Transmembrane Polyproline Helix - ACS Publications - American

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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

A Transmembrane Polyproline Helix Vladimir Kubyshkin, Stephan L. Grage, Jochen Bürck, Anne S. Ulrich, and Nediljko Budisa J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b00829 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 11, 2018

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A Transmembrane Polyproline Helix Vladimir Kubyshkin*†, Stephan L. Grage,‡ Jochen Bürck,‡ Anne S. Ulrich*‡,§, Nediljko Budisa*† †

Institute of Chemistry, Technical University of Berlin, Müller-Breslau-Strasse 10, Berlin

10623, Germany ‡

Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology (KIT), P.O.B.

3640, Karlsruhe 76021, Germany §

Institute of Organic Chemistry, KIT, Fritz-Haber-Weg 6, Karlsruhe 76131, Germany

AUTHOR INFORMATION Corresponding Authors * Vladimir Kubyshkin, e-mail: [email protected] * Anne S. Ulrich, e-mail: [email protected] * Nediljko Budisa, e-mail: [email protected]

ABSTRACT The third most abundant polypeptide conformation in nature, the polyproline-II helix, is a polar, extended secondary structure, with a local organization stabilized by intercarbonyl interactions within the peptide chain. Here we design a hydrophobic polyproline-II helical peptide based on an oligomeric octahydroindole-2-carboxylic acid scaffold, and demonstrate its transmembrane alignment in model lipids by means of solid-state 19F NMR. As

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result, we provide a first example of a purely artificial transmembrane peptide with a structural organization that is not based on hydrogen-bonding.

TOC Graphic

There is a number of secondary structure elements, which natural polypeptide sequences can adopt. This set is restricted by internal factors, such as the conformational preferences of certain amino acids, and external factors, for example, the presence of denaturing agents, solvents, temperature, etc. In structured proteins, the most favored elements are the ones based on hydrogen bonding, -helices and -sheets. However, the third most abundant structure is not based on H-bonding.1 It has been named polyproline-II helix (PII helix) due to its original discovery in polymeric proline;2,3 and possesses an extended helical structure with a remarkable geometrical simplicity: 3.0 residues per turn and about 3 Å linear increment per amino acid residue.4,5 Even though, the PII helix is a generic structure (i.e., all coded amino acids can adopt it), in a historical perspective it took relatively long time to recognize its widespread presence. Arguably,

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the main reason for this is, that, unlike H-bond-/salt bridge-/hydrophobic motif-based structures, the PII helix lacks denaturing agents/conditions able to eliminate the forces behind its structural organization. Compared to an -helix with an i ↔ i+4 hydrogen bonding pattern, the PII structural organization is more localized,6 and the main interaction responsible for its stability occurs between the carbonyl groups of residues i and i+1. This interaction has been deciphered relatively recently with the help of collagen studies,7 and the main reason behind its structural integrity was attributed to the n→* orbital interaction between the adjacent carbonyl groups in the peptide chain.8 The energy of this attractive interaction is usually quite low (≤ 3 kJ mol−1),9,10 nonetheless, when competitive, stronger structures are eliminated, the forces leading to a PII helix inevitably become dominant. This, for example, may occur in the denatured state11,12 or in proline-rich segments,13 where proline lacks the ability to donate an H-bond. Effectively, the extended PII helix can be well featured by ‘unstructured’ and exposed segments that are essentially polar,14 whereas nature has not developed hydrophobic PII helical sequences. We were intrigued by this fact, and decided to construct an entirely hydrophobic P II helix. We thus aimed to create a completely new-to-nature motif based on this generic, natural secondary structure. The most common hydrophobic environment in biological settings is the lipid membrane, with membrane proteins constituting about ⅓ of the genome sequences.15 In these proteins, hydrophobic -helical or -sheet-adopting segments are designed to transverse the membrane.16 In special cases, the transmembrane orientation can be adopted by other structures, for example, H-bond based -helical pores formed by the antimicrobial peptide gramicidin A.17 In contrast to these existing membrane-spanning peptide structures, herein we report on a construction of an artificial hydrophobic PII helix, and demonstrate its transmembrane orientation in model lipids.

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Figure 1. Design of the study. Several aspects were taken into account in the design of a hydrophobic PII helix, such as suitable constructing amino acid (A), a peptide length matching the hydrophobic thickness of the lipid bilayer (B), suitable charged terminal groups to anchor the peptide in the bilayer (C), orientation-sensitive 19F NMR labels (D), and a sufficient lipophilicity of the residues (E). In order to create a PII helix which would be affine to the hydrophobic core of a lipid membrane, we decided to employ the oligoproline scaffold, as proline restricts the set of possible dihedral angles, and is naturally optimized for the PII helical fold (Figure 1A). However, since proline is not sufficiently hydrophobic, it is necessary to further decorate the oligoproline sequence with strongly lipophilic side chains. Only two entirely hydrophobic P II helices have been reported in the literature so far, oligomeric (2S)-silaproline (Sip)18,19 and oligomeric

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(2S,3aS,7aS)-octahydroindole-2-carboxylic acid (Oic).20,21 We previously demonstrated the solubility of oligo-Oic peptides in alkanes,21 solvents resembling the hydrophobic core of membranes. Importantly, these peptides are also featuring the enhanced stability of the PII fold due to the fixed C4-exo envelope conformation of the proline ring,20 which is optimal for the establishment of the PII stabilizing forces.7 Therefore, we decided to employ an oligo-Oic sequence as the core construction element that should be immersed in the membrane surroundings. According to the previously reported crystal structure,20 a 9-residue (3 turn) sequence should result in a peptide with a linear length of 26 Å, which exactly matches the hydrophobic thickness of a model membrane of the C14-based phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC or 14:0/14:0 PC),22 as illustrated in Figure 1B. The next necessary design elements are the terminal groups, which should anchor the model helix in the polar regions of the membrane. Perhaps, the easiest way to create a polar anchor would be to leave the C- and N-terminal parts unmodified (non-capped), and thus to make use of the intrinsic terminal charges. Nonetheless, we expected a somewhat reduced P II helix stability when the charged termini are directly located at the peptide backbone.23,24 Therefore, we decided to introduce two positive charges on C6-linkers at both termini (Figure 1C). The linkers in this case resemble lysine side chains, and thus this construction principle is reminiscent to that of the KALP peptide, an -helical model peptide consisting of a repetitive alanine-leucine sequence flanked with charged lysine residues on both ends.25 For the read-out we needed a reporter element, which would indicate the expected transmembrane alignment.

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F NMR provides an ideal technique to study the membrane

alignment of a polyproline structure, as it does not rely on N-H moieties in the backbone, which

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are used in the most common solid-state NMR methods such as PISEMA.26 We have previously demonstrated the use of a few proline analogues for the determination of peptide orientation in membranes from solid-state 19F NMR orientational constraints (Figure 1D).27,28,29,30 From this set of amino acids we chose 4-trifluoromethylproline (4TfmPro), since we expected minimal structural perturbance from this amino acid. As evident from the measured octan-1-ol / water partition coefficients (logP values) obtained for the used amino acids in model compounds (Figure 1E),21 both Oic and 4TfmPro are hydrophobic, therefore, these amino acids should be compatible with a membrane environment.

Chart 1. Peptide structures. The final construct was peptide 1 (Chart 1), having (a) a hydrophobic core of 3 turns of a P II helix matching the DMPC bilayer hydrophobic core thickness, (b) terminal charges and (c) the reporter amino acid 4TfmPro in the middle of the sequence. In addition, we prepared a single turn peptide (peptide 2 in Chart 1) for referencing purposes. The peptides were synthesized using a combination of solid and solution phase synthesis as shown in Scheme 1.

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Scheme 1. Synthesis of the peptides. We then analyzed peptide 1 by circular dichroism (CD). The spectra were recorded in SDS micelles in a temperature series (Figure 2A), as well as in DMPC, which is a lipid matching the length of the peptide, in a shorter (DLPC) and in a longer (POPC) lipid (Figure 2B). Overall, the observed CD lineshapes were consistent with a PII helix, while the unusual, pronounced red shift of the main negative band can be attributed to several effects associated with the CD spectrum of the tertiary amide bond in a nonpolar environment.21,31 Notably, the results of the temperature series (Figure 2A) may indicate some loss of the ordered conformation upon heating. The minor differences in the peptide CD curves observed in the lipid series (Figure 2B) may be attributed, for example, to changes in conformational equilibria due to the adaptation of the transmembrane alignment in DMPC and DLPC.

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Figure 2. Circular dichroism spectra of the peptide 1: temperature series in SDS micelles (A); in unilamellar vesicles formed by model lipids at 303 K (B). To further demonstrate that a transmembrane alignment was indeed achieved, peptide 1 was reconstituted in oriented model lipid membranes for solid-state NMR measurements. In this method, hydrated multilayer stacks of lamellar lipid bilayers are uniformly aligned with the help of glass plates. These samples are placed in the magnetic field of an NMR spectrometer under a defined angle. When the bilayer normals are parallel to the magnetic field, a transmembrane helix should be aligned parallel to the magnetic field. As a result, the CF3-group in 1 will adopt a defined alignment of ~90° with respect to the field vector B0, assuming a C4-exo pucker of the side chain.30 In a

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F NMR spectrum the CF3-group will be observed as a triplet with a dipolar

splitting value FF expressed according to Eq. 1.32 Assuming FFmax is 16 kHz33,34 and a molecular order parameter Smol=1, the spectrum should exhibit a dipolar coupling of about −8 kHz. This resonance is very characteristic, because it should occur at the very right (highfield) edge of the spectrum.

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 FF   FF max S mol

Figure 3.

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3 cos 2   1 2

(1)

F NMR spectra of the peptide 1 in oriented lipid samples with their membrane

normal aligned parallel with respect to the magnetic field (A) and proposed interpretation (B). The samples were measure at a peptide/lipid ratio of 1/40 and 308 K. For the hammock state an arbitrary CF3-label orientation is depicted. Yellow arrows indicates free rotation about the membrane normal. As a next step we measured solid state 19F NMR spectra in several model lipids (Figure 3A). Our conclusions regarding the peptide alignment are summarized in Figure 3B. The spectrum in DMPC demonstrates the presence of a triplet with −7.5 kHz splitting, close to the value of −8 kHz indicating the presence of the transmembrane state. The transmembrane state becomes the only state in the short-chain lipid DLPC, where a small decrease in the coupling value (to −6.8 kHz) may be attributed to a small tilting of the helix caused by the so-called hydrophobic mismatch.35 Interestingly, the spectrum in DMPC contained two further triplets, with dipolar splitting values inconsistent with a transmembrane alignment (see Figures S1 and S2 in the

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Supporting Information). We speculate that these splittings correspond to peptides which are immersed only from one side of the membrane, with their charged termini reaching out to the polar bilayer region of the bilayer on the same side in a hammock-like fashion (referred to as hammock state in Figure 3B). The hammock state becomes dominant in POPC, where the peptide may be too short to span the membrane. The exact reason for the occurrence of two hammock states is unknown. We presume that this happens because the charged C6-anchors point into different directions, resulting into two peptide alignment possibilities with similar energetic preference. Thus the CF3-probe is as well oriented differently, giving rise to the observed two triplets. Our conclusions regarding the co-existence of transmembrane and hammock-like alignment states was further confirmed by samples of the single turn reference peptide 2 (Figure 4). Due to the geometric structure of the PII helix (3.0 residues per one turn) deletion of one turn from each terminus should maintain the relative label/anchor orientation, whereas the transmembrane state becomes impossible because the peptide is too short to span the membrane. As a result, the experimentally observed spectra of 2 (Figure 4) do not depend on the lipid thickness, and the splitting pattern is similar to the one observed for the hammock state of peptide 1. We thus conclude that the competition of the hammock and transmembrane alignments of model peptide 1 can be driven by the lipid hydrophobic thickness. Interestingly we found that addition of approx. ⅓ lyso-lipids to the POPC and DMPC membranes was unable to increase the transmembrane state signal (see Supplementary information). This is in contrast to the behavior of amphipathic, membrane associated peptides36 and membrane proteins,37 which are usually sensitive to membrane spontaneous curvature modulation, and insert into the membrane in a tilted or transmembrane alignment upon the addition of lyso-lipids.

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Figure 4. 19F NMR spectra of the single turn reference peptide 2 under same conditions (above) with proposed interpretation (below). In summary, we, for the first time, designed and observed a transmembrane polyproline-II helix. The structure was designed as an oligomeric sequence of a hydrophobic proline analogue, (2S,3aS,7aS)-octahydroindole-2-carboxylic acid, Oic. Using solid-state 19F NMR, and employing (4S)-trifluoromethylproline as a reporter amino acid, we confirmed the transmembrane alignment, and observed a response of the peptide orientation to the bilayer thickness. As a result we provide the first example of a transmembrane peptide structure endowed with stability not based on hydrogen bonding.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Synthesis of the peptides and details of their characterization (PDF file).

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Notes The authors declare no competing financial interests. ACKNOWLEDGMENT VK acknowledges DFG research group FOR 1805 for postdoctoral position. SG, JB and AU acknowledge financial support by the DFG grant INSTR 121384/58-1 FFUG and the Helmholtz Association program “BIF-TM”. We thank the Synchrotron Light Source KARA at KIT for providing beam time at the UV-CD12 beamline, and Bianca Posselt (KIT) for technical assistance in the CD lab and at the beamline. Dr Oleg Babii (KIT) is acknowledged for his kind assistance in the laboratory settings. Dr Erik Strandberg (KIT) is acknowledged for valuable discussions. REFERENCES

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(36) Strandberg, E.; Zerweck, J.; Wadhwani, P.; Ulrich, A. S. Synergistic Insertion of Antimicrobial Magainin-family Peptides in Membranes Depends on the Lipid Spontaneous Curvature. Biophys. J. 2013, 104, L9-L11. (37) Milovanovic, D.; Honigmann, A.; Koike, S.; Göttfert, F.; Pähler, G.; Julius, M.; Müllar, S.; Diederichsen, U.; Janshoff, A.; Grubmüller, H.; Risselada, H. J.; Eggeling, C.; Hell, S. W.; van den Bogaart, G.; Jahn, R. Hydrophobic Mismatch Sorts SNARE Proteins into Distinct Membrane Domains. Nat. Commun. 2015, 6, 5984.

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