-N was investigated. Cp3K-N is a non-polymerizing model for the polymerizing peptide Cp3K, allowing for quantitative experiments that would be impossible in an aggregating system. Cu(II)TPPS4, Ni(II)TPPS4, Pd(II)TPPS4, and Zn(II)TPPS4 were studied using circular dichroism (CD) spectroscopy, UV-Visible spectroscopy, and sedimentation velocity analytical ultracentrifugation (SV-AU). CD and UV-Vis titrations of the ratio of Cp3K-N to porphyrin were performed, and binding constants for the complexes were determined. A blue-shifted Soret Band absorbance in the porphyrin, concomitant with induction of Cp3K-N alpha-helicity, was observed for all metals but zinc, which demonstrated a red-shifting of its Soret Band. Such spectroscopic characteristics suggest specific porphyrin-peptide binding. Titration experiments for the copper, nickel, and palladium porphyrins indicated equal peptide:porphyrin stoichiometry, while SV-AU experiments suggest higher order complex formation. These results confirm that metalled TPPS4 can bind Cp3K-N with the specificity and tightness of TPPS4, suggesting that such porphyrins have potential for use in polymerizing systems, towards the goal of designing novel photoelectronically active biomaterials.

Lattice model simulations of the effects of the position of a peptide trigger segment on helix folding and dimerization

The folding and dimerization of proteins is greatly facilitated by the presence of a trigger site, a segment of amino acids that has a higher propensity for forming α-helix structure as compared to the rest of the chain. In addition to the helical propensity of each chain, dimerization can also be facilitated by interhelical interactions such as saltbridges, and interfacial contacts of different strengths. In this work, we are interested in understanding the interplay of these interactions in a model peptide system. We investigate how these different interactions influence the kinetics of dimer formation and the stability of the fully formed dimer. We use lattice model computer simulations to investigate how the effectiveness of the trigger segment and its saltbridges depends on the location along the protein primary sequence. For different positions of the trigger segment, heat capacity and free energy of unfolded and folded configurations are calculated to study the thermodynamics of folding and dimerization. The kinetics of the process is investigated by calculating characteristic folding times. The thermodynamic and kinetic data from the simulations combine to show that the dimerization process of the model system is faster when the segment with high helical propensity is located near either end of the peptide, as compared to the middle of the chain. The dependence of the stability of the dimer on the trigger segment's position is also studied. The stability can play a role in the ability of the dimer to perform a biological function that involves partial unzipping. The results on folding and dimer stability provide important insights for designing proteins that involve trigger sites.

Minimalist probes for studying protein dynamics: thioamide quenching of selectively excitable fluorescent amino acids.

Fluorescent probe pairs that can be selectively excited in the presence of Trp and Tyr are of great utility in studying conformational changes in proteins. However, the size of these probe pairs can restrict their incorporation to small portions of a protein sequence where their effects on secondary and tertiary structure can be tolerated. Our findings show that a thioamide bond-a single atom substitution of the peptide backbone-can quench fluorophores that are red-shifted from intrinsic protein fluorescence, such as acridone. Using steady-state and fluorescence lifetime measurements, we further demonstrate that this quenching occurs through a dynamic electron-transfer mechanism. In a proof-of-principle experiment, we apply this technique to monitor unfolding in a model peptide system, the villin headpiece HP35 fragment. Thioamide analogues of the natural amino acids can be placed in a variety of locations in a protein sequence, allowing one to make a large number of measurements to model protein folding.

Peptide Bond Distortions from Planarity: New Insights from Quantum Mechanical Calculations and Peptide/Protein Crystal Structures

By combining quantum-mechanical analysis and statistical survey of peptide/protein structure databases we here report a thorough investigation of the conformational dependence of the geometry of peptide bond, the basic element of protein structures. Different peptide model systems have been studied by an integrated quantum mechanical approach, employing DFT, MP2 and CCSD(T) calculations, both in aqueous solution and in the gas phase. Also in absence of inter-residue interactions, small distortions from the planarity are more a rule than an exception, and they are mainly determined by the backbone ψ dihedral angle. These indications are fully corroborated by a statistical survey of accurate protein/peptide structures. Orbital analysis shows that orbital interactions between the σ system of Cα substituents and the π system of the amide bond are crucial for the modulation of peptide bond distortions. Our study thus indicates that, although long-range inter-molecular interactions can obviously affect the peptide planarity, their influence is statistically averaged. Therefore, the variability of peptide bond geometry in proteins is remarkably reproduced by extremely simplified systems since local factors are the main driving force of these observed trends. The implications of the present findings for protein structure determination, validation and prediction are also discussed.

Nanotubes and bilayers in a model peptide system

The trifluoroacetate (tfa) salt of the peptide (ala)6lys self-assembles in water into very long, hollow nanotubes with a radius R = 26 nm, above a critical aggregation concentration (volume fraction), ϕcac = 0.10. The peptides carry a net positive charge that ensures colloidal stability of the self-assembly structures through a long-range electrostatic repulsion. There is only a weak temperature dependence of ϕcac from which an enthalpy of aggregation of −kBT per molecule is estimated. SAXS data show that the thickness of the nanotube wall, δ, is less than 1 nm indicating that the peptides form a monolayer in the nanotube wall. The nanotubes have a very large aspect ratio and form an ordered nematic or hexagonal phase. Because of the low δ/R ratio, the nanotube volume fraction grows very rapidly with increasing peptide concentration, ϕ, and reaches close packing already at ϕ = 0.15. When increasing the concentration further, there is a phase transition to a novel lamellar phase where the peptide molecules form bilayers consisting of two, presumably oppositely oriented, monolayers.

How Post‐Translational Modifications Influence Amyloid Formation: A Systematic Study of Phosphorylation and Glycosylation in Model Peptides

A reciprocal relationship between phosphorylation and O-glycosylation has been reported for many cellular processes and human diseases. The accumulated evidence points to the significant role these post-translational modifications play in aggregation and fibril formation. Simplified peptide model systems provide a means for investigating the molecular changes associated with protein aggregation. In this study, by using an amyloid-forming model peptide, we show that phosphorylation and glycosylation can affect folding and aggregation kinetics differently. Incorporation of phosphoserines, regardless of their quantity and position, turned out to be most efficient in preventing amyloid formation, whereas O-glycosylation has a more subtle effect. The introduction of a single beta-galactose does not change the folding behavior of the model peptide, but does alter the aggregation kinetics in a site-specific manner. The presence of multiple galactose residues has an effect similar to that of phosphorylation.

Molecular Recognition at the Membrane−Water Interface: Controlling Integral Peptide Helices by Off-Membrane Nucleobase Pairing

The aggregation and organization of membrane proteins and transmembrane peptides is related to the interacting molecular species itself and strongly depends on the lipid environment. Because of the complexity and dynamics of these interactions, they are often hardly traceable and nearly impossible to predict. For this reason, peptide model systems are a valuable tool in studying membrane associated processes since they are synthetically accessible and can be readily modified. To control and study the aggregation of peptide transmembrane domains (TMDs) the interacting interfaces of the TMDs themselves can be altered. A second less extensively studied approach targets the TMD assembly by using interaction and recognition of domains at the membrane outside as frequently found in the membrane protein interplay and protein assembly. In the present study, double helical transmembrane domains were designed and synthesized on the basis of a recently reported d,l-alternating peptide pore motif derived from gramicidin A. The highly hydrophobic and aromatic transmembrane peptide was covalently functionalized with a short peptide nucleic acid (PNA) used as specific outer-membrane recognition unit. The PNA sequences were chosen with high polarity to ensure localization within the aqueous phase. To estimate the impact of the membrane adjacent recognition on the TMD assembly by Förster resonance energy transfer (FRET), fluorescence probes were covalently attached to the side chains of the membrane spanning peptide helices. Dimerization of the TMD-peptide/PNA conjugates within unilamellar lipid vesicles was observed. The dimer/monomer ratio of TMDs can be controlled by temperature variation.

Non-peptide entry inhibitors of HIV-1 that target the gp41 coiled coil pocket

The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide 1 is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3–41 μM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.

The inherent flexibility of peptides and protein fragments quantitized by CD in conjunction with CCA+

ECD spectroscopy is traditionally used for rapid, non-atomic level structure analysis of natural products such as peptides and proteins. Unlike globular proteins, peptides less frequently adopt a single 3D-fold in a time average manner. Moreover, they exhibit an ensemble of conformers composed of a multitude of substantially different structures. In principle, both ECD- and vibrational circular dichroism (VCD)-spectroscopy are sensitive enough to pick up structural information on these dynamic ensembles. However, the interpretation of the raw spectral data of these highly dynamic molecular systems can be cumbersome. The herein presented Convex Constraint Analysis Plus method, or CCA+ for short (http://www.chem.elte.hu/departments/protnmr/cca/), provides a unique opportunity for spectral ensemble analysis of peptides, glycopeptides, peptidomimetics, and other foldamers. The precision and accuracy of the approach is presented here through different peptide model systems. An interesting temperature and pH dependent folding and unfolding of a miniprotein (e.g. Tc5b variant) is also described. Analysis of CD spectra sets strongly affected by solvent and ion type is also introduced to account for severe environmental-induced structure influencing effect(s). The deconvolution makes always possible the quantitative data analysis even when the interpretation of the deconvolution resulted in pure CD curves is complex.