Peptides on the Surface. PELDOR Data for Spin-Labeled

Varun Pratap Singh , Nalli Yedukondalu , Vandana Sharma , Manoj Kushwaha , Richa Sharma , Asha Chaubey , Anil Kumar , Deepika Singh , and Ram A...
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Peptides on the Surface. PELDOR Data for Spin-Labeled Alamethicin F50/5 Analogues on Organic Sorbent Alexander D. Milov,† Rimma I. Samoilova,† Yuri D. Tsvetkov,*,† Cristina Peggion,‡ Fernando Formaggio,‡ and Claudio Toniolo‡ †

V.V. Voevodsky Institute of Chemical Kinetics and Combustion, 630090 Novosibirsk, Russian Federation Institute of Biomolecular Chemistry, Padova Unit, CNR, Department of Chemistry, University of Padova, 35131 Padova, Italy

‡

ABSTRACT: The PELDOR technique was used to obtain the spectra of distances between spin labels for mono and double TOAC substituted analogues of [Glu(OMe)7,18,19] alamethicin F50/5 (Alm′) peptaibiotic on the surface of the organic sorbent Oasis HLB and in ethanol solution at 77 K. For the double-labeled Alm′, the free radical probes are at positions 1 and 16 (Alm′1,16). The intra- and intermolecular contributions to the PELDOR time traces were separated, with regard to the fractality of the system studied. We established that on HLB the labeled Alm′ molecules are prone to aggregation. The distance spectra for Alm′1,16 show that, in both adsorbed state and in ethanol solution, the peptaibiotic is predominantly folded in the α-helix conformation. We assign the asymmetry of the distance spectrum in both cases to the occurrence of an admixture of more elongated α/310-helical conformers. The portion of these conformers is higher for the peptide adsorbed on HLB. We speculate that both the broadening of the basic spectrum line at rmax = 2.0 nm and the increase in the contribution of elongated conformers might be associated with the spread of the peptaibiotic adsorption sites on HLB as compared with the more uniform Alm′1,16 trap structure in frozen ethanol solution. The aggregates of mono-labeled Alm′1 and Alm′16 also studied. The intermolecular distance spectrum for Alm′1 on HLB is shifted toward longer distances as compared with those of Alm′16. This result suggests that in the aggregates Alm′ molecules are preferentially oriented with their C-terminal regions in the vicinity.

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INTRODUCTION

Several works were carried out on more complex organic surfaces. Here, either a Au or SiO2 surface was covered with a monolayer of organic molecules or with several polymer layers on which the peptide was adsorbed.13−16 These multilayer systems serve as models for studying and creating materials for medical applications.17 The studies involved various physical methods to extract information both on the kinetics and mechanisms of peptide adsorption (microcrystal quartz balance method),15,16 and on the effect of the surface structure on peptide adsorption and orientation on organic monolayers (atomic force microscopy and spectroscopic methods).13,14,16 Similar to NMR, EPR studies of dipole−dipole interactions between unpaired electrons in spin-labeled peptides provide information on peptide conformations and aggregation motifs. Data for both frozen peptide solutions in organic solvents and peptides on lipid surfaces were extracted using the pulsed electron−electron double-resonance technique (PELDOR).18 In this work, we tried to assess the potentialities of PELDOR in investigating peptide conformation and aggregation upon adsorption on organic surfaces. Mono and double spin-labeled analogues of [Glu(OMe)7,18,19] (OMe, methoxy) alamethicin F50/5 (Alm′), the secondary structure of which is sensitive to the properties of the molecular environment, were used as adsorbates. The conformation and aggregation properties of

Studying the properties of peptides adsorbed on inorganic or organic surfaces is of interest for many interdisciplinary areas of academic science and applications, such as bionanotechnology, biocatalysis, bionanosensorics, etc. The characteristics of these systems are determined by the primary and secondary (backbone conformation) structures of the peptides, the nature of the surface, and the type of the peptide−surface interaction. This information can hardly be analyzed in detail by physical methods. For instance, circular dichroism data testify of conformational changes in peptides upon adsorption on inorganic surfaces.1,2 However, their structural interpretations are not straightforward. Numerous works were performed by calculations involving molecular dynamics methods of peptide adsorption on metals,3,4 their oxides,5−7 and quartz.8 However, these methods, in most of the cases, fail to extract details of peptide conformation upon adsorption.9,10 Remarkable success, however, was achieved by NMR studies of such systems.11,12 Structural investigations, using the solidphase NMR technique, of dipolar interactions between C13 and N15 atoms in adsorbate nuclei made it possible to establish the conformation and orientation of the statherin peptide upon its adsorption on hydroxyapaptite.11 To ascertain the relationship between the structure of the peptide and its ability to adsorb on an inorganic surface, the conformations and orientations of 12mer peptides of various sequences and 3D structures were studied by NMR on TiO2 and SiO2 oxide surfaces.12 © 2014 American Chemical Society

Received: April 15, 2014 Revised: May 29, 2014 Published: June 13, 2014 7085

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these spin-labeled Alm′ have been previously studied by PELDOR.19−23 Alamethicin F50/5 is characterized by a linear sequence of 19 amino acid residues, a high percentage of the Cα-tetrasubstituted, highly helicogenic α-aminoisobutyric acid (Aib), an N-terminal acetyl (Ac) group, and a C-terminal 1,2aminoalcohol (phenylalaninol, Phl).24 To determine the conformation of Alm′ accurately, the choice of the type of spin label is extremely important. In our approach, we replaced two helicogenic Aib residues25−27 by their equally helicogenic 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) residues28,29 at well-chosen positions in the peptide sequence. The six-membered ring of this spin-labeled amino acid is rigidly connected to the peptide backbone, and as a result, any change in the backbone conformation of the peptide is reflected in a predictable different location of the nitroxide electron spin. The synthetic alamethicin F50/5 analogue Alm′ was used,30 wherein the Aib residues at positions 1 (near the Nterminus) and/or 16 (near the C-terminus) of the peptide chain were substituted by TOAC spin labels (Scheme 1).

Scheme 2. Chemical Structure of the Oasis HLB Organic Sorbent (m-Divinylbenzene-N-vinylpyrrolidone Copolymer)31

and then covered with the solution of the Alm′ analogue in methylene chloride. The amount of the Alm′ analogue in solution did not exceed the sorbent capacity. The solution flow rate was about 1.5 mL/min. The deposition of the Alm′ analogues was controlled by EPR signal intensity. At room temperature, the column with the adsorbent was placed in a resonator of the EPR spectrometer. A CuCl2·2H2O single crystal was employed as a paramagnetic standard for calibrating the amount of the deposited spin-labeled Alm′ analogue. The residual methylene chloride was removed without changing the amount of the Alm′ analogue on the sorbent. To this end, the column was blown out with dry nitrogen for 1−2 min until drying. To perform measurements at 77 K, the samples were sealed to avoid condensation of the moisture. EPR and PELDOR Measurements. PELDOR data and CW EPR spectra were obtained using an X-band Bruker ELEXSYS E580 EPR spectrometer. For EPR measurements, the sample was located in the finger of a quartz Dewar vessel placed in the spectrometer resonator. EPR spectra were recorded at a modulation frequency of 100 kHz and a modulation amplitude of 0.1 mT and for lack of spectrum saturation. In pulse experiments, we used the following methods: three pulses (two detecting pulses and one pumping pulse), 3p PELDOR, and four pulses (three detecting pulses and one pumping pulse), 4pPELDOR. The PELDOR experiments were carried out using a split ring Bruker ER 4118 XMS-3 resonator and an Oxford Instruments CF-935 cryostat. The resonator was cooled with gaseous nitrogen. The sample temperature was kept near 77 K. According to ref 33 in the 3p PELDOR sequence, the pumping pulse scan starts at time d0 before the first detecting pulse. This makes it possible to detect the PELDOR signal independently of the pumping pulse effect in the T time scale from d0 up to the first detecting pulse. The delay d0 of the first detecting pulse relative to the beginning of the pumping pulse sweep amounted to 328 ns to allow the pumping pulse to go through the first detecting pulse. All detecting pulses at frequency νA are of equal duration (24 ns). The rotation angles of the A spins at frequency νA under the action of the first and second detecting pulses were 90 and 180, respectively, and were controlled by the echo signal shape and intensity. The pumping pulse duration at the νB frequency was 24 ns. The rotation angle of the B spins under the action of the pumping pulse was measured by comparing the experimental and calculated shapes of the echo signal of the A spins provided that νA = νB. In this case, the pumping pulse was applied at the beginning of the

Scheme 1. Chemical Structures of Aib and TOAC and Amino Acid Sequences of Alamethicin F50/5 Spin-Labeled Analogues

Alamethicin F50/5 (Alm). Ac-Aib-Pro-Aib-Ala-Aib-AlaGln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Gln-Gln-Phl Alm′. Ac-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-Aib-Val-AibGly-Leu-Aib-Pro-Val-Aib-Aib-Glu(OMe)-Glu(OMe)-Phl Alm′1. Ac-TOAC-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-Aib-ValAib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(OMe)-Glu(OMe)-Phl Alm′16. Ac-Aib-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-Aib-ValAib-Gly-Leu-Aib-Pro-Val-TOAC-Aib-Glu(OMe)-Glu(OMe)Phl Alm′1,16. Ac-TOAC-Pro-Aib-Ala-Aib-Ala-Glu(OMe)-AibVal-Aib-Gly-Leu-Aib-Pro-Val-TOAC-Aib-Glu(OMe)-Glu(OMe)-Phl The patented, polymeric, reversed-phase organic sorbent Oasis HLB (HLB) was employed as the organic surface.31 It manifests both hydrophilic and lipophilic properties, is unique in its purity, reproducibility, and stability, and is widely applied in the chromatographic practice. The chemical structure of its surface is shown in Scheme 2.

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EXPERIMENTAL SECTION Materials and Methods. Syntheses and characterizations of the TOAC-labeled Alm′ peptaibiotics used here were reported elsewhere.32 In our work, the Oasis HLB sorbent was used. The mean diameter of the sorbent pores was 8.2 nm, and the specific surface area was 831 m2/g.31 The samples were prepared as follows. The sorbent (30−50 mg in methanol solution) was transferred to small columns, 2.8−3 mm in diameter, and kept there until application. Before usage, the columns were washed sequentially with 1 mL of methanol and 1 mL of water and activated again with methanol. Prior to peptide deposition, the column was freed of methanol 7086

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PELDOR Data. Figure 2 presents the PELDOR signal traces for the double- and mono-labeled Alm′ on the HLB surface and in ethanol at 77 K.

scan (at d0 = 328 ns). The turning angle of the B spins was 102 ± 2°. In all experiments, the pumping pulse frequency was set at the maximum of the EPR spectrum. To minimize the orientation selectivity effects, the difference (νA − νB) was equal to 65 MHz.34 The pumping pulse sweeping was performed with steps of 8 ns. The spin echo signal was integrated completely in a gate of 104 ns. The changes in the PELDOR signal upon passage of the pumping pulse through the first detecting pulses were corrected using the method already described.35 Expressions presented in ref 33 for obtaining the spectra of distances between the labels in the 3p PELDOR experiments hold for the case where the pumping mw pulse does not overlap the detecting pulses. Thus, to analyze the 3p PELDOR time T traces, the starting time T 0 was determined experimentally as the time at which the falling edge of the first detecting pulse coincides with the rising one of the pumping pulse.33 To study the fractal dimensionality of the HLB sorbent, the initial position of the pumping pulse for 3p PELDOR was found experimentally using an approach already reported36 for comparing the 3p and 4p PELDOR time traces obtained under the same experimental conditions. To get the point T = 0, we compared the 3p and 4p PELDOR time traces for Alm′1,16 solutions in ethanol at 77 K by minimizing the difference between the oscillating parts of these curves depending on the relative position of the curves on the T axis. The position of the spin echo maximum for the 4p time trace upon complete coincidence of the 3p and 4p PELDOR traces was taken as T = 0 for the 3p PELDOR.

Figure 2. PELDOR time traces of spin-labeled Alm′ analogues on HLB and in ethanol at 77 K. Curves 1 and 3 are obtained for Alm′1,16 on HLB at peptide concentrations of 5 × 10−4 and 1.2 × 10−3 M, respectively. Curve 2 is obtained for Alm′1,16 in ethanol at a peptide concentration of 8 × 10−4 M. Curves 4 and 5 refer to Alm′1 and Alm′16 on HLB, respectively. For an easy comparison, curves 4 and 5 are shifted down by 0.4. V0 is the PELDOR signal value in the absence of the pumping pulse.

Curves 1 and 3 refer to Alm′1,16 on HLB, and curve 2 belongs to an Alm′1,16 solution in ethanol. The PELDOR time traces 1−3 exhibit a fast decay at low T followed by a slow decay due to the intermolecular dipole−dipole interactions of spin labels (background line). In the case of Alm′1,16 in ethanol, the trace is modulated owing to the intramolecular interactions of labels. The modulation depth λ, obtained from the crossing point of the background line and the vertical axis, increases with increasing amount of peptide Alm′1,16 on the HLB surface. This finding indicates the occurrence of Alm′1,16 aggregates under these conditions. In this connection, the intramolecular interactions of spin labels in Alm′1,16 were studied for the lower peptide concentration on the HLB surface, where the λ values are close to each other on the HLB surface and in ethanol (curves 1 and 3). This result allows the assumption that for curve 1 the content of aggregated molecules of Alm′1,16 on the HLB surface is low enough to neglect the influence of aggregation on the PELDOR time trace. A comparison of curves 1, 2, and 3 in Figure 2 indicates that in ethanol the intramolecular dipolar interactions of spin labels in Alm′1,16 lead to a PELDOR time trace dipolar modulation, whereas for the Alm′1,16 molecules adsorbed on the HLB surface the dipolar modulation is absent. This difference in the behavior of the PELDOR signal suggests a wider spectrum of distances between the spin labels for the Alm′1,16 molecules on the HLB surface than that in ethanol. Curves 4 and 5 in Figure 2 represent the PELDOR time traces for Alm′1 and Alm′16 on the HLB surface. As for Alm′1,16 on HLB (curves 1 and 3), for curves 4 and 5, we also note a fast initial decay of the PELDOR time traces followed by a slow decay. PELDOR time traces of this type support the view that aggregation of both Alm′1 and Alm′16 molecules does take place on the HLB surface. To determine the spectra of distances between labels from the PELDOR time traces, we selected the part, VINTRA, which corresponds to either intramolecular (for Alm′1,16) or intraaggregate (for Alm′1 and Alm′16) interaction of spin labels, by

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RESULTS AND DISCUSSION CW EPR Spectra. Figure 1 shows both the CW EPR spectra of peptides Alm′1, Alm′16, and Alm′1,16 on HLB and the spectrum of Alm′1,16 in ethanol.

Figure 1. CW EPR spectra of spin-labeled Alm′ analogues in ethanol and on HLB at 77 K. Curves 1 and 2 are the spectra of Alm′1 and Alm′16 on the HLB surface, respectively. Curves 3 and 4 are the spectra of Alm′1,16 on HLB and in ethanol, respectively.

The shape of the lines is the same for the adsorbed peptides and the peptide in solution. However, the spectra of Alm′1,16 on the HLB surface and in ethanol are more broadened. This effect for the double-labeled Alm′ analogue is likely to be due to the contribution to the spectral width originated by the intramolecular dipole−dipole interactions between the spin labels. 7087

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removing from the complete PELDOR time trace the background line arising from intermolecular interactions of spin labels. The resulting λ values (Figure 2) were used to derive the normalized VINTRA time traces, Vn(T), using the relationship Vn(T ) = =

VINTRA(T ) − VINTRA(∞) 0 V INTRA − VINTRA(∞) 0 VINTRA(T )/V INTRA − (1 − λ) λ

(1)

Here VINTRA is the usual intramolecular part of the experimental PELDOR trace, VINTRA(∞) is the VINTRA value at T high enough when dipole oscillations are damped, and V0INTRA is the VINTRA value in the absence of the mw pumping pulse. The Vn(T) time trace is proportional to the oscillating part of VINTRA and can be used for obtaining the distance spectrum.33 It is significant that eq 1 is based on the theory that ignores cross-pumping spin at frequencies A and B, which may increase with decreasing pulse duration. However, our estimations show that at the conditions of our experiments eq 1 is applicable to the analysis of experimental data. It is worth noting that, for sorbents with a well developed surface, the mutual space arrangement of the adsorbed molecules depends on parameter d, the fractal dimension of the sorbent. In the case of spin-labeled molecules, the fractal dimension of the sorbent manifests itself in the intermolecular, magnetic dipolar interaction of spin labels that provides a PELDOR signal trace of the type V (T )/V0 = exp( −αT d /3)

Figure 3. 3p PELDOR time traces of normalized intramolecular dipolar contributions. Curves 1 and 2 refer to Alm′1,16 in ethanol and on HLB, respectively. Curves 3 and 4 correspond to aggregates of Alm′16 and Alm′1 adsorbed on HLB, respectively. Points denote experimental data, and solid lines are calculated using the spectra distances shown in Figure 4. Curves 2−4 are shifted up by 0.3, 0.6, and 0.9, respectively. Here, T0 is the point of the beginning of the PELDOR time trace analysis.

boundaries of the range of r variations). We used the expressions for F̃(r) and K(r, T) given in ref 33. Equation 3 is a Fredholm integral equation of the first type. Its solution will afford the F̃ (r) function, which after normalization will allow one to calculate the desired distance distribution F(r) (its integral over r should be equal to unity) or the distance spectrum. The F̃(r) function was refined using the Tikhonov regularization method.39,40 From Figure 3, it appears that the range of the T variations is limited from above by the maximal experimental time Tlim = 0.55 μs. The limitation of T imposes the upper boundary for the distances between spin labels, rlim. From the relation rlim = 5(Tlim/(2μs))1/3 in ref 41, rlim was estimated to be 3.2 nm. The part of the spectrum at distances exceeding rlim can be used only to determine the fraction of these distances in the spectrum. Figure 4 illustrates the spectra of distances between labels obtained by solving eq 2 in terms of the Vn(T) time traces presented in Figure 3. Curves 1 and 2 belong to Alm′1,16 in ethanol and on HLB, respectively. Both spectra of distances are shaped asymmetrically with the maxima position, rmax, close to 2 nm. In ethanol, rmax = 2.0 ± 0.02 nm and the line width at half-height is Δ =

(2)

Here α depends on the concentration of the spin labels.37,38 Note that for the uniform distribution in a volume d = 3, on a plane d = 2, and along a line d = 1. A decrease in d leads to the increase in the portion of spins with short interspin distances, as compared with the case of uniformly distributed spin-labels in a volume. This phenomenon causes a decrease in the PELDOR signal at short T and a subsequent passage to a slow decay with increasing T, i.e., a drastic difference from a simple exponential signal time trace. In this case, determining VINTRA from the experimental data may suffer from some inaccuracies. Therefore, we measured the fractal dimensionality d of the sorbent HLB. To this end, we initially obtained the V(T)/V0 time traces for the stable nitroxyl radical TEMPO adsorbed on HLB under the same conditions as for the spin-labeled Alm′1,16. The d value, calculated from experimental Vn(T)/ V0 dependence using eq 2, was found as 2.6 ± 0.1. The use of d = 2.6 to determine VINTRA for the spin-labeled Alm′1,16 on HLB, taking into account the fractal sorbent dimensionality, has no effect on the behavior of Vn(T) within the experimental error. Figure 3 shows the Vn(T) time traces for the peptides studied. The method of analysis has been described in detail for the 3p PELDOR.33 The theoretical expression for Vn(T) for two spin dipoles interacting at distance r obeys the equation Vn(T ) =

∫r

1

r2

F (̃ r )K (r , T ) dr

Figure 4. Spectra of intramolecular distances between TOAC spin labels in Alm′1,16 and of intermolecular distances between the TOAC labels in aggregates of the Alm′ analogues. Curves 1 and 2 refer to Alm′1,16 in ethanol and on HLB, respectively. Curves 3 and 4 refer to the aggregates of Alm′16 and Alm′1 on HLB, respectively. Curves 1 and 2 are shifted up by 0.6.

(3)

where F̃ (r) dr is proportional to the fraction of spin pairs with an interspin distance varying from r to r + dr (r1 and r2 are the 7088

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0.25 ± 0.03 nm. For the maximum position, rmax = 2.0 ± 0.03 nm, the spectrum of distances for adsorbed Alm′1,16 is much wider, Δ = 0.75 ± 0.1 nm. The maximum position rmax = 2 nm corresponds to the α-helix conformation.22 The spectrum asymmetry is probably due to an admixture of more elongated α/310-helix25,42−45 conformers with characteristic distances within the range 2.0 nm < r < 2.7 nm. 3D structures of this type are possible for Alm′ due to the concomitant presence of a 310-helix in the N-terminal region.22 The fraction of these conformers increases substantially for the peptide adsorbed on HLB. Probably, both the broadening of the basic spectrum line at rmax = 2.0 nm and the increase in the contribution of elongated conformers are due to the spread of peptide adsorption sites as compared with the more uniform Alm′1,16 trap structure in frozen ethanol solution. It should be noted that for Alm′1,16 in methanol the distance spectrum exhibited similar parameters with the maximum position rmax = 2.08 nm and the line width at half-height Δ = 0.72 nm.22 In Figure 4, curves 3 and 4 represent the spectra of distances between the labels in the aggregates of the mono spin-labeled Alm′ on HLB. This figure shows that the spectrum maximum for Alm′1 (rmax = 3.0 ± 0.1 nm) is shifted toward longer distances as compared with the related maximum for Alm′16 (rmax = 2.1 ± 0.1 nm). This result suggests that in the aggregates Alm′ molecules are preferentially oriented with their C-terminal regions in the vicinity. For both types of aggregates, the spectrum of distances between spin labels is remarkably broad, Δ = 1.6 ± 0.1 nm.

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AUTHOR INFORMATION

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We are grateful to Dr. Alexander Maryasov for valuable discussions. This work was supported by Grant No. 5.6.3 of divisional RAS Project 5.6.

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REFERENCES

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CONCLUSIONS In this work, the PELDOR technique was used to study the intra- and intermolecular interactions between spin labels for both the mono and double TOAC spin-labeled Alm′ analogues at positions 1 and/or 16 adsorbed on the organic sorbent HLB and in the frozen glassy ethanol solution at 77 K. The distance spectra determined for Alm′1,16 indicate that, in both the adsorbed state and ethanol solution, the peptaibiotic analogue is folded in the α-helix conformation. The asymmetry of the distance spectra is likely to be caused by admixture also involving more elongated α/310-helix conformers. The fraction of these conformers increases for the peptide adsorbed on HLB. Probably, both the broadening of the basic spectrum line at rmax = 2.0 nm and the increase in the contribution of elongated conformers associated with the spread of structure parameters of the peptaibiotic adsorption sites as compared with the more uniform Alm′1,16 trap structure in frozen ethanol solution. We demonstrated that the Alm′ analogues studied here are prone to aggregation on HLB. Spectra of intermolecular distances between labels were obtained for both the Alm′1 and Alm′16 aggregates on HLB. The spectrum of Alm′1 was found to be shifted toward longer distances as compared with that of Alm′16. This result suggests that in the aggregates Alm′ molecules are preferentially oriented with their C-terminal regions in the vicinity. For both types of peptides, the spectra of distances between spin labels are markedly broad (about 1.6 nm) in aggregates. This result specifies the friable structure of the Alm′ aggregates on HLB. We conclude that the PELDOR method, in combination with site-directed spin labeling, can offer detailed structural information on peptides adsorbed on surfaces. 7089

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dx.doi.org/10.1021/jp503691n | J. Phys. Chem. B 2014, 118, 7085−7090