Semi-Rigid Nitroxide Spin Label for Long-Range EPR Distance Measurements of Lipid Bilayer Embedded β-Peptides

β-Peptides are an interesting new class of transmembrane model peptides based on their conformationally stable and well-defined secondary structures. Herein, we present the synthesis of the paramagnetic β-amino acid β³-hTOPP (4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-d -β³-homophenylglycine) that enables investigations of β-peptides by EPR spectroscopy. This amino acid adds to the, to date, sparse number of β-peptide spin labels. Its performance was evaluated by investigating the helical turn of a 3₁₄-helical transmembrane model β-peptide. Nanometer distances between two incorporated β³-hTOPP labels in different environments were measured by using pulsed electron/electron double resonance (PELDOR/DEER) spectroscopy. Due to the semi-rigid conformational design, the label delivers reliable distances and sharp (one-peak) distance distributions even in the lipid bilayer. The results indicate that the investigated β-peptide folds into a 3.25₁₄ helix and maintains this conformation in the lipid bilayer.     

Aggregation modes of the spin mono-labeled tylopeptin B and heptaibin peptaibiotics in frozen solutions of weak polarity as studied by PELDOR spectroscopy

The X band PELDOR spectroscopy was used to investigate the magnetic dipole-dipole interactions in glassy solutions of nitroxide mono-labeled tylopeptin B and heptaibin peptaibiotics at 77 K. Specifically, a study was performed of the tylopeptin B peptides labeled at either position 3, 8, or 13, denoted as T3, T8, and T13, respectively. The heptaibin analogs labeled at either position 2 or 14, denoted as H2 and H14, respectively, were also investigated. It was shown that in frozen glassy peptide solutions in methanol, the spin labels are randomly distributed over the solvent volume. This result points to the absence of specific dipolar interactions between the peptides under these conditions. However, peptide aggregation was detected in weakly polar methanol/toluene environments. To study the properties of the resulting aggregates, we examined the depth of modulation for the PELDOR traces as a function of the concentration of the peptides in solution and the distances between the spin labels in the aggregates. Based on the concentration dependencies, the number of peptide molecules in the aggregates was estimated. We find that this value ranges from 2 to 3, depending on the position of the spin label in the peptide sequence. The combined analysis of the distance spectra and the number of peptide molecules in the aggregates allows us to suggest that dimer formation is the prevailing mode of self-association. In the case of spin-labeled tylopeptin B, the molecules in the dimer are head-to-head oriented. In addition, the distance spectra of the aggregates show that the C-termini of the molecules in the tylopeptin B dimer are more mobile than the Ntermini. This phenomenon leads to an increase in the spread of the distances between the nitroxides as the label position is approaching the peptide C-terminus. For heptaibin, we show that two forms of dimerization (head-to-head and head-to-tail) occur. Finally, in addition to dimers, aggregates containing 3 or 4 peptide molecules, which give broad lines in the distance spectra, are seen in solution.     

Selective High‐Resolution Detection of Membrane Protein–Ligand Interaction in Native Membranes Using Trityl–Nitroxide PELDOR

The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero‐oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro–electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter‐subunit interaction and the intra‐subunit conformational changes in hetero‐oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein–ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment.     

A Bis‐Manganese(II)–DOTA Complex for Pulsed Dipolar Spectroscopy

High‐spin gadolinium(III) and manganese(II) complexes have emerged as alternatives to standard nitroxide radical spin labels for measuring nanometric distances by using pulsed electron–electron double resonance (PELDOR or DEER) at high fields/frequencies. For certain complexes, particularly those with relatively small zero‐field splitting (ZFS) and short distances between the two metal centers, the pseudosecular term of the dipolar coupling Hamiltonian is non‐negligible. However, in general, the contribution from this term during conventional data analysis is masked by the flexibility of the molecule of interest and/or the long tethers connecting them to the spin labels. The efficient synthesis of a model system consisting of two [Mn(dota)]^2? (MnDOTA; DOTA^4?=1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetate) directly connected to the ends of a central rodlike oligo(phenylene–ethynylene) (OPE) spacer is reported. The rigidity of the OPE is confirmed by Q‐band PELDOR measurements on a bis‐nitroxide analogue. The Mn^II?Mn^II distance distribution profile determined by W‐band PELDOR is in reasonable agreement with one simulated by using a simple rotamer analysis. The small degree of flexibility arising from the linking MnDOTA arm appears to outweigh the contribution from the pseudosecular term at this interspin distance. This study illustrates the potential of MnDOTA‐based spin labels for measuring fairly short nanometer distances, and also presents an interesting candidate for in‐depth studies of pulsed dipolar spectroscopy methods on Mn^II?Mn^II systems.     

The susceptibility of α-helical secondary structure to steric strain: Coarse-grained simulation of dendronized polypeptides

The propensity of a peptide chain for adopting helical secondary structure can be modulated not only through the solvation properties of its side chains but also through their size and shape. Here we examine a coarse-grained model for dendronized polypeptides that focuses on the susceptibility of α-helical structure to the steric strain exerted by hydrophilic pendant groups. Undecorated molecules exhibit a pronounced transition from random coil to helix upon cooling [J. P. Kemp and J. Z. Y. Chen, Biomacromolecules 2, 389 (2001)]. As gauged by specific heat and by order parameters characterizing helicity at several length scales, this transition is quite robust to the introduction of first- and second-generation dendron side chains. More highly branched side chains, however, reduce the entropy of compact states so severely that helical ordering is undetectable over the entire temperature range accessible to our importance sampling methods. Consistent with experimental observations for side chains comparable to those of our model in volume-excluding size and shape, we find the backbone of these third-generation molecules to assume a distended rodlike state that is both stiff and achiral.     

Ring-closing metathesis of olefinic peptides: design, synthesis, and structural characterization of macrocyclic helical peptides

Heptapeptides containing residues with terminal olefin-derivatized side chains (3 and 4) have been treated with ruthenium alkylidene 1 and undergone facile ring-closing olefin metathesis (RCM) to give 21- and 23-membered macrocyclic peptides (5 and 6). The primary structures of peptides 3 and 4 were based upon a previously studied heptapeptide (2), which was shown to adopt a predominantly 3(10)-helical conformation in CDCl(3) solution and an alpha-helical conformation in the solid state. Circular dichroism, IR, and solution-phase (1)H NMR studies strongly suggested that acyclic precursors 3 and 4 and the fully saturated macrocyclic products 7 and 8 also adopted helical conformations in apolar organic solvents. Single-crystal X-ray diffraction of cyclic peptide 8 showed it to exist as a right-handed 3(10)-helix up to the fifth residue. Solution-phase NMR structures of both acyclic peptide 4 and cyclic peptide 8 in CD(2)Cl(2) indicated that the acyclic diene assumes a loosely 3(10)-helical conformation, which is considerably rigidified upon macrocyclization. The relative ease of introducing carbon-carbon bonds into peptide secondary structures by RCM and the predicted metabolic stability of these bonds renders olefin metathesis an exceptional methodology for the synthesis of rigidified peptide architectures.     

Conformational flexibility of DNA

Pulsed Electron-Electron Double Resonance (PELDOR) on double-stranded DNA (ds-DNA) was used to investigate the conformational flexibility of helical DNA. Stretching, twisting, and bending flexibility of ds-DNA was determined by incorporation of two rigid nitroxide spin labels into a series of 20 base pair (bp) DNA duplexes. Orientation-selective PELDOR experiments performed at both X-band (9 GHz/0.3 T) and G-band (180 GHz/6.4 T) with spin label distances in the range of 2-4 nm allowed us to differentiate between different simple models of DNA dynamics existing in the literature. All of our experimental results are in full agreement with a dynamic model for ds-DNA molecules, where stretching of the molecule leads to a slightly reduced radius of the helix induced by a cooperative twist-stretch coupling.     

PELDOR measurements on a nitroxide-labeled Cu(II) porphyrin: orientation selection, spin-density distribution, and conformational flexibility

Metal ions are functionally or structurally important centers in metalloproteins or RNAs, which makes them interesting targets for spectroscopic investigations. In combination with site-directed spin labeling, pulsed electron-electron double resonance (PELDOR or DEER) could be a well-suited method to characterize and localize them. Here, we report on the synthesis, full characterization, and PELDOR study of a copper(II) porphyrin/nitroxide model system. The X-band PELDOR time traces contain besides the distance information a convolution of orientational selectivity, conformational flexibility, exchange coupling, and spin density distribution, which can be deconvoluted by experiments with different frequency offsets and simulations. The simulations are based on the known experimental and spin Hamiltonian parameters and make use of a geometric model as employed for structurally similar bis-nitroxides and spin density parameters as obtained from density functional theory calculations. It is found that orientation selection with respect to dipolar angles is only weakly resolvable at X-band frequencies due to the large nitrogen hyperfine coupling of the copper porphyrin. On the other hand, the PELDOR time traces reveal a much faster oscillation damping than observed for structurally similar bis-nitroxides, which is mainly assigned to a small distribution in exchange couplings J. Taking the effects of orientation selectivity, distribution in J, and spin density distribution into account leads finally to a narrow distance distribution caused solely by the flexibility of the structure, which is in agreement with distributions from known bis-nitroxides of similar structure. Thus, X-band PELDOR measurements at different frequency offsets in combination with explicit time trace simulations allow for distinguishing between structural models and quantitative interpretation of copper-nitroxide PELDOR data gives access to localization of copper(II) ions.     

Light‐Induced Porphyrin‐Based Spectroscopic Ruler for Nanometer Distance Measurements

We present a novel pulsed electron paramagnetic resonance (EPR) spectroscopic ruler to test the performance of a recently developed spin‐labeling method based on the photoexcited triplet state (S=1). Four‐pulse electron double resonance (PELDOR) experiments are carried out on a series of helical peptides, labeled at the N‐terminal end with the porphyrin moiety, which can be excited to the triplet state, and with the nitroxide at various sequence positions, spanning distances in the range 1.8–8 nm. The PELDOR traces provide accurate distance measurements for all the ruler series, showing deep envelope modulations at frequencies varying in a progressive way according to the increasing distance between the spin labels. The upper limit is evaluated and found to be around 8 nm. The PELDOR‐derived distances are in excellent agreement with theoretical predictions. We demonstrate that high sensitivity is acquired using the triplet state as a spin label by comparison with Cu(II)–porphyrin analogues. The new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the properties of the porphyrin triplet state.     

Intramolecular chiral communication in peptide-dendron hybrids

The conformational properties of a series of peptide-dendron hybrids progressively incorporating 1-4 dendritic side chains were investigated by circular dichroism. Although the presence of multiple adjacent dendrons along the peptide backbone precluded the formation of α-helical or β-sheet secondary structure, intramolecular packing of the dendrons mediated efficient peptide → dendron chirality transfer in both organic and aqueous media.     

Secondary structures of short peptide chains in the gas phase: double resonance spectroscopy of protected dipeptides

The conformational structure of short peptide chains in the gas phase is studied by laser spectroscopy of a series of protected dipeptides, Ac-Xxx-Phe-NH(2), Xxx=Gly, Ala, and Val. The combination of laser desorption with supersonic expansion enables us to vaporize the peptide molecules and cool them internally; IR/UV double resonance spectroscopy in comparison to density functional theory calculations on Ac-Gly-Phe-NH(2) permits us to identify and characterize the conformers populated in the supersonic expansion. Two main conformations, corresponding to secondary structures of proteins, are found to compete in the present experiments. One is composed of a doubly gamma-fold corresponding to the 2(7) ribbon structure. Topologically, this motif is very close to a beta-strand backbone conformation. The second conformation observed is the beta-turn, responsible for the chain reversal in proteins. It is characterized by a relatively weak hydrogen bond linking remote NH and CO groups of the molecule and leading to a ten-membered ring. The present gas phase experiment illustrates the intrinsic folding properties of the peptide chain and the robustness of the beta-turn structure, even in the absence of a solvent. The beta-turn population is found to vary significantly with the residues within the sequence; the Ac-Val-Phe-NH(2) peptide, with its two bulky side chains, exhibits the largest beta-turn population. This suggests that the intrinsic stabilities of the 2(7) ribbon and the beta-turn are very similar and that weakly polar interactions occurring between side chains can be a decisive factor capable of controlling the secondary structure.     

Conformational structure of the tetrapeptide Boc–Aib–Leu–Leu–Aib–OMe–the central fragment of the nonapeptide antibiotic leucinostatin A

The conformational structure of the tetrapeptide Boc–Aib–Leu–Leu–Aib–OMe has been investigated by the PCILO method. The computational results show the formation of two closed β-turns, both of which are of type III, and the peptide backbone folds into a right-handed 3₁₀-helical conformation stabilized by two intramolecular 4 → 1 hydrogen bonds. The helix thus formed generates a pore of ～3 Å along helix axis with hydrophobic amino acid side chains located on the outside of the helix, and this tendency of leucine side chains may enable leucinostatin A to fit into the membrane bilayer. The pore thus formed is cation-selective, and through this pore, the cation can pass only in a single file.     

Theoretical study of the Raman optical activity spectra of 3(10)-helical polypeptides

Raman optical activity (ROA) spectroscopy is a promising analytical method for studying the structure and conformation of polypeptides and proteins in solution. However, the structural information obtained from such vibrational spectra is only indirect and theoretical studies are often necessary to identify how the structure determines the observed spectra. One particular target is the identification and discrimination of different helical secondary structure elements. Herein, a theoretical investigation of the ROA spectra of a series of 3(10)-helical polypeptides is presented. In particular, the effect of chain length, C(α)-substitution pattern, the introduction of larger aliphatic side chains, and the variation of their conformation on the ROA spectra is studied. To extract general principles from these calculations, the positions, intensities, and shapes of the ROA bands are analyzed in terms of localized modes, which makes it possible to identify possible ROA signatures of 3(10) -helical structures, but also provides fundamental insight into the generation of ROA signals in complex polypeptides. Finally, the calculated spectra can be compared to the previously reported ROA spectrum of a specifically designed 3(10) -helical heptapeptide. This allows most of the features in the experimental spectrum to be assigned.     

Conformationally Restricted Isoindoline‐Derived Spin Labels in Duplex DNA: Distances and Rotational Flexibility by Pulsed Electron–Electron Double Resonance Spectroscopy

Three structurally related isoindoline‐derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation‐dependent pulsed electron–electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, ^ExIm U , was synthesized and incorporated into DNA oligonucleotides. ^ExIm U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N?O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline‐based spin label to a uridine base. Continuous‐wave (CW) EPR measurements of ^ExIm U confirm a very high rotational mobility of the spin label in duplex DNA relative to the structurally related spin label ^Im U , which has restricted mobility due to an intramolecular hydrogen bond. The X‐band CW‐EPR spectra of ^ExIm U can be used to identify mismatches in duplex DNA. PELDOR distance measurements between pairs of the spin labels ^Im U , ^Ox U , and ^ExIm U in duplex DNA showed a strong angular dependence for ^Im U , a medium dependence for ^Ox U , and no orientation effect for ^ExIm U . Thus, precise distances can be extracted from ^ExIm U without having to take orientational effects into account.     

The Double‐Histidine Cu2+‐Binding Motif: A Highly Rigid,Site‐Specific Spin Probe for Electron Spin Resonance Distance Measurements

The development of ESR methods that measure long‐range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein‐backbone structure. Herein we present the double‐histidine (dHis) Cu²⁺‐binding motif as a rigid spin probe for double electron–electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X‐ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 Å of the experimental value. Cu²⁺ DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein‐backbone structure and flexibility.     

The Double‐Histidine Cu2+‐Binding Motif: A Highly Rigid,Site‐Specific Spin Probe for Electron Spin Resonance Distance Measurements

The development of ESR methods that measure long‐range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein‐backbone structure. Herein we present the double‐histidine (dHis) Cu²⁺‐binding motif as a rigid spin probe for double electron–electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X‐ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 Å of the experimental value. Cu²⁺ DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein‐backbone structure and flexibility.     

A pulsed EPR study of surfactant layer structure in composites of a synthetic layered silicate with polystyrene and polycaprolactone

Double electron electron resonance (DEER), deuterium electron spin-echo envelope modulation (ESEEM) spectroscopy and 31P electron nuclear double resonance (ENDOR) spectroscopy were applied to site-specifically spin-labeled surfactants in the organically modified layered silicate magadiite and its composites with polystyrene (PS) and polycaprolactone (PCL). The organomagadiite consist of stacks of silicate platelets with surfactant layers between these platelets. In PS composites the stacks are dispersed in the polymer matrix as a whole, while melt processing with PCL leads to intercalation of polymer chains into the galleries between the platelets. The DEER data prove that even in the case of the non-intercalated PS composites the density of surfactant molecules changes drastically during composite formation on length scales of a few nanometers. Deuterium ESEEM data demonstrate that spin labels attached both in the middle and at the end of the alkyl chain have contact with the headgroups of neighboring surfactant molecules. By analysis of the 31P ENDOR spectra, two characteristic distances are found between the spin labels and the headgroups of phosphonium surfactants. The shorter, proximal distance can be assigned to headgroups in the same surfactant layer. By comparison with the basal spacing between consecutive silicate platelets the longer, distal distance is assigned to a layer of surfactants that is not attached to the surface of the next platelet but rather located between platelets. Altogether the data support a picture of trilayers of disordered surfactant molecules with their alkyl chains oriented nearly parallel to the surface.     

Stapling of a 3(10)-helix with click chemistry

Short peptides are important as lead compounds and molecular probes in drug discovery and chemical biology, but their well-known drawbacks, such as high conformational flexibility, protease lability, poor bioavailability and short half-lives in vivo, have prevented their potential from being fully realized. Side chain-to-side chain cyclization, e.g., by ring-closing olefin metathesis, known as stapling, is one approach to increase the biological activity of short peptides that has shown promise when applied to 3(10)- and α-helical peptides. However, atomic resolution structural information on the effect of side chain-to-side chain cyclization in 3(10)-helical peptides is scarce, and reported data suggest that there is significant potential for improvement of existing methodologies. Here, we report a novel stapling methodology for 3(10)-helical peptides using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction in a model aminoisobutyric acid (Aib) rich peptide and examine the structural effect of side chain-to-side chain cyclization by NMR, X-ray diffraction, linear IR and femtosecond 2D IR spectroscopy. Our data show that the resulting cyclic peptide represents a more ideal 3(10)-helix than its acyclic precursor and other stapled 3(10)-helical peptides reported to date. Side chain-to-side chain stapling by CuAAC should prove useful when applied to 3(10)-helical peptides and protein segments of interest in biomedicine.     

Salt-specific stability and denaturation of a short salt-bridge-forming alpha-helix

The structure of a single alanine-based Ace-AEAAAKEAAAKA-Nme peptide in explicit aqueous electrolyte solutions (NaCl, KCl, NaI, and KF) at large salt concentrations (3-4 M) is investigated using approximately 1 mus molecular dynamics (MD) computer simulations. The peptide displays 71% alpha-helical structure without salt and destabilizes with the addition of NaCl in agreement with experiments of a somewhat longer version. It is mainly stabilized by direct and indirect (" i + 4")EK salt bridges between the Lys and Glu side chains and a concomitant backbone shielding mechanism. NaI is found to be a stronger denaturant than NaCl, while the potassium salts hardly show influence. Investigation of the molecular structures reveals that consistent with recent experiments Na (+) has a much stronger affinity to side chain carboxylates and backbone carbonyls than K (+), thereby weakening salt bridges and secondary structure hydrogen bonds. At the same time, the large I (-) has a considerable affinity to the nonpolar alanine in line with recent observations of a large propensity of I (-) to adsorb to simple hydrophobes, and thereby "assists" Na (+) in its destabilizing action. In the denatured states of the peptide, novel long-lived (10-20 ns) "loop" configurations are observed in which single Na (+) ions and water molecules are hydrogen-bonded to multiple backbone carbonyls. In an attempt to analyze the denaturation behavior within the preferential interaction formalism, we find indeed that for the strongest denaturant, NaI, the protein is least hydrated. Additionally, a possible indication for protein denaturation might be a preferential solvation of the peptide backbone by the destabilizing cosolute (sodium). The mechanisms found in this work may be of general importance to understand salt effects on protein secondary structure stability.     

Epr Spectroscopy of Adsorbed Spin-Labeled Peptides: Immobilization of Trichogin on the Titanium Oxide

The TOAC-spin-labeled peptide Trichogin GA IV adsorbed on the TiO₂ surface is studied. It is shown that the continuous wave (CW) electron paramagnetic resonance (EPR) spectrum does not depend on temperature in a wide range of 77–300 K. A pulsed EPR method of electron spin echo (ESE) utilizing a two-pulse sequence (π/2-τ-π) is used to study temperature dependence of the phase relaxation time, T_F. The T_F values are found to change from 750 ns to 100 ns in the interval of 77–300 K. The pulsed electronelectron double resonance (PELDOR) measurements utilizing the pulse sequence((π/2)_A,-T-π_B,-(τ-T)-π_A) show that the space distribution of spin labels on the surface remains uniform irrespective of the temperature, and provide the fractal dimension of the surface of 2.7±0.1. The obtained results testify that EPR pulse experiments can be used to study adsorbed spin-labeled molecules at room temperatures, i.e. not only at cryogenic temperatures.