Supramolecular Assembly of His-Tagged Fluorescent Protein Guests within Coiled-Coil Peptide Crystal Hosts: Three-Dimensional Ordering and Protein Thermal Stability

Abstract

The use of biomaterials for the inclusion and stabilization of biopolymers is an ongoing challenge. Herein, we disclose three-dimensional (3D) coiled-coil peptide crystals with metal ions that include and overgrow His-tagged fluorescent proteins within the crystal. The protein guests are found within two symmetry-related growth sectors of the crystalline host that are associated with faces of the growing crystal that display ligands for metal ions. The fluorescent proteins are included within this "hourglass" region of the crystals at a notably high level, display order within the crystal hosts, and demonstrate sufficiently tight packing to enable energy transfer between a donor−acceptor pair. His-tagged fluorescent proteins display remarkable thermal stability to denaturation over extended periods of time (days) at high temperatures when within the crystals. Ultimately, this strategy may prove useful for the prolonged storage of thermally sensitive biopolymer guests within a 3D crystalline matrix.

Recent advances in coiled-coil peptide materials and their biomedical applications

“…31 Chmielewski and coworkers have successfully used metalligand interactions for assembly using a trimeric coiled coil building block based on the GCN4 sequence. [32][33][34] This design is different from the above work of the Ogawa in that the ligands for metal ions are at the termini of the peptide to promote head-to-tail assembly, rather than at a central heptad of the coiled-coil, and a trimeric module was used. 32 The peptide (p2L) contained nitrilotriacetic acid (NTA) and di-histidine ligands at the N-and C-termini, respectively.…”

“…8A). 34 In an alternate strategy, Clark and coworkers stabilized citrate synthase (CS) by incorporating the enzyme covalently in coiled-coil filaments. 60 These filaments were composed of the coiled-coil peptide building blocks EE and KK derived from the γPFD protein.…”

Spatially Patterned, Porous Protein Crystals as Multifunctional Materials

“…The design of crystalline substances with new structural, chemical, and physical attributes is a major goal in materials science and engineering. − Although the primary utility of protein crystals has historically been in crystallographic structure determination, they are now also being recognized as functional solid-state materials. − Protein crystals typically have high porosities with a typical water content of 30–80%, sometimes even exceeding 90%. ,, Crystallization generally increases the stability of the proteins, , and with proper modifications (e.g., chemical crosslinking , ), protein crystals can even be rendered resistant to denaturation in organic solvents. ,, Importantly, protein (or peptide) crystals can be readily modified with organic, inorganic, or biological functionalities to augment their inherent functions, enabling diverse applications in biocatalysis, separation, and therapeutics. ,,− …”