Secondary Structure Formation and LCST Behavior of Short Elastin

Biomacromolecules 2008, 9, 2755–2763

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Secondary Structure Formation and LCST Behavior of Short Elastin-Like Peptides Harald Nuhn and Harm-Anton Klok* Ecole Polytechnique Fe´de´rale de Lausanne (EPFL), Institut des Mate´riaux, Laboratoire des Polyme`res, Baˆtiment MXD, Station 12, CH-1015 Lausanne, Switzerland Received May 13, 2008

This contribution investigates the effects of chain length and chemical composition on the secondary structure and LCST behavior of a library of short, elastin-like peptides based on the GVGVP motif. CD experiments revealed that most of the investigated peptides showed the typical elastin conformational behavior with a decrease in random coil and an increase in β-turn character with increasing temperature. For several peptides, LCST behavior was observed in aqueous NaCl solutions containing 10 mg/mL peptide. By extrapolation of the LCSTs measured at different NaCl concentrations to zero-salt concentration, apparent LCSTs were determined. The apparent LCST was found to decrease with increasing peptide chain length, which correlated well with the trend in the predicted partition coefficients. The apparent LCST of the peptides could be manipulated by successive replacement of the valine residues by more hydrophobic isoleucine, leucine, or phenylalanine residues. Within a particular series of variants, the apparent LCST was found to decrease with an increasing number of valine replacements, which also correlated well with the predicted evolution of the partition coefficient. Although the relative importance of the overall peptide hydrophobicity and the conformational preferences of the constituent amino acids on the LCST behavior still remains an open question, the results described in this contribution clearly demonstrate that short, elastin-like peptides are potentially attractive building blocks for a range of materials applications in biomedicine and engineering.

Introduction Elastin-like polypeptides are artificial biopolymers composed of the pentapeptide repeat Xaa-Pro-Gly-Yaa-Gly, which is derived from the hydrophobic domain of tropoelastin.1,2 A characteristic feature of elastin-like polypeptides is their lower critical solution temperature (LCST) behavior.3 In the elastin literature, this is also referred to as inverse transition behavior and the LCST is referred to as inverse temperature transition (Tt).3 Above the LCST, elastin-like polypeptides coacervate and ultimately precipitate. This process is reversible and can take place in a temperature window that can be as narrow as 3 °C.3 Upon heating above the LCST, the secondary structure of the polypeptide changes from an extended to a collapsed state.3 The LCST of elastin-like polypeptides depends on the pH and the ion strength of the medium in which they are dissolved. Furthermore, for a given set of solvent conditions, the LCST of an elastin-like polypeptide can be tailored over a broad temperature range by adjusting the chain length and by varying the nature of the amino acid at positions Xaa or Yaa in the pentad repeat.3 Due to their LCST behavior, elastin-like polypeptides have attracted interest as smart biomaterials, for example, for drug delivery and release4-6 as molecular switches,7,8 to fabricate temperature responsive protein pores,9 and as purification tools for recombinant protein expression.10 In contrast to the wealth of literature that is available on elastin-like polypeptides, only relatively few studies on short, elastin-like peptides composed of 1-6 pentapeptide repeats have been published.11-19 In most of these studies, these short peptides were used as models to gain more detailed insight into the conformational behavior of the corresponding high molecular * To whom correspondence should be addressed. Fax: +41 (0)21 693 56 50. E-mail: [email protected].

weight analogues. Most of the reports on short, elastin-like peptides published so far have focused exclusively on the conformational properties of these peptides and did not investigate their possible LCST behavior. In one example, Urry et al. reported on the LCST behavior of a cyclododecapeptide analogue of elastin.11 In a more recent report, Pechar et al. described the thermoresponsive behavior of two short, elastinlike peptides, (VPGVG)4 and (VPAGV)4, and the corresponding poly(ethylene glycol) (PEG) conjugates.13 Using light scattering to determine the onset of turbidity, these authors observed LCST behavior for the Fmoc end-functionalized (VPGVG)4 peptide as well as for the Fmoc end-functionalized (VPGVG)4-PEG and (VPAGV)4-PEG conjugates. Van Hest et al. have demonstrated that triblock copolymers composed of a central PEG block flanked by two methacrylate segments containing a single VPGVG pentapeptide side chain show LCST behavior.20 In a related, more recent, report it was shown that methacrylate functionalized VPGVG can be homopolymerized under reversible addition fragmentation chain transfer (RAFT) conditions to afford elastin-based, side-chain homopolymers that display LCST behavior.21 Systematic studies, which investigate the effects of chain length and composition on the LCST behavior of short, elastin-like peptides, however, are not available to the best of our knowledge. In this report, we describe the results of a comprehensive study on the effects of chain length and amino acid composition on the conformational properties and LCST behavior of a series of short, elastin-like peptides. Short, elastin-like peptides are potentially attractive building blocks for the development of smart biomaterials or biological microelectromechanical systems (bioMEMS) as they are easily prepared via (solid phase) peptide synthesis, which also allows to introduce nonnatural functional groups that cannot be incorporated using protein expression.

10.1021/bm800784y CCC: $40.75  2008 American Chemical Society Published on Web 08/29/2008

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The results described in this paper provide a first insight into the feasibility of short, elastin-like peptides as building blocks for smart biomaterials and demonstrate how the LCST behavior of these peptides can be manipulated by adjusting the length and composition of the peptides.

Experimental Section Materials. Preloaded Fmoc-L-proline-trityl resin (loading: 0.5-0.6 mmol/g) was purchased from PepChem (Reutlingen, Germany). Fmocglycine-Wang resin (loading: 0.6 mmol/g) and amino acids were obtained from NovaBiochem (Switzerland) or Advanced Chemtech (U.K.). N-Methylpyrrolidone (NMP) was obtained from BASF (Ludwigshafen, Germany) and Schweizer Hall (Basel, Switzerland). 2-(6Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) and 1-hydroxybenzotriazole (HOBt) were purchased from NovaBiochem (Switzerland) and Iris Biotech (Marktredwitz, Germany). 4-Methylmorpholine (NMM), 1,8-diazabicyclo[5.4.0]undec7-ene (DBU), piperidine, dichloromethane (DCM), trifluoroacetic acid (TFA), trimethylsilyl bromide (TMSBr), diethylether, methanol (HPLC grade), calcium hydride, hydrochloric acid, sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride (NaCl) were obtained from Fluka (Buchs, Switzerland). Acetonitrile (ACN) was purchased from Reactolab (Lausanne, Switzerland) and VWR (Dietikon, Switzerland). Methods. Electron-Spray Quadrupole Mass Spectrometry. ESI-MS was carried out using a Finnigan SSQ 710C instrument. Samples were prepared by dissolving 0.5 mg of the crude peptide in 500 µL of methanol (MeOH) containing 0.1 vol % TFA. ReVerse Phase High Performance Liquid Chromatography. Preparative RP-HPLC was performed on a Waters Delta 600 System equipped with a dual-wavelength UV-vis detector (Waters 2487; set to 214 and 220 nm) and autosampler (Waters WTC-III). A flow rate of 20 mL/ min was selected and a retention time of 9 s was set to bridge the internal system delay between detector and fraction collector. Waters (Atlantis dC18 ODB 5 µm, 30 × 150 column) or Grace Vydac HPLC columns (238TP15202215, C18 monomeric) were used. A linear gradient from 5 to 100% ACN with a slope of 4.75% ACN/min (v/v) was performed. The 214 nm signal was used to trigger the fraction collector, and the threshold was set between 150000 and 600000 AU. The collected fractions were lyophilized and analyzed by ESI-MS. Finally, all fractions containing peptides of the correct molecular weight were pooled. Residual TFA was removed by dissolving the peptide in 0.03 mM hydrochloric acid (HCl), followed by lyophilization.22 Circular Dichroism Spectroscopy. Circular dichroism (CD) spectra were recorded on a Jasco J-715 spectrophotometer under a nitrogen atmosphere with a gas flow of 5 L/min. The temperature-controlled measuring cell (Jasco PTC-348WI) was counter-cooled by a Julabo cryostat (Tset ) 20 °C). CD spectra were recorded using the temperature/ wavelength scan module offered by Jasco. Scans were performed in the range between 5 and 95 °C with increments of 10 °C. Before each scan, the system was equilibrated for 5 min. Spectra were recorded from 300 to 190 nm at a scan speed of 50 nm/min, a step size of 0.2 nm, a bandwidth of 2 nm, and a response time of 1 s. Usually 10 scans were averaged. Far UV/vis quartz cells (Starna, type 21/Q/1) were used, with a path length of 0.1 cm. Unless indicated otherwise, all peptides were dissolved in degassed (sonicated for 15 min) phosphate buffer (10 mM; pH 7) and measured at a concentration of 100 µM. Data were processed using an automated software, which allowed for simultaneous processing of the recorded spectra. Each spectrum was corrected by subtraction of a corresponding background spectrum recorded at the same temperature. The resulting spectra were smoothed by a moving window filter (width ) 10) and, afterward, converted into mean molar residue ellipticity (MMRE; [θ], in mdeg · cm2 · dmole-1) as described in the CD-spectrophotometer manual.23 Turbidimetry. LCSTs were determined by measuring the absorbance of peptide solutions at 300 nm as a function of temperature on a Cary 100 UV-visible spectrophotometer equipped with a Peltier 1 × 1 cell

Nuhn and Klok holder temperature controller (Varian Instruments, Walnut Creek, CA). Aqueous solutions were degassed by sonication (15 min) prior to sample preparation. Unless indicated otherwise, peptide concentrations were 10 mg/mL. Samples were dissolved in MilliQ water. Experiments were carried out in far UV/vis quartz cells (Starna, type 21/Q/1) with a path length of 0.1 cm, sealed by a Teflon-stopper. Data were collected in 1.5 °C increments over a temperature range from 10 to 96 °C. The obtained turbidity profiles were normalized and the LCST was defined as half-maximal turbidity.24 The LCSTs could be determined with an accuracy of (1 °C. If the LCST was above the maximal measurable temperature, it was decreased by the addition of sodium chloride (NaCl). The salt concentration in the sample solution was adjusted by adding the appropriate volume of a 5 M stock solution. In this case, at least three turbidity profiles at different, but equidistant, NaCl concentrations were recorded. The reported LCST values are apparent LCSTs that were obtained by extrapolation to zero-salt concentration. In a plot of the experimentally determined cloud point versus the NaCl concentration, the point of intersection affords the apparent LCST of the peptide, while the slope of the linear fit through the data points reflects the change in the LCST per mole NaCl (δNaCl). Procedures. Solid Phase Peptide Synthesis. Solid phase peptide synthesis (SPPS) was performed on a PSW1100 automated peptide synthesizer (Chemspeed, Switzerland) using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Fmoc-glycine-Wang-resin and Fmoc-L-prolinetrityl-resin were used as the solid supports. Typically, peptide synthesis was carried out on a 0.11 mmol scale. Fmoc-protected amino acids were used as free base (2.5 equiv relative to resin loading) and dissolved in NMP (c ) 0.5 M), which was freshly distilled from calcium hydride. Peptide coupling was mediated by 2.5 equiv HCTU and 1.25 equiv HOBt relative to resin loading. HCTU/HOBt were dissolved together in NMP (c ) 0.6 M) and the vial was constantly purged with dry nitrogen and protected against light. NMM was used as base (5 equiv relative to resin loading). Each coupling step was performed for 30 min. Fmoc groups were cleaved by treating the resin-bound peptides with a mixture of DBU and piperidine in NMP (3:2:95 vol %) for 5 min. After each coupling and deprotection step, excessive reagents were removed by three washing steps with NMP for 5 min. After the last coupling and deprotection step, excessive reagents were removed by a single NMP wash (5 min), followed by six washing steps using DCM for 5 min. Peptides were cleaved from the resin by adding 3 mL of an ice-cold mixture of TFA and TMSBr (95:5 vol %). The mixture was stirred at room temperature for 15 min, filtered, and poured into icecold diethylether. The resulting precipitate was centrifuged for 5 min at 2 °C at 8500 rpm. Afterward the supernatant was decanted and the pellet was kept for further usage. The cleavage was repeated by adding 100% TFA to the resin (5 min). The filtrate of this step was pooled with the first pellet, and ice-cold diethylether was added. The tube was sealed and vortexed to resuspend the pellet. Afterward the tube was centrifuged at 2 °C for 5 min at 8500 rpm. The TFA cleavage step was repeated two times. Finally the pellet was dissolved in MilliQ water and lyophilized. The crude peptides were purified using reverse phase HPLC, as described above in Methods. Calculation of the Partition Coefficient (Log P). Log P values were calculated using the residue addition model.25 Assuming that the partition coefficient of a peptide can be considered the sum of the contributions of the individual amino acids, this model allows to calculate log P using the following equation

Log P )

∑

n

anRnp+bBp+uUp

(1)

where an describes the occurrence of the nth kind of amino acid and Rnp describes the log P contribution of each nth kind of amino acid. Rnp values for the different amino acid residues were taken from ref 25. For a blocked peptide, b ) 1 and u ) 0. For an unblocked peptide, b ) 0 and u ) 1. Bp and Up are the corrections for log P values of blocked and unblocked peptides, respectively (Bp ) -1.19; Up ) -3.25).25

Short Elastin-Like Peptides


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Table 1. Investigated Short, Elastin-Like Peptidesa name

sequence

M [Da]

log P [-]

A1 A1ext A2 A2ext A3 A4 A5 A3(I2) A3(I13) A3(I123) A3(I123)(I2) A3(I123)(I13) A3(I123)(I123) A3(L2) A3(L13) A3(L123) A3(L123)(L2) A3(L123)(L13) A3(L123)(L123) A3(F2) A3(F13) A3(F123) A3(F123)(F2) A3(F123)(F13) A3(F123)(F123)

GVGVP GVGVPGVG GVGVPGVGVP GVGVPGVGVPGVG GVGVPGVGVPGVGVP GVGVPGVGVPGVGVPGVGVP GVGVPGVGVPGVGVPGVGVPGVGVP GVGVPGIGVPGVGVP GIGVPGVGVPGIGVP GIGVPGIGVPGIGVP GIGVPGIGIPGIGVP GIGIPGIGVPGIGIP GIGIPGIGIPGIGIP GVGVPGLGVPGVGVP GLGVPGVGVPGLGVP GLGVPGLGVPGLGVP GLGVPGLGLPGLGVP GLGLPGLGVPGLGLP GLGLPGLGLPGLGLP GVGVPGFGVPGVGVP GFGVPGVGVPGFGVP GFGVPGFGVPGFGVP GFGVPGFGFPGFGVP GFGFPGFGVPGFGFP GFGFPGFGFPGFGFP

427.51 640.75 837.00 1050.24 1246.49 1655.98 2065.47 1260.52 1274.54 1288.57 1302.60 1316.62 1330.65 1260.52 1274.54 1288.57 1302.60 1316.62 1330.65 1294.53 1342.58 1390.62 1438.66 1486.71 1534.75

-2.90 -3.02 -2.55 -2.67 -2.20 -1.85 -1.50 -1.82 -1.44 -1.06 -0.68 -0.30 0.08 -1.72 -1.24 -0.76 -0.28 0.20 0.68 -1.36 -0.52 0.32 1.16 2.00 2.84

LCST [°C]

δLCST [°C/MNaCl]

not observed not observed not observed not observed not observed b 151 ( 9.8 /201 ( 18c -36 ( 3.5b/-51 ( 6.7c 124 ( 6 -31 ( 2.5 176 ( 13 -38 ( 3.7 186 ( 23 -48 ( 7.1 136 ( 13 -34 ( 4.7 186 ( 18 -64 ( 8.2 118 ( 9.8 -48 ( 7.1 122 ( 3.6 -68 ( 3.6 279 ( 34 -72 ( 11 211 ( 14 -64 ( 5.5 176 ( 18 -60 ( 9.0 136 ( 8.3 -54 ( 4.7 121 ( 6.3 -44 ( 3.5 80 ( 5.2/42 ( 1d -40 ( 4.1 187 ( 22 -48 ( 7.1 122 ( 17 -55 ( 12 88 ( 9.6 -471 ( 136 not observed not observed not observed

a M, molecular weight; LogP, partition coefficient; LCST, apparent lower critical solution temperature; δLCST, change of the LCST per added mole NaCl; G, glycine; I, isoleucine; L, leucine; F, phenylalanine; P, proline. b Measured at c ) 10 mg/mL ≡ 6.04 mM. c Measured at c ) 8 mg/mL ≡ 4.84 mM. d “Cloud point” measured at c ) 100 mg/mL in deionized water.

Results and Discussion Peptide Design. The peptides investigated in this study are based on the elastin pentad GVGVP, a sequence which also forms the basis for numerous elastin-like polypeptides.3 The primary structures and molecular weights of the investigated peptides are summarized in Table 1. To investigate the influence of chain length on secondary structure formation and LCST behavior, peptides composed of one to five GVGVP pentads were synthesized (samples A1-A5). In addition, two peptides with an additional C-terminal GVG sequence were prepared to study the influence of partial pentads (A1ext and A2ext). To study the influence of amino acid composition, the valine residues in peptide A3 were successively substituted by the more hydrophobic amino acids isoleucine (I), leucine (L), and phenylalanine (F). Variants of the A3 peptide in which the valine residues are successively replaced by isoleucine, leucine, and phenylalanine are referred to as A3I, A3L, and A3F, respectively. For each variant, six peptides were prepared with increasing levels of substitution. Within the sequence (GXGYP)3 the substitution started at the X-position of the central pentad, followed by substitution of the valines of the first and third pentad and finally at all three X-positions. Afterward, all valines at position Y were successively substituted as described for the X-positions. The rationale for the design of these variants is based on earlier work on poly([(VPGVG)n(VPGXG)m] elastinlike polypeptides, which revealed a direct relationship between the hydrophobicity of the amino acid at position X and the LCST of the polypeptide.3,26,27 Peptide Synthesis. Peptides were prepared by Fmoc solid phase peptide synthesis (SPPS) on preloaded Fmoc-glycineWang and Fmoc-L-proline-trityl resins. The former was used for the synthesis of the peptides A1ext and A2ext. The latter resin was used for all other peptides, because it suppresses diketopiperazine formation, which can occur with C-terminally positioned prolines and lead to premature cleavage of the peptide from the resin.28 Furthermore, deprotection was performed using a mixture of piperidine and DBU. DBU decreases the depro-

tection time while piperidine acts as a scavenger for the cleaved dibenzofulvene.28 Generally, peptides were obtained in 50-80% yield and purities of 95% after purification. Partition Coefficients. Investigations on elastin-like polypeptides have revealed a direct relationship between their hydrophobicity and the LCST.29 A common parameter to describe the lipophilicity/hydrophobicity and hydrophilicity of substances is the logarithm of the partition coefficient (log P).30 The partition coefficient is defined as the molar equilibrium concentration ratio of a single species between two phases, usually 1-octanol and water.31 Log P can be determined experimentally or predicted using different methods.30-33 In this report, the residue addition model25 was used to predict log P and estimate the effects of chain length and composition on the hydrophobicity of the peptides. The residue addition model is a relatively simple model that allows to calculate peptide partition coefficients by summing the contributions of the individual amino acids. This model has been shown to produce log P values that correlate well with the experimentally determined values for a large set of peptides.25 The predicted log P values for the peptides studied in this contribution are listed in Table 1 and represented in Figure 1. The data in Figure 1A indicate an increase in hydrophobicity with increasing number of pentads. The log P values for A1ext and A2ext indicate that the addition of a C-terminal GVG sequence results in a decrease in hydrophobicity. For these two peptides, the contribution of the two hydrophilic glycine residues to log P cannot be compensated by the more hydrophobic valine. Figure 1B plots the predicted log P values for the A3I, A3L, and A3F peptides. Within each group, each substitution of valine by isoleucine, leucine, or phenylalanine shifts log P to more positive values, that is, to higher hydrophobicity. The different slopes of the imaginary lines that connect the data points for each of these peptide series reflect the different hydrophobicity of each amino acid (isoleucine < leucine < phenylalanine).

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Figure 1. (A) Predicted log P values for (GVGVP)n peptides with n ) 1-5. (B) Predicted log P values for the A3I, A3L, and A3F peptides. The degree of substitution reflects the number of valine residues that have been replaced by isoleucine, leucine, or phenylalanine residues.

Figure 2. Temperature/wavelength CD spectra of the peptides A1-A5: (A) A1; (B) A1ext; (C) A2; (D) A3; (E) A4; (F) A5 (c ) 100 µM).

Influence of Peptide Chain Length on Secondary Structure Formation and LCST Behavior. CD spectra of the peptides A1-A5 are shown in Figure 2. To facilitate a comparison of the temperature response of the peptides, all spectra in Figure 2 were recorded at a sample concentration of 100 µM. Additional temperature-wavelength CD spectra of peptides A1-A5 can be found in the Supporting Information. With the exception of peptide A1, all samples show the typical

elastin CD spectra34 and also resemble those reported from other short, elastin-like peptides.14 The spectra are characterized by a high proportion of random coil and a low proportion type II β-turns at low temperatures. This distribution is gradually inverted with increasing temperature. These structural features are reflected in the measured CD spectra by a distinct minimum at 197 nm (ππ*-transition) and a less-pronounced minimum at 218 nm (nπ*-transition) at low temperatures, which are the



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Figure 3. Temperature-dependence of the mean molar residue ellipticity at (A) 196 nm and (B) 218 nm for peptides A1-A5. The legend in (A) also applies to (B).

Figure 4. (A) Turbidimetry profiles of peptide A5 (c ) 10 mg/mL) recorded in 3.0, 2.5, and 2.0 M aqueous NaCl solutions; (B) determination of the apparent LCST of A5 by extrapolation to zero-salt concentration (LCST ) 124 °C, δLCST ) -31 °C).

Figure 5. Apparent LCST (9) and δLCST (0) for the peptides A4 and A5 (c ) 10 mg/mL).

characteristics for random coil and β-turn structures, respectively.34 With increasing temperature, the random coil signal becomes less pronounced, while the β-turn increases, indicating a transition from a less-ordered state toward a more ordered secondary structure. The CD results for peptide A1 are anomalous in that they already reveal a high proportion of β-structures at low temperatures. This suggests that this peptide, in contrast to the other homologues, already adopts an ordered conformation at low temperatures. The spectra recorded for A1 at low temperatures are similar to those obtained for high molecular weight elastinlike polypeptides in trifluoroethanol (TFE).34 With increasing temperature, the random coil signal at 197 nm decreases, while the β-signal becomes more pronounced, indicating that A1, just as all other peptides in this series, adopts a more ordered structure at higher temperatures. In contrast to A1, the CD spectra obtained for A1ext resemble those typically obtained for elastin-like polypeptides. Apparently, the addition of the C-terminal GVG triad with two additional glycines allows the peptide to adopt unordered conformations at low temperatures,

while the valine and proline residues promote the formation of β-turns at high temperatures. Figure 3 plots the mean molar residue ellipticity at 196 and 218 nm versus temperature for peptides A1-A5. Figure 3A illustrates the decrease in random coil content with increasing temperature that is observed for all peptides. With increasing number of GVGVP pentads, the data points are shifted toward more negative ellipticity, indicating that the peptides become more unordered at low temperatures with increasing chain length. This somewhat surprising result is in agreement with earlier observations by Rees et al., who also found that shorter elastin-like peptides are less-disordered at low temperatures than the longer peptides.14 The data points for the three longest peptides (A3, A4, A5) can be fitted by two straight lines, which intersect at approximately 45 °C. Interestingly, plots of [θ]196 versus temperature for the other groups of peptides also revealed the presence of two linear regimes that intersect at approximately 45 °C (vide infra). At the moment, we can only speculate about the origin of this transition point and more experiments will be necessary to explain this observation. When studying the effect of trifluoroethanol (TFE) on [θ]198 of another short, elastinlike peptide, Rees et al., however, also observed two linear regimes that intersected at ∼30% (v/v) TFE.14 Below 30% (v/ v) TFE, hydrophobic interactions within the peptides were thought to dominate, whereas above 30% (v/v) TFE electrostatic interactions were proposed to become more dominant. Figure 3B shows that the exception of peptides A4 and A5, the β-turn character increases with increasing temperature. LCSTs were determined by recording turbidimetry profiles of 10 mg/mL peptide solutions over a temperature range between 10 and 96 °C at 300 nm. Initial experiments, carried out in deionized water as the solvent, did not reveal any coacervation within the investigated temperature range at this sample concentration. In subsequent experiments, aqueous NaCl solu-

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Figure 6. Temperature-dependence of the mean molar residue ellipticity at (A) 196 and (B) 218 nm for the A3I peptides (c ) 150 µM).

Figure 7. Apparent LCST (9) and δLCST (0) of the A3I peptides (c ) 10 mg/mL).

tions were used as the solvent to decrease the LCST.35 For each peptide, turbidimetry profiles were recorded at different NaCl concentrations. The reported, apparent LCSTs refer to zerosalt concentration and were obtained by extrapolation of the cloud points determined at different salt concentrations. As an example, Figure 4A shows turbidimetry profiles of peptide A5 recorded in aqueous solutions of three different NaCl concentrations. Figure 4B plots the experimentally determined cloud points versus salt concentration and illustrates how the apparent LCST is obtained via extrapolation. The slope of the linear fit through the data points (δLCST) is a measure for the change in apparent LCST per mole NaCl added to the peptide solution. The results of the turbidimetry experiments for peptides A4 and A5 are summarized in Figure 5 and Table 1. Unfortunately, no turbidimetry profiles could be obtained for the peptides A1-A3, even at high NaCl concentrations (c ) 6 M). It should be pointed out here that the turbidimetry experiments were carried out at peptide concentrations that were much higher than those that were used for the CD experiments and that it was not possible to perform CD experiments using the relatively highly concentrated peptide solutions that were used for the turbidimetry studies. The data in Figure 5 and Table 1 show that the apparent LCST of 10 mg/mL peptide solutions decreases from 151 to 124 °C upon increasing the length of the peptide with one additional GVGVP pentad from A4 to A5. Similar chain length dependencies of the LCST have been observed previously for high molecular weight elastin-like polypeptides,36,37 and the results presented here provide a first indication that this structureproperty relationship also applies to short elastin-like peptides. The log P values in Table 1 indicate that the decrease in apparent LCST from A4 to A5 correlates with an increase in the hydrophobicity of the peptide. For peptide A4, Table 1 also lists a second apparent LCST, which was determined at the same molar concentration as A5 (10 mg/mL A5 ≡ 4.84 mM). Decreasing the concentration of A4 from 6.04 to 4.84 mM

results in an increase in the apparent LCST of 50 °C and a decrease in δLCST of -15 °C, respectively. A similar concentration dependence of the LCST has been observed previously for high molecular weight elastin-like polypeptides and indicates the importance of considering sample concentration when comparing the results of different studies.36,37 At a sample concentration of 10 mg/mL, the δLCST decreases from -36 °C/mol NaCl for A4 to -31 °C/mol NaCl for A5. These numbers are significantly larger than the -14 °C/mol NaCl, which has been reported for high molecular weight elastin-like polypeptides.35 This demonstrates that the apparent LCSTs of short, elastin-like peptides are more sensitive toward the addition of NaCl compared to those of their high molecular weight polypeptide analogues. Influence of Peptide Composition on Secondary Structure Formation and LCST Behavior. A3I Peptides. The CD spectra of all peptides of the A3I series indicate typical elastinlike polypeptide behavior and illustrate a change from predominantly random coil to β-turn structures with increasing temperature (Supporting Information). The changes in the mean molar residue ellipticity at 196 and 218 nm with temperature, which can be deduced from the CD spectra, are shown in Figure 6. Figure 6A indicates a nonlinear decrease in the random coil content of the A3I peptides with increasing temperature. As for peptides A3-A5, the data points for the A3I peptides can be fitted with two straight lines, which intersect at approximately 45 °C. The data in Figure 6 suggest that the A3I series can be subdivided in two groups: one group comprises A3(I2), A3(I13), and A3(I123) and the other group comprises A3(I123)(I2), A3(I123)(I13), and A3(I123)(I123). Figure 6A shows that the random coil character of the peptides decreases with increasing the number of isoleucine substitutions. In contrast to the random coil content, the changes in the relative amount of β-turn structures are small. At any given temperature, the β-turn content of A3(I123)(I2), A3(I123)(I13), and A3(I123)(I123) is slightly lower than that of the lower substituted A3I variants. There is a small increase in β-turn content with increasing temperature for all peptides. The subdivision of the A3I peptides in two groups, as suggested by the data in Figure 6, may be related to a difference in the substitution pattern of A3(I2), A3(I13), and A3(I123) on the one hand and A3(I123)(I2), A3(I123)(I13), and A3(I123)(I123) on the other hand. The three higher substituted homologues contain at least one pentad repeat in which two isoleucine residues are positioned in close proximity. This may affect the hydrophobic character as well as the backbone flexibility of these peptides compared to the A3I homologues that contain at most one isoleucine residue per pentad. Figure 7 plots the apparent LCST values of the A3I peptides as a function of the number of isoleucine substituents. Also, in



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Figure 8. Temperature-dependence of the mean molar residue ellipticity at (A) 196 and (B) 218 nm for the A3L peptides (c ) 150).

Figure 9. Apparent LCST (9) and δLCST (0) of the A3L peptides (c ) 10 mg/mL).

this case, no coacervation was observed in 10 mg/mL aqueous solutions and the addition of NaCl was necessary to determine the cloud points. The apparent LCST values plotted in Figure 7 and summarized in Table 1 were obtained by extrapolation of the experimentally determined cloud points to zero NaCl concentration. In contrast to the other peptides discussed in this contribution, the LCSTs and δLCSTs of the A3I peptides do not change continuously with increasing degree of substitution but show a zigzag pattern. The origin of this zigzag pattern of the apparent LCST and δLCST is not clear at the moment and will require further experimental work. Overall, however, the data in Figure 7 indicate a decrease in the apparent LCST with increasing number of isoleucine substitutions, which correlates with the increase in the predicted log P values of the peptides. Furthermore, the evolution of δLCST with an increasing number of isoleucine substitutions indicates that the sensitivity of the peptide’s LCST toward changes in the NaCl concentration increases with increasing degree of substitution. A3L Peptides. CD spectroscopic investigations of the peptides of the A3L family revealed the typical elastin-like CD spectra (see Supporting Information). Figure 8 shows the evolution of the mean molar residue ellipticities at 196 and 218 nm of each of the peptides as a function of temperature. The data in Figure 8 indicate that with increasing number of leucine substitutions the random coil character increases, while the β-turn character decreases. The observed decrease in β-turn content may be attributed to the fact that leucine in contrast to valine and isoleucine does not favor β-sheet formation, but rather promotes the formation of R-helical secondary structures.38 As was observed for some of the other peptides earlier, the evolution of [Θ]196 with temperature for the A3L peptides can also be fitted by two straight lines that intersect at ∼45 °C. Similar to the A3I peptides, the A3L peptides can be subdivided in two groups. This becomes most apparent by considering the [Θ]218 data in Figure 8 at high temperature. The first group consists

of peptides A3(L2), A3(L13), and A3(L123). The second group comprises A3(L123)(L2), A3(L123)(L13), and A3(L123)(L123). The apparent LCST and δLCST values determined for the A3L peptides at a sample concentration of 10 mg/mL are shown in Figure 9 and summarized in Table 1. With an increasing number of leucine substitutions, the apparent LCST monotonously decreases from 279 to 80 °C. This decrease is in agreement with the increase in the predicted log P values of the peptides (Table 1). In contrast to the A3I and A3F peptides, the δLCST of the A3L peptides does not increase with an increasing level of substitution, but continuously decreases from -72 °C/MNaCl to -40 °C/MNaCl upon increasing the number of leucine substituents. This indicates that the LCSTs of the variants containing a high number of leucine substituents are less sensitive toward the addition of NaCl, as compared to analogues that contain a small number of leucine substituents. The apparent LCST of 80 °C for peptide A3(L123)(L123) is the lowest value for all of the peptides discussed in this paper so far. Turbidimetry experiments with 10 mg/mL solutions of this peptide in deionized water, however, did not reveal any coacervation. LCST behavior of this peptide in salt-free aqueous solution could be observed by increasing the peptide concentration. For A3(L123)(L123) an LCST of 42 °C was determined at a sample concentration of 100 mg/mL in deionized water (Supporting Information). CD experiments with this peptide in deionized water revealed a secondary structural behavior that was identical to that observed in phosphate buffer (Supporting Information). The discrepancy between the experimentally determined and apparent LCST of peptide A3(L123)(L123) may be attributed to the fact that the apparent LCSTs were obtained by extrapolation of cloud points determined at relatively high NaCl concentrations. Unfortunately, extrapolation to zero-salt concentration from cloud points determined at smaller NaCl concentrations was not feasible because relatively high NaCl concentrations were required to be able to measure cloud points within the temperature range that could be accessed during the turbidimetry experiments. A3F Peptides. CD analysis of the phenylalanine variants (A3F) is complicated by the UV-absorbance of the aromatic side chain of this amino acid, which shows three absorption maxima at 260, 210, and 180 nm.39 These UV-absorptions can complicate and lead to additional signals in the CD spectra. CD spectra of the A3F peptides are included in the Supporting Information. The CD spectra of A3(F2) are very similar to those obtained of the other elastin-like peptides discussed in this contribution and show a gradual transition from a random coil dominated secondary structure into a type II β-turn spectrum with increasing temperature. The mean molar residue ellipticities at 196 and 218 nm in the CD spectra of A3(F2) are not very

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Figure 10. Temperature-dependence of the mean molar residue ellipticity at (A) 196 and (B) 218 nm for the A3F peptides (c ) 150 µM). The legend in (A) also applies to (B).

Figure 11. Apparent LCST (9) and δLCST (0) of the A3F peptides (c ) 10 mg/mL).

Whereas 10 mg/mL solutions of the three highest substituted phenylalanine variants did not show any LCST behavior (not even at high salt concentrations), coacervation was observed for the three lowest substituted peptides A3(F2), A3(F13), and A3(F123). The resulting apparent LCST and δLCST values are plotted in Figure 11 and summarized in Table 1. The successive substitution of valine residues by phenylalanine residues leads to a very rapid decrease in the apparent LCST, which is in agreement with the increase in the predicted log P value for these peptides. The increase of the δLCST with increasing number of phenylalanine residues indicates a higher sensitivity of these peptides toward the addition of NaCl.

Conclusions different from those measured for peptide A3. With an increasing number of phenylalanine substituents, however, the shape of the CD spectra changes with a distortion appearing at 196 nm and a positive maximum around 218 nm. At higher degrees of substitution (A3(F123)(F2), A3(F123)(F13), A3(F123)(F123)), these new signals dominate the CD spectra and mask the characteristic random coil and β-turn signatures. Nevertheless, it is interesting to observe that the shape of the CD spectra of all phenylalanine containing peptides continuously changes with temperature, which suggests that this series of peptides also undergoes temperature-induced conformational changes. Figure 10 plots the temperature dependence of [Θ]196 and [Θ]218. It is important to note here that in this case these ellipticities do not only reflect the contribution of random coil and β-turn structures but also include that of the aromatic side chain of the phenylalanine residues. As a consequence, these ellipticities may be dominated by the contribution of the phenylalanine side chains and mask any changes in the random coil or β-turn structures (especially at high degrees of substitution). If changes in [Θ]196 or [Θ]218 are observed, these are not necessarily due to changes in random coil or β-turn structures, but may also be due to changes in the CD spectrum of the phenylalanine side chain. Second, because all peptides in the A3F series contain different numbers of phenylalanine residues, it is difficult to compare data of different peptides within this series. For low degrees of substitutions (A3(F2), A3(F13), A3(F123)), the data in Figure 10 qualitatively resemble those of the other elastin-like peptides with decreasing apparent random coil contents and increasing fractions of apparent β-turn structures with increasing temperature. For the higher substituted phenylalanine variants, however, changes in [Θ]196 or [Θ]218 are less obvious, which is probably due to the fact that the corresponding CD spectra are largely dominated by the contribution of the phenylalanine chains.

This contribution presents the results from a comprehensive study on the secondary structure and LCST behavior of a library of short, elastin-like peptides. This work was motivated by the LCST behavior of elastin and elastin-like polypeptides, which makes these proteins attractive as smart biomaterials, and was driven by the question whether short, elastin-like peptides would also be useful building blocks for such applications. The peptides discussed in this contribution are based on the GVGVP pentad repeat sequence. The first group of peptides consisted of seven samples in which the peptide chain length was systematically varied from one to five pentapeptide repeats. The other three groups of peptides were composed of three pentapeptide repeat units. Each of these three groups consisted of six peptides in which the valine residues of the original sequence were successively replaced by an increasing number of more hydrophobic isoleucine, leucine, or phenylalanine repeat units. Most of the investigated peptides showed typical elastin CD spectra with a decrease in random coil and an increase in β-turn structure content with increasing temperature. The LCST behavior of the peptides was investigated with turbidimetry experiments. Apparent LCSTs were determined by extrapolation of the cloud points of aqueous peptide solutions containing different amounts of NaCl to zero-salt concentration. For two of the investigated peptides, apparent LCSTs of less than 100 °C were determined. For the highest substituted leucine variant (A3(L123)(L123)), an LCST of 42 °C was determined at a sample concentration of 100 mg/mL in deionized water. It should be noted that the apparent LCSTs reported in this paper were recorded using samples containing mM concentrations of the peptides, whereas turbidimetry experiments with high molecular weight elastin-like polypeptides can be carried out at µM concentrations.36,37 Within a given series of peptides, the apparent LCST was observed to decrease with increasing chain length and with increasing number of substitutions of


valine for isoleucine, leucine, or phenylalanine. The observed trends in the apparent LCSTs of the isoleucine, leucine, and phenylalanine variants with increasing degree of substitution were found to correlate well with the predicted log P values. In contrast, for a given family of peptide variants there seems to be no obvious correlation between the evolution of secondary structure formation and apparent LCST with increasing number of substitutions. This may suggest that within one homologous series of peptides, the LCST behavior is primary, determined by the peptides’ hydrophobicity. In contrast, comparison of the apparent LCST of peptides from different groups but with identical chain length (e.g., A3, A3(I2), A3(L2) and A3(F2)) with the predicted log P values does not reveal a clear correlation. This suggests that hydrophobicity, although important, is probably not the only factor determining the LCST behavior of short, elastin-like peptides and other factors such as the conformational preferences of the constituent amino acids may also play a role. The precise contributions of these different factors are an interesting subject for further studies. In spite of these open questions, the results reported in this contribution for the first time systematically demonstrate the LCST behavior of short, elastin-like peptides and show that the apparent transition temperature of these peptides can be controlled by adjusting their composition and chain length. Supporting Information Available. HPLC and mass spectra of the synthesized peptides. Additional CD and turbidimetry data. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) Foster, J. A.; Bruenger, E.; Gray, W. R.; Sandberg, L. B. J. Biol. Chem. 1973, 248, 2876–2879. (2) Mithieux, S. M.; Weiss, A. S. AdV. Protein Chem. 2005, 70, 437– 461. (3) Urry, D. W. J. Phys. Chem. B 1997, 101, 11007–11028. (4) Massodi, I.; Bidwell, G. L., III; Raucher, D. J. Controlled Release 2005, 108, 396–408. (5) Herrero-Vanrell, R.; Rincón, A. C.; Alonso, M.; Reboto, V.; MolinaMartinez, I. T.; Rodrı´guez-Cabello, J. C. J. Controlled Release 2005, 102, 113–122. (6) Betre, H.; Liu, W.; Zalutsky, M. R.; Chilkoti, A.; Kraus, V. B.; Setton, L. A. J. Controlled Release 2006, 115, 175–182. (7) Hyun, J.; Lee, W.-K.; Nath, N.; Chilkoti, A.; Zauscher, S. J. Am. Chem. Soc. 2004, 126, 7330–7335. (8) Sallach, R. E.; Wei, M.; Biswas, N.; Conticello, V. P.; Lecommandoux, S.; Dluhy, A.; Chaikof, E. L. J. Am. Chem. Soc. 2006, 128, 12014– 12019. (9) Jung, Y.; Bayley, H.; Movileanu, L. J. Am. Chem. Soc. 2006, 128, 15332–15340.


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(10) Lim, D. W.; Trabbic-Carlson, K.; MacKay, J. A.; Chilkoti, A. Biomacromolecules 2007, 8, 1417–1424. (11) Urry, D. W.; Long, M.; Sugano, H. J. Biol. Chem. 1978, 253, 6301– 6302. (12) Cook, W. J.; Einspahr, H.; Trapane, T. L.; Urry, D. W.; Bugg, C. E. J. Am. Chem. Soc. 1980, 102, 5502–5505. (13) Pechar, M.; Brus, J.; Kostka, L.; Konˇa´k, C.; Urbanova´, M.; Sˇlouf, M. Macromol. Biosci. 2007, 7, 56–69. (14) Reiersen, H.; Clarke, A. R.; Rees, A. R. J. Mol. Biol. 1998, 283, 255– 264. (15) Flamia, R.; Lanza, G.; Salvi, A. M.; Castle, J. E.; Tamburro, A. M. Biomacromolecules 2005, 6, 1299–1309. (16) Yao, X. L.; Hong, M. J. Am. Chem. Soc. 2004, 126, 4199–4210. (17) Schreiner, E.; Nicolini, C.; Ludolph, B.; Ravindra, R.; Otte, N.; Kohlmeyer, A.; Rousseau, R.; Winter, R.; Marx, D. Phys. ReV. Lett. 2004, 92, 148101. (18) Arad, O.; Goodman, M. Biopolymers 1990, 29, 1651–1668. (19) Baer, M.; Schreiner, E.; Kohlmeyer, A.; Rousseau, R.; Marx, D. J. Phys. Chem. B 2006, 110, 3576–3587. (20) Ayres, L.; Koch, K.; Adams, P. J. H. M.; van Hest, J. C. M. Macromolecules 2005, 38, 1699–1704. (21) Fernańdez-Trillo, F.; Dureáult, A.; Bayley, J. P. M.; van Hest, J. C. M.; Thies, J. C.; Michon, T.; Weberskirch, R.; Cameron, N. R. Macromolecules 2007, 40, 6094–6099. (22) Cornish, J.; Callon, K. E.; Lin, C. Q.-X.; Xiao, C. L.; Mulvey, T. B.; Cooper, G. J. S.; Reid, I. R. Am. J. Physiol. 1999, 277, E779–783. (23) CD-Spectropolarimeters Manual for J-715; JASCO Inc.: Easton, MD. (24) Urry, D. W. Angew. Chem., Int. Ed. 1993, 32, 819–841. (25) Tao, P.; Wang, R; Lai, L. J. Mol. Model. 1999, 5, 189–195. (26) Urry, D. W.; Luan, C.-H.; Parker, T. M.; Gowda, D. C.; Prasad, K. U.; Reid, M. C.; Safavy, A. J. Am. Chem. Soc. 1991, 113, 4346–4348. (27) Urry, D. W.; Gowda, D. C.; Parker, T. M.; Luan, C.-H.; Reid, M. C.; Harris, C. M.; Pattanaik, A.; Harris, R. D. Biopolymers 1992, 32, 1243– 1250. (28) Fmoc solid phase peptide synthesis; Chan, W. C., White, P. D. , Eds.; Oxford University Press: New York, 2000. (29) Urry, D. W. Biopolymers 1998, 47, 167–178. ¨ rfi, L.; Takaćs-Novak, K.; Acsa´dy, G.; Keri, (30) Ero¨s, D.; Ko¨vesdi, I.; O G. Curr. Med. Chem 2002, 9, 1819–1829. (31) Buchwald, P.; Bodor, N. Proteins 1998, 30, 86–99. (32) Leo, A. J. Chem. ReV. 1993, 93, 1281–1306. (33) Bodor, N.; Buchwald, P. J. Phys. Chem. B 1997, 101, 3404–3412. (34) Urry, D. W.; Long, M. M.; Cox, B. A.; Ohnishi, T.; Mitchell, L. W.; Jacobs, M. Biochim. Biophys. Acta 1974, 371, 597–602. (35) Luan, C.-H.; Parker, T. M.; Prasad, K. U.; Urry, D. W. Biopolymers 1991, 31, 465–475. (36) Meyer, D. E.; Chilkoti, A. Biomacromolecules 2004, 5, 846–851. (37) Meyer, D. E.; Chilkoti, A. Biomacromolecules 2002, 3, 357–367. (38) Stryer, L. Biochemistry, 4th ed.; W.H. Freeman and Company: New York, 1996. (39) Woody, R. W.; Dunker, A. K. Aromatic and cystine side-chain circular dichroism in proteins In Circular Dichroism and the Conformational Analysis of Biomolecules, 1st ed.; Fasman, G. D., Ed.; Plenum Press: New York, 1996; Chapter 4.

BM800784Y

Secondary Structure Formation and LCST Behavior of Short Elastin

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