Molecular tandem repeat strategy for elucidating mechanical properties of high-strength proteins

Abstract

Many globular and structural proteins have repetitions in their sequences or structures. However, a clear relationship between these repeats and their contribution to the mechanical properties remains elusive. We propose a new approach for the design and production of synthetic polypeptides that comprise one or more tandem copies of a single unit with distinct amorphous and ordered regions. Our designed sequences are based on a structural protein produced in squid suction cups that has a segmented copolymer structure with amorphous and crystalline domains. We produced segmented polypeptides with varying repeat number, while keeping the lengths and compositions of the amorphous and crystalline regions fixed. We showed that mechanical properties of these synthetic proteins could be tuned by modulating their molecular weights. Specifically, the toughness and extensibility of synthetic polypeptides increase as a function of the number of tandem repeats. This result suggests that the repetitions in native squid proteins could have a genetic advantage for increased toughness and flexibility.tandem repeat | high strength | protein | thermoplastic | squid ring teeth P roteins are heteropolymers that provide a variety of building blocks for designing biological materials (1). Proteins have several advantages as natural materials: (i) their chain length, sequence, and stereochemistry can be easily controlled, (ii) the molecular structure of proteins is well-defined (e.g., secondary, tertiary, and quaternary structures), (iii) they provide a variety of functional chemistries for conjugation to other biomolecules or polymers, and (iv) they can be designed to exhibit a variety of physical properties (2). Proteins are diverse but often display substantial similarity in sequence and 3D structure. Duplication of structural units is a natural evolutionary strategy for increasing the complexity of both globular and fibrous/ structural proteins (3). For example, collagen has polyproline-and glycine-rich helices, whereas silk and elastin have β-spiral [GPGXX], linker [GP(S,Y,G)], and 3 10 -helix [GGX] repeats. These repetitions are advantageous because of the intrinsic promotion of stability through the periodic recurrence of favorable interactions (4-7).A new family of repetitive structural proteins was recently identified in the tentacles of several squid species (8, 9). Squid have teeth-like structures inside their suckers that allow the animals to grip tightly on a diverse array of objects (10). Using the tools of molecular biology and proteomics, it has been shown that these squid ring teeth (SRT) proteins have segmented semicrystalline morphology with repetitive amorphous and crystalline domains. SRT-based materials were shown to have high elastic modulus: 4-8 GPa in air and 2-4 GPa underwater below the glass transition temperature (11). However, a clear relationship between the molecular structure and the mechanical properties of this material remains elusive. This problem is complex, because SRT proteins are polydispersed i...

Programmable molecular composites of tandem proteins with graphene oxide for efficient bimorph actuators

“…In light of these studies, it has been recently demonstrated that it is possible to control and improve the physical properties (i.e., mechanical and thermal) of SRT-based biopolymers using a specific polypeptide sequence for both crystalline and amorphous regions. [21] Heterologous expression of these tandem repeat proteins in bacteria has enabled bulk synthesis of these unique biopolymers ( Figure 1B). Genes for these tandem-repeat SRT proteins are synthesized via a modified rolling-circle-amplification method.…”

“…Tandem repeat proteins was prepared following protocol reported by Jung et al [21] Protein expression is initiated by a single colony, which was inoculated and grown overnight in 5mL of LB with ampicillin (100 µg/mL). The overnight culture were scaled up to 2 L (i.e., four 500mL LB media) and was grown on a shaker at 210 rpm and 37°C for 5 hours.…”

“…Gaussian curve fitting was performed as described elsewhere. [21] Thermogravimetric analysis (TGA) was performed on a TA instruments Q50 coupled to a Pfeiffer vacuum mass spectrometer. For each measurement samples were initially left for equilibration for 30 minutes.…”

“…The crystal dimensions of these repetitive TR proteins are relatively independent of the number of repeat units. [21] The crystal domains of these TR proteins consist of anti-parallel β-sheets formed by 4 strands of the crystalline polypeptide sequence. This βsheet orientation results in a crystal 3 nm long in the polypeptide-backbone direction and 2 nm wide in the hydrogen-bonding direction.…”

Bioinspired Load‐Bearing Hydrogel Based on Engineered Sea Anemone Skin‐Derived Collagen‐Like Protein

“…This manipulability enables the mechanical properties of protein‐based materials to be improved in a manner analogous to synthetic polymers. Many repeat sequences have been chosen as building blocks and are repeated to generate high molecular weight and enhance mechanical properties . Genetically engineered silk‐like proteins (GAGAS of silkworm and GPGXX of spider), elastin‐like protein (GVGVP), and resilin (GGRPSDSYGAPGGGN) are representative examples of repeat sequences that have been engineered for scaffolding biomaterials based on high tensile strength and/or deformability.…”