An official website of the United States government
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.
more »
« less
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.
more »
« less
- 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
More Like this
-
-
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
-
ABSTRACT A tetrameric coiled‐coil peptide,TetNL, is used herein as a building block for hierarchical assembly into higher order structures. Assembly within phosphate buffer (pH 7.4) led to the rapid formation of micron‐sized fibers and cuboid structures, a process that could be shifted toward cuboid formation with agitation during the assembly process. Investigation of the packing of the cuboid assemblies by TEM demonstrated a regular banding pattern (4.6 nm) within the structures that was perpendicular to the length of the cuboids, a value that supports an end‐to end organization of the tetrameric coiled coils along the blocks. SWAXS analysis supports that the internal packing of the tetrameric coiled coil building blocks is a close‐packed hexagonal structure. These data represent an interesting comparison with a trimeric coiled coil peptide,TriNL, that forms hollow nanotubes with the same internal hexagonal packing. ModifiedTriNLhas been used to generate numerous unique morphologies, and the data presented herein provide a distinct tetrameric building block that can also be exploited in this manner.more » « less
-
Fluorescent protein biomaterials have important applications such as bioimaging in pharmacological studies. Self-assembly of proteins, especially into fibrils, is known to produce fluorescence in the blue band. Capable of self-assembly into nanofibers, we have shown we can modulate its aggregation into mesofibers by encapsulation of a small hydrophobic molecule. Conversely, azobenzenes are hydrophobic small molecules that are virtually non-fluorescent in solution due to their highly efficient photoisomerization. However, they demonstrate fluorogenic properties upon confinement in nanoscale assemblies by reducing the non-radiative photoisomerization. Here, we report the fluorescence of a hybrid protein-small molecule system in which azobenzene is confined in our protein assembly leading to fiber thickening and increased fluorescence. We show our engineered protein Q encapsulates AzoCholine, bearing a photoswitchable azobenzene moiety, in the hydrophobic pore to produce fluorescent mesofibers. This study further investigates the photocontrol of protein conformation as well as fluorescence of an azobenze-containing biomaterial.more » « less
-
Labeled protein-based biomaterials have become popular for various biomedical applications such as tissue-engineered, therapeutic, and diagnostic scaffolds. Labeling of protein biomaterials, including with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, has enabled a wide variety of imaging and therapeutic techniques. These USPIO-based biomaterials are widely studied in magnetic resonance imaging (MRI), thermotherapy, and magnetically-driven drug delivery, which provide a method for direct and non-invasive monitoring of implants or drug delivery agents. Where most developments have been made using polymers or collagen hydrogels, shown here is the use of a rationally designed protein as the building block for a meso-scale fiber. While USPIOs have been chemically conjugated to antibodies, glycoproteins, and tissue-engineered scaffolds for targeting or improved biocompatibility and stability, these constructs have predominantly served as diagnostic agents and often involve harsh conditions for USPIO synthesis. Here, we present an engineered protein–iron oxide hybrid material comprised of an azide-functionalized coiled-coil protein with small molecule binding capacity conjugated via bioorthogonal azide–alkyne cycloaddition to an alkyne-bearing iron oxide templating peptide, CMms6, for USPIO biomineralization under mild conditions. The coiled-coil protein, dubbed Q, has been previously shown to form nanofibers and, upon small molecule binding, further assembles into mesofibers via encapsulation and aggregation. The resulting hybrid material is capable of doxorubicin encapsulation as well as sensitive -weighted MRI darkening for strong imaging capability that is uniquely derived from a coiled-coil protein.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D2ME00153E
