Self-assembly of the leucine chain domains of the AC10A protein leads to a network, in which the oligomeric bundles serve as attachment points10. The hydrophilic middle block lacks regular secondary structure and prevents chain precipitation under conditions favoring leucine chain aggregation. The dissolution of AC10A hydrogels is a consequence of the structural and dynamic properties of the leucine chain aggregates and the topological states of the artificial protein chains.
When the two end groups of a given chain participate in different junctions of the network, the middle block of the chain forms a bridge. Second, replacement of the a-position or d-position by a cysteine residue should cause minimal disruption of the secondary and tertiary structures of the leucine chain domains. Primers were synthesized at the Beckman Institute Biopolymer Synthesis Center at the California Institute of Technology.
The sequence of pQE9AC10Acys(L11C) was verified at the Beckman Institute DNA Sequencing Core Facility of the California Institute of Technology. The surfaces of the vials were aminated by cleaning with 98% sulfuric acid (EM Science, Gibbstown, NJ) and . The thickness of the gel pad was determined by marking the first and last Z-stacks where the embedded fluorescent beads were observed.
The degree of overall swelling was calculated based on changes in dimensions or weight.
Results and Discussion 1. Protein biosynthesis
Images of the gel pads before and after rehydration were recorded in tapping mode on a Nanoscope III atomic force microscope (Digital Instruments, Inc.) to examine the changes in size and shape. Identical CD spectra were recorded for AC10Acys(L11C) solutions under both oxidizing and reducing conditions, suggesting that it is the replacement of the leucine residue with a cysteine residue, rather than the formation of disulfide bonds, that causes this modest disruption of secondary structure causes. If such linkages were significant, the secondary structure of the leucine zipper domain should be highly disrupted but recoverable under reducing conditions.
At pH 7.6, none of the fluorescent beads trapped in AC10Acys(L11C) gels appeared to escape into the surrounding buffer after 2 days. The release rates of embedded beads were quantified by measuring the fluorescence intensity of the surrounding buffer as a function of time. This result confirms the important role of disulfide bonds in maintaining the stability of the AC10Acys(L11C) network in open solutions.
Although alkaline cleavage of disulfide bonds can occur at pH 12.2, only about 1% of the cleavage occurs within one hour31. The predominance of linear bonds is most likely due to the redundancy of the disulfide bonds formed from C-terminal cysteine residues. The increased valence of the building units improves the stability of AC10Acys(L11C) hydrogels, while the noncovalent nature of the nodes confers reversibility to the network.
A solution of the resulting protein was prepared in 8 M urea (pH 7.6) at a concentration of 6% w/v, and the free thiol concentration was titrated at successive time points. As shown in Figure 8, disulfide bond formation was significantly slowed in the absence of the template provided by the self-assembly of the leucine zipper. 30% in the first hour after preparation, while that of the AC10Atrp hydrogel remained constant.
Since the AC10Acys(L11C) middle block carries 10 glutamic acid residues and is therefore negatively charged at pH 7.6, anchoring of the gels is likely to occur through electrostatic attraction. The reversibility of gelation in response to pH in closed systems, the stability of gels in open solutions, and the anisotropic swelling of gels on aminated surfaces allowed us to create stable micropatterns of the AC10Acys(L11C) hydrogel by soft lithography. The diameter of the gel pads was consistent with the diameter of the stamp wells (30 µm) and remained unchanged when the gel pads were placed in an excess of buffer for 48 h, as examined by light microscopy.
The retention of the original micropatterns is facilitated by the electrostatic attraction between the gel pads and the aminated surface, which not only immobilizes the gel pads but also suppresses their swelling in the lateral direction. The ability of hydrogel pads to maintain shape and registration is important for their potential application in array technologies.
Conclusions
MRGSHHHHHGSDDDDKWA SGDLENE VAQLERE VRSLEDE AAELEQK VSRLKNE IEDLKAE IGDHVAPRDTSYRDPMG (AGAGAGPEG)10ARMPT SGDLENE VAQLERE VRSLEDE AAELEQK VSRLKNE IEDLKAE IGDHVAPRDTSMGGC AC10Acys. MRGSHHHHHGSDDDDKA SGDLENE VAQLERE VRSLEDE AAELEQK VSRLKNE IEDLKAE IGDHVAPRDTSYRDPMG (AGAGAGPEG)10ARMPT SGDLENE VAQLERE VRSLEDE AAELEQK VSRLKNE IEDLKAE IGDHVAPRDTSW AC10Atrp. MRGSHHHHHGSDDDDKWA SGDLENE VAQCERE VRSLEDE AAELEQK VSRLKNE IEDLKAE IGDHVAPRDTSYRDPMG (AGAGAGPEG)10ARMPT SGDLENE VAQCERE VRSLEDE AAELEQK VSRLKNE IEDLKAE IGDHVAPRDTSMGGC AC10Acys (L11C).
Loop configurations mainly involve antiparallel association of leucine zipper end groups; asymmetric placement of the cysteine residue is unfavorable for disulfide bonds in this configuration. Release of embedded fluorescent beads (20 nm) from hydrogel films made of different proteins (5.5% w/v, pH 7.6, room temperature, 100 mM phosphate buffer). CD spectra of 50 µM AC10Acys(L11C) solutions at different pH values (room temperature, 100 mM phosphate buffer).
The α-helix proportions at pH 7.6 and 12.2 are estimated as 29% and 3%, respectively, by a Ridge Regression Analysis (CONT)30 program. The Debye plot of polygonal light scattering signals from a 2.42×10-4 mg/L solution (room temperature, pH=11.0) reveals that the average molecular weight of clusters disassembled from an AC10Acys (L11C) hydrogel at pH 12.2 is about. Percentage of reduced cysteine residues in a 6% w/v solution of AC10Acys(L11C) in 8 M urea as a function of time (pH 7.6, room temperature).
The dry protein was pretreated so that the leucine zipper domains were denatured and the majority of the cysteine residues were reduced. Storage moduli of protein hydrogels as determined by rheological oscillatory frequency sweep tests (7% w/v, 100 mM phosphate buffer, pH 7.0, 22 °C, 1% strain). Lateral and vertical swelling ratios of AC10Acys(L11C) gels on aminated and untreated surfaces. the diameter/thickness aspect ratio of the dry protein pillow is approximately 150).
