Reversible Hydrogels from Self-Assembling Artificial Proteins

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Science  17 Jul 1998:
Vol. 281, Issue 5375, pp. 389-392
DOI: 10.1126/science.281.5375.389


Recombinant DNA methods were used to create artificial proteins that undergo reversible gelation in response to changes in pH or temperature. The proteins consist of terminal leucine zipper domains flanking a central, flexible, water-soluble polyelectrolyte segment. Formation of coiled-coil aggregates of the terminal domains in near-neutral aqueous solutions triggers formation of a three-dimensional polymer network, with the polyelectrolyte segment retaining solvent and preventing precipitation of the chain. Dissociation of the coiled-coil aggregates through elevation of pH or temperature causes dissolution of the gel and a return to the viscous behavior that is characteristic of polymer solutions. The mild conditions under which gel formation can be controlled (near-neutral pH and near-ambient temperature) suggest that these materials have potential in bioengineering applications requiring encapsulation or controlled release of molecular and cellular species.

Thermally reversible network formation in polymeric systems is a well-known phenomenon (1–3). In aqueous solutions, synthetic polymers such as poly(vinyl alcohol) (4), proteins such as gelatin (5), and polysaccharides such as carrageenan (6) exhibit reversible gelation within prescribed limits of concentration and temperature. Despite the utility of this behavior (1), the molecular origins of gel formation in such systems are poorly understood, and opportunities for systematic engineering of gel properties are limited. Gel formation demands two seemingly contradictory kinds of behavior: Interchain interactions must be strong enough to form junction points in the molecular network, yet at the same time the chain cannot exclude solvent, or it will precipitate from solution rather than forming a swollen gel. Assignment of these two roles to different portions of a repetitive polymer such as poly(vinyl alcohol) is far from straightforward. Furthermore, the ability to tune the gel-solution (gel-sol) transition conditions is limited in such systems.

The approach to gel design presented here circumvents that problem through creation of multidomain (“triblock”) artificial proteins in which the interchain binding and solvent retention functions are engineered independently (7). The triblock architecture3, illustrated in Fig. 1, consists of relatively short “leucine zipper” end blocks flanking a water-soluble polyelectrolyte domain. The modular nature of the design allows independent investigation of the behavior of the individual terminal 1 and polyelectrolyte 2 blocks as well.

Figure 1

Amino acid sequences of the three proteins studied. Protein 1 consists of 76 amino acids, 42 of which make up the leucine zipper Helix. Protein2 consists of 122 amino acids, 90 of which make up the alanylglycine-rich repeat [(AG)3PEG]10. Protein 3 consists of 230 amino acids, 84 of which make up the Helix repeat and 90 of which make up the alanylglycine-rich repeat. The acidic or basic amino acids occupying the e and g positions of the Helix heptad repeat abcdefg are in bold. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and W, Trp.

Gelation of triblock proteins such as 3 is driven by the formation of dimers (and higher order aggregates) of the terminal leucine zipper peptide domains (Fig. 2). The leucine zipper motif is characterized by a heptad periodicity generally designated abcdefg, where a and d are hydrophobic amino acids (frequently Leu, especially at position d) and the residues at positions e and g are usually charged (8). Under appropriate conditions of pH and temperature, such peptides adopt helical conformations that place the hydrophobic a and d residues on a single face of the helix. Aggregation, most often in the form of coiled-coil dimers, is then promoted by the formation of hydrophobic interhelical interfaces, whereas the pH-dependent interactions between e and g residues modulate the stability of the coiled-coil aggregates. Aggregation number (9), dimerization specificity (10), aggregate stability (11), and aggregate structure (12) can be manipulated within wide limits through the control of chain length and amino acid sequence. The results of these studies suggest that the network junctions in gels of proteins such as 3 should be subject to similar control.

Figure 2

Proposed physical gelation of monodisperse triblock copolymer 3. The chains are drawn as disulfide-linked dimers, joined through their COOH-terminal cysteine residues. Analysis with Ellman's reagent confirmed the absence of free thiols under the conditions used for spectroscopic and scattering studies.

The putative leucine zipper domain (designated Helix in Fig. 1) comprises six heptad repeats. The choice of residues at positions a and d was based on the a/d residue pattern of the Junoncogene product (13); and a database developed by Lupaset al. (14), which identifies the most probable amino acids at positions a through g in naturally occurring coiled-coil proteins, was used to select residues occupying the b, c, and f positions. Nine of the 12 e and g positions were populated by Glu residues in order to destabilize the coiled-coil structure in basic solutions and thereby to facilitate pH control of gelation and viscoelastic behavior. The polyelectrolyte domain of 3, based on the Ala-Gly–rich sequence [(AG)3PEG]10, was chosen because of its water solubility and absence of regular secondary structure (15)—features believed to be desirable in the design of highly swollen hydrogels (16).

Proteins 1 through 3 were prepared by bacterial expression of the corresponding artificial genes (17,18). Circular dichroism (CD) spectroscopy confirmed the helical secondary structures of 1 and 3 at room temperature, as well as the random coil character of 2 (Fig. 3A) (19). In dilute solution,1 and 3 exhibited thermal unfolding transitions, with the temperature of the transition midpoint (T m) rising in each case from ∼30oC at pH 11 to ≥80°C at pH 6 (Fig. 3B) because of progressive neutralization of the predominantly acidic charged residues with decreasing pH (20). The abrupt increase inT m between pH 7.5 and 6 is characteristic of coiled-coil peptides in which positions e and g are occupied predominantly by acidic residues (13). At pH values of 5 and below, no transitions were observed for either protein at temperatures below 100oC, and the helical content decreased only slightly over the temperature range from 0o to 100oC. Acidic conditions stabilize the helix sufficiently that no thermal denaturation of 1 or 3 was observed.

Figure 3

(A) Secondary structural analyses of proteins 1 (•) (5 μM), 2 (▴) (5 μM), and3 (○) (5 μM), using CD spectroscopy. Spectra were recorded at 25°C in 10 mM NaH2PO4 and 150 mM NaCl (pH 7.4 upon addition of 1 N NaOH). (B) Thermal denaturation T m of proteins 1 (•) (5 μM) and 3 (○) (5 μM), monitored at 222 nm as a function of pH. Measurements were made in 10 mM NaH2PO4 and 150 mM NaCl (pH 7.6).T m was determined by taking the first derivative of the CD signal [θ]222 with respect to temperature (K−1).

The gel-sol behavior of 3 in concentrated solution may be anticipated from the dilute solution behavior presented above. In a low-pH solution, the Glu side chains are protonated and the stability of the coiled-coil aggregates increases. Furthermore, protonation of the Glu residues in the polyelectrolyte linker domain causes the linker to collapse to a hydrophobic globule. Hence, precipitation of3 should occur at low pH, and this in fact was observed. With increasing pH, progressive deprotonation of the Glu residues occurs, resulting first in swelling of the collapsed spacer block and eventually, at higher pH, in the dissociation of coiled-coil domains because of repulsive electrostatic interactions between predominantly negatively charged end blocks. Thus, in a certain range of pH, the protein should form a thermoreversible physical gel, where the reversibility of the gelled state results from thermal dissociation of the coiled-coil domains. At sufficiently high pH, a viscous solution of predominantly nonassociated proteins results. This proposed scenario is shown schematically in Fig. 2 and is borne out by simple visual observation: Aqueous solutions of 3, which flow readily at pH 10, exhibit no visible flow on a time scale of hours upon acidification to pH 7. Aqueous solutions of either 1 or2 do not gel at any pH, indicating that both aggregating terminal groups 1 and a spacer block 2 are necessary constituents of a gel-forming protein of this type.

In order to investigate the pH and temperature dependence of gelation, we used diffusing wave spectroscopy (21), a noninvasive optical technique based on dynamic light scattering in the multiple scattering limit, to deduce the viscoelastic behavior of3 by monitoring the thermally induced fluctuations of a dilute suspension of added scattering particles (22). The time-averaged mean-square displacement (MSD) of the tracer particles as a function of time, <r 2(t)>, may be obtained from the dynamic intensity autocorrelation function of light that is multiply scattered from these tracer particles (23). The MSD of the tracer particles contains essential information about the viscoelastic properties of the embedding medium. In particular, it can be shown that the MSD of the tracer particles is related to the shear creep compliance J(t) of an incompressible embedding medium by J(t) = (πa/k B T) <r 2(t)>, wherea is the radius of a tracer particle andk B T is the thermal energy. Equivalently, the complex shear modulus G*(ω) may be obtained from G*(ω) =k B T/(iωπa< 2(ω)>), where < 2(ω)> is the Fourier transform of <r 2(t)>. In a viscous liquid, the tracer particles may freely diffuse, resulting in a MSD that increases linearly with time; whereas in an elastic medium, the amplitude of the tracer particle fluctuations is limited, resulting in a plateau in the MSD (23). In a viscoelastic medium, the MSD will show some aspects of each limiting behavior. The MSDs measured for protein 3 (5% w/v in 10 mM tris buffer at 23oC) at pH 8.0, 8.8, and 9.5 are shown in Fig. 4A. At pH 8.0, the MSD exhibited a plateau that is characteristic of an elastic gel with a plateau modulusGp ≅ 200 Pa near ω = 100 s−1. Such gel behavior was observed for protein concentrations above about 4% w/v. At pH 8.8, this plateau region was reduced to an inflection point, whereas at pH 9.5, the MSD increased with time as in a viscous liquid. These data show a progressive transformation from elastic to viscoelastic to viscous behavior and suggest that the gel point lies between pH 8.0 and 9.5.

Figure 4

(A) Log-linear plot of the MSD [<r 2(t)>] as a function of time for protein 3 (5% w/v in 10 mM tris buffer at 23oC) for pH 8.0 (bottom curve), pH 8.8 (middle curve), and pH 9.5 (top curve). (B) Log-linear plot of the MSD as a function of time for protein 3 (5% w/v in 10 mM tris at pH 7.8) collected upon heating from 23oC (bottom solid curve) to 55oC (top solid curve) and upon cooling to 23oC (dashed curve).

The thermal dependence of gelation for 3 was investigated by cycling a 5% gel through a series of temperatures ranging from 23o to 55oC at pH 7.8. The MSD versus time at various temperatures is plotted in Fig. 4B. The solid curves in Fig. 4B show the data obtained during the heating portion of the cycle; the dashed curve shows the MSD after cooling from 55o back to 23oC. These data show that as the temperature was increased to 55oC, the gel progressively became a fluid. Furthermore, the gel state was recovered upon cooling back to 23oC from the fluid state at 55°C. However, a slight hysteresis occurred after a complete cycle of heating to 55oC and returning to 23oC. This hysteresis suggests that there is a curing time associated with the refolding of the coiled-coil network junctions into the associated state. The 2- to 3-hour equilibration time allotted between 6-hour experiments was insufficient for the gel to recover its original state.

The gelation behavior of polymers such as 3illustrates the advantages of biological synthesis and assembly processes used in combination with concepts drawn from macromolecular materials science. The triblock protein design introduced here preserves the dimerization function of the leucine zipper domain but turns that function toward entirely new objectives. Protein-protein recognition, which in nature might lead to DNA binding, now results in the formation of switchable hydrogels. The biosynthetic approach allows precise and independent control of the length, composition, and charge density of the polyelectrolyte domain and of each of the relevant architectural features of the associative end blocks. For instance, modified versions of 3 in which one acidic leucine zipper domain is replaced by a basic one undergo the gel-sol transition at much higher temperature than does unmodified 3(24). Such control is valuable in designing hydrogels of predetermined physical and biological properties (such as strength, porosity, and sensitivity to enzymatic degradation) and makes these triblock copolymer systems attractive candidates for use in molecular and cellular encapsulation and in controlled reagent delivery.


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