Investigating the Role of (2S,4R)-4 ... - American Chemical Society

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INVESTIGATING THE ROLE OF (2S,4R)-4HYDROXYPROLINE IN ELASTIN MODEL PEPTIDES Brigida Bochicchio, Alessandro Laurita, Andrea Heinz, Christian E.H. Schmelzer, and Antonietta Pepe Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm4011529 • Publication Date (Web): 15 Oct 2013 Downloaded from http://pubs.acs.org on October 20, 2013

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Investigating the role of (2S,4R)-4-hydroxyproline in elastin model peptides Brigida Bochicchio1, Alessandro Laurita1, 2, Andrea Heinz3, Christian E. H. Schmelzer3, Antonietta Pepe1*. 1

Department of Science, University of Basilicata, 85100 Potenza Italy

2

CIGAS, University of Basilicata, 85100 Potenza, Italy

3

Institute of Pharmacy, Martin Luther University Halle-Wittenberg, 06120 Halle (Saale), Germany.

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ABSTRACT. Post-translational modifications play a key role in defining the biological functions of proteins. Among them, the hydroxylation of proline producing the (2S,4R)-4-hydroxyproline (Hyp) is one of the most frequent modifications observed in vertebrates, being particularly abundant in the proteins of the extracellular matrix. In collagen, hydroxylation of proline plays a critical role, conferring the correct structure and mechanical strength to collagen fibres. In elastin, the exact role of this modification is not yet understood. Here, we show that Hyp-containing elastin polypeptides have flexible molecular structures, analogously to proline-containing polypeptides. In turn, the self-assembly of the elastin peptides is significantly altered by the presence of Hyp, evidencing different supramolecular structures. Also the in vitro susceptibility to protease digestion is changed. These findings give a better insight into the elastic fibre formation and degradation processes in the extracellular matrix. Furthermore, our results could contribute in defining the subtle role of proline structural variants in the folding and selfassembly of elastin-inspired peptides, helping the rational design of elastin biomaterials.

Keywords: elastin, hydroxyproline, self-assembly, molecular structure, protease susceptivity, coacervation.


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Introduction Protein post-translational modifications (PTMs) are chemical modifications decorating proteins at different locations along their amino acid chains. PTMs play a key role in the function of proteins, regulating activity, localization and interaction with other cellular molecules such as proteins, nucleic acids, lipids, and cofactors1. Consequently, the analysis of proteins and their PTMs is particularly important for the study of their functional role. In vertebrates the elasticity of tissues and organs is ensured by elastic fibres that are localized in the extracellular space and are mainly composed of elastin and fibrillin. In order to form mature elastin, human tropoelastin, the 70 kDa soluble monomer, undergoes a series of events resulting into a highly cross-linked elastin polymer2,3. The newly synthesized protein is secreted out of the cell, where it accumulates and undergoes a peculiar phenomenon of self-assembly, called coacervation4. Before final maturation, tropoelastin undergoes a series of PTMs. The most studied modification occurs in the extracellular space and envisages the deaminative oxidation of lysine residues catalyzed by the lysyloxidase (LOX) or LOX-like enzymes. This step is necessary for the formation of allysines, responsible together with lysines, of the peculiar crosslinks of elastin. Cross-linking provides elastic fibres with structural integrity and durability, contributing to their high insolubility5. Another less studied PTM observed for elastin is hydroxylation of some of the about 90 proline residues in tropoelastin. The extent of hydroxylation of prolines in elastin is variable and in the range of 0-33% of the total proline content6. The enzyme prolyl-4-hydroxylase (P4H) catalyzes the introduction of a hydroxyl group at position 4 of proline forming the (2S,4R)-4-hydroxyproline (Hyp) (Figure 1). This reaction occurs in the endoplasmic reticulum, where the same enzyme catalyzes also the hydroxylation of collage7. Hydroxylation of proline for collagen plays a critical role in stabilizing the triple helix structure thus conferring the correct structure, mechanical strength and thermal stability to 3 ACS Paragon Plus Environment

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the collagen fibres. The stabilizing effect of Hyp was attributed to the ability of the hydroxyl group to form hydrogen bonds8.

Figure 1. Chemical structures of proline and analogue. (2S)-proline with R1=H and (2S,4R)-4hydroxyproline with R1=OH. Further studies have shown the importance of a stereo-electronic effect generated from electronwithdrawing substituents on the proline ring9. The 4R configuration is fundamental in order to promote a preorganization of the polypeptide chain, thus favoring the folding into the triple helix. In elastin in turn the possible role of this modification has not yet been elucidated. Some authors speculated that hydroxylation of proline in elastin is a coincidental feature, due to the co-localization of procollagen and tropoelastin in the same cellular district, the endoplasmatic reticulu10. The action of P4H in this cellular compartment could occur on Pro-Gly segments of both proteins11. However, unlike collagen, hydroxylation is not fundamental for the secretion of tropoelastin12. In recent studies the sites of proline hydroxylation in skin elastin were identified. The mapping of the Hyp sites was possible thanks to the identification of proteolytic fragments obtained by enzymatic digestion of skin elastin13-15. These studies confirmed also that hydroxylation is partial, occurring at sub-stoichiometric abundance, in some, but not all, copies of tropoelastin. Few studies have examined the effect of the proline hydroxylation on elastin. Urry et al. have shown that the presence of Hyp in the (VGVHypG)n elastin-like polypeptide sequences requires a higher 4 ACS Paragon Plus Environment

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temperature for coacervation16. Elastin polypeptides containing the fluorine electron-withdrawing group in the 4R configuration were synthesized and analyzed by Conticello and colleagues17. Their studies infer a great importance to the stereoelectronic effect in driving the coacervation process. Recently, small-sized elastin peptides containing the elastin-repeated sequences VGVXGVG, where X corresponds to proline (Pro), (2S,4R)-4-hydroxy-proline (Hyp) and (2S,4R)-4-methoxy-proline (Mop), were synthesized and gave preliminary insights in the effect of the introduction of electronwithdrawing substituents on the conformational structure of elastin peptides18. In order to investigate the effect of Hyp introduction in the molecular and supramolecular structure of elastin, we chemically synthesized by solid-phase peptide synthesis (SPPS) two medium-sized elastin–related model peptides containing Hyp. We investigated by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies the conformations adopted by Hyp-containing elastin polypeptides and compared them to the parent proline-containing polypeptides. Furthermore, we examined their self-assembling behaviour by defining the coacervation propensities and observing the ultrastructural morphologies of the aggregates by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Finally, the susceptibility to protease digestion was evaluated. Our results show that hydroxylation of proline has a reduced effect on the conformations adopted by the polypeptides. On the contrary, the presence of Hyp in elastin polypeptides altered the selfassembling properties as evidenced by a reduced propensity to coacervate and a remarkable difference in the supramolecular pattern as shown by TEM and AFM microscopy.


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Experimental Section Peptide synthesis. The polypeptides were synthesized by solid phase methodology using an automatic synthesizer Applied Biosystem model 431A. Fmoc/DCC/HOBT chemistry was used, starting from (0.25 mmol) Wang resin (Nova Biochem, Laufelfingen, Switzerland) for E50, E50H polypeptide syntheses. In the case of E18 and E18H polypeptides having a proline at the C-terminus, the amidated polypeptides were synthesized by using (0.25 mmol) Rink-amide resin (Nova Biochem, Laufelfingen, Switzerland) to avoid side reactions. The Fmoc-amino acids were purchased from Nova Biochem (Laufelfingen, Switzerland) and from Inbios (Pozzuoli, Italy). The cleavage of the peptides from resin was achieved by using an aqueous mixture of 95% trifluoroacetic acid (TFA). The peptides were lyophilized and purified by reversed-phase HPLC (High Performance Liquid Chromatography). A binary gradient was used and the solvents were H2O (0.1% TFA) and CH3CN (0.1% TFA). The purity of peptides was assessed by electrospray or MALDI–TOF mass spectrometry (MS). CD spectroscopy. Far-UV CD spectra were acquired with a Jasco J-815 Spectropolarimeter equipped with a temperature controller by using quartz cells of 0.1 cm. The concentrations of polypeptides were 0.1 mg mL-1 in aqueous solution or trifluoroethanol (TFE) solution. Spectra at different temperatures were acquired in the range 190–250 nm by taking points every 0.1 nm, with 100 nm min-1 scan rate, an integration time of 2 s, and a 1 nm bandwidth. The data are expressed in terms of [θ], the molar ellipticity in units of degree cm2 dmol-1. NMR spectroscopy. All 1H NMR experiments were performed on a Varian Unity INOVA 500 MHz spectrometer equipped with a 5mm triple-resonance probe and z-axial gradients. The purified peptides were dissolved in 700 µl of H2O/D2O (90/10, v/v) and TFE-d3/H2O (80/20), containing 0.1 mM of 3(trimethyl-sily1)-1-propane sulfonic acid (DSS) as internal reference standard at 0 ppm. 3 mM peptide solutions were used. One-dimensional spectra were acquired in Fourier mode with quadrature 6 ACS Paragon Plus Environment

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detection. The residual HDO signal was suppressed by double-pulsed field-gradient spin-echo19. Twodimensional TOCSY20 and NOESY21 spectra were collected in the phase-sensitive mode using the States method. Typical data were 2048 complex data points, 16 or 32 transients and 256 increments. Relaxation delays were set to 2.5 s and spinlock (MLEV-17) mixing time was 80 ms for TOCSY while 100-200 ms mixing time was applied to NOESY experiments. Shifted sine bell squared weighting and zero filling to 2K x 2K was applied before Fourier transformation. Amide proton temperature coefficients were usually measured from 1D 1H NMR spectra recorded in 5°C increments from 20 °C to 45 °C. Data were processed and analyzed by VnmrJ software (Agilent, Palo Alto). Sequential resonance assignments were made by the approach described by Wüthrich22. Great overlap of the signals precluded a complete resonance assignment of E18 and E18H peptides (Figure S3). Coacervation studies. Coacervation experiments were performed using a Varian Cary 5 spectrophotometer equipped with a temperature controller. The peptides were dissolved in coacervation buffer (50 mM Tris, 1.5 M NaCl, 1 mM CaCl2, pH 7.5) to a concentration of 1 mM, 2 mM and 3 mM. For experiments aiming to study the effect of TFE on the coacervation 7.5% (v/v) of TFE was added to the coacervation buffer. Coacervation studies were performed by increasing the solution temperature at a rate of 1°C min-1, monitoring the absorbance at 440 nm. The coacervation temperature (Tc) was determined as the temperature value at which 50% of coacervation took place. TEM microscopy. The samples were solubilized in ultrapure water (concentration of 1mg mL-1). 10 µL of the solution was placed on formvar and carbon-coated copper grids, negatively stained with a few drops of 1% uranyl acetate in bidistilled water, air dried and observed by a ZEISS EM10C transmission electron microscope. An aliquot of the samples were incubated at 50°C for 48h, while another aliquot was incubated at 37°C for 9 days and analyzed as previously described.


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AFM microscopy. The samples were solubilized in ultrapure water (concentration of 1mg mL-1) and 5 µL aliquots were deposited as drops on Si (100) substrate. After water evaporation the AFM images were obtained by XE-120 microscope (Park Systems) in air and at room temperature. Data acquisition was carried out in intermittent contact mode at scan rates between 0.4 and 0.8 Hz, using rectangular Si cantilevers (NCHR, Park Systems) having the radius of curvature less than 10 nm and with the nominal resonance frequency and force constant of 330 kHz and 42 N/m, respectively. The same samples were incubated at 50°C for 48h and analyzed as previously described. Proteolysis of peptides. E18 and E18H peptides were dissolved in 50 mM Tris buffer pH 7.5 at concentrations of 1 mg mL-1 by shaking in a Thermomixer (Eppendorf, Hamburg, Germany). Enzymatic digestions were carried out using porcine pancreatic elastase (EC 3.4.21.36; Elastin Products Company, Owensville, MO, USA). The incubation temperature was 37 °C and the enzyme-tosubstrate ratio 1:500 (w/w). Samples were taken at 6 time points between 10 min and 31 h and proteolysis was immediately stopped by acidification with TFA. Control experiments were performed by incubating the samples under identical conditions but without adding the enzyme Mass spectrometry and peptide sequencing. NanoHPLC/MALDI-TOF/TOF MSexperiments were carried out using an Ultimate 3000 RSLCnano system coupled to a Probot microfraction collector (Thermo Fisher, Idstein, Germany). After spotting the samples were measured on a 4800 MALDITOF/TOF Analyzer (AB Sciex, Darmstadt, Germany) as described in detail previously23. Complementary nanoHPLC-nanoESI MS and tandem MS experiments were conducted using an Ultimate 3000 nanoHPLC system (Thermo Fisher, Idstein, Germany) coupled online to a QqTOF mass spectrometer Q-TOF-2 (Waters, Manchester, UK). In brief, chromatographic separation was carried out using a binary reversed-phase gradient that was identical to the one used for LC/MALDI experiment


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and eluting peptides in the m/z range 300 to 2500 were selected for MS/MS either using an inclusion list or in data directed acquisition mode. All fragment spectra were pre-processed using proprietary software and the resultant peak lists were subjected to automated de novo sequencing using Peaks Studio 6.0 (Bioinformatics Solutions, Waterloo, ON, Canada) with subsequent database matching using a modified SwissProt database. The peptide score threshold was adjusted until a false discovery rate below 1% was achieved. Furthermore, some short peptides were identified manually by their precursor and fragment masses.

Results Peptide sequences. The synthesized elastin model polypeptides are listed in Table 1. The E50 and E50H polypeptides contain a ten-fold repeat of the consensus sequence –VGVPG– and –VGVHypG–, respectively. Chemically synthesized as well as recombinantly produced polypeptides containing the – VPGVG– motif have been commonly used as a simplified model of elastin to such an extent that they have been widely explored for drug delivery applications, tissue engineering and many other biomedical applications24-26. Table 1: Sequences and monoisotopic molecular weights of the synthesized polypeptides. Peptide

Sequence

MW (Da)

E50

(VGVPG)10

4110.34

E50H

(VGVHypG)10

4270.28

E18

GAAAGLVPGGPGFGPGVVGVPGAGVPGVGVPGAGIP

4002.22

VVPGAGIPGAAVPa E18H

GAAAGLVPGGHypGFGPGVVGVPGAGVHypGVGVHyp GAGIHypVVHypGAGIHypGAAVPa

a

amidated peptide.


4098.19

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The main reason is due to the particular abundance of the VGVPG sequence in the bovine and porcine tropoelastin sequences. In the human tropoelastin the pentapeptide VGVPG, or any of its permutation is present once in exon 7-, 20-, 24, and 30-encoded domains27. Only in the exon18-encoded domain (E18) it is present in a not perfectly repeated pattern (underlined in Table 1). According to studies from Getie et al.13 this domain is also the one with the highest degree of hydroxylation. This observation prompted us to investigate the conformational and supramolecular behavior of modified E18 domain with the introduction of Hyp instead of Pro in position 327, 342, 347, 352, 355, and 360 of the human tropoelastin protein sequence (Swiss Prot. accession number P15502, isoform 3). Conformational analysis. In order to examine the influence of hydroxyl group in 4R configuration of the pyrrolidine ring on the conformation of elastin model peptides we employed CD and NMR spectroscopy. CD spectroscopy is a very sensitive tool, able to elucidate the secondary structures of proteins and peptides. CD spectra were recorded at different temperatures and in two solvent conditions in order to highlight the differences in secondary structures adopted by the peptides under various experimental conditions, showing the conformations adopted by the polypeptide chains. This approach has been successfully employed for the studies of elastomeric proteins, in general, and for elastinrelated polypeptides in particular28-30. The CD spectrum of E50 polypeptide in water at 0°C shows a negative band at 198 nm, usually assigned to unordered and/or PPII conformation (Figure 2a)31,32. The intensity of the negative band is considerably reduced and slightly red-shifted on heating. This temperature-induced behavior is commonly observed in elastin peptides and has been assigned to a conformational transition between random coil/unordered conformations and a more ordered type II βturn18,33,34. For E50H a higher intensity of the negative band at 198 nm with respect to E50 is observed at 0 °C (Figure 1 b). This more distinct negative band could infer a higher PPII content, for the Hypcontaining E50H polypeptide. Analogously to E50, the increase of the temperature induces a reduction 10 ACS Paragon Plus Environment

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of the intensity of the negative band. In TFE the CD spectra of E50 and E50H are very similar and indicate high contribution of type II β-turn conformations as evidenced by the positive band at 205 nm.

Figure 2. Temperature–dependent CD spectra. CD spectra recorded in acqueous and TFE solutions of (a) E50, (b) E50H, (c) E18 and (d) E18H polypeptides.


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The negative band of comparable intensity at 225 nm suggests the co-presence of significant amount of unordered conformations (Figure 2a and 2b). The analysis of CD spectra of E18 (Figure 2c) and E18H (Figure 2d) polypeptides reveals that E18H polypeptide has a higher content of PPII in water as shown by the stronger intensity of the negative band at 195 nm. On increasing the temperature, both peptides show a reduction of the negative band, indicative of the conformational transition from PPII and/or unstructured conformation to β-turn conformations. In TFE the spectra suggest a more complex situation with evidence of a mixture of different types of β-turns and unordered conformations35. NMR studies were performed on E50 and E50H polypeptides in H2O/D2O (90/10, v/v) and in TFEd3/H2O (80/20, v/v) at 25 °C. The presence of the repeated unit, -VGVP/HypG- in the sequence of E50 and E50H polypeptides simplified the interpretation of the spectra by assuming that each of the repeating pentapeptidyl subunit has a structure that is identical to the others29,36 (Figure 3a). 1D 1H-NMR spectra of E50 and E50H peptides recorded in H2O/D2O (90/10 v/v) showed, as expected, 4 main signals (2 doublets for the two valines and 2 triplets for the two glycines) with a high degeneracy of the signals belonging to the same amide protons present in the V1nG2nV3nX4nG5n (X= Pro, Hyp) repeat. In particular in the low field region, four predominant signals at 8.51, 8.44, 8.01, and 7.96 ppm for E50 and at 8.59, 8.52, 8.06 and 7.97 ppm for E50H are observed (Figure 3b and 3c). The intensities of the integrated peaks are consistent with the structures in Figure 3a. Some smaller signals are also observed, probably belonging to the N- and C-terminal residues or to a minor extent to the cis conformers. The simplicity of the observed spectra could be interpreted in two ways: (i) the polypeptides adopt very stable and rigid secondary structures that are common to all the pentapeptide units, or (ii) the polypeptides adopt highly flexible structures, that exhibited multiple conformations interconverting rapidly in solutions on the NMR time scale. In the second case a set of averaged 12 ACS Paragon Plus Environment

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Figure 3. Structural formulas of the elastin peptides and 1H NMR spectra of the amide region. Chemical structure of the pentapeptide repeat units corresponding to E50 (R1=H) and E50H (R1=OH) polypeptides (a). The amide regions of the 1H NMR spectra of (b) E50 and (c) E50H recorded in H2O/D2O (90/10, v/v) at 25°C and of (d) E50 and (e) E50H polypeptides recorded in TFE-d3/H2O (80/20, v/v) at 25 °C. experimental NMR parameters should be observed corresponding to weighted values belonging to the different conformations37. As suggested by the CD spectra, we interpreted the NMR data in term of highly unstructured conformations, in which residues belonging to different repeats have a homogeneous microenvironment populating similar highly dynamic conformations. This is confirmed also by other NMR data, such as NOE cross-peaks pattern or temperature coefficients (-∆δ/∆τ > 5.0 13 ACS Paragon Plus Environment

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ppb/K), consistent with unstructured polypeptides (Tables S1-S3 and Figure S1a and S2a). When comparing the chemical shifts of the amide protons of E50 and E50H polypeptides in aqueous solution, a significant difference is only observed for G5n (∆δ=0.15 ppm) and in a minor extent to V3n ( ∆δ=0.05 ppm). The presence of 4R-hydroxyl group on the pyrrolidine ring determines a downfield shift of the following amide protons. This behaviour could be assigned to an inductive electron-withdrawing effect of the OH group, and was recently observed also for two TYPN and TAPN tetrapeptides38. 1D 1H-NMR spectra of E50 and E50H recorded in TFE-d3/H2O (80/20, v/v) showed a more complex situation (Tables S2-S4). In particular the signals belonging to the glycine residues show a certain heterogeneity, suggesting that in this case different structures are adopted in the different pentapeptide subunits the polypeptides chain (Figure 3d and 3e). By analyzing the temperature coefficients of the main amide signals, we observe the presence of low values (-4.5 ppb/K for E50 and -3.8 ppb/K for E50H) for V1n amide protons, suggesting the presence of β-turns. Further evidence of β-turn conformations arise from the great differences in the chemical shift of the enantiotopic protons of glycine G5n (∆δ= 0.35 ppm for E50 and ∆δ= 0.36 ppm for E50H) involved in the 10-membered C10 ring of a type II β-turn. The presence of NOE cross-peaks typical for type II β-turns are also observed, in particular daN(i, i+2) between Pro4n/Hyp4n and V1n , a strong daN(i, i+1) between Pro4n/Hyp4n and G5n, and intense dNN between G5n and V1n (Figure S1b and S2b)22. The lineshapes of the spectra recorded in TFE-d3 of E50 and E50H are consistent with a more complex structural ensemble, nevertheless aggregation induced by the organic solvent, TFE, should be considered. While coacervation could be ruled out, being the sample clear during the whole set of experiments, some small-sized aggregates, such as dimers or oligomers could not be excluded, and could be responsible for the broadening of the signals (Figure 3d and e).


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On the whole, the results of the conformational studies suggest that in aqueous solutions the contribution of PPII to the conformational ensemble is higher for the Hyp-containing polypeptides than for the Pro-containing polypeptides. Analogous findings were observed for collagen mimetic peptide9. The presence of the hydroxyl group in the 4R position of proline affects the pucker of its pyrrolidine ring, favoring the Cγ-exo conformation, whereas Pro has a slight preference for the Cγ-endo pucker9,39. The conformation of the pyrrolidine ring exerts an influence also on the local dihedral angles (φ, ψ, ω) of the proline residues defining the conformational thermodynamics of the polypeptide chain. However, the presence of beta–branched amino acids, such as valine and isoleucine in the peptide sequences with reduced PPII conformation propensities, restricted the presence of PPII conformation mainly to the PG sequences. This is the case for E50 and E50H polypeptides as well as for the alaninerich sequences present in E18 and E18H polypeptides40-42. In TFE, a solvent that stabilizes folded structures, such as α-helix and β-turns and destabilizes PPII conformation, we did not observe any difference in the propensity to fold into β-turn conformations between the Hyp-containing and the Procontaining polypeptides. Coacervation studies. In aqueous solution several elastin-related polypeptides undergo a reversible, temperature-dependent hydrophobic self-assembly, process that is analogous to the in-vivo coacervation of native tropoelastin occurring prior to cross-linking43. The coacervation process is experimentally measured by spectrophotometry as a sharp increase in the apparent absorbance value concomitant with heating to a critical temperature. Coacervation proceeds as a cooperative reaction that is highly dependent on solution conditions such as temperature, pH and salt and co-solvent concentration and on the concentration, molecular weight and sequence5,43,44. In order to investigate the effect of Hyp on the coacervation propensities of the polypeptides E50H and E18H with respect to the Pro-containing complements, E50 and E18, respectively, the peptides 15 ACS Paragon Plus Environment

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Figure 4. Turbidimetry as a function of temperature. Coacervation studies by turbidimetry of (a) E50 and E50H and (b) E18 and E18H polypeptides. were studied in different experimental conditions (Figure 4). The turbidimetry was monitored by recording the apparent absorbance (TAA) at 440 nm as a function of temperature. In Figure 4a the coacervation profile of E50 recorded at 1mM shows a coacervation temperature (Tc) of 45 °C. In the same conditions E50H does not coacervate. Previous studies have shown that TFE was able to favor coacervation45,46. Consequently, addition of 7.5% TFE to the 1mM E50 polypeptide solution shifted the transition temperature Tc to 26 °C. The addition of 7.5 % of TFE to the 1mM E50H peptide solution was not able to promote coacervation. Only increasing the concentration of E50H to 2mM and 3mM with the concomitant presence of 7.5% TFE induced the coacervation of the polypeptide with a Tc of 58 °C and 56 °C, respectively. Increasing the concentration of the polypeptide 16 ACS Paragon Plus Environment

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did not induce a significant effect on the temperature of transition, however it had a great effect on the apparent absorbance, showing an increase from 0.18 OD to 0.56 OD at higher concentrations. This in turn suggests the formation of aggregates of higher dimensions. Coacervation of E18 and E18H showed differences that are less pronounced because of the minor number of Hyp residues in the polypeptide sequence (Figure 4b). The observed Tc of E18 at 1mM was 62 °C. In the same experimental conditions E18H is not able to coacervate. After addition of 7.5% TFE to the coacervation solution we observed for E18 a great reduction of the Tc to 28 °C and a pronounced increase in the apparent absorbance. In the same conditions the E18H polypeptide coacervates with Tc at 37 °C. The coacervation transition of E18H shows a less cooperative behavior witnessed by the absence of a sharp transition. Furthermore aggregation is less effective as deduced from the reduced value of the apparent absorbance (Figure 4b). TFE is particularly effective in favoring the coacervation. The role of TFE in facilitating coacervation has been previously commented46 and may consist in a coating effect of the solvent on the polypeptide molecules. TFE is a protic solvent, able to form hydrogen bonds with the peptide backbone, while the hydrophobic CF3-CH2 favors hydrophobic interactions47. It has been shown by molecular dynamics simulations that in a mixture TFE/H2O, TFE molecules are able to interrupt the strong water-peptide bonds, and can substitute them with less strong peptide-TFE interactions48. Our results are in agreement with previous studies by Urry et al. on elastinlike polymers containing Hyp in different ratios (1%, 10%, 100%). The polymers obtained by (VXGVG) (X=Pro, Hyp) pentamer polymerization, have shown that the transition temperature of the polymers is strictly related to the Hyp ratio, with an increased Tc and a reduced apparent absorbance value on increasing the Hyp content16. Supramolecular characterization. The initial assumption that elastin is an amorphous protein arose from investigations with conventional electron microscopy. This picture was changed with the 17 ACS Paragon Plus Environment

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observation of filamentous structures of elastin in the pioneering work of Gotte49,50. Subsequent studies have shown that also other elastin-derived samples (α-elastin, tropoelastin51,52) and elastin-related polypeptides have a peculiar self-assembling behavior that is common to many peptides with elastinlike sequences regardless of their lengths. The ultrastructural organization of negatively stained preparations of elastin, α-elastin and elastin-like peptides observed by TEM exhibits highly organized filamentous structures (53

and reference therein

). The filaments commonly show 5-7 nm diameters and are

associated in highly oriented bundles of fibres of various dimension and length. In order to examine the influence of Hyp on the morphology of the aggregates formed by E50 and E50H polypeptides, an investigation by TEM was conducted. The samples were deposited on the grids immediately after preparation and after 2 days of incubation at 50°C. The images of E50 showed electron-dense aggregates from which rod-like structures of 15-18 nm width emerged (Figure 5a). Close examination of these structures revealed an inner fine-structure constituted by finer filaments of 3-4 nm. However, these filaments are not discernible in all the structures. After 2 days of incubation at 50°C the E50 polypeptide assembles into more defined filamentous structures forming bundles of fibres, that are similar to those observed for elastin and elastin-like structures (Figures 5b and 5c). Highly oriented filaments parallel to the main fibre axis are discernible. The average diameter of the filaments is in the range of 4-6 nm. The aggregation of E50H was monitored in the same conditions described for E50 polypeptide and showed globular aggregates observed immediately after preparation (Figure 5d), while after 2 days of incubation at 50°C a more complex aggregation morphology evolved. Rod-like structures were observed, showing an inner fine structure with some filaments (Figure 5e). The incubation was performed also at physiological temperature (37°C). In this case, after 2 days of incubation E50 showed a tendency to form film structures together with small globules that started to align, while after a longer period of 9 days a complex network was observed. In the same conditions 18 ACS Paragon Plus Environment

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Figure 5. Time-dependent self-assembly studied by TEM. Electron micrographs, negatively stained with uranyl acetate, scale bar are 50 nm (a,c,d) or 100 nm (e) or 200 nm (b). E50 (a) and E50H (d) immediately after preparation. E50 (b,c) and E50H ( e) after 2 days of incubation at 50°C. after 2 days of incubation, E50H revealed grape-like structures previously observed, that evolved after 9 days in a more densely-packed assemblies (Figure S4). While elastin samples were commonly observed by TEM, the possibility that the strong dehydration 19 ACS Paragon Plus Environment

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Figure 6. Time-dependent AFM images. E50 polypeptide immediately after preparation (a), and after 2 days of incubation at 50°C (b,e). E50H polypeptide immediately after preparation (c), and after 2days of incubation at 50°C (d). Height profiles of some fibrils (f) taken from Figure 6e as indicated.


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due to low pressure conditions of the analysis could introduce artifacts has to be considered. Therefore we analyzed the same samples also by AFM. AFM has the advantage of investigating the sample at ambient temperature and pressure conditions. Figures 6 shows representative AFM images of aliquots of E50 and E50H peptide solutions, taken immediately after preparation and after 2 days of incubation at 50 °C. E50 polypeptide solution deposited on silicon substrate immediately after preparation covers the substrate uniformly with sparse presence of developing fibrils (Figure 6a). After 2 days of incubation at 50 °C a canvas of aligned fibrils oriented in different directions is observable (Figure 6 b and 6e). The fibrils have uniform diameters in the range of 15-18 nm (Figure 6f), with no smaller aggregates associated with them. E50H polypeptide deposits show a different morphology. After preparation the polypeptides deposits show leopard-skin feature (Figure 6 c), previously observed for the seven residue-long Hyp-containing elastin peptides18. After 2 days of incubation more dense and polymorphic aggregates are visible (Figure 6 d). No evidence of fiber formation was observed. Enzymatic degradation studies. In order to investigate the susceptibilities of E18 and E18H, both substrates were incubated with pancreatic elastase. The serine protease was chosen since it is a very aggressive, low specific and effective digestive enzyme which hydrolyzes most proteins of the human body including the enzymatically rather resistant mature elastin54. Although the substrate peptides are only 49 residues in length and the enzyme-to-substrate ratio used was relatively high, the proteolysis did not proceed rapidly. Even small, non-hydrolyzed portions of both peptides were detectable after 6 h of incubation and it took about 24 h until no further changes in the peptide patterns were recognizable. Hence, it was possible to follow the progress of the hydrolysis. For this purpose, samples were taken at several time points and their peptides were separated by nanoHPLC and analyzed by tandem mass spectrometry on ESI and MALDI instruments. Based on the fragment spectra acquired, peptide sequences and thus 21 ACS Paragon Plus Environment

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cleavages sites were identified. Figure 7a shows the determined cleavage sites in both substrate peptides after 3 h and 31 h of incubation. Interestingly, although the hydrolysis was performed under identical conditions, many more cleavage sites were found for the non-hydroxylated polypeptide E18 as compared to the six times hydroxylated E18H. This can be further seen from the individual peptides that were released within the first 3 h of proteolysis and which are highlighted with solid lines below the respective precursors in Figure 7b.

Figure 7. Enzymatic degradation studies. (a) Cleavage sites identified after elastase digestion of E18 (marked with arrows above the sequences) and E18H (arrows below) for 3 h and 31 h, respectively. Pro residues marked with an asterisk denote Hyp residues in E18H. All identified peptides after 3 h of incubation are displayed by solid lines are shown underneath the respective sequences (b). The modifications oxidation of proline and amidation of the C-terminus are indicated by small letter o and a, respectively.


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Although after longer incubation additional cleavage sites could be verified for E18H, these results suggest that the non-modified peptide is more readily catabolized and that the hydroxylation of Pro in elastin could have an impact on its proteolytic susceptibility. The reason for this behavior most likely lies in a modified backbone orientation of the peptides induced by the electron-withdrawing nature of the additional OH-groups and this could lead to differences in the peptide’s accessibility to proteases which in turn affects the degradability.

Discussion The PTM converting the natural amino acid Pro into Hyp significantly influences the chemical structure and properties of the elastin peptides including proteolytic resistance. The hydroxyl group, compared to the H atom has a different dimension and electronegativity and is susceptible to hydrogenbonding. However, molecular spectroscopic studies of the polypeptides have shown that the presence of Hyp did not dramatically alter the conformations of the elastin peptides. The presence of multi-conformational equilibria among extended (PPII, β-strand) and folded conformations (β-turns) has been ascertained for all the analyzed peptides and could account for the high entropy of the relaxed state at the basis of the elasticity mechanism55,56. The highly flexible nature of the elastin polypeptide chain, observed previously for tropoelastin57, is fundamental for the entropically driven elasticity mechanism58. The introduction of Hyp in the elastin peptides did not alter the flexibility of the polypeptide chain, populating only locally some fluctuating ordered structures such as PPII and β-turns. Previously, elastin-like polypeptides containing proline analogue with an electronegative group on the pyrrolidine ring at 4 position, the (2S, 4R)- 4F-proline (Flp) were studied. These studies revealed a more evident temperature-dependent conformational transition from random coil to β-turn structures17. 23 ACS Paragon Plus Environment

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This observation was ascribed to stereo-electronic effects of the fluorine atom on the conformation of the pyrrolidine ring, affecting the secondary structure of the polypeptide. In particular their investigation suggests that the stereoelectronic effect may pre-organize the conformation of the Flp in elastin-mimetic peptides for the transition to a type II β-turn, thereby facilitating the self-assembly process. This does not seem to be the case for Hyp-containing elastin polypeptides where the more hydrophilic nature of the amino acid did not induce a stereo-electronic bias toward β-turn conformation as observed for Flp-containing elastin polypeptides. Probably, different solvent-peptide interactions could be responsible for the dissimilar behaviour. As a matter of facts covalently bound fluorine atoms, despite their high electronegativity, are very weak hydrogen bond acceptors, rendering low the possibility to form H-bonds with water molecules. Hyp, in turn has a high tendency to form H-bonds with water molecules59. From a supramolecular point of view, Hyp has a dramatic effect on the self-assembly of elastin peptides, by increasing the temperature of the phase transition, reducing the coacervation propensity. The main reason for this could be attributed to different hydration shells of the elastin polypeptides, thus supporting the observation that elastin self-assembly was triggered mainly by hydrophobic interactions, with hydrophobic hydration having a predominant role60. The hydrophobic hydration shell that surrounds the polypeptide chain is constituted by water molecules that tend to align themselves parallel to the nonpolar side chains and interact with each other rather than with the protein atoms shielding the hydrophobic side-chains from self-interactions43. On the other hand, waters surrounding polar side chains did not flatten out in this way; instead, they formed hydrogen bonds with the polar side-chain atoms, giving rise to


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hydrophilic hydration61. The presence of the hydroxyl-group of Hyp in the polypeptide chain, that can undergo hydrogen-bonding with water molecules, alters the equilibrium

Figure 8: Schematic representation of the hydration shell around Pro and Hyp residues. Water molecules interact exclusively among themselves forming the hydrophobic hydration shell surrounding Proline residue (blue dotted lines), while no interactions with the proline side-chain are observed(left). Water molecules interact closely to the hydroxyl group

of the Hyp sidechain, determining the

formation of the hydrophilic hydration shell around Hyp (red dotted lines, right). among the hydrophobic and hydrophilic hydration (Figure 8). As a consequence a higher energy is required to destabilize the hydration shield in order to direct the exposure of the hydrophobic residues and thus promote hydrophobic interactions. The reduced hydrophobicity of the Hypcontaining peptides altered the ultrastructural features of the formed aggregates. While Pro-containing peptides have the tendency to form fibrillar structures, the Hyp-containing peptides show a more polymorphic aggregation pattern with lower tendency to form fibres and fibrils. Also the spatial arrangements of the hydrophobic interactions necessary for higher order structures observed in the fibrils and fibres could be altered by Hyp thus supporting the formation of less regular polymorphic aggregates.


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Our results on elastin model peptides were the first to give significant clues on the biological role of elastin hydroxylation. The E50H peptide, and in a minor extent the E18H peptide, represent elastin models where the introduction of Hyp is significantly high. The high number of Hyp in both peptides rendered them examples of over-hydroxylation. Nevertheless, our data do infer that the presence of a “physiological” degree of hydroxylation could induce subtle modification in the fibre assembly by modulating the coacervation of tropoelastin. In vivo, the coacervation is triggered by an increase in the tropoelastin concentration due to monomer secretion at the cell surface. When the

critical

concentration is reached, globules are formed and added to the microfibrillar scaffold, through a mechanism mediated also by cell motions. In this regard, the most striking consequence of hydroxylation is probably the production of more dense globules. Considering different hydroxylation degrees in different organs, it is attempting to assume that Hyp content is related to tailored structural and mechanical properties. The observation of Sandberg et al. that hydroxylation is higher in lung parenchyma than in ligamentum nuchae could be significantly related to different functions and mechanical stresses of the organs62. As a matter of fact, over-hydroxylation was demonstrated to induce an incorrect elastin assembly63. Furthermore, our results suggest that the hydroxylation of Pro in elastin, having an impact on the proteolytic susceptibility, could delay the degradation pathway of elastin

Conclusions In this work we investigated the effect of proline hydroxylation in elastin model peptides. The experimental data provide evidence that the presence of Hyp in elastin-model peptides has a striking effect on the self-assembly, that is not correlated to variations in the local conformations. More


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probably a difference in the hydration shell surrounding the polypeptides could account for the observed differences in coacervaton and in supramolecular aggregation. Altogether, the data give a better insight into the role played by hydroxylation of Pro in elastin involved in elastic fibre formation and degradation in different tissues. Furthermore, our findings could contribute in defining the subtle role of proline structural variants in the folding and self-assembly of elastin-inspired peptides, helping the rational design of elastin biomaterials.

ASSOCIATED CONTENT Supporting Information. 1H-NMR chemical shift assignments of E50 and E50H polypeptides; NOE summaries, 1D 1H NMR spectra of E18 and E18H, TEM images of E50 and E50H incubated at 37°C. “This material is available free of charge via the Internet at http://pubs.acs.org AUTHOR INFORMATION Corresponding Author *[email protected]

Acknowledgements The financial support from MIUR (PRIN 2010-Project 2010L(SH3K)) is gratefully acknowledged. We thank Dr. S. Panariello for preliminar experiments, Dr. M. A. Crudele for technical assistance and Dr. N. Ibris for AFM images (CIGAS-University of Basilicata). The Deutsche Forschungsgemeinschaft (DFG) is thanked for financial support in the scope of project HE 6190/1-1 (AH).

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Table of Content:


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Figure 1. Chemical structures of proline and analogue. (2S)-proline with R1=H and (2S,4R)-4hydroxyproline with R1=OH. 34x35mm (300 x 300 DPI)

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Figure 2. Temperature–dependent CD spectra. CD spectra recorded in acqueous and TFE solutions of (a) E50, (b) E50H, (c) E18 and (d) E18H polypeptides.


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Figure 3. Structural formulas of the elastin peptides and 1H NMR spectra of the amide region. Chemical structure of the pentapeptide repeat units corresponding to E50 (R1=H) and E50H (R1=OH) polypeptides (a). The amide regions of the 1H NMR spectra of (b) E50 and (c) E50H recorded in H2O/D2O (90/10, v/v) at 25°C and of (d) E50 and (e) E50H polypeptides recorded in TFE-d3/H2O (80/20, v/v) at 25 °C. 102x66mm (300 x 300 DPI)


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Figure 4. Turbidimetry as a function of temperature. Coacervation studies by turbidimetry of (a) E50 and E50H and (b) E18 and E18H polypeptides.


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Figure 5. Time-dependent self-assembly studied by TEM. Electron micrographs, negatively stained with uranyl acetate, scale bar are 50 nm (a,c,d) or 100 nm (e) or 200 nm (b). E50 (a) and E50H (d) immediately after preparation. E50 (b,c) and E50H ( e) after 2 days of incubation at 50°C.


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Figure 6. Time-dependent AFM images. E50 polypeptide immediately after preparation (a), and after 2 days of incubation at 50°C (b,e). E50H polypeptide immediately after preparation (c), and after 2days of incubation at 50°C (d). Height profiles of some fibrils (f) taken from Figure 6e as indicated.


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Biomacromolecules

Figure 7. Enzymatic degradation studies. (a) Cleavage sites identified after elastase digestion of E18 (marked with arrows above the sequences) and E18H (arrows below) for 3 h and 31 h, respectively. Pro residues marked with an asterisk denote Hyp residues in E18H. All identified peptides after 3 h of incubation are displayed by solid lines are shown underneath the respective sequences (b). The modifications oxidation of proline and amidation of the C-terminus are indicated by small letter o and a, respectively. 127x86mm (300 x 300 DPI)


Biomacromolecules

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Figure 8: Schematic representation of the hydration shell around Pro and Hyp residues. Water molecules interact exclusively among themselves forming the hydrophobic hydration shell surrounding Proline residue (blue dotted lines), while no interactions with the proline side-chain are observed(left). Water molecules interact closely to the hydroxyl group of the Hyp sidechain, determining the formation of the hydrophilic hydration shell around Hyp (red dotted lines, right).


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Investigating the Role of (2S,4R)-4 ... - American Chemical Society

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