Peptide Model System Research Articles

Solid State NMR Studies Of Structural And Motional Complexity In Amyloid-Like Fibrils Of The Peptide GNNQQNY

Solid state NMR allows the site-specific characterization of structure and dynamics in a variety of immobilized biomolecules, and thus allows a unique structural view on amyloid fibrils. These fibrils are common to various human disorders and appear to share a number of characteristic features, both in terms of their structure and formation. In the hope of delineating the biophysical details of the fibrillization process and the fibrils themselves, various groups have focused on the experimental and theoretical study of small peptide fragments of amyloid-forming proteins. One prominent system is the GNNQQNY7-13 fragment of the yeast prion protein Sup35p, since it was found to form not just amyloid-like fibrils, but also seemingly amyloid-like microcrystals. X-ray diffraction based structures from the latter have inspired numerous theoretical analyses and generalizations regarding the biophysics and structures of amyloid fibrils.We have instead applied biological solid state NMR methods to characterize the GNNQQNY fibrillar aggregates. Magic angle spinning (MAS) solid state NMR was used for various structural measurements, aimed at both the intramolecular as well as intermolecular structural motifs of the fibrils (as well as the crystalline aggregates). Our studies have revealed a remarkable complexity in these fibrils, despite the relatively small size of the peptide building blocks. This is in marked contrast with the rigid and homogeneous nature of the crystalline structures, as revealed by X-ray crystallography and solid state NMR. These observations provide further insights into the structure of the fibrils of this peptide model system and should also be of importance as input to numerous theoretical studies that rely on the crystal structure data.

Charge-Pairing Mechanism of Phosphorylation Effect upon Amyloid Fibrillation of Human Tau Core Peptide

Phosphorylation of a fibrillogenic protein, human tau, is believed to play crucial roles in the pathogenesis of Alzheimer's disease. For elucidating molecular mechanisms of the phosphorylation effect on tau fibrillation, we synthesized a peptide, VQIVY 310K (PHF6) and its phosphorylated derivative (PHF6pY). PHF6 is a partial peptide surrounding a plausible in vivo phosphorylation site Tyr310 and forms amyloid-type fibrils similar to those generated by full-length tau. Fibrillation of PHF6 and PHF6pY were studied by spectroscopic and microscopic methods, and the critical concentration of the fibrillation was determined for comparing the fibril stability. The results showed that the phosphorylation strongly influenced the fibrillation propensity of PHF6 by changing its dependency on pH and ionic strength. On the basis of the observations, we suggested that charged sites on the phosphate group and its electrostatic pairing with the neighboring charged residues were physical origins of the phosphorylation effect. To verify this charge-pairing mechanism, we conducted experiments using a series of PHF6 derivatives with non-native charge distributions. The electrostatic interaction in an intermolecular mode was also demonstrated by the system composed of two different peptide species, which found that fibrillation of nonphosphorylated PHF6 was drastically enhanced when a trace amount of phosphorylated PHF6 molecules coexisted. A simulation analysis utilizing crystal coordinates of the PHF6 fibril was also performed for interpreting the experimental results in a molecular level. The present study using the model peptide system gave us a microscopically insightful view on the roles of tau phosphorylation in amyloid-related diseases.

Femtosecond pulse shaping as analytic tool in mass spectrometry of complex polyatomic systems

An additional dimension to mass spectrometric studies on building blocks of proteins is discussed in this paper. The present approach is based on tailored femtosecond laser pulses, using the concept of strong-field pulse shaping in an adaptive feedback loop. We show that control strategies making use of coherent properties of the electromagnetic wave allow one to break pre-selected backbone bonds in amino acid complexes that may be regarded as peptide model systems. Studies on different chromophores, such as phenylalanine and alanine, while keeping the backbone structure unchanged elucidates the effect of the excitation dynamics on the relaxation pathways. The observation of protonated species in the corresponding mass spectra indicates that optimal control of ultrafast laser pulses may even be useful to study intramolecular reactions such as hydrogen- or proton-transfer in particular cases. This opens new perspectives for biophysical and biochemical research, since these photochemical reactions are suggested to explain, e.g. photostability of DNA.

Atomic Models of De Novo Designed ccβ-Met Amyloid-Like Fibrils

The common characteristics of amyloid and amyloid-like fibrils from disease- and non-disease-associated proteins offer the prospect that well-defined model systems can be used to systematically dissect the driving forces of amyloid formation. We recently reported the de novo designed ccβ peptide model system that forms a native-like coiled-coil structure at low temperatures and which can be switched to amyloid-like fibrils by increasing the temperature. Here, we report a detailed molecular description of the system in its fibrillar state by characterizing the ccβ-Met variant using several microscopic techniques, circular dichroism spectroscopy, X-ray fiber diffraction, solid-state nuclear magnetic resonance, and molecular dynamics calculations. We show that ccβ-Met forms amyloid-like fibrils of different morphologies on both the macroscopic and atomic levels, which can be controlled by variations of assembly conditions. Interestingly, heterogeneity is also observed along single fibrils. We propose atomic models of the ccβ-Met amyloid-like fibril, which are in good agreement with all experimental data. The models provide a rational explanation why oxidation of methionine residues completely abolishes ccβ-Met amyloid fibril formation, indicating that a small number of site-specific hydrophobic interactions can play a major role in the packing of polypeptide-chain segments within amyloid fibrils. The detailed structural information available for the ccβ model system provides a strong molecular basis for understanding the influence and relative contribution of hydrophobic interactions on native-state stability, kinetics of fibril formation, fibril packing, and polymorphism.

Molecular Recognition and Screening Using a 15N Group Selective STD NMR Method

We present a novel saturation transfer difference (STD) experiment where group selective (GS) saturation of amide protons in (15)N labeled hosts is achieved. It is demonstrated that a train of BIRD(d) pulses that inverts only protons attached to (15)N indeed results in saturation of the amide protons, while the background proton magnetization is much less affected. The undesired effect of partial saturation of the unlabeled protons can be completely cancelled out in difference spectra by switching the (15)N carrier between the on- and the off-resonance frequencies. As a result, clean and artifact-free STD spectra are obtained without the need of time-consuming optimization of experimental parameters and acquiring control spectra in the absence of the host. The use of the (15)N-GS STD experiment is demonstrated for the case of a glycopeptide antibiotic (dimeric eremomycin)-cell-wall analogue peptide (N-Ac-D-Ala) model system where the host and guest (1)H signals overlap. The application seems feasible for ligand screening against proteins without the prerequisite of a clean on-resonance frequency or defined ligand library. The new experiment can be used as the basis for studying intermolecular interactions where the standard STD experiment is difficult to optimize.

Recognition of trimethyllysine by a chromodomain is not driven by the hydrophobic effect.

Posttranslational modifications of histone proteins regulate gene expression via complex protein-protein and protein-DNA interactions with chromatin. One such modification, the methylation of lysine, has been shown to induce binding to chromodomains in an aromatic cage [Nielsen PR, et al. (2002) Nature 416:103-107]. The binding generally is attributed to the presence of cation-pi interactions between the methylated lysine and the aromatic pocket. However, whether the cationic component of the interaction is necessary for binding in the aromatic cage has not been addressed. In this article, the interaction of trimethyllysine with tryptophan is compared with that of its neutral analog, tert-butylnorleucine (2-amino-7,7-dimethyloctanoic acid), within the context of a beta-hairpin peptide model system. These two side chains have near-identical size, shape, and polarizabilities but differ in their charges. Comparison of the two peptides reveals that the neutral side chain has no preference for interacting with tryptophan, unlike trimethyllysine, which interacts strongly in a defined geometry. In vitro binding studies of the histone 3A peptide containing trimethyllysine or tert-butylnorleucine to HP1 chromodomain indicate that the cationic moiety is critical for binding in the aromatic cage. This difference in binding affinities demonstrates the necessity of the cation-pi interaction to binding with the chromodomain and its role in providing specificity. This article presents an excellent example of synergy between model systems and in vitro studies that allows for the investigation of the key forces that control biomolecular recognition.

Role of Aromatic Side Chains in the Folding and Thermodynamic Stability of Integral Membrane Proteins

Aromatic residues are frequently found in helical and beta-barrel integral membrane proteins enriched at the membrane-water interface. Although the importance of these residues in membrane protein folding has been rationalized by thermodynamic partition measurements using peptide model systems, their contribution to the stability of bona fide membrane proteins has never been demonstrated. Here, we have investigated the contribution of interfacial aromatic residues to the thermodynamic stability of the beta-barrel outer membrane protein OmpA from Escherichia coli in lipid bilayers by performing extensive mutagenesis and equilibrium folding experiments. Isolated interfacial tryptophanes contribute -2.0 kcal/mol, isolated interfacial tyrosines contribute -2.6 kcal/mol, and isolated interfacial phenylalanines contribute -1.0 kcal/mol to the stability of this protein. These values agree well with the prediction from the Wimley-White interfacial hydrophobicity scale, except for tyrosine residues, which contribute more than has been expected from the peptide models. Double mutant cycle analysis reveals that interactions between aromatic side chains become significant when their centroids are separated by less than 6 A but are nearly insignificant above 7 A. Aromatic-aromatic side chain interactions are on the order of -1.0 to -1.4 kcal/mol and do not appear to depend on the type of aromatic residue. These results suggest that the clustering of aromatic side chains at membrane interfaces provides an additional heretofore not yet recognized driving force for the folding and stability of integral membrane proteins.

Electrostatically tuned rate of peptide self-assembly resolved by multiple particle tracking

Hydrogels formed from the self-assembly of oligopeptides are being extensively studied for biomedical applications. The kinetics of their gelation, as well as a quantitative description of the forces controlling the rate of assembly has not yet been addressed. We report here the use of multiple particle tracking to measure the self-assembly kinetics of the model peptide FKFEFKFE (KFE8). KFE8 forms well-defined β-sheet intermediates and is often used as a model peptide system that forms a fibrous network in aqueous solvent. We find that increasing the pH of this system from 3.5 to 4.0 decreases the time of KFE8 gelation by almost hundredfold, from hours to minutes. A remarkable self-similarity between measurements performed at different pH suggests that, although accelerated by the pH increase, gelation follows an invariable mechanism. We propose a semi-quantitative interpretation for the order of magnitudes of gelation time using a simple model for the interaction driving the self-assembly in terms of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Such understanding is important for the development of current and future therapeutic applications ( drug delivery).

Evaluation of a carbohydrate–π interaction in a peptide model system

A carbohydrate-pi interaction contributes -0.8 kcal mol(-1) to the stabilization of a beta-hairpin peptide.

Conformational Preference of Polyglycine in Solution to Elongated Structure

Do polypeptide chains ever behave like a random coil? In this report we demonstrate that glycine, the residue with the fewest backbone restrictions, exhibits a strong preference for an extended conformation in solution when polymerized in short segments of polyglycine. A model peptide system comprised of two unique tripeptide units, between which 1 to 18 glycine residues are inserted, is characterized by NMR and by small-angle X-ray scattering (SAXS). The residual dipolar coupling (RDC) values of the two tripeptide units are insensitive to changes in number of intervening glycines, suggesting that extension of the linker does not alter the average angular relationship between the tripeptides. Polyglycine segments longer than nine residues form insoluble aggregates. SAXS measurements using synchrotron radiation provide direct evidence that polyglycine peptides adopt elongated conformations. In particular, the construct with a linker with six glycines showed a scattering profile indicative of a monomeric state with a radius of gyration and the maximum dimension of 9.1 A and approximately 34 A, respectively. The ensemble averaged global structure of this 12-mer peptide can best be approximated by a cylinder with a radius of 4 A and a length of approximately 33 A, making it intermediate in extension between a beta strand and an alpha helix.

Contribution of Cation−π Interactions to Protein Stability

Calculations predict that cation- interactions make an important contribution to protein stability. While there have been some attempts to experimentally measure strengths of cation-pi interactions using peptide model systems, much less experimental data are available for globular proteins. We have attempted to determine the magnitude of cation-pi interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP, and Trx). In each case, Lys was replaced with Gln and Met. In a separate series of experiments, the aromatic amino acid in each cation-pi pair was replaced by Leu. Stabilities of wild-type (WT) and mutant proteins were characterized by both thermal and chemical denaturation. Gln and aromatic --> Leu mutants were consistently less stable than corresponding Met mutants, reflecting the nonisosteric nature of these substitutions. The strength of the cation-pi interaction was assessed by the value of the change in the free energy of unfolding [DeltaDeltaG(degrees) = DeltaG(degrees)(Met) - DeltaG(degrees)(WT)]. This ranged from +1.1 to -1.9 kcal/mol (average value -0.4 kcal/mol) at 298 K and +0.7 to -2.6 kcal/mol (average value -1.1 kcal/mol) at the Tm of each WT. It therefore appears that the strength of cation-pi interactions increases with temperature. In addition, the experimentally measured values are appreciably smaller in magnitude than calculated values with an average difference /DeltaG(degrees)expt - DeltaG(degrees)calc/av of 2.9 kcal/mol. At room temperature, the data indicate that cation-pi interactions are at best weakly stabilizing and in some cases are clearly destabilizing. However, at elevated temperatures, close to typical Tm's, cation-pi interactions are generally stabilizing.

Ion-Binding Study by17O Solid-State NMR Spectroscopy in the Model Peptide Gly-Gly-Gly at 19.6 T

Li(+) and Ca(2+) binding to the carbonyl oxygen sites of a model peptide system has been studied by (17)O solid-state NMR spectroscopy. (17)O chemical shift (CS) and quadrupole coupling (QC) tensors are determined in four Gly-(Gly-(17)O)-Gly polymorphs by a combination of stationary and fast magic-angle spinning (MAS) methods at high magnetic field, 19.6 T. In the crystal lattice, the carbonyl oxygen of the central glycyl residue in two gly-gly-gly polymorphs form intermolecular hydrogen bonds with amides, whereas the corresponding carbonyl oxygens of the other two polymorphs form interactions with Li(+) and Ca(2+) ions. This permits a comparison of perturbations on (17)O NMR properties by ion binding and intermolecular hydrogen bonding. High quality spectra are augmented by density functional theory (DFT) calculations on large molecular clusters to gain additional theoretical insights and to aid in the spectral simulations. Ion binding significantly decreases the two (17)O chemical shift tensor components in the peptide plane, delta(11) and delta(22), and, thus, a substantial change in the isotropic chemical shift. In addition, quadrupole coupling constants are decreased by up to 1 MHz. The effects of ion binding are found to be almost an order of magnitude greater than those induced by hydrogen bonding.

Membrane Binding and Structure of De Novo Designed α‐Helical Cationic Coiled‐Coil‐Forming Peptides

We introduce a de novo designed peptide model system that enables the systematic study of 1) the role of a membrane environment in coiled-coil peptide folding, 2) the impact of different domains of an alpha-helical coiled-coil heptad repeat on the interaction with membranes, and 3) the dynamics of coiled-coil peptide-membrane interactions depending on environmental conditions. Starting from an ideal alpha-helical coiled-coil peptide sequence, several positively charged analogues were designed that exhibit a high propensity toward negatively charged lipid membranes. Furthermore, these peptides differ in their ability to form a stable alpha-helical coiled-coil structure. The influence of a membrane environment on peptide folding is studied. All positively charged peptides show strong interactions with negatively charged membranes. This interaction induces an alpha-helical structure of the former random-coil peptides, as revealed by circular dichroism measurements. Furthermore, vesicle aggregation is induced by a coiled-coil interaction of vesicle-bound peptides. Dynamic light scattering experiments show that the strength of vesicle aggregation increases with the peptide's intrinsic ability to form a stable alpha-helical coiled coil. Thus, the peptide variant equipped with the strongest inter- and intra-helical coiled-coil interactions shows the strongest effect on vesicle aggregation. The secondary structure of this peptide in the membrane-bound state was studied as well as its effect on the phospholipids. Peptide conformation within the peptide-lipid aggregates was analyzed by (13)C cross-polarization magic-angle spinning NMR experiments. A uniformly (13)C- and (15)N-labeled Leu residue was introduced at position 12 of the peptide chain. The (13)C chemical shift and torsion angle measurements support the finding of an alpha-helical structure of the peptide in its membrane-bound state. Neither membrane leakage nor fusion was observed upon peptide binding, which is unusual for amphiphatic peptide structures. Our results lay the foundation for a systematic study of the influence of the alpha-helical coiled-coil folding motif in membrane-active events on a molecular level.

De novo design of a two-stranded coiled-coil switch peptide

The properties and characteristics shared by amyloid fibrils formed from disease and non-disease associated proteins that are unrelated in sequence and structure offer the prospect that model systems can be used to systematically assess the factors that predispose a native protein to form amyloid fibrils. Based on a de novo design approach, we recently reported a unique switch peptide model system, ccβ, that forms a three-stranded coiled-coil structure at low temperatures and which can be easily converted to amyloid fibrils by increasing the temperature. To simplify the system further, we describe here the redesign of a two-stranded ccβ coiled-coil variant and its detailed analysis by a variety of biophysical methods. Compared with the original design, the characteristics of the peptide make it even simpler to elucidate and validate fundamental principles of amyloid fibril-formation.

A Conformational Two-State Peptide Model System Containing an Ultrafast but Soft Light Switch

Combining an azobenzene chromophore with the bis-cysteinyl active-site sequence of the protein disulfide isomerase (PDI) we constructed a simple but promising model for allosteric conformational rearrangements. Paralleling cellular signaling events, an external trigger, here absorption of a photon, leads to a structural change in one part of the molecule, namely the azobenzene-based chromophore. The change in geometry translates to the effector site, in our case the peptide sequence, where it modifies covalent and nonbonded interactions and thus leads to a conformational rearrangement. NMR spectroscopy showed that the trans-azo and cis-azo isomer of the cyclic PDI peptide exhibit different, but well-defined structures when the two cystine residues form a disulfide bridge. Without this intramolecular cross-link conformationally more variable structural ensembles are obtained that again differ for the two isomeric states. Ultrafast UV/Vis spectroscopy confirmed that the rapid isomerization of azobenzene is not significantly slowed down when incorporated into the cyclic peptides, although the amplitudes of ballistic and diffusive pathways are changed. The observation that most of the energy of an absorbed photon is dissipated to the solvent in the first few picoseconds when the actual azo-isomerization takes place is important. The conformational rearrangement is weakly driven due to the absence of appreciable excess energy and can be described as biased diffusion similar to natural processes.

Double quantum coherence electron spin resonance on coupled Cu(II)–Cu(II) electron spins

We demonstrate for the first time the ability to generate double quantum coherences (DQCs) for the case of Cu(II). We show that small splittings (∼7 MHz) from the Cu(II)–Cu(II) electron–electron magnetic dipolar interaction can be reliably resolved even though the inhomogeneously broadened Cu(II) linewidth is ∼2 GHz. A Cu(II)–Cu(II) distance of 2.0 nm was measured on a model peptide system, thus, demonstrating that distances on the nanometer scale may be measured using DQC electron spin resonance (ESR).

Distinguishing and Quantifying Peptides and Proteins Containingd-Amino Acids by Tandem Mass Spectrometry

Tandem mass spectrometry (MS/MS) utilizing both electron capture dissociation (ECD) and collisionally activated dissociation (CAD) was used to develop a qualitative and quantitative analytical method for chiral analysis of individual amino acid residues in polypeptides. ECD produced a more distinct chiral recognition than CAD, which is attributed to the smaller degree of vibrational excitation in ECD. Several peptide and protein model systems were used in this study, including the smallest known protein, tryptophan cage, a lactoferrin peptide, and the biologically relevant opioid peptide, dermorphin. An adaptation of the kinetic method was used to quantify the degree of separation between fragmentation patterns of stereoisomeric peptides as a function of fragment ion abundances. The obtained calibration scale for relative abundances of d-amino acids in diastereomeric peptide mixtures was accurate to 1% for ECD and to 3-5% for CAD. It was found that separation and quantification of stereoisomers could be advantageously performed by nanoflow reversed-phase liquid chromatography, with the objective of on-line MS/MS limited to stereoisomer identification. This technique shows promise for the analysis of chiral substitution in peptides and proteins, broadening the application area for tandem mass spectrometry.

The influence of phospholipid membranes on bovine calcitonin secondary structure and amyloid formation

Calcitonin, a peptide hormone associated with medullary carcinoma of the thyroid, has the potential to form amyloid fibrils and may be a valuable model for investigating the role of peptide-membrane interactions in beta-sheet and amyloid formation. Via a new model peptide system, bovine calcitonin, we found that the exposure of peptide to phospholipid membranes altered its structure relative to the structures formed in aqueous solutions. Of particular relevance to the amyloidoses, incubation of calcitonin with cholesterol-rich and ganglioside-containing membranes resulted in significant enrichment in the beta-sheet and amyloid content of the peptide. The formation of amyloid was also accelerated in these systems. A correlation between the phospholipid-induced structural alterations and calcitonin binding affinities to phospholipid membranes was evident. Bovine calcitonin has considerably higher binding affinity for the phospholipid systems that enhanced its beta-sheet and amyloid structure. Electrostatic forces were not the governing forces behind the observed behavior, as supported by the fact that the ionic strength did not affect the peptide structures or binding affinities. A Van't Hoff analysis of the temperature-dependent peptide binding affinities indicated that binding led to an increase in enthalpy and possibly an increase in entropy of the peptide-membrane systems. Experiments with other amyloid-forming peptides such as beta-amyloid of Alzheimer's disease have also shown similar results and may indicate the need to manipulate peptide-membrane interactions in order to control amyloid formation and its associated disease.

Design of a Coiled-Coil-based Model Peptide System to Explore the Fundamentals of Amyloid Fibril Formation

Protein deposition as amyloid fibrils underlies more than twenty severely debilitating human disorders. Interestingly, recent studies suggest that all peptides and proteins possess an intrinsic ability to assemble into amyloid fibrils similar to those observed in disease states. The common properties and characteristics of amyloid aggregates thus offer the prospect that simple model systems can be used to systematically assess the factors that predispose a native protein to form amyloid fibrils and understand the origin and progression of fatal disorders associated with amyloid formation. Here, we report the de novo design of a 17-residue peptide model system, referred to as ccβ, which forms a protein-like coiled-coil structure under ambient solution conditions but can be easily converted into amyloid fibrils by raising the temperature. Oxidation of methionine residues at selected hydrophobic positions completely abolished amyloid fibril formation of the peptide while not interfering with its coiled-coil structure. This finding indicates that a small number of site-specific hydrophobic interactions can play a major role in the packing of polypeptide chain segments within amyloid fibrils. The simplicity and characteristics of the ccβ system make it highly suitable for probing molecular details of the assembly of amyloid structures.

Reversibility of conformational switching in light-sensitive peptides

Reversible photo-control of protein structure and function is used by Nature to direct complex biological response to light. Reversibility is a feature that has been difficult to reliably achieve in analogous biomimetic systems. We report here measurement of the thermal and photoisomerization rates for peptide model systems in which isomerization of an intramolecularly linked azobenzene unit causes a helix-coil transition in the peptide. Reversibility is maintained and indeed the quantum yield for cis-to- trans photoisomerization is enhanced in these systems compared to an unstructured glutathione adduct of the chromophore alone. Reversibility can be understood by considering the intrinsic conformational preferences of the peptides to which the linkers are attached and how these preferences impact the stabilities of the transition states for isomerization.