The ability to engineer a solvent-exposed surface of self-assembling coiled coils allows one to achieve a higher-order hierarchical assembly such as nano- or microfibers. Currently, these materials are being developed for a range of biomedical applications, including drug delivery systems; however, ways to mechanistically optimize the coiled-coil structure for drug binding are yet to be explored. Our laboratory has previously leveraged the functional properties of the naturally occurring cartilage oligomeric matrix protein coiled coil (C), not only for its favorable motif but also for the presence of a hydrophobic pore to allow for small molecule binding. This includes the development of Q, a rationally designed pentameric coiled coil derived from C. Here, we present a small library of protein microfibers derived from the parent sequences of C and Q bearing various electrostatic potentials with the aim to investigate the influence of higher-order assembly and encapsulation of candidate small molecule, curcumin. The supramolecular fiber size appears to be well-controlled by sequence-imbued electrostatic surface potential, and protein stability upon curcumin binding is well correlated to relative structure loss, which can be predicted by in silico docking.
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Tuning a coiled-coil hydrogel via computational design of supramolecular fiber assembly
The previously reported Q is a thermoresponsive coiled-coil protein capable of higher-order supramolecular assembly into fibers and hydrogels with upper critical solution temperature (UCST) behavior. Here, we introduce a new coiled-coil protein that is redesigned to disfavor lateral growth of its fibers and thus achieve a higher crosslinking density within the formed hydrogel. We also introduce a favorable hydrophobic mutation to the pore of the coiled-coil domain for increased thermostability of the protein. We note that an increase in storage modulus of the hydrogel and crosslinking density is coupled with a decrease in fiber diameter. We further fully characterize our α-helical coiled-coil (Q2) hydrogel for its structure, nano-assembly, and rheology relative to our previous single domain protein, Q, over the time of its gelation demonstrating the nature of our hydrogel self-assembly system. In this vein, we also characterize the ability of Q2 to encapsulate the small hydrophobic small molecule, curcumin, and its impact on the mechanical properties of Q2. The design parameters here not only show the importance of electrostatic potential in self-assembly but also provide a step towards predictable design of electrostatic protein interactions.
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- Award ID(s):
- 1728858
- PAR ID:
- 10414711
- Date Published:
- Journal Name:
- Molecular Systems Design & Engineering
- Volume:
- 8
- Issue:
- 2
- ISSN:
- 2058-9689
- Page Range / eLocation ID:
- 217 to 226
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)Owing to their tunable properties, hydrogels comprised of stimuli-sensitive polymers are one of the most appealing scaffolds with applications in tissue engineering, drug delivery and other biomedical fields. We previously reported a thermoresponsive hydrogel formed using a coiled-coil protein, Q. Here, we expand our studies to identify the gelation of Q protein at distinct pH conditions, creating a protein hydrogel system that is sensitive to temperature and pH. Through secondary structure analysis, transmission electron microscopy, and rheology, we observed that Q self-assembles and forms fiber-based hydrogels exhibiting upper critical solution temperature behavior with increased elastic properties at pH 7.4 and pH 10. At pH 6, however, Q forms polydisperse nanoparticles, which do not further self-assemble and undergo gelation. The high net positive charge of Q at pH 6 creates significant electrostatic repulsion, preventing its gelation. This study will potentially guide the development of novel scaffolds and functional biomaterials that are sensitive towards biologically relevant stimuli.more » « less
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Abstract Structure‐based engineering starting from the antiparallel coiled coil domain of the Bcr‐Abl oncoprotein (BCR30‐65) afforded a new homodimeric antiparallel coiled coil whose folding is responsive to solution pH. The BCR30‐65homodimer contains two glutamic acids (Glu52) buried in its hydrophobic core which one would expect to impart pH responsive folding behavior. Surprisingly, BCR30‐65is resistant to thermal denaturation over a wide pH range. This behavior arises from stabilizing salt bridges formed between solvent‐exposed Arg55 and Glu52 that passivates the glutamates’ negative charge within the protein’s core. Replacing Arg55 with alanine imparts pH‐responsive folding behavior, but also results in the loss of directional specificity between the coiled coil’s helices. We find that an Ile42Glu mutation preserves the salt bridges and imparts the desired pH responsive folding behavior. The Ile42Glu protein forms an antiparallel homodimeric coiled coil under acidic conditions but unfolds under basic conditions and is only marginally less thermally stable compared to BCR30‐65.
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Abstract Swallow, a 62 kDa multidomain protein, is required for the proper localization of several mRNAs involved in the development of Drosophila oocytes. The dimerization of Swallow depends on a 71‐residue self‐association domain in the center of the protein sequence, and is significantly stabilized by a binding interaction with dynein light chain (LC8). Here, we detail the use of solution‐state nuclear magnetic resonance spectroscopy to characterize the structure of this self‐association domain, thereby establishing that this domain forms a parallel coiled‐coil and providing insight into how the stability of the dimerization interaction is regulated.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D2ME00153E
