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Effects of Temperature and Pressure on the Aggregation Properties of an Engineered Elastin Model Polypeptide in Aqueous Solution T. Tamura,† T. Yamaoka,† S. Kunugi,*,† A. Panitch,‡ and D. A. Tirrell§ Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan; Department of Polymer Science and Engineering, University of Massachusetts, Amherst, Massachusetts 01003; and Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125 Received September 5, 2000; Revised Manuscript Received October 10, 2000
The pressure and temperature dependence of the cloud point transition of an aqueous solution of an elastinlike polypeptide (MGLDGSMG(VPGIG)40VPLE), prepared by bacterial expression of the corresponding artificial gene, was measured. A temperature-pressure diagram was constructed over a wide range of conditions. The (VPGIG)40 solution exhibited a well-defined pressure-induced cloudpoint (Pc), as well as a temperature-induced transition (Tc). From near atmospheric pressure up to 100 MPa, Tc increased with increasing pressure, but decreased with further increases in pressure above 200 MPa. The maximum Tc was reached at 100-200 MPa. Between 10 and 25 °C, the Pc decreased with increasing temperature, and a broad maximum in Pc was observed in the range -10 to 0 °C. These results are compared with our previous results on synthetic thermoresponsive vinyl polymers. Introduction Elastin, which forms the core protein of elastic fibers in the body, has been widely investigated for its unique properties in solution. For R-elastin, a fragmentation product formed upon hot oxalic acid treatment, coacervation has been reported by Partridge et al.1 Similar properties have been observed in tropoelastins isolated from the aortas of copperdeficient chicks2 or prepared by recombinant DNA methods.3 Elastin contains several repetitive sequences, including VPGVG, APGVGV, VPGFGVGAG, and VPGG; the most frequently occurring repetitive pentapeptide sequence in porcine and bovine elastins, VPGVG,4,5 is known to exhibit an inverse temperature transition. Urry and co-workers have reported extensive studies of the properties of a chemically prepared elastin-like polypeptide, poly(VPGVG). This polymer forms homogeneous aqueous solutions at or below room temperature, but becomes turbid upon heating to body temperature, in a reversible manner.6,7 This inverse temperature transition is accompanied by a temperature-driven conformational change of the elastin-like polypeptide. As the temperature rises, the polymer collapses to a β-spiral structure with three VPGVG units per turn, with each pentamer adopting a type II β-turn conformation.8,9 This structural change is believed to give rise to molecular aggregation which provides a regular arrangement of tropo* To whom correspondence should be addressed. Telephone: +81-75724-7836. Fax: +81-75-724-7800. E-mail:
[email protected]. † Kyoto Institute of Technology. ‡ University of Massachusetts. Present address: Chemical, Bio and Materials Engineering, College of Engineering and Applied Sciences, Arizona State University Main, Tempe, AZ 85287. § California Institute of Technology.
elastin molecules for cross-linking by lysyl oxidase to form insoluble elastic fibers. Moreover, the elastomeric matrix cross-linked by γ-ray irradiation of poly(VPGVG) exhibited a force-temperature relationship.10 These important features can be modeled by the temperature-dependent phase separation of an aqueous solution containing elastin-like polypeptides,1 and has potential applications in protein purification, protein engineering, and biomaterial design.12-14 The behavior of elastin-like polypeptides is derived from their hydrogen-bonding and hydrophobic properties, which are similar to those several synthetic polymers, e.g., poly(N-isopropylacrylamide) (PNIPAM)15,16,17 and poly(N-vinylisobutyramide) (PNVIBA),18,19 which exhibit lower critical solution temperatures (LCSTs). The LCSTs of these polymers are influenced by several physical and chemical factors, such as pressure,20-22 salt concentration,23 surfactant addition,24 solvent,25 etc.; the folding temperature of elastin-like polypeptides is also influenced by these factors.3 Changes in the composition and sequence of poly(VPGVG) also affected these properties in response to changes in pH,10 ionic strength,26 oxidation/reduction potential,27 photomodulation,28 and enzymatic phosphorylation,29 which can be used for energy conversion via the elastomeric matrix. Among these factors, pressure has been found to cause significant changes in the phase transition temperature of the polypeptide solution, especially for sequences containing aromatic residues such as Phe, Tyr, and Trp (instead of Val) flanked by two Gly residues. An increase in pressure up to approximately 10 MPa caused a substantial increase in the temperature of the transition.30 In the low-pressure region, similar effects of pressure on the LSCT of PNIPAM have been reported by Lee et al.31
10.1021/bm005606u CCC: $19.00 © 2000 American Chemical Society Published on Web 11/01/2000
An Engineered Elastin Model Polypeptide
and Ohta et al.20 At higher pressures, however, the LCST of PNIPAM exhibited a decrease with increasing pressure.21,22,32 Furthermore, we have shown that the LCSTs of these thermoresponsive polymers (PNIPAM and PNVIBA) increase with increasing pressure at subzero temperatures,32 and that addition of salts or incorporation of ionic comonomers strongly influences the observed pressure-temperature transition properties.32-34 Our main focus in the present study is the temperature and pressure responsive behavior of elastin-like polypeptides, which would lead to useful information on the mechanism responsible for the solution properties of amphiphilic polymers. To avoid complications arising from the possible molecular weight dependence35 of the phase separation temperatures of the polypeptide solutions, we prepared an elastin-like polypeptide with a repeating pentapeptide ValPro-Gly-Ile-Gly sequence (1) by bacterial expression of the corresponding artificial gene36 in the present study. The pressure dependence of the thermal transition of these solutions obtained from structurally homogeneous polypeptides down to subzero temperatures was then measured to obtain temperature-pressure diagrams over a wide range of conditions. MGLDGSMG(VPGIG)40VPLE 1
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thermostated water containing ethylene glycol was circulated through the cell block, and the temperature of the solution inside the cell (just outside of the inner cell) was detected by a Cu-constantan thermocouple. In both cases, the output was monitored by a change in either the temperature (ca. 0.5 °C /min) at constant pressure or by the pressure (ca. 0.1 MPa/s) at constant temperature. Extraneous pressure was applied by a high-pressure hand pump equipped with an intensifier (ratio 8:5:1) (Teramecs Co.), and the pressure medium was deionized water. The pressure was measured by a Bourdon tube-type pressure gauge. Samples for the cloud-point measurements were prepared from 1 to 5.0 mg/mL stock solutions of 1 dissolved at 4 °C in distilled, deionized water. A 1.0 mL aliquot of the polypeptide stock solution was diluted to a total volume of 2.0 mL with cold distilled and deionized water. For each measurement, a fresh sample was prepared. The temperatureand pressure-induced cloud points of the polypeptide solutions were defined as the temperature and pressure corresponding to the intersection of a line drawn through the steepest portion of the curve and the baseline. Preliminary calorimetric measurements were performed on a highly sensitive differential scanning calorimeter, the nanoDSC model 5100 (Calorimetry Science Co., UT). The temperature scanning rate was 1 °C/min. The reference was the solution used in the overnight dialysis of each sample.
Experimental Section Results and Discussion The expression vector for the elastin-like polypeptide (VPGIG)40 was prepared as described in a previous report.36 Briefly, double-stranded oligonucleotides encoding (VPGIG)5 domains were synthesized, inserted into a cloning vector, amplified, excised from the vector, and then polymerized by an in vitro ligation method. The resulting DNA fragment, which encodes (VGPGIG)40, was inserted into plasmid pET28ap, giving rise to pET28ap(VPGIG)40. pET28ap(VPGIG)40 was used to transform the Escherichia coli expression host BL21(DE3)pLysS. Protein expression was induced by adding isopropyl β-thiogalactopyranoside (IPTG) at a concentration of 1.0 mM when the cultures had grown to OD600 ) 0.8 at 37 °C in 2xYT medium. The cells were harvested after 20 h of culture, and 1 was purified by centrifugation above its critical solution temperature. To obtain a pure sample, this purification process was repeated five times. The cloud point for the polypeptide solutions was determined by observing the apparent absorbance or light scattering. In the former case, a high-pressure optical cell with two sapphire windows (Teramecs Co., Kyoto, Japan) was set inside a MultiSpec-1500 spectrophotometer (Shimadzu Co., Kyoto, Japan), and the apparent absorbance at 500 nm was recorded. The temperature of the cell was controlled by a Peltier-type thermoregulator, and detected by a Pt resistance thermometer. In the light-scattering experiment, a highpressure optical cell with three sapphire windows and a quartz inner optical cell (Teramecs Co.), was set inside a RF-5300PC spectrofluorophotometer (Shimadzu Co.), and the apparent light scattering was recorded with excitation and emission wavelengths set at 400 nm. In this apparatus,
Figure 1 a shows the change in light-scattering intensity of a 0.5 mg/mL solution of 1 observed during heating at atmospheric pressure (0.1 MPa). The solution exhibited a well-defined temperature-induced cloud point, and the observed changes in light-scattering intensity were reversible. Similar microphase separation of the solution was caused by increasing the pressure. Figure 1b shows the absorbance change observed upon increasing the pressure at a constant temperature of 10 °C. The solution exhibited a well-defined cloud point pressure, and the changes in absorbance were reversible. The temperature- and pressure-induced cloudpoints were measured over wide ranges of pressure and temperature. The temperature-induced cloud points (Tc) depended on the pressure, and the pressure-induced cloud point (Pc) depended on the temperature. The former was measured over a range 0.1-250 MPa, and the latter was measured from -12.5 to +30 °C. The data obtained were then plotted in the form of a T-P diagram, as shown in Figure 2. From near atmospheric pressure to 100 MPa, Tc increased with increasing pressure. However, above 200 MPa, Tc decreased with increasing pressure, and a maximum in Tc was observed between 100 and 200 MPa. In the temperature range 10-25 °C, Pc decreased with increasing temperature, and a maximum in Pc was observed at -10 to 0 °C. The T-P curve contained two turning points (extrema), similar to the results obtained previously for PNIPAM and PNVIBA which have similar molecular weight to 1.21,32,33 With these extrema, as shown in the example in Figure 1c, a pressure increase from 0.1 MPa (along the arrow in Figure
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Figure 1. Changes in the scattering intensity (a) and the apparent absorbance (b, c) of the (VPGIG)40 solution (0.05 w/v %) during temperature scanning at 0.1 MPa (a) or pressure scanning at 10 (b) or 30 °C (c).
Figure 2. T-P diagrams for the cloud-point of the (VPGIG)40 (0.05 w/v %) solution: open symbols, temperature scanning; closed symbols, pressure scanning; dashed line, freezing point of water. Arrow: pressure increasing process at 30 °C (see Figure 1c). The solid curve was drawn arbitrarily.
2) made the turbid solution clear at medium pressure and then cloudy again at much higher pressures, which was completely reversible. These characteristic changes were strongly dependent on the concentration of 1 in the range of 0.01-0.2 (wt/v) % (data not shown), in contrast to the behavior of thermoresponsive vinyl polymers, for which such changes are independent of concentration over a wide range.37,38 This implies that different aggregation processes are involved in solutions of 1 vs PNIPAM. In the PNIPAM solution, intermolecular aggregation of compact globular polymer molecules is dominant, whereas intermolecular interactions coupled with conformational (intramolecular) change drive the transition of (VPGIG)40. On the other hand the consequent aggregation following these transitions seems to be based on the similar hydrophobic interaction and dehydration in both cases. These polymers therefore revealed a similar LCST change depending on the temperature and pressure when detected by the absorbance and/or scattering intensity. The transitions of thermoresponsive vinyl polymers involve dehydration of the amide groups and strengthening of the hydrophobic interactions among the side chains, as well as structural reorganization of water around the hydrophobic groups. The latter effect is essentially similar in the present
case. Hydrophobic interactions among alkyl side chains have been found to result in slightly positive volume changes,39 and thus an antagonism of the temperature and pressure effects was observed for both of PNIPAM and 1 solutions at lower pressures.21,29,30 This hypothesis is also supported by calorimetric measurements. The enthalpy changes of similar transitions of poly(pentapeptide)s have been reported as 5-13 kJ/ mole of pentamer and the accompanying entropy changes were estimated as 16-40 J/mol-pentamer/K.39 Our preliminary calorimetric measurements on solutions of 1 indicate that ∆H and ∆S are approximately 12 kJ/(mol pentamer) and 30 J/(mol pentamer/K), respectively. The positive ∆S values correspond to a positive volume change with a positive dP/dT slope near atmospheric pressure, according to the Clapeyron-Clausius equation. The compressibility of bulk water is greater than that of the water surrounding the hydrophobic groups, and thus a further increase in pressure can invert the situation. Above 200 MPa, hydrophobic interactions among the alkyl chains are volumetrically favored,40 and thus the extremum in the T and P dependence of the cloud point was observed. In the case of thermoresponsive vinyl polymers, the concentration independence allowed us to analyze the T-P diagrams with Hawley’s general equation41 of a two-state transition in order to evaluate several thermodynamic parameters.22,32 However, such an exact analysis for the present polymer showing a concentration dependence is rather difficult to perform at this stage of research. On the basis of the present results, the apparent compressibility ∆β at above room temperature is predicted to be negative, and the apparent ∆Cp at high pressures and low temperatures should be positive. Thermodynamic standard values can be obtained only after an evaluation of the concentration-independent transition diagram and the cross-term (expansibility). References and Notes (1) Partridge, S. M.; Davis, H. F. Adair, G. S. Biochem. J. 1955, 61, 11. (2) Betty, A. C.; Barry, C. S.; Urry, D. W. J. Biol. Chem. 1974, 249, 997. (3) Vrhovski, B.; Jensen, S.; Weiss, A. S. Eur. J. Biochem. 1997, 250, 92. (4) Sandberg, L. B.; Leslie, J. G.; Leach, C. T.; Alvares, V. L.; Torres, A. R.; Smith, D. W. Pathol. Biol. 1985, 33, 266.
An Engineered Elastin Model Polypeptide (5) Yeh, H.; Ornstein-Goldstein, N.; Indik, Z.; Sheppard, P.; Anderson, N.; Rosenbloom, J. C.; Cicila, G.; Yoon, K. G.; Rosenbloom, J. Collagen Relat. Res. 1987, 7, 235. (6) Urry, D. W. Angew. Chem., Int. Ed. Engl. 1993, 32, 819. (7) Urry, D. W. J. Phys. Chem. B 1997, 101, 11007. (8) Urry, D. W. Polym. Mater. Sci. Eng. 1990, 62, 587. (9) Cook, W. J.; Einspahr, H. M.; Trapane, T. L.; Urry, D. W.; Bugg, C. E. J. Am. Chem. Soc. 1980, 102, 5502. (10) Urry, D. W.; Haynes, B.; Zhang, H.; Harris, R. D.; Prasad, K. U. Proc. Nat. Acad. Sci. U.S.A. 1988, 85, 3407. (11) Kaibara, K.; Okamoto, K.; Miyakawa, K. New Funct. Mater., B 1993, 281. (12) Meyer, D. E.; Chilkoti, A. Nature Biotechnol. 1999, 17, 1112. (13) Reiersen, H.; Rees, A. R. Biochemistry 1999, 38, 14897. (14) Urry, D. W.; Pattanaik, A.; Xu, J.; Woods, T. C.; McPherson, D. T.; Parker, T. M. J. Biomater. Sci. Polym. Ed. 1998, 9, 1015. (15) Schild, H. G. Prog. Polym. Sci. 1992, 17, 163. (16) Heskins, M.; Guillet, J. E. Makromol. Sci., A2 1968, 1441. (17) Bae, Y. H.; Okano, T.; Hsu, R.; Kim, S. W. Makromol. Chem., Rapid Commun. 1987, 8, 481. (18) Akashi, M.; Nakano, S.; Kishida, A. J. Polym. Sci., Part A: Polym. Chem. Ed. 1996, 34, 301. (19) Suwa, K.; Morishita, K.; Kishida, A.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. Ed. 1997, 35, 3377. (20) Ohta, H.; Ando, I.; Fugishige, S.; Kubota, K. J. Polym. Sci., Part B 1991, 29, 963. (21) Otake, K.; Karaki, R.; Ebina, T.; Yokoyama, C.; Takahashi, S. Macromolecules 1993, 26, 2194. (22) Kunugi, S.; Takano, K.; Tanaka, N.; Suwa, K.; Akashi, M. Macromolecules 1997, 15, 44499. (23) Park, T. G.; Hoffman, A. S. Macromolecules 1993, 26, 5045. Schild, H. G.: Tirrell, D. A. J. Phys. Chem. 1990, 94, 4352. (24) Kokufuta, E.; Nakaizumi, S.; Ito, S.; Tanaka, T. Macromolecules 1995, 28, 1704.
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