Lipid-Specific β-Sheet Formation in a Mussel Byssus Protein Domain

Article pubs.acs.org/Biomac

Lipid-Specific β‑Sheet Formation in a Mussel Byssus Protein Domain Markus Heim, Martina B. Elsner, and Thomas Scheibel*,†,‡,§,∥ Fakultät für Ingenieurwissenschaften, Lehrstuhl Biomaterialien, †Bayreuther Zentrum für Kolloide und Grenzflächen (BZKG), ‡ Institut für Bio-Makromoleküle (bio-mac), §Bayreuther Zentrum für Molekulare Biowissenschaften (BZMB), ∥Bayreuther Materialzentrum (BayMAT), Universität Bayreuth, Universitätsstraße 30, 95440 Bayreuth, Germany S Supporting Information *

ABSTRACT: Intrinsically disordered proteins (IDP) or regions (IDR) can adopt multiple conformational states, depending on the interaction partners they encounter. This enables proteins or individual domains to fulfill multiple functions. Here, we analyzed the flank sequences of preCol-NG, one of three collagenous proteins of a mussel byssus thread governing its mechanical performance. preCol-NG comprises a collagen domain and nonrepetitive termini enclosing specific flank regions characterized by tandem repeats known from silk proteins, protein elastomers, and plant cell wall-associated proteins. We recombinantly produced a protein mimicking the M. galloprovincialis preCol-NG Cterminal flank region. The protein was intrinsically unfolded in solution, even at elevated temperatures. However, upon contact with small unilamellar vesicles (SUVs) reversible β-structure formation occurred, reminiscent of partitioning-folding coupling. This behavior of preCol-NG flank domains likely impacts byssogenesis and sheds new light on a distinct mechanism of how fibrous protein materials might be achieved by lipid-induced self-assembly in nature.

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INTRODUCTION Intrinsically disordered proteins (IDP) or disordered regions (IDR) often change conformation or gain defined secondary structure upon contact with other molecules or surfaces. Depending on the interaction partner, different conformations can be adopted, thereby enabling the protein or domain to fulfill more than one distinct task.1,2 Here, we investigated an intrinsically disordered protein constituent of a mussel’s pivotal anchorage system. Mussels of the family Mytilidae are sessile bivalve mollusks. They are perfectly adapted to their ecological niche, such as intertidal and near-shore coastal areas. To withstand the impact of waves, water currents, and tides upon anchorage, mussels (like all other bivalve mollusks) developed a protein-based anchoring structure known as the byssus.3 The byssus is perfectly tailored to meet the mechanical requirements determined by the habitat, shows excellent self-healing properties and usually comprises several threads that serve to tether the mussel to a broad variety of different substrates, like piles, rocks, or other mussels. On its molecular level, each byssal thread represents a complex composite structure involving several different structural proteins and enzymes.4 The core of each thread is formed by putative homotrimers of three collagenous proteins named preCol-D, preCol-P, and preCol-NG embedded in a matrix comprising additional proteins.5 Two of these collagens, preCol-D and preCol-P, are gradually distributed along the thread axis having their highest concentration in the distal (preCol-D) and proximal (preCol-P) portion of the thread, respectively. The third core protein, preCol-NG, is distributed © 2013 American Chemical Society

along the whole axis without a gradual change in concentration.5,6 preCol-NG has a block copolymer-like primary structure. The major part is a central collagen domain with characteristic Gly-Xaa-Yaa repeats (with common occurrence of Pro and hydroxyproline (Hyp) in positions Xaa and Yaa, respectively), which is flanked by noncollagenous domains with a high content of histidine and 3,4-dihydroxyphenylalanine (DOPA) residues, as well as preCol type-specific repetitive amino acid stretches referred to as “flank regions” (Figure 1).5 PreCol-NG’s flank domains are dominated by Xaa-(Gly)n repeats (Xaa usually being a hydrophobic residue)5 with homology to Gly-rich proteins (GRPs) found in the cell walls of plants and to abductin, which is the main protein of the inner hinge ligament in mollusks.7,8 The Xaa-(Gly)n repeats are sandwiched by either poly-Ala runs and (Gly-Xaa)n motifs (with Xaa being mostly Ala or Ser), both typically found in silk proteins, or Gly-Gly-Xaa repeats known to act as the pliable part within protein elastomers.9,10 The characteristic flank sequences of each preCol suggest a role in determining the macroscopic mechanical features of byssal threads.11−13 The flank domains of preCol-P are thought to confer elasticity to the threads,5,10 while those of preCol-D and -NG could act as load-bearing elements under byssus tension.12 Further, the latter are likely involved in byssal selfhealing.14 preCol-NG additionally might act as a mediator between preCol-P and -D within their molecular gradients.10 Received: June 11, 2013 Revised: July 23, 2013 Published: August 15, 2013 3238

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lyophilized spider silk proteins eADF4(C16) and Sfl were dissolved in 6 M guanidinium thiocyanate at room temperature, followed by two subsequent dialysis steps (each 2 h at room temperature) against 10 mM 2-amino-2-hydroxymethyl-propane-1,3-diol/HCl (Tris/HCl), pH 7.5, or 5 mM MOPS, pH 7.5, to completely remove the denaturing agent. BSA was directly dissolved in 10 mM Tris/HCl, pH 7.5 at room temperature. Preparation of Small Unilamellar Vesicle (SUV) Emulsions. 1,2-Dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DLPG), 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, U.S.A.). Lipids were dissolved in CHCl3 (30 min at room temperature in a sealed glass vessel). CHCl3 was completely removed under vacuum using a rotary evaporator (1 h at 20 °C and additional 3 h at 40 °C). Finally, preheated MQ (70 °C) was added to the dried lipid cake to yield a final lipid monomer concentration of 10 mM. After rotating the sample in a water bath for 1 h at 70 °C, samples were sonicated for 10 min at 70 °C in a bath sonicator (USC300T, VWR, Radnor, PA, U.S.A.). To avoid oxidative degradation, prepared SUV emulsions were stored at 4 °C, and storage time never exceeded 7 days. Prior to use, the emulsions were sonicated for 10 min in a 70 °C hot water bath. Assuming an average diameter of 75 nm, a lipid bilayer thickness of 6 nm,15 and an average lipid monomer volume ratio (v/a0lc) of 0.74,16 the aggregation number (i.e., lipid molecules per vesicle) of DSPGSUVs was calculated to be ∼40000. Therefore, 10 mM of the respective lipid monomers theoretically correspond to an equivalent of 0.25 μM SUVs (used as stock solution for all experiments). Preparation of Hydrophobic Polystyrene Particles. Polystyrene Nanospheres with 30 ± 1 nm diameter were purchased from Thermo Scientific (Waltham, MA, U.S.A.). Based on the specific density and concentration (as determined by Thermo Scientific) of the spheres in aqueous solution, the molarity was 1.12 μM at 25 °C. The spheres were centrifuged using an OPTIMA MAX XP ultracentrifuge (Beckman Coulter, Inc., Brea, CA, U.S.A.) for 45 min at 186000g and 4 °C. The spheres were resuspended on ice using a Bandelin SONOPULS GM 3200 homogenizer (probe MS 73 with 20% amplitude for 1 min) and washed three times with 10 mM HEPES, pH 7.5/150 mM NaCl. UV absorbance below 260 nm was checked before use to ensure that substances potentially impairing far-UV CD spectroscopy were completely removed. CD Spectroscopy. Far-UV CD spectra were recorded using a Jasco J-815 spectrophotometer (Jasco, Tokyo, Japan). cwCT(F34W), either dissolved in MQ, 5 mM MOPS, pH 7.5, or ASW, was used at a final concentration of 15 μM in quartz glass cuvettes with 0.1 cm path length. If not otherwise stated, data points were taken every 0.2 nm with 1 nm bandwidth. The scanning speed for cwCT(F34W) in aqueous solutions was 50 nm/min with 2 s integration time. Five spectra in presence of SDS micelles, SUVs, or polystyrene particles were accumulated with 4 s integration time at 20 nm/min. The heating rate for the temperature transition assays was 1 °C/min, and the molar ellipticity at 218 nm was recorded in 1 °C steps. For baseline corrections, all buffers and solutions were analyzed separately using identical parameters. For time course measurements, samples were kept at constant temperature while directly remaining within the cuvettes. Structure Transition Assays in Presence of SDS, SUVs, and Polystyrene Nanospheres. BSA (fraction V) was purchased from Roth (Karlsruhe, Germany). Secondary structure changes of cwCT(F34W), eADF4(C16), Sfl, and BSA were monitored in presence of SUVs by far-UV CD spectroscopy using a Jasco J-815 spectrophotometer (Jasco, Tokyo, Japan). cwCT(F34W), 15 μM, eADF4(C16), 2.1 μM, Sfl, 20.9 μM, and BSA, 3.6 μM, were employed to ensure a peptide bond concentration between 1 and 2 mM, suitable for far-UV CD spectroscopy. Appropriate volumes of a 100 mM SDS solution or of the SUV stock solutions were added to the buffered protein solutions directly before the assay.

Figure 1. Schematic of the investigated protein. Mussels use a byssus to tether themselves to substrates in their habitat. The fibrous core of each byssal thread consists mainly of the proteins preCol-D, preCol-P, and preCol-NG. While preCol-D and preCol-P are gradually distributed, preCol-NG is homogenously distributed along the thread. The C-terminal flank region of preCol-NG from M. galloprovincialis (I) shows sequence characteristics of an intrinsically unstructured protein domain and was used as a template to design an optimized sequence for bacterial production (II), which is fused to cleavable tags (SUMO and CBD) for stability and purification purposes (III). The scale bar represents 1 cm.

Here, we investigated the role of the C-terminal flank region of preCol-NG by characterizing a recombinantly produced and purified protein domain mimicking that of Mytilus galloprovincialis preCol-NG. It is shown that the intrinsically unstructured protein domain is able to undergo induced, reversible structural transitions in presence of small unilamellar phospholipid vesicles (SUVs), which is reminiscent of partitioning-folding coupling. Based on our results, preColNG flank regions likely play an important role during byssal protein assembly and potentially self-healing.

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MATERIALS AND METHODS

Preparation of cwCT(F34W). All experiments were performed with tag-free, purified cwCT(F34W) (101 amino acids, 7178.5 Da, theoretical pI 4.0): SMGAGEGGAG GAGGGAGGAG GLGGGAGGAG GLGGGWGGLG GGAGGLGGLGGGAGGAGGVG GLGGGVGGLG GVGGLGFGGA GASAGAGANA GAGGAGGSAS A (the underlined sequence displays favorable water-to-membrane partitioning free energies with the dashed subsequence being prone to adopt a β-hairpin conformation). Lyophilized cwCT(F34W) was dissolved in Milli-Q water (MQ), 5 mM 3-morpholinopropane-1-sulfonic acid (MOPS), pH 7.5, or artificial seawater (ASW; 480 mM NaCl/28 mM Mg2SO4×7 H20/24 mM MgCl2×6 H20/16 mM CaCl2×6 H20/2.4 mM NaHCO3)12 at room temperature by gently inverting the sample. After 1 h of incubation, the protein solution was centrifuged to remove aggregated cwCT(F34W) using an OPTIMA MAX XP ultracentrifuge (Beckman Coulter, Inc., Brea, CA, U.S.A.) at 186000g and 4 °C for 45 min. The 3239

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Figure 2. Structural characterization of cwCT(F34W). (A) Far-UV CD spectra of cwCT(F34W) dissolved in Milli-Q water (MQ) and artificial seawater (ASW), showing a stable random coil conformation of cwCT(F34W). (B) Far-UV CD spectra of cwCT(F34W) at 5 °C before (black curve) and after heating to 95 °C (red curve). The inset shows molar ellipticity at 218 nm and the corresponding UV absorbance during heating (black squares) and cooling (red circles) of cwCT(F34W) in MQ. Polystyrene particles were added to yield a final concentration of 0.056 μM. After recording of the initial far-UV CD spectra, the particles were removed by centrifugation (45 min, 186000 g, 4 °C), and residual protein in the supernatant was again analyzed by CD spectroscopy indicating the lack of protein binding to the particles. Fluorescence Spectroscopy Assay. Fluorescence spectra of 8.4 μM cwCT(F34W) were taken in 5 mM MOPS, pH 7.5 in presence and absence of an equivalent of 0.125 μM DSPG-SUVs (corresponding to 5 mM DSPG monomer). cwCT(F34W) fluorescence was monitored using a Jasco FP-6500 spectrophotometer (Jasco, Tokyo, Japan). Trp emission was excited at λ = 280 nm (slit width 10 nm) and emission spectra were recorded between 300 and 450 nm (slit width 5 nm). Data points were taken every 0.2 nm at a scanning speed of 50 nm/min and a response time of 1 s. Finally, each spectrum was normalized between 0 and 1.

helical) conformation. Nevertheless, the absence of strongly hydrophobic amino acid residues or larger numbers of charged amino acids might favor a backbone driven compaction of cwCT(F34W)’s conformation in solution,20,21 thereby rendering it similar to a conformation determined for the glycine-rich snow flea antifreeze protein.22 Furthermore, the observed structure was persistent over time and a wide range of temperatures (Figure 2B). A compact structure devoid of clear α-helical and β-sheet conformations is in good agreement with both recently published experimental data on native byssi showing that the flank regions of M. californianus preCol-NG display an amorphous structure with low order14 and with AFM studies, suggesting an overall globular morphology for the M. galloprovincialis preCol-NG flanks.23 Structure of cwCT(F34W) in the Presence of SDS Micelles and Small Unilamellar Vesicles (SUVs). Peptides and proteins being devoid of a defined conformation often gain secondary structure and function upon contact with specific molecules, interaction partners, or surfaces. Especially intrinsically disordered proteins (IDPs) or regions (IDR; comprehensively reviewed in refs 24−26) can adopt multiple conformational states, including folding into defined secondary structures such as α-helices or β-sheets upon contact with lipid vesicles or membranes.27−31 The secondary structure of cwCT(F34W) was, therefore, investigated in presence of amphiphilic SDS micelles and small unilamellar phospholipid vesicles (SUVs) regarding putative interactions with lipid (membrane) structures. Apart from its role as a protein denaturing agent, SDS can induce the formation of α-helices in random coil proteins at concentrations above its critical micelle concentration (cmc).32,33 cwCT(F34W) was incubated in the presence of SDS micelles that represent a simplified but widely used lipid micelle mimicry,32 and the secondary structure was determined by CD spectroscopy. As controls, two engineered, random coil spider silk proteins, eADF4(C16)34 and Sfl35 (the sequences were derived from A. diadematus dragline silk (ADF4) and N. clavipes flagelliform silk (Flag) proteins, respectively) and natively folded bovine serum albumin (BSA)36 were analyzed. The two engineered spider silk proteins were chosen because they, like cwCT(F34W), are representatives of a class of extracorporally used fibrous proteins, showing highly repetitive primary structures and glycine-rich consensus motifs. Beyond that, the naturally occurring variants of both proteins encounter lipid contacts during vesicular secretion and, in case of ADF4,

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RESULTS AND DISCUSSION Recombinant Production of a preCol-NG Flanking Domain. Based on sequence information of preCol-NG from M. galloprovincialis (GenBank Accession Number KC793982), a sequence based on its C-terminal flank region was designed which was optimized for Escherichia coli’s codon usage additionally comprising a tryptophan residue (Trp) for spectroscopic analysis, resulting in protein cwCT(F34W) (cell wall-like C-terminal flank region; GenBank Accession Number KC793983). In order to protect the intrinsically unstructured protein against degradation in the bacterial host, it was hybridized with an amino-terminal small ubiquitin-like modifier (SUMO)17 and a carboxyl-terminal chitin binding domain (CBD) (Figure 1).18 After production, SUMO-cwCT(F34W)-CBD was affinity purified and the tags were cleaved, yielding pure and tag-free cwCT(F34W) (Figure 1) as shown by SDS-PAGE and MALDI-TOF analysis (see Figure S1 in the Supporting Information (SI)). Solution Structure and Stability of cwCT(F34W). Lyophilized cwCT(F34W) is completely resolvable in Milli-Q water (MQ), aqueous buffered solutions, and artificial seawater (ASW).12 To determine the protein’s overall secondary structure in solution, far-UV CD spectra of cwCT(F34W) dissolved in MQ as well as ASW were recorded, indicating a stable random coil structure19 under the given conditions (Figure 2A). However, because CD spectroscopy does not unambiguously allow to differentiate between archetypal random coil or 31-helical conformations, it could not be definitely said whether cwCT(F34W) existed as an extended random coil or in a more compact polyglycine II helix-like (313240

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conformation in eADF4(C16) and Sfl (Figure 3B,C), because signal changes above 215 nm are qualitatively reliable, even if vesicle-related scattering may occur.37 Further, the isosbestic point at 205 nm suggested that the random coil to α-helix transition in eADF4(C16) took place without involvement of structural intermediates (Figure 3B). Further, this conformational change was very fast and already occurred within the mixing time during sample preparation. As expected, the native α-helical conformation of BSA was not affected in the presence of SDS micelles (Figure 3D). Next, different SUVs representing more complex membrane mimicking systems were tested concerning their influence on the structure of our intrinsically unstructured proteins. Lipids were employed varying in length of their acyl chains and charge of their head groups, and conformational effects of the respective SUVs on cwCT(F34W), eADF4(C16), Sfl, and BSA were monitored by CD spectroscopy. Among negatively charged phospholipids comprising different acyl chains, DLPGSUVs had no influence on the secondary structure of any tested protein (Figure 4A−D, magenta curves). The cwCT(F34W) spectra in the presence of DMPG-SUVs slightly differed from those recorded in either buffer or the presence of DLPG-SUVs (Figure 4A, blue curve). However, a similar trend in the obtained spectra infers that the observed effect was more likely the result of DMPG-SUV related light scattering than of distinct secondary structure transitions. In contrast to DLPG and DMPG, clear minima around 218 nm showed that DSPG-SUVs induced β-sheet conformation in cwCT(F34W) and eADF4(C16) (Figure 4A,B, red curves), further accompanied by a hypsochromic (blue) shift of the fluorescence emission maximum of the Trp residue of cwCT(F34W), indicating its translocation to a less polar environment (Figure 4E). Although the presence of SUVs yielded a weak, light scattering-related decrease in the signal-to-

also within the mature silk threads. BSA, in contrast, was chosen because of its characteristics as well-established, stably folded, nonfibrous protein. In our assay, SDS micelles had no detectable influence on cwCT(F34W)’s secondary structure (Figure 3A). In contrast,

Figure 3. Influence of SDS on the secondary structure of selected proteins. Far-UV CD spectra of (A) cwCT(F34W), (B) eADF4(C16), (C) Sfl, and (D) BSA in the absence and presence of 1, 5, 10, and 50 mM SDS (as depicted by the different colors).

the spectra of the two engineered spider silk protein analogues showed conformational changes in the presence of SDS micelles, as indicated by two minima at around 222 and 208 nm. Although the SDS micelles caused some light scattering decreasing the signal-to-noise ratio, especially the minimum at around 222 nm was indicative for the formation of α-helical

Figure 4. Induction of secondary structure formation in dependence of SUV lipid composition. Far-UV CD spectra of (A) cwCT(F34W), (B) eADF4(C16), (C) Sfl, and (D) BSA before and after contact with SUVs made of either 5 mM DSPG, DMPG, or DLPG. (E) Trp emission spectra of cwCT(F34W) in MOPS buffer, pH 7.5 (black curve), and in presence of DSPG-SUVs (red curve). (F) Far-UV CD spectra of cwCT(F34W) after incubation with SUVs comprising lipids with differently charged head groups but identical acyl chains. 3241

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Table 1. Effects of SUVs Made of Different Lipids on the Secondary Structures of cwCT(F34W), eADF4(C16), Sfl, and BSAa

a SUV-induced structural transitions correlate with the lipid composition: (−) means that no structural transition was observable, while (+) denotes the induction of secondary structure in the investigated protein. n.d. states that the secondary structure was not determined under the respective conditions.

noise ratios, spectral changes above 215 nm were shown to be nevertheless qualitatively reliable.37 Zwitterionic DSPC (only differing from DSPG in the charge of the headgroup) caused stronger, light scattering-related spectral perturbations than the phospholipid-SUVs did. However, considering the molar ellipticities around 218 nm, it is reasonable to assume that DSPC-SUVs did not cause induction of β-sheet conformation in cwCT(F34W) (Figure 4F). Strikingly, the secondary structures of the artificial flagelliform silk protein Sfl and of BSA remained unchanged upon contact with any of the SUVs tested (Figure 4C,D). The chemical structures of the phospholipids forming the SUVs and their effects on the secondary structures of the proteins are summarized in Table 1. Next, we investigated the impact of lamellarity and morphology of DSPG-vesicles on the induction of β-sheet formation in cwCT(F34W). Upon hydration of reconstituted lipid without sonication, multilamellar lipid structures were formed, which were unable to influence the secondary structure of cwCT(F34W) (Figure 5A). As a further control, we tested polystyrene nanospheres, providing a simple, curved hydrophobic surface. In their presence, cwCT(F34W) also retained its original conformation and showed no unspecific hydrophobic interactions with the hydrophobic polystyrene surface (Figure 5B). In summary, neither inert hydrophobic surfaces nor very simplified lipid mimics in form of SDS micelles32 had the potential to induce β-sheet formation in cwCT(F34W). Secondary structure transitions were only observable in the presence of SUVs resembling a lipid bilayer membrane, but not in the presence of multilamellar systems. Interestingly, both acyl chain length and headgroup charge of the lipids

Figure 5. Induced secondary structure formation of cwCT(F34W) depends on the physicochemical properties of the SUVs. (A) Unilamellarity of the lipid structure is important to induce secondary structure transitions. (B) Polystyrene nanospheres (ns) had no impact on the cwCT(F34W) structure. β-sheet formation of cwCT(F34W) upon contact with DSPG-SUV correlates with ionic strength (C) and is completely reversible upon heating the SUVs above the transition temperature of DSPG (55 °C), which destroys the micellar structure of the lipid (D).

constituting the SUVs were equally important, and a conformational switch in cwCT(F34W) was only detectable in the case of negatively charged phospholipids with saturated acyl chains comprising more than 14 carbon atoms. Thus, on one hand, the observed SUV-mediated secondary structure transitions 3242

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to-membrane partitioning free energy of −3.66 kcal/mol (Table S1). Further, as a control, we also performed a Wimley-White whole-residue analysis of the engineered spider silk protein eADF4(C16), displaying 16 hydrophobic segments (one per C-module). The calculated water-to-membrane partitioning free energy was about 30 times lower than for cwCT(F34W), and neither membrane spanning nor β-hairpin forming sequences were predicted by MPEx. In the case of the other tested spider silk protein Sfl, hydrophobic segments (in terms of membrane bilayer partitioning) were not predicted at all. Nevertheless, MPEx revealed three putative β-hairpin segments with a score twice that of cwCT(F34W) (Table S1). In summary, cwCT(F34W) was the only tested protein with a sequence showing a favorable water-to-membrane partitioning free energy and simultaneously having the intrinsic potential to fold into a β-hairpin structure. The combination of these two features renders cwCT(F34W) the only tested protein with a behavior typical for partitioning-folding coupling. In case of eADF4(C16), the formation of β-sheets in presence of SUVs is more likely to be caused by polymerization processes at hydrophobic−hydrophilic interfaces than by partitioning−folding coupling. This less specific effect was already previously described for the formation of eADF4(C16) capsules in biphasic solvent systems.45 Taken altogether, recombinant cwCT(F34W), mimicking a flank region of preCol-NG, clearly shows characteristics of an IDP in aqueous solution, adopting a defined β-sheet structure upon contact with phospholipid vesicles. Based on our experimental results and Wimley-White whole-residue hydrophobicity analysis,39,40 we developed a model in which a hydrophobic stretch within cwCT(F34W) interacts with the interfacial region of a vesicle bilayer. Subsequently, a rapid secondary structure transition toward a β-hairpin conformation occurs. In a last step, the hydrogen bonds formed between backbone atoms upon β-hairpin formation allow the penetration of the β-hairpin into the SUV membrane (Figure 6),15 with the β-hairpin being now transiently stabilized by side chain-mediated hydrophobic interactions between the involved β-strands and between the β-strands and the lipid molecules’ aliphatic tails. Strikingly, the predicted 32 amino acid long βhairpin segment of cwCT(F34W) (Figure 6, green segment) comprises an amino acid stretch long enough to form a lipid bilayer-inserted β-hairpin structure consisting of two antiparallel β-strands.46 However, the β-hairpin could also just interact with the SUV’s surface area. The magnitude of the hypsochromic fluorescence emission shift of the Trp, which resides at the first position of the predicted β-hairpin segment, suggests a translocation to a less polar but not completely hydrophobic environment (Figure 4E). This agrees both with an insertion of the β-hairpin with Trp remaining at the polar surface of the interfacial region or with a nonpenetrating, superficial interaction of cwCT(F34W) with the SUV. However, the fact that membrane-spanning segments within transmembrane proteins often contain Trp located in the polar areas of the interfacial region close to the water-membrane interface43,44 combined with the favorable length of the predicted β-hairpin segment in cwCT(F34W) strengthens the theory of β-hairpin insertion. Interestingly, compared to Trp or Tyr, the Phe, natively occurring in the preCol-NG C-terminal flank (that was replaced by Trp in cwCT(F34W)), could even facilitate the insertion of the βhairpin. This is based on its smaller and less polar side chain

strongly depend on the overall physicochemical parameters of the SUVs. On the other hand, the intrinsic properties of the proteins are equally important because DSPG only displayed a secondary structure inducing effect on distinct proteins. In conclusion, the reversible induction of β-sheet structures in cwCT(F34W) resulted from a very specific and complex interplay between its characteristic primary structure and the physicochemical properties of the lipids, therefore, not being simply the result of classic hydrophobic surface interactions. In this context, the ionic strength also had an impact on βsheet formation which was apparently increased in presence of NaCl (Figure 5C). The variation of the ionic strength directly correlated with alterations of the accessible vesicle surface area. Various thermodynamic models describe that an increased ionic strength lowers the critical micelle concentration (cmc) of double-chained lipids and leads to higher aggregation numbers (average number of monomers per vesicle).38−40 The reduced cmc and higher aggregation numbers favor the formation of larger vesicles and promote alterations in vesicle curvature, as does vesicle fusion promoted by counterion shielding of the SUVs’ negative surface potential. Therefore, one can infer that both the increased vesicle surface area and the altered (decreased) curvature correlated with the degree of induced β-sheet structure. Importantly, the formation of β-sheet structure of cwCT(F34W) upon contact within DSPG-SUVs is completely reversible. Upon heating the DSPG-SUV-cwCT(F34W) mixtures above the transition temperature of the lipid (i.e., destroying the vesicle structure), cwCT(F34W) readopted its original conformation (Figure 5D). SUV-Induced Formation of β-Sheets of preCol-NG Flanks is Reminiscent of Partitioning−Folding Coupling. The connection between the SUVs’ accessible interfacial area and the apparent amount of β-sheet formed in cwCT(F34W) upon contact, as well as the thermal reversibility of the induced structural transitions are altogether reminiscent of a process referred to as partitioning−folding coupling.41−43 Therefore, we performed a Wimley-White whole-residue analysis, which can be used to describe propensities of amino acid sequences to interact with the interfacial area of lipid bilayers, taking into account the unfavorable partitioning free energies of the polar peptide bonds.43 The Membrane Protein Explorer (MPEx) software allows calculating thermodynamic probabilities of a protein to interact with lipid bilayer interfacial areas and predicts the protein’s further tendency to adopt α-helical or βhairpin-like conformations as commonly found in membrane spanning proteins.44 The results of the Wimley-White whole residue hydrophobicity analysis are summarized in Table S1 (see SI). A stretch of 61 amino acids (with Trp at position 6) of the cwCT(F34W) sequence revealed a water-to-membrane partitioning free energy of −4.23 kcal/mol. Interestingly, the predicted stretch was nearly perfectly flanked by Trp and Phe, with both large hydrophobic residues interrupting the otherwise repetitive character of the primary structure. Within this stretch, a 32 amino acid sequence (starting with Trp) is theoretically able to adopt a β-hairpin conformation during water-to-membrane partitioning (Table S1). The same applied for cwCT comprising the native Phe instead of Trp. 32 amino acids (starting with the Phe) were predicted to adopt a βhairpin conformation upon partitioning-folding coupling. In this case, the β-hairpin forming segment was part of a stretch comprising 71 amino acids that reveals a slightly lower water3243

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +49 921 557360. Fax: +49 921 557346. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful for Deutsche Forschungsgemeinschaft, DFG SCHE 603/7−1, which funded this work. We kindly acknowledge Adrian Golser, Michael Suhre, and Anja Hagenau for providing cDNA-derived sequence information on M. galloprovincialis preCol-NG. We further thank Claudia Blüm for the donation of purified eADF4(C16) and Christian Ackerschott for purified Sfl. We thank Martin Humenik for MALDI-TOF sample preparation and Christian Borkner for assisting in the graphical design of the lipids’ structural formulas.

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Figure 6. Model of partitioning−folding coupling in cwCT(F34W). An amino acid stretch within cwCT(F34W) (green characters) favorably interacts with the interfacial area of negatively charged phospholipid SUVs. The interaction induces partitioning−folding coupling of cwCT(F34W), yielding a β-hairpin structure. Upon hairpin formation, hydrogen bonding reduces the polarity of peptide bonds, thus, enabling integration of the β-hairpin into the lipid bilayer.

not strictly limiting Phe’s localization to the most polar regions of the water−membrane interfacial area.43,44

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CONCLUSION

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ASSOCIATED CONTENT

REFERENCES

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A recombinantly produced protein domain mimicking the carboxyterminal flank of M. galloprovincialis preCol-NG (cwCT(F34W)) is intrinsically disordered in aqueous environments. Upon contact with small unilamellar vesicles (SUVs), cwCT(F34W) undergoes reversible structural transitions toward a β-sheet dominated fold. Strikingly, this behavior is in accordance with bioinformatical analysis predicting a partitioning−folding coupling resulting in the formation of a β-hairpin. The lipid-triggered formation of a reversible β-hairpin structure in preCol-NG flanks could play an important role during molecular self-assembly upon byssal fibril formation which, in vivo, is initiated in lipid-membrane coated vesicles called granulae.4,47,48 Further, a lipid-dependent structural variability of preCol-NG flanks could be of potential importance considering byssal self-healing after mechanical yield of the thread,49 because lipids and waxes are also implemented in mature byssus threads.50,51

S Supporting Information *

SDS-PAGE and MALDI-TOF spectrum of purified cwCT(F34W) (Figure S1), and summary of MPEx whole-residue hydrophobicity analyses (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org. 3244

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