Structure analysis of the membrane-bound dermcidin-derived peptide SSL-25 from human sweat

Accepted Manuscript Structure analysis of the membrane-bound dermcidin-derived peptide SSL-25 from human sweat

Philipp Mühlhäuser, Parvesh Wadhwani, Erik Strandberg, Jochen Bürck, Anne S. Ulrich PII: DOI: Reference:

S0005-2736(17)30278-X doi: 10.1016/j.bbamem.2017.09.004 BBAMEM 82577

To appear in: Received date: Revised date: Accepted date:

28 April 2017 11 August 2017 5 September 2017

Please cite this article as: Philipp Mühlhäuser, Parvesh Wadhwani, Erik Strandberg, Jochen Bürck, Anne S. Ulrich , Structure analysis of the membrane-bound dermcidinderived peptide SSL-25 from human sweat, (2017), doi: 10.1016/j.bbamem.2017.09.004

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ACCEPTED MANUSCRIPT Structure analysis of the membrane-bound dermcidin-derived peptide SSL-25 from human sweat Philipp Mühlhäusera, Parvesh Wadhwania, Erik Strandberga, Jochen Bürcka and Anne S. Ulricha,b,*

Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2), POB

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a

3640, 76021 Karlsruhe, Germany

Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, Fritz-Haber-Weg

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b

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6, 76131 Karlsruhe, Germany

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* Corresponding Author (email: [email protected], phone: +49-(0)721-608-23222)

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Keywords

Amphipathic α-helical peptide dermcidin; 19

F-L-Bpg scan;

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solid-state NMR data analysis;

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25 amino acid fragment SSL-25;

oriented circular dichroism (OCD);

Highlights

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membrane selectivity

- SSL-25 was studied in lipid bilayers by oriented CD and solid-state 19F- and 15N-NMR - The N-terminal half of SSL-25 forms an amphiphilic α-helix in the membrane-bound state - The glycine-rich C-terminal half is disordered in the membrane-bound state - The SSL-25 helix lies flat on the membrane surface, regardless of the lipid system - The peptide binds to bacterial membranes but not to eukaryotic model membranes

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ACCEPTED MANUSCRIPT Abstract SSL-25 (SSLLEKGLDGAKKAVGGLGKLGKDA) is one of the shortest peptides present in human sweat and is produced after the proteolytic processing of the parent peptide dermcidin. Both peptides are reported to have antimicrobial function. To determine the structure of SSL25 in lipid bilayers, a series of

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F-labeled SSL-25 analogs were synthesized. Circular

dichroism (CD) analysis showed that SSL-25 and all of its analogs formed α-helices in the

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presence of lipid vesicles, thus allowing a detailed analysis via oriented CD and solid-state NMR. The results suggest that SSL-25 resides on the membrane surface with a slight helix tilt 19

F-NMR analysis revealed that SSL-25 does not form a continuous helix.

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angle. A detailed

The α-helical structure of the N-terminal part of the peptide was preserved in membranes of

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different lipid compositions and at various peptide-to-lipid molar ratios, but the C-terminus was disordered and did not fold into a well-defined α-helical conformation. Furthermore, the

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NMR results showed that SSL-25 resides on the membrane surface and does not re-orient into the membrane in response to changes in either peptide concentration or membrane composition. SSL-25 does not aggregate and remains fully mobile within the membrane

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bilayer, as shown by 19F-NMR. SSL-25 has a high binding affinity toward bilayers mimicking bacterial lipid compositions, but does not bind to mammalian model membranes containing

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cholesterol. These observations may explain the selectivity of this peptide for bacterial

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membranes, but they are also in line with basic biophysical considerations on spontaneous

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lipid curvature and the general effect of cholesterol on peptide/lipid interactions.

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ACCEPTED MANUSCRIPT

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DMPC/DMPG 7:3

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Graphical Abstract

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DMPC/Cholesterol 3:1

ACCEPTED MANUSCRIPT 1. INTRODUCTION

An intriguing protein, dermcidin, has recently been identified in human sweat. It is supposed to have an antimicrobial function, and its active 48-mer form is known as DCD-1L [1]. The structure of DCD-1L has been previously investigated by circular dichroism spectroscopy (CD), which indicated that the peptide is unstructured in solution but forms an α-helix in the

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presence of lipid vesicles [2]. Solution-state NMR experiments in trifluoroethanol have indicated peptide oligomerization. Atomic force microscopy has revealed morphological

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changes in bacterial cells after treatment with DCD-1L and a destabilization of POPC bilayers, thus indicating that DCD-1L is membrane-active [2]. Electrophysiology studies have

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shown signals indicating pores in DPhPC black lipid bilayers with a conductance of 40-60 pS. It has been speculated that at high concentrations the peptides may insert into the membrane

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according to a charge zipper motif [2, 3].

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by forming a transmembrane helix hairpin [2] that is stabilized by internal salt bridges

Another study [4] has determined the 3D structure of DCD-1L by using X-ray crystallography of crystals grown without any lipids or detergents. A trimeric complex has

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been identified, consisting of antiparallel dimers of straight α-helices. The complex is 8 nm

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long with a 4 nm diameter and contains a central pore along the entire length. Also in this study, electrophysiological measurements indicated pores in DPhPC/cholesterol (9:1) model membranes with a conductance of 80 pS. It was assumed that the complex might be

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positioned in a transmembrane orientation and forms a pore (even though it is much too long to fit into the membrane, which has a thickness of approximately 4 nm including the lipid

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head group region). MD simulations have indicated that such a pore may be stable. The ion conduction determined from the simulation was compatible with the electrophysiological results. In this model, the entire length of the peptide forms one continuous α-helix with no charge zipper motif [4].

Through oriented CD (OCD), the helical DCD-1L has been found to be oriented flat on the membrane surface in POPG and DMPC/DMPG bilayers [2]. Solid-state

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N-NMR

spectroscopy has been used to study the orientation of this 48-mer in POPE/POPG (3:1) bilayers and revealed a flat orientation on the surface [4]. These results do not support the proposed pore models. 4

ACCEPTED MANUSCRIPT SSL-25 (sequence SSLLEKGLDGAKKAVGGLGKLGKDA), is one of the shortest peptides produced after proteolytic processing of dermcidin [5]. It has been reported that SSL-25 retains the antimicrobial activity of full-length DCD-1L against both Gram-positive and Gram-negative bacteria [5-8]. The short peptide carries six cationic and four anionic groups, giving it a net charge of +2. It can fold as an amphipathic helix with one polar face and one hydrophobic side (Fig. 1A). The length of SSL-25 as an α-helix would fit the thickness of a typical membrane quite well. It has been proposed that SSL-25 induces membrane

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permeabilization in bacteria [6, 8]; however, its structure within membranes has not been

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investigated.

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Here, we present a biophysical study of the structure and orientation of SSL-25 in its proposed functionally relevant membrane-bound form. CD was used to determine the

state

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secondary structure of the peptide in solution and in the presence of lipid membranes. SolidN-NMR [9] was used along with OCD [10] to determine the membrane-bound

alignment of the peptide. In addition, solid state 19F-NMR on selectively 19F-labeled peptides

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was applied to resolve the secondary structure and orientation within the membrane with a higher accuracy [11-13]. We utilized 3-(trifluoromethyl)-L-bicyclopent-[1.1.1]-1-ylglycine

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(CF3-L-Bpg, structure shown in Fig. 1B) as a 19F-label which is especially suitable for solidstate NMR analysis of membrane-bound peptides [14-19]. This label was incorporated into

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the SSL-25 sequence at eight different positions one-by-one. These methods also allow the monitoring of membrane binding and the aggregation behavior of membrane-bound peptides [16, 20]. The peptide behavior was studied in several lipid systems mimicking bacterial or

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eukaryotic membranes. These results suggest that this peptide selectively binds to bacterial

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rather than eukaryotic cell membranes.

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ACCEPTED MANUSCRIPT

Fig. 1. (A) Helical wheel projection of SSL-25. Hydrophobic residues are shown in yellow, polar

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residues are shown in light blue, glycine residues are shown in green, positively charged residues are shown in deep blue and negatively charged residues are shown in red. All hydrophobic residues except

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Ala-25 were labeled with 3-(CF3)-bicyclopent-[1.1.1]-1-ylglycine (CF3-L-Bpg), whose chemical

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structure is shown in (B).

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ACCEPTED MANUSCRIPT 2. MATERIALS AND METHODS

2.1. Materials The

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F-labeled amino acid used for the

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F-NMR measurements, 3-(trifluoromethyl)-L-

bicyclopent-[1.1.1]-1-ylglycine (CF3-L-Bpg), was obtained from Enamine (Kiev, Ukraine). 15

N-labeled leucine was purchased from Cambridge Isotope Laboratories (Andover, USA).

All other amino acids and coupling reagents were either purchased from Novabiochem

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(Merck Chemicals Ltd., Nottingham, UK) or Iris Biotech GmbH (Marktredwitz, Germany). The solvents used for synthesis and purification were purchased from Biosolve BV

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(Valkenswaard, The Netherlands) or from Fisher Scientific GmbH (Schwerte, Germany).

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UV-grade chloroform and methanol used for the NMR and OCD sample preparation were purchased from VWR International (Bruchsal, Germany). The lipids 1,2-dimyristoyl-sn-

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glycero-3-phosphatidylcholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE), 1-palmitoyl2-oleoyl-sn-glycero-3-phosphatidylglycerol sodium salt (POPG), 1',3'-bis[1,2-dioleoyl-sn-

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glycero-3-phospho]-sn-glycerol sodium salt (TOCL), and cholesterol were purchased from Avanti Polar Lipids (Alabaster, USA). Sodium dodecyl sulfate (SDS) was purchased from

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Carl Roth (Karlsruhe, Germany).

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Merck (Darmstadt, Germany), and n-dodecyl-β-D-maltoside (DDM) was purchased from

2.2. Peptide synthesis and purification SSL-25 was synthesized with a single CF3-L-Bpg at one of eight different positions (Leu-3,

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Leu-4, Leu-8, Ala-11, Ala-14, Val-15, Leu-18 or Leu-21), or with a single

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N-isotope

labeled leucine (15N-Leu) either at position Leu-8 or Leu-18. The synthesized peptides are

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listed in Table 1. Standard Fmoc solid-phase peptide synthesis protocols were used as previously described [21]. Labeled amino acids were coupled manually and the rest of the synthesis was performed on an automated MultiSynTech Syro II multiple peptide synthesizer (Witten, Germany). The peptides were purified with a C18 reverse phase HPLC column with water/acetonitrile gradients supplemented with 5 mM HCl. The purity of the peptides was confirmed by using an LC/MS system consisting of an 1100 Series LC system from Agilent (Santa Clara, USA) and a connected ESI micro-TOF mass spectrometer from Bruker Daltonics (Bremen, Germany). The peptide purity was determined to be above 95%.

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ACCEPTED MANUSCRIPT Table 1. Sequences of SSL-25 and analogs used for NMR studies. Peptide

labeled

sequence

position none

SSLLEKGLDGAKKAVGGLGKLGKDA

SSL-25 L3CF3-L-Bpg

Leu-3

SS-CF3-L-Bpg-LEKGLDGAKKAVGGLGKLGKDA

SSL-25 L4CF3-L-Bpg

Leu-4

SSL-CF3-L-Bpg-EKGLDGAKKAVGGLGKLGKDA

SSL-25 L8CF3-L-Bpg

Leu-8

SSLLEKG-CF3-L-Bpg-DGAKKAVGGLGKLGKDA

SSL-25 A11CF3-L-Bpg

Ala-11

SSLLEKGLDG-CF3-L-Bpg-KKAVGGLGKLGKDA

SSL-25 A14CF3-L-Bpg

Ala-14

SSLLEKGLDGAKK-CF3-L-Bpg-VGGLGKLGKDA

SSL-25 V15CF3-L-Bpg

Val-15

SSLLEKGLDGAKKA-CF3-L-Bpg-GGLGKLGKDA

SSL-25 L18CF3-L-Bpg

Leu-18

SSLLEKGLDGAKKAVGG-CF3-L-Bpg-GKLGKDA

SSL-25 L21CF3-L-Bpg

Leu-21

SSLLEKGLDGAKKAVGGLGK-CF3-L-Bpg-GKDA

SSL-25 L8-15N

Leu-8

SSLLEKG-15N-Leu-DGAKKAVGGLGKLGKDA

SSL-25 L18-15N

Leu-18

SSLLEKGLDGAKKAVGG-15N-Leu-GKLGKDA

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SSL-25

2.3. Circular dichroism (CD) spectropolarimetry Sample preparation. The lipid powders were dissolved in chloroform/methanol (1:1) to obtain

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5 mg/ml stock solutions. Aliquots of the stock solutions were mixed in glass vials and

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subsequently vortexed to yield the desired mixtures (molar ratio: DMPC/DMPG 7:3). The organic solvent was evaporated with a gentle stream of nitrogen, and samples were then placed under vacuum for at least 4 h. The lipid film at the bottom of the vials was further

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resuspended in 10 mM phosphate buffer (PB, pH 7.0) and homogenized by 10 freeze-thaw cycles. After each cycle, the sample was thoroughly vortexed. Small unilamellar vesicles (SUVs) were generated by sonication of the suspension for 15 min in a strong ultrasonic

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device (UTR 200, Hielscher, Germany). A constant sample temperature of 30°C was maintained by circulating water from a thermostat connected to the sonicator, thus preventing overheating of the sample during sonication. To prepare the final CD samples, an aliquot of peptide stock solution (380 µM) dissolved in water was added to either 10 mM PB, TFE (2,2,2-trifluoroethanol), 100 mM SDS in 10 mM PB, 10 mM DDM in 10 mM PB or to the DMPC/DMPG (7:3) liposome dispersion in 10 mM PB. These samples were diluted with 10 mM PB to yield the respective concentrations: 10 mM PB, 50% TFE, 30 mM SDS in 10 mM PB, 2 mM DDM in 10 mM PB and a peptide to lipid ratio (P/L) of 1:50. The peptide concentration ranged from 50 to 120 µM. Because there are no W or Y residues in SSL-25 for concentration determination on the basis of UV absorption, the concentration was based on 8

ACCEPTED MANUSCRIPT the weight of the added peptide and the volume of the solution and was used for the conversion of ellipticities into mean residue ellipticities.

Experimental details. Measurements were performed on a J-815 spectropolarimeter (JASCO, Groß-Umstadt, Germany) in quartz glass cells (Hellma, Müllheim, Germany) with a 1 mm path length between 260 and 180 nm at 0.1 nm intervals. The spectra were recorded at 30°C using a thermostated cell holder. Three scans with a 10 nm/min scan rate, 1 nm bandwidth

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and 8 s response time were averaged for each sample as well as for the baseline measurements of the corresponding peptide-free samples. The baseline was subtracted from

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the spectra of the respective peptide-containing sample to obtain the pure peptide spectra. The

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spectra were further processed by using an adaptive smoothing algorithm incorporated in the

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JASCO analysis software.

CD spectra deconvolution. Secondary structure estimation from the CD spectra converted into mean residue ellipticity (MRE) units was performed using the CDSSTR program

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implementing the singular value decomposition (SVD) algorithm [22, 23], the CONTIN-LL program based on the ridge regression algorithm [24, 25], and the SELCON-3 program,

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which incorporates the self-consistent method together with the SVD algorithm to assign protein secondary structure [26, 27]. These three algorithms were provided by the

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DICHROWEB on-line server [28, 29]. In each program, an appropriate reference protein dataset provided by DICHROWEB (in this case, reference set 7) was used. The reference protein dataset was selected on the basis of suitability for the expected secondary structure

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and the wavelength range used. The lowest data point used in the analysis was 190 nm (below that value, the signal acquired by the spectrometer was not reliable because of saturation of

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the detector). The quality of the fit between the experimental and back-calculated spectra corresponding to the estimated secondary structure fractions was assessed from the normalized root mean square deviation (NRMSD). Values <0.1 (for CONTIN-LL and CDSSTR) and <0.25 (for SELCON-3) were considered to be good fits [28]. Finally, the secondary structure element fractions of each sample were calculated as the average value of the individual data obtained with the three algorithms. Individual values were not included in the average if the sum of all structural elements fractions was <0.98 or >1.02, or when the NMRSD between the experimental and back-calculated CD spectrum exceeded the threshold (0.1 for CONTIN-LL and CDSSTR, and 0.25 for SELCON-3).

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ACCEPTED MANUSCRIPT 2.4. Oriented circular dichroism (OCD) spectropolarimetry For the OCD experiments, mixtures of DMPC, DMPG and SSL-25 were deposited from the organic solvents (chloroform/methanol) onto 20 mm diameter quartz glass plates, as described previously [30]. After the solvents were evaporated, the glass plates were placed in a vacuum for 3-4 h and hydrated overnight in a hydration chamber built in-house. The experiments were performed with a JASCO J-810 spectropolarimeter fitted with a dedicated

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computer-controlled OCD sample cell described previously [30].

2.5. Solid-state NMR

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Sample preparation. Oriented samples for solid-state NMR were prepared as previously

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described, following procedures similar to those used for OCD [20, 31]. Typically, 0.1-0.4 mg of the CF3-Bpg labeled peptides or 1 mg of the

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N-labeled peptides were used. The

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amount of lipid was calculated in each case to obtain the desired peptide-to-lipid molar ratio (P/L). Briefly, the lipids were dissolved in chloroform/methanol 1:1 (v/v), and the peptides were dissolved in water/methanol 1:10 (v/v). The dissolved lipids were added to the peptide

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solution. The resulting clear solution was vortexed, sonicated and uniformly spread over several thin glass plates (9 mm × 7.5 mm × 0.08 mm in size; Marienfeld Laboratory

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Glassware, Lauda-Königshofen, Germany). The solvent was allowed to evaporate under vacuum overnight. The glass plates covered with the dried lipid-peptide thin film were

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hydrated 16-24 h in a humidity chamber at 96 % relative humidity at 321 K to obtain well oriented bilayers, as previously described [31].

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Experimental details. All NMR measurements were carried out on a Bruker Avance 500 MHz spectrometer (Bruker Biospin, Karlsruhe, Germany) at 308 K. The

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were performed on a flat-coil

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F-NMR experiments

F/1H probe head built in-house by using an “anti-ringing”

sequence (to reduce background signals from the probe) [32], a 3.9 s 90° pulse, a 1 s relaxation delay time, 500 kHz spectral width, 4096 data points and 24 kHz proton decoupling with a SPINAL-64 sequence [33]. Typically between 10,000 and 40,000 scans were collected. The spectra were referenced to a 100 mM NaF solution for which the

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signal was set to -119.5 ppm. 1H-15N cross-polarization experiments using a CP-MOIST pulse sequence [34] were performed using a double-tuned probe with a low-E flat-coil resonator (3 mm × 9 mm cross section), typically using 1H and kHz during cross-polarization, and 23 kHz

1

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N radiofrequency field strengths of 42

H SPINAL-16 decoupling [33] during

acquisition. Approximately 20,000 scans were collected with a mixing time of 1000 µs. The 10

ACCEPTED MANUSCRIPT acquisition time was 10 ms, and the recycle time was 3 s. The

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N chemical shift was

referenced using a dry ammonium sulfate powder sample, for which the

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N chemical shift

was set to 26.8 ppm. To assess the sample quality and the degree of orientation,

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P-NMR

measurements were performed using a Hahn echo sequence [35] with a typical 90° pulse of 4.2 µs, a 30 µs echo time and 13 kHz 1H SPINAL-64 decoupling during acquisition. Typically, 256 scans were collected. The acquisition time was 10 ms, and the recycle time was 1 s. Samples were measured with the glass plates oriented so that the membrane normal

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was parallel or perpendicular to the external magnetic field; in figures, the orientation is

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indicated next to NMR spectra.

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2.6. NMR data analysis

The orientation of SSL-25 within the lipid bilayer was calculated from the experimentally

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determined 19F-19F dipolar couplings (within the estimated experimental error of 0.5 kHz) of the CF3-Bpg-labeled SSL-25 analogs. On the basis of the CD results, the backbone was modeled as an ideal -helix, in which the orientation relative to the helix axis of the C-C 19

F-labeled side chain was described by the angles  = 121.1° and  =

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bond vector of the

53.2° [36]. The alignment of the helix in the bilayer is described by the tilt angle () with

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respect to the membrane normal, and by the azimuthal rotation angle () around the helix.

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The overall effect of motional averaging is taken into account by the Gaussian distribution parameters  and , or by the molecular order parameter Smol, as previously described [37]. The orientational parameters , , , and  were determined by using a least-squares fit, in

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which the sum of the squared deviations of the experimentally determined dipolar couplings was minimized to find the best-fit parameters. The details of this method have been published

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previously [36-38].

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ACCEPTED MANUSCRIPT 3. RESULTS

The replacement of hydrophobic residues in a given amphiphilic peptide with CF3-L-Bpg is usually well tolerated. Several studies have reported that the replacement of these residues with CF3-L-Bpg does not cause any structural or functional changes [14-16, 39, 40]. The natural peptide, eight SSL-25 analogs with CF3-L-Bpg in place of the hydrophobic amino acid at positions Leu-3, Leu-4, Leu-8, Ala-11, Ala-14, Val-15, Leu-18 and Leu-21 (Table 1), as 15

N-label on the backbone amide of Leu-8 or Leu-18 were

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well as two analogs with an

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successfully synthesized and characterized.

Fig. 2. (A) Circular dichroism spectra of SSL-25 in different environments. (B) OCD spectra of SSL25 in DMPC/DMPG (7:3) at different peptide-to-lipid molar ratios. In the OCD experiment, the sample is oriented with the membrane normal (n) parallel to the incident UV beam, as indicated by the inset figure. 12

ACCEPTED MANUSCRIPT 3.1. CD spectroscopy CD spectroscopy was used to study the secondary structure of SSL-25 wt (Fig. 2A). In 10 mM phosphate buffer (PB), a line shape typical of disordered peptides was found with negative ellipticities over the full spectral range from 185 to 260 nm and a pronounced negative CD band at approximately 198 nm. A similar line shape was found at concentrations of peptide between 50 µM and 3.33 mM (Fig. S1 in Supplementary Material). Additionally, a mostly random coil signal was observed in the presence of 2 mM uncharged DDM micelles.

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In PB/trifluoroethanol (TFE) (1/1 v/v) a helical line shape evolved, which became more pronounced in 30 mM anionic SDS micelles. Even more pronounced α-helical spectra were

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obtained in the presence of anionic DMPC/DMPG (7:3 molar ratio) lipid vesicles at

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P/L=1:50. Some 50% and 59% helical conformation were measured in SDS and DMPC/DMPG vesicles, respectively, according to the deconvolution of the CD data (Table

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S1 in Supplementary Material). It can be concluded that SSL-25 binds to SDS micelles and lipid vesicles and thereby adopts an α-helical fold. However, at higher concentrations of peptides in DMPC/DMPG, P/L=1:25 and 1:10, the helicity is lower (Fig. S2), which indicates

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a reduced binding under these conditions; at 1:10 the signal corresponds to mostly

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unstructured peptides.

N-NMR labels do not affect the chemical properties, whereas

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F-labeling

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introduces non-natural amino acids in the peptide sequence which might influence the secondary structure compared with that of the natural peptide. CD spectra were measured also for all

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F-labeled analogs to determine their conformation and for comparison with the

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natural peptide. As seen from the CD spectra in Fig. S3, the secondary structures of the SSL25 mutants and the natural peptide in vesicles did not differ significantly, thus indicating that

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labeling with CF3-L-Bpg did not change the secondary structure of the peptide in its membrane-bound state. The results of the deconvolution of the CD data revealed a helix fraction of 51-66% for all analogs (Table S2). It can therefore safely be concluded that all labeled analogs fold into an α-helical structure with a helix content similar to the natural peptide when they are membrane-bound. This assumption allowed for further structural analysis of the membrane-bound peptide.

3.2. OCD spectroscopy The orientation of an α-helical peptide within a membrane can be determined from the CD spectra of the oriented samples [10]. OCD experiments were performed at varying P/L ratios 13

ACCEPTED MANUSCRIPT from 1:200 to 1:25 (see spectra in Fig. 2B). The spectra show that SSL-25 folded into an αhelix at all peptide concentrations tested and did not aggregate even at high peptide concentrations of up to P/L = 1:25. In all spectra, there was a pronounced negative CD band at 208 nm, thus indicating that the orientation of the helical peptides was almost flat on the membrane surface at all concentrations [10, 30]. The small differences between the spectra at

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different P/L values do not indicate any significant difference in orientation.

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N-NMR spectra of SSL-25 L8-15N and SSL-25 L18-15N. (A) Dry peptide powder. (B) In

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Fig. 3.

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oriented lipid bilayers composed of DMPC/DMPG (7:3). The samples were oriented with the membrane normal (n) parallel to the external magnetic field (B0). (C) In oriented bilayers composed of POPE/POPG/TOCL (72:23:5) at P/L=1:50. For SSL-25 L8-15N a sharp signal was found at 91 ppm in

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both lipid systems, whereas in SSL-25 L18-15N, the peak was at 71 ppm. These values indicate that

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the peptide is oriented with the helix axis parallel to the membrane surface

3.3. Solid-state 15N-NMR Another method to determine the approximate orientation of an α-helical peptide within the membrane is to label the backbone amide of the peptide with 15N at one position in the helical portion and to measure the

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N-NMR spectrum in oriented membranes [41-43]. SSL-25 was

labeled at two positions in the sequence, in the N-terminal half at Leu-8, and in the C-terminal half at Leu-18. Both labels are in the central part of the peptide, far from the ends that might not form a well-defined helix.

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N-NMR spectra of SSL-25 L8-15N and L18-15N were

measured in oriented lipid bilayers composed of DMPC/DMPG (7:3) and of POPE/POPG/TOCL (72:23:5) at a P/L=1:50. DMPC/DMPG was used since previously DCD1L was studied in this lipid system [2]. POPE/POPG/TOCL is a model system similar to the 14

ACCEPTED MANUSCRIPT lipid composition of E. coli membranes [44] which has previously been used in our group [42, 45]. All four

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N-NMR spectra showed a single sharp peak at 70-90 ppm (Fig. 3),

indicating that the peptide lies flat on the membrane surface. This orientation is fully consistent with the OCD results described above. The two labeled positions however, showed different chemical shifts. The peak in both lipid systems was found at 90 ppm for the labeled position 8 whereas for the labeled position 18 the peak was at 71 ppm. If both labeled positions are assumed to be part of one continuous a-helix, this finding could be explained by 15

N CSA tensors in relation to the magnetic field,

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a difference in the orientation of the two

Fig. 4. Solid-state

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which were located at different positions around the helical wheel (Fig. 1).

P- and

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F-NMR spectra of SSL-25 labeled with CF3-L-Bpg at the indicated

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positions, in DMPC/DMPG (7:3) at varying P/L ratios. The dashed lines in the 19F-NMR spectra (here and also in Figs. 5 and 8 below) indicate the isotropic chemical shift (-72 ppm). The samples were

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oriented with the membrane normal (n) parallel to the external magnetic field (B0).

3.4. Solid-state 19F-NMR 19

F-NMR was used to obtain a more accurate picture of the conformation and orientation of

SSL-25 in the membrane. This method has been used by our group on various membraneactive peptides [15, 16, 19, 20, 36, 41, 46-49], because it provides a more exact tilt angle and azimuthal rotation angle of the peptide in the membrane than

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N-NMR [12, 13, 50, 51].

Compared to other NMR nuclei used in studies of peptides in membranes, like 2

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N, 13C, and

H, 19F has several advantages; the most important are the high sensitivity (the gyromagnetic

ration is 94% of that of 1H) and the lack of a natural background. This means that it is possible to study low concentrations of peptides in membranes, down to P/L=1:3000 [52], 15

ACCEPTED MANUSCRIPT and/or to use smaller amount of labeled peptides in the samples. SSL-25 was labeled with CF3-L-Bpg at eight different positions. Oriented samples of the peptides in DMPC/DMPG (7:3) bilayers were prepared and then measured with 19F-NMR.

The concentration-dependent behavior of SSL-25 was studied in a series of samples prepared with

19

F-labeled peptides at different P/L. The NMR spectra of two selectively labeled

peptides are shown in Fig. 4. At P/L=1:25, a large isotropic peak and a lower intensity triplet

PT

were observed for both, SSL-25 L8CF3-L-Bpg, and for SSL-25 L18CF3-L-Bpg. At a lower peptide concentration of P/L=1:50, the triplets became more pronounced; however, the

RI

isotropic peak was still observed, and it overlapped with the triplet. At a very low P/L of

SC

1:500, mainly the triplet was observed with a minor residual isotropic peak. The sizes of the splittings did not change with P/L, thus indicating that the peptide orientation did not change

Table 2. Homonuclear

19

NU

with concentration.

F-19F dipolar couplings of the respective labeled peptide in

MA

DMPC/DMPG (7:3) at P/L=1:500 and POPE/POPG/TOCL (72:23:5) at P/L=1:25, at 0° and 90° sample orientation. The error in the measured couplings is estimated to be ± 0.5 kHz.

0°

SSL-25 L3CF3-L-Bpg SSL-25 L4CF3-L-Bpg

0°

90°

+4.1 kHz

-1.9 kHz

+5.7 kHz

-2.7 kHz

-2.6 kHz

+1.2 kHz

0 kHz

0 kHz

-5.9 kHz

+2.9 kHz

-4.0 kHz

+1.7 kHz

CE

SSL-25 L8CF3-L-Bpg

POPE/POPG/TOCL (72:23:5)

90°

PT E

Peptide

D

DMPC/DMPG (7:3)

+4.0 kHz

-1.9 kHz

+5.7 kHz

-2.8 kHz

SSL-25 A14CF3-L-Bpg

+8.1 kHz

-4.0 kHz

+7.8 kHz

-4.1 kHz

SSL-25 V15CF3-L-Bpg

0 kHz

0 kHz

+2.4 kHz

-0.8 kHz

SSL-25 L18CF3-L-Bpg

+9.6 kHz

-4.8 kHz

+9.5 kHz

-4.9 kHz

SSL-25 L21CF3-L-Bpg

+10.0 kHz -5.0 kHz

+9.6 kHz

-5.4 kHz

AC

SSL-25 A11CF3-L-Bpg

16

31

P- and

AC

Fig. 5. Solid-state

CE

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

19

F-NMR spectra of SSL-25 labeled with CF3-L-Bpg at the indicated

positions, in DMPC/DMPG (7:3) at P/L=1:500.

31

P-NMR spectra before (A) and after (D) the

19

F-

19

NMR experiments measured with 0° sample tilt (membrane normal parallel to B0); F-NMR spectra measured with 0° sample tilt (B) and 90° sample tilt (membrane normal perpendicular to B 0) (C). In all cases, the

31

P-NMR spectra show well oriented samples. Well-resolved

19

F-NMR splittings are

seen upon tilting the sample orientation by 90°, and they are scaled by a factor of -0.5 as expected.

The 19F-NMR splittings were resolved best at a low P/L. Therefore, a P/L of 1:500 was used in the following set of experiments to ensure that the

19

F-19F dipolar splittings were clearly

visible. As seen in Fig. 5, all eight CF3-L-Bpg-labeled analogs of SSL-25 produced wellresolved triplets, except SSL-25 V15CF3-L-Bpg where only a single peak is observed, which 17

ACCEPTED MANUSCRIPT we assume is because the splitting is too small to be resolved; we assume that the splitting in this case is zero. These splittings depend on the orientation of the C-CF3-vectors in the labeled amino acids relative to the magnetic field [36]. Samples were measured with the membrane normal parallel (Fig. 5B) or perpendicular (Fig. 5C) to the membrane normal. The fact that the splittings in the perpendicular orientation are scaled a factor -1/2 compared to the parallel orientation shows that peptides are rotating fast around the membrane normal [53, 54]. The splittings are listed in Table 2 and were used to calculate the peptide orientation, as

RI

peptide dynamics [37] produced essentially identical results.

PT

described in Materials and Methods. Two different methods used to take into account the

SC

An explicit dynamical model comprising all 8 labeled data points produced a poor fit with a very large root-mean-square deviation (RSMD) between experimental and calculated

19

F-

NU

NMR splittings of 2.3 kHz (Fig. 6A-C and Table 3). The best fit values were 38° for the tilt angle (τ), and 3° for the azimuthal angle (ρ). The dynamical wobble of the helix is described by the width of the τ and ρ angle distributions, which are referred to as στ and σρ, respectively.

MA

The best-fit values of στ = 28° and σρ = 28° indicated high peptide mobility. In an alternative approach, using an implicit model of dynamics, the motion of the peptide was assumed to reduce the splittings by a fixed scaling factor, the so-called order parameter Smol. The best fit

PT E

D

produced a result with an RMSD of 2.3 kHz, τ = 52°, ρ = 3°, and Smol = 0.64. Table 3. Results of the 19F-NMR data analysis of SSL-25 analogs in DMPC/DMPG (7:3) at parameters.a

CE

P/L=1:500. Best-fit values are given of tilt angle (τ), rotation angle (ρ), and dynamic ρ

στ

σρ

RMSD

8 positions labeled

38°

3°

28°

28°

2.3 kHz

5 positions labeled

96°

178°

5°

21°

0.5 kHz

Smol model analysis

τ

ρ

Smol

RMSD

8 positions labeled

51°

3°

0.60

2.3 kHz

5 positions labeled

98°

178°

0.76

0.5 kHz

AC

Explicit dynamical model analysis τ

a

The error in the parameters for the good fit using 5 data points is estimated to be ± 10° for τ,

± 5° for ρ, ± 10° for στ and ± 5° σρ. For the poor fit using 8 data points, the RMSD is very high and the estimated errors are much larger.

18

19

F-NMR data analysis to determine the peptide orientation in DMPC/DMPG (7:3) lipid

PT E

Fig. 6.

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

bilayers with a P/L=1:500. (A-C) Fit of all 8 positions using the explicit dynamical model. (D-F) Fit of 5 positions only. (G-I) Fit of 5 positions using the Smol model. (A, D, G) The experimental data points

CE

(black filled squares) fitted to helical curves, shown as a function of the labeled position. (B, E, H) Best-fit helical curves with all data points projected to one turn around the helix. Data points marked by red open squares were not included in the analysis. (C, F, I) RMSD plots showing the RMSD for

AC

the best fit as a function of τ and ρ angles, color-coded at each point. The best fit values are indicated by dashed lines. The RMSD plots as a function of στ and σρ are given in the insets in panels (C) and (F).

This analysis assumes an ideal α-helical structure, and thus the splittings should be repeated after each full number of helix turns. Specifically for SSL-25, the splittings from position 3 and 21 (1800° or 5 full turns apart) should produce the same splittings; however, this was not the case, as seen in Table 2. Therefore, the labels were grouped to carry out a more local analysis, in case different conformational regions should be present. Labels in the N-terminal stretch from positions 3 to 14 produced a good fit with an RMSD of 0.5 kHz, but when 19

ACCEPTED MANUSCRIPT position 15 was included, the fit became much worse and the orientation found was not reliable (Fig. 7, Table S3). This finding indicated that the peptide forms an almost ideal αhelix throughout positions 3-14. Next, we wondered whether the peptide could form a kinked helix. We thus attempted to fit the C-terminal part of the peptide, but it was not possible to obtain a useful fit (RMSD was 1.6 kHz or more) when four or more labeled positions starting from the C-terminal were used, and even trying to fit only positions 15-21 did not give a good fit (Fig. 7, Table S3). Thus, it seems that the C-terminal region of SSL-25 does not form a

PT

well-defined α-helical structure. The CD analysis revealed a helicity of approximately 55%,

PT E

D

MA

NU

SC

RI

corresponding to approximately 14 residues, which fits well with the NMR analysis.

Fig 7. Best-fit RMSD for fitting of different stretches of the peptide from the N- or C-terminal using the explicit dynamical model. Only data from positions 3-14 gave a good fit, indicating that the

19

F-NMR data from positions 3-14 using the explicit dynamical model

AC

Analysis of the

CE

peptide forms an essentially ideal helix only in this region.

produced a low RMSD of 0.5 kHz, a tilt angle of 96°, an azimuthal angle of 178°, and reduced dynamics (στ = 8° and σρ = 20°). Here, the helical curve fit the data well, as seen in Fig. 6D-F. The Smol model also yielded a very similar best-fit, with an RMSD = 0.5, τ = 98°, ρ = 178°, and Smol = 0.64 (Fig. 6G-I). As seen in Fig. 6I, the minimum in this case was somewhat better defined. The tilt angle of approximately 90° was consistent with the OCD and

15

N-NMR results. Notably, the azimuthal angle did not differ significantly from the

azimuthal angle obtained by using all of the data points. (Owing to symmetry in the system, the ρ values of 0° and 180° are equivalent; therefore, a seemingly large change from 3° to 178° also corresponds to a small change from 183° to 178° [37].) This result corresponds to 20

ACCEPTED MANUSCRIPT peptides lying flat on the membrane surface, such that charged residues point away from the hydrophobic interior of the membrane. This orientation is in agreement with the OCD and 15

N-NMR results described above in the same lipid system. Because splittings did not change

with peptide concentration, and the surface orientations were determined at all concentrations using OCD, the observed orientation at a P/L=1:500 was also valid for higher peptide concentrations (up to P/L=1:25) in this lipid system.

PT

NMR samples were also prepared in POPE/POPG/TOCL (72:23:5) lipids, a system more similar to E. coli membranes [44]. In this case, the sample quality and spectral resolution at

RI

high peptide concentration were not problematic; therefore, the samples could be prepared

SC

with a P/L=1:25. For each labeled analog, the spectra showed splittings similar to those in DMPC/DMPG. The spectra are shown in Fig. 8, and splittings are given in Table 2. Some

NU

samples were also prepared at P/L=1:500 in POPE/POPG/TOCL, and the splittings were found to be almost identical to those at P/L=1:25 (see Table S4). The results produced by the analysis of the

19

F-NMR data using the explicit dynamical model were similar to those in

MA

DMPC/DMPG. Including all data points produced a poor fit with RMSD = 1.9 kHz. However, including data from only positions 3-14 produced a low RMSD of 0.7 kHz, a tilt

D

angle of 106°, an azimuthal angle of 178°, and rather small dynamics (see Fig. S4 and Table S5 for more details). Owing to some ambiguity in the tilt angle in this analysis (as seen from

PT E

the quite large region of tilt angles in the RMSD plot giving similar RMSD values), the change of 10° in the tilt angle between DMPC/DMPG and POPE/POPG/TOCL was within the margin of error. Considering the identical position of the

15

N-NMR peaks (Fig. 3), the

CE

helix orientation is nevertheless also probably very similar in both lipid systems.

AC

Similarly to bacterial membranes, the DMPC/DMPG (7:3) and POPE/POPG/TOCL (72:23:5) lipids also have a high negative charge; however, they may not be a good model of eukaryotic plasma membranes. To investigate whether SSL-25 interactions with bacterial and eukaryotic cell membranes differ, samples were also prepared in DMPC/cholesterol (3:1) at P/L=1:500. For these samples (spectra are shown in Fig. 8) and for all labeled positions, there was only a single sharp peak at -71 ppm, the isotropic chemical shift position. After a 90° tilt of the sample orientation, the signals did not move, thus indicating that the peptide was not bound to the membrane, but was probably located in the water phase (possibly in an unfolded state). The presence of cholesterol in the lipid bilayer or the lack of charged lipids may explain the lack of binding. Additional samples were prepared in anionic DMPC/DMPG/cholesterol 21

ACCEPTED MANUSCRIPT (2:1:1), with 25% charged lipids. Isotropic signals were also found in this case (Fig. S5). It can thus be concluded that the presence of cholesterol in the membrane reduces peptide

AC

CE

PT E

D

MA

NU

SC

RI

PT

binding.

Fig. 8. Solid-state 19F-NMR spectra of SSL-25 labeled with CF3-L-Bpg at the indicated positions and measured in oriented bilayers with 0° or 90° sample tilt. (A, B) In POPE/POPG/TOCL (72:23:5) with a P/L = 1:25; splittings are similar to those found in DMPC/DMPG (7:3) with a P/L = 1:500. (C, D) In DMPC/cholesterol (3:1) with a P/L=1:500; in all cases, 19F-NMR spectra contain only one sharp line at the isotropic chemical shift that does not move after a 90° tilt of the sample. 22

ACCEPTED MANUSCRIPT The

15

N-labeled peptides were also studied in DMPC/cholesterol. Many different mixing

times were used in the 50-1000 µs range; however, no signal was detected. This result would be expected for isotropically tumbling peptides, because the cross-polarization scheme used in the

15

N-NMR experiments depends on dipole-dipole interactions, which are averaged to

zero in such cases [55]. A small peak at approximately 128 ppm (using a 100 µs mixing time) was observed only when the samples were allowed to dry out. For even drier samples, a strong peak was observed at 90 ppm (data not shown). Thus, SSL-25 has a very low binding

PT

affinity for DMPC/cholesterol membranes and therefore stays in the water phase of the sample. However, when the sample dries out, the bulk water phase disappears, and the

AC

CE

PT E

D

MA

NU

SC

RI

peptide is forced to bind to the membrane.

23

ACCEPTED MANUSCRIPT 4. DISCUSSION

SSL-25 is a membrane-active peptide found in human sweat. It contains a total of 10 charges including five positively and three negatively charged residues in the sequence and additional charges on the N- and C-termini. A common feature of antimicrobial peptides is the presence of many cationic amino acids in the sequence. SSL-25, like most AMPs, is overall positively charged, with a net charge of +2. The longer parent peptide DCD-1L is one of only a few

PT

known anionic AMPs [56]. It might be possible that the positive and negative charges in the peptide form salt-bridges, which would reduce the total charge and facilitate peptide insertion

RI

into the membrane, as has previously been suggested for the TatA membrane protein [3]. Like

SC

many AMPs, SSL-25 is unstructured in solution but folds into an amphipathic α-helix after

NU

binding to a membrane, as shown here by CD analysis (Fig. 2A).

In many previous studies, amphipathic helical AMPs have been found to be oriented flat on the membrane surface at low concentrations but to become more tilted at higher

MA

concentrations [20, 30, 31, 38, 54, 57]. SSL-25, in contrast, appears to have the same surfacebound orientation at all concentrations ranging from a P/L=1:200 to 1:25 according to our

D

OCD results (Fig. 2B). The 19F-NMR splittings of the labeled peptides in the DMPC/DMPG lipids did not change across P/L ratios ranging from 1:500 to 1:25 (Fig. 4). From these results

PT E

it can be concluded that the orientation does not change with concentration over this wide range. A few positions were also measured at a P/L=1:500 in POPE/POPG/TOCL. This analysis revealed that a P/L=1:500 produced almost the same splittings as a P/L=1:25, thus

CE

indicating that the peptide orientation was very similar within this P/L range (1:500 to 1:25) in this lipid system. Previous studies have reported similar behaviors in the highly charged

AC

BP100 peptide, which has a net charge of +6 and only 11 amino acids, which provide a very high charge density. This peptide is only slightly more tilted at very high P/L ratios [40, 58]. Similarly to SSL-25, magainin 2, a 23-mer peptide charged at both termini and with one anionic residue in its sequence, is less prone to membrane insertion than PGLa, a 21-mer peptide with five positive charges but no negative charge [42]. The three peptides, SSL-25, BP100, and magainin 2, have a very large polar sector of almost 180°, whereas PGLa and MSI-103, peptides which insert more easily into membranes, have a smaller polar sector of only 100° [40, 59]. All results mentioned above have been found in DMPC/DMPG lipids, with a small positive spontaneous curvature that has been found to be favorable for peptide insertion [42, 60]. SSL-25 also was found to lie flat on the membrane surface in 24

ACCEPTED MANUSCRIPT POPE/POPG/TOCL (72:23:5) at all tested concentrations, a result in line with those from previous studies; thus, all studied amphipathic helices are found in this orientation in lipid systems with a negative spontaneous curvature [60]. From this comparison, it is not entirely unexpected that SSL-25 was found to lie flat on the membrane surface, even at high concentrations in DMPC/DMPG, because many other AMPs with similar characteristics

MA

NU

SC

RI

PT

exhibit the same behavior.

Fig. 9. Orientation of SSL-25 in DMPC/DMPG bilayers (gray box) determined from the

19

F-NMR

D

data analysis. (A) The N-terminal region up to position 14 forms a regular α-helix (green) which is

PT E

oriented almost flat on the surface with a tilt angle of 96°. The C-terminal part (red) is rather unstructured, and most likely also flat on the membrane surface. (B) The helical part has an azimuthal

CE

rotation angle of 178°, with charged and polar groups pointing out of the membrane interior.

The detailed analysis of the peptide orientation using 19F-NMR data indicated that the peptide

AC

forms a regular α-helix from position 3 to position 14, whereas labels in the C-terminal part of the peptide did not fit well to a helical curve. The CD deconvolution shows 55% helicity in the presence of membranes, which is compatible with a stable helix of 14 residues. From position 16 to 22 there are four glycine residues, which are known to give an increased flexibility to peptides and to disfavor helix formation. It is therefore not surprising that this part of the peptide is less helical. The situation is somewhat similar to TP10, a peptide previously studied with 19F-NMR, where the glycine-rich N-terminal region is unordered and the C-terminal region forms a regular α-helix [16]. The N-terminal helix is found to be oriented with a tilt angle of 96° and an azimuthal rotation angle of 178° in DMPC/DMPG bilayers, and a similar orientation in POPE/POPG/TOCL (Table 3). The situation is illustrated in Fig. 9. The C-terminus does not form an ideal helix, but the presence of several 25

ACCEPTED MANUSCRIPT hydrophobic residues should bind this region to the membrane, and the large splittings at positions 18 and 22 also indicates that this part of the peptide is bound to the membrane and not freely moving in the water phase, as high mobility would lead to motional averaging of the splittings. We therefore assume that the C-terminal part is bound to the membrane surface as shown in Fig. 9.

An unusual behavior was found in the

19

F-NMR study of SSL-25 in DMPC/DMPG with a

PT

high concentration, in which a large isotropic signal was observed together with low intensity splittings from membrane-bound peptides (Fig. 4). This behavior has not been observed 19

F-NMR on other 19F-labeled peptides. The isotropic signal found at different

RI

previously in

SC

labeled positions indicated that the peptide was not bound to the membrane, but was probably located in the water phase outside the membrane, where fast isotropic motion would average

NU

out the dipolar couplings. This is also supported by CD experiments showing less helicity of SSL-25 in DMPC/DMPG vesicle environment at high P/L ratios (Fig. S2). An isotropic signal would also be observed if the peptide induced micellization of the membrane, but this

MA

possibility can be excluded because there were no isotropic peaks in the corresponding

P-

D

NMR spectra (Fig. 4).

31

At lower peptide concentrations, the peptide was mostly bound to the membrane, and the

PT E

isotropic signal disappeared from the 19F-NMR spectra at a P/L=1:500 (Fig. 5). The reduced binding at higher peptide concentrations appears to indicate saturation of the membrane. It has previously been observed that the antimicrobial cationic peptide KIGAKI does not bind to

CE

lipid vesicles when the number of negative charges on the lipids is less than the number of positive charges in the peptides; i.e., when a sufficient number of peptides have bound to a

AC

vesicle to neutralize the negative charges, no more peptide can be bound [19]. However, in the present study, there were always more negative charges on the membrane than positive charges on the peptides, even at a P/L=1:25. Even if the total number of positive charges on the peptide was used, rather than the net charge, there was still more negative charge on the membrane. Thus, it appears that the low binding affinity of SSL-25 is not due to a lack of electrostatic interactions.

In comparison to the 30 negative charges found in DMPC/DMPG (7:3), POPE/POPG/TOCL (72:23:5) includes 33 negative charges per 100 lipids and results in much stronger SSL-25 binding. This finding indicated that charge was not the main factor accounting for the lack of 26

ACCEPTED MANUSCRIPT peptide binding in DMPC/DMPG. There were no isotropic peaks in the

19

F-NMR spectra of

POPE/POPG/TOCL (72:23:5) at a P/L=1:25 (Fig. 8).

It has been proposed that cholesterol in the membrane provides protection against AMPs, such as PGLa, gramicidin S [61], magainin [62], MSI-103 [63] and model AMPs [64]. This effect may partly explain the selectivity of AMP-action toward bacterial cell membranes that lack cholesterol, rather than the cholesterol-rich eukaryotic membranes. In this study, SSL-25

the

19

PT

did not bind to the DMPC/cholesterol membranes. At a P/L=1:500 only an isotropic peak in F-NMR was observed for all labeled positions (Fig. 8). In DMPC/DMPG/cholesterol,

RI

also only an isotropic peak was observed (Fig. S5), thus indicating that the lack of binding

SC

was due to cholesterol and not due to a lack of charged lipids. CD also showed mostly unstructured peptides in the presence of vesicles containing cholesterol (Fig. S2), most likely

was found both with

19

F-NMR on

19

NU

because of reduced binding to these membranes containing cholesterol. The lack of binding F-labeled peptides and with

15

N-NMR on the natural

peptide. In 15N-NMR no signal was observed in hydrated samples, which is to be expected for

MA

unbound peptides, since the cross polarization scheme used will not work for isotropically moving peptides. Only the dried samples produced a signal in the

15

N-NMR. The dried

D

samples did not contain any bulk water phase, and consequently the peptide was forced into the membrane. However, this is not a realistic situation in a biological system, in which water

PT E

is always available. We conclude that in nature, SSL-25 does not bind to cholesterolcontaining hydrated membranes. Strong binding to POPE/POPG/TOCL (72:23:5), a membrane model with a similar lipid composition as that in E. coli, and a lack of binding to

CE

DMPC/cholesterol, which more closely resembles eukaryotic membranes, indicates that SSL-

AC

25 is highly selective toward bacterial membranes.

The fact that SSL-25 shows less isotropic signals in POPE/POPG/TOCL than in DMPC/DMPG can be due to the difference in spontaneous curvature of the lipid systems. As discussed previously [42, 60, 63], lipids with a negative spontaneous curvature, like PE or CL, prefer to form structures with a negative physical curvature, like hexagonal phase. This is not possible in the oriented samples, where membranes are forced to be flat, and packing of the lipids with small head groups will lead to a stressed membrane with some “potential empty space” in the head group region. Binding of peptides to the head group region can reduce this stress, and this can contribute to the binding affinity. On the other hand, binding to lipid systems with a positive spontaneous curvature, like DMPC, can be expected to be 27

ACCEPTED MANUSCRIPT reduced since the head groups are more tightly packed in this case [42, 60, 63]. Cholesterol also induces a tighter packing of lipids [65], and can therefore also be expected to reduce peptide binding.

A previous study of vesicle leakage induced by the AMP maculatin 1 has also shown a very low amount of leakage in POPC/sphingomyelin/cholesterol vesicles, but a high amount of leakage in both E. coli-mimicking (POPE/POPG) and S. aureus-mimicking (POPG/TOCL)

PT

membranes [66]. However, maculatin 1 has been found to bind to cholesterol-containing membranes [67], in contrast to our findings for SSL-25. One reason for this difference may be

RI

that maculatin is a strongly hydrophobic peptide with a mean residue hydrophobicity of 1.45

SC

on the Kyte-Doolittle scale [68], whereas SSL-25 is much less hydrophobic, with a mean residue hydrophobicity of -0.36. Owing to its lower hydrophobicity, the water solubility of

NU

SSL-25 is much higher, and SSL-25 is less likely to bind to the membrane. Therefore, binding of SSL-25 is weak to membranes containing lipids with a positive spontaneous curvature (to which binding is less favorable than to lipids with a negative spontaneous curvature) or to

MA

membranes containing cholesterol. However, maculatin will be drawn into the membrane by

D

its hydrophobic residues and will bind even if cholesterol is present.

PT E

5. Conclusions

SSL-25 is an antimicrobial peptide that is found in human sweat and contains many charged residues. SSL-25 binds only weakly to model lipid bilayers mimicking eukaryotic membranes

CE

but binds strongly to model membranes mimicking bacterial membranes. When bound to either membrane, our results using OCD,

15

N-NMR and

19

F-NMR show that the orientation

AC

of this peptide is flat on the membrane surface at all concentrations examined (P/L ratios from 1:25 to 1:500). From the 19F-NMR analysis, the N-terminal part of the peptide forms a regular α-helix, whereas the glycine-rich C-terminal part does not show a helical structure. Although differences in binding affinity cause the peptide to be strongly selective for bacteria, as is characteristic of AMPs, the flat orientation does not explain its mechanism of action. It cannot be excluded that the peptide may form short-lived transient pores, as has been previously suggested in DCD-1L [2, 4]. It is also possible that another mechanism, such as the carpet model, is responsible for its action.

28

ACCEPTED MANUSCRIPT Acknowledgements We acknowledge financial support for NMR hardware from the DFG project "INST 121384/58-1 FUGG". We thank Andrea Eisele and Kerstin Scheubeck for help with peptide synthesis and purification, Markus Schmitt and Dr Stephan Grage for help with the NMR infrastructure, and Bianca Posselt for help with the CD measurements.

PT

Appendix. Supplementary material

SC

RI

Supplementary figures and tables to this article can be found online at …

[4]

[5]

[6]

[7]

[8]

MA

D

PT E

[3]

CE

[2]

B. Schittek, R. Hipfel, B. Sauer, J. Bauer, H. Kalbacher, S. Stevanovic, M. Schirle, K. Schroeder, N. Blin, F. Meier, G. Rassner, C. Garbe, Dermcidin: a novel human antibiotic peptide secreted by sweat glands, Nat. Immunol., 2 (2001) 1133-1137. M. Paulmann, T. Arnold, D. Linke, S. Özdirekcan, A. Kopp, T. Gutsmann, H. Kalbacher, I. Wanke, V.J. Schuenemann, M. Habeck, J. Bürck, A.S. Ulrich, B. Schittek, Structure-activity analysis of the dermcidin-derived peptide DCD-1L, an anionic antimicrobial peptide present in human sweat, J. Biol. Chem., 287 (2012) 8434-8443. T.H. Walther, C. Gottselig, S.L. Grage, M. Wolf, A.V. Vargiu, M.J. Klein, S. Vollmer, S. Prock, M. Hartmann, S. Afonin, E. Stockwald, H. Heinzmann, O.V. Nolandt, W. Wenzel, P. Ruggerone, A.S. Ulrich, Folding and self-assembly of the TatA translocation pore based on a charge zipper mechanism, Cell, 152 (2013) 316-326. C. Song, C. Weichbrodt, E.S. Salnikov, M. Dynowski, B.O. Forsberg, B. Bechinger, C. Steinem, B.L. de Groot, U. Zachariae, K. Zeth, Crystal structure and functional mechanism of a human antimicrobial membrane channel, Proc. Natl. Acad. Sci. U.S.A., 110 (2013) 4586-4591. D. Baechle, T. Flad, A. Cansier, H. Steffen, B. Schittek, J. Tolson, T. Herrmann, H. Dihazi, A. Beck, G.A. Mueller, M. Mueller, S. Stevanovic, C. Garbe, C.A. Mueller, H. Kalbacher, Cathepsin D is present in human eccrine sweat and involved in the postsecretory processing of the antimicrobial peptide DCD-1L, J. Biol. Chem., 281 (2006) 5406-5415. I. Senyurek, M. Paulmann, T. Sinnberg, H. Kalbacher, M. Deeg, T. Gutsmann, M. Hermes, T. Kohler, F. Gotz, C. Wolz, A. Peschel, B. Schittek, Dermcidin-derived peptides show a different mode of action than the cathelicidin LL-37 against Staphylococcus aureus, Antimicrob. Agents Chemother., 53 (2009) 2499-2509. I. Senyurek, G. Doring, H. Kalbacher, M. Deeg, A. Peschel, C. Wolz, B. Schittek, Resistance to dermcidin-derived peptides is independent of bacterial protease activity, Int. J. Antimicrob. Agents, 34 (2009) 86-90. H. Steffen, S. Rieg, I. Wiedemann, H. Kalbacher, M. Deeg, H.G. Sahl, A. Peschel, F. Gotz, C. Garbe, B. Schittek, Naturally processed dermcidin-derived peptides do not

AC

[1]

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References

29

ACCEPTED MANUSCRIPT

[11]

[12] [13]

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ACCEPTED MANUSCRIPT Structure analysis of the membrane-bound dermcidin-derived peptide SSL-25 from human sweat Philipp Mühlhäusera, Parvesh Wadhwania, Erik Strandberga, Jochen Bürcka and Anne S. Ulricha,b,*

Karlsruhe Institute of Technology (KIT), Institute of Biological Interfaces (IBG-2), POB

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3640, 76021 Karlsruhe, Germany

Karlsruhe Institute of Technology (KIT), Institute of Organic Chemistry, Fritz-Haber-Weg

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6, 76131 Karlsruhe, Germany

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* Corresponding Author (email: [email protected], phone: +49-(0)721-608-23222)

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Conflict of interest statement

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The authors declare no conflicts of interest.

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