Programmable viscoelastic matrices from artificial proteins

Extracellular matrix compliance influences cellular adhesion and migration, proliferation and apoptosis, and differentiation. Much of our current knowledge of the effects of substrate stiffness on cellular behavior is based on elastic substrates, in particular cross‐linked polyacrylamide hydrogels. Biological tissues, however, are viscoelastic and exhibit stress relaxation and energy dissipation on physiologically relevant timescales. While emerging evidence suggests that these physical properties also influence cellular behavior, materials in which viscoelasticity can be precisely engineered are currently lacking. Here, we describe programmable hydrogel matrices assembled from artificial recombinant proteins designed to be cross‐linked by covalent bonds involving cysteine residues, by association of helical domains as coiled coils, or by both mechanisms. Using these proteins, we construct chemical, physical, and chemical‐physical hydrogel networks that deform elastically or viscoelastically depending on the type of cross‐linking (Dooling et al., Adv. Mater., 2016, 28, 4651–4657). In viscoelastic networks, the amount of stress relaxation is tuned by controlling the ratio of physical cross‐linking to chemical crosslinking, and the timescale for stress relaxation is tuned over five orders of magnitude by single point mutations to the coiled‐coil physical cross‐linking domain (Dooling and Tirrell, ACS Cent. Sci., 2016, 2, 812–819). The genetic engineering approach also allows biological activity to be encoded directly within the protein sequence in the form of cell‐adhesive domains and proteolytic cleavage sites. The capacity to program the viscoelasticity and biological activity of hydrogel matrices is anticipated to have applications in studying and engineering cell‐matrix interactions

Identification of an expanded set of translationally active methionine analogues in Escherichia coli

Amino acid incorporation into proteins in vivo is controlled most stringently by the aminoacyl-tRNA synthetases. Here we report the incorporation of several new methionine analogues into protein by increasing the rate of their activation by the methionyl-tRNA synthetase (MetRS) of Escherichia coli. cis-Crotylglycine (4), 2-aminoheptanoic acid (7), norvaline (8), 2-butynylglycine (11), and allylglycine (12) will each support protein synthesis in methionine-depleted cultures of E. coli when MetRS is overexpressed and the medium is supplemented with the analogue at millimolar concentrations. These investigations suggest important opportunities for protein engineering, as expansion of the translational apparatus toward other amino acid analogues by similar strategies should also be possible

New Goals for Polymer Synthesis [translated from Japanese]

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Artificial Protein Hydrogel Materials

Recombinant DNA methods were used to create a new class of artificial proteins that undergo reversible gelation in response to changes in pH or temperature. These proteins consist of terminal a-helical "leucine zipper" domains flanking a central, water-soluble polyelectrolyte segment. The formation of coiled-coil aggregates of the terminal domains in near-neutral pH solution triggers formation of a polymer hydrogel, with the central polyelectrolyte segment retaining solvent and preventing precipitation of the chains. Dissociation of the coiled-coil aggregates through elevation of pH or temperature causes dissolution of the gel and a return to the viscous behavior characteristic of a polymer solution. The pH and temperature range of the hydrogel state and its viscoelastic properties may be systematically varied through precise changes of the length, composition and charge density of the terminal and central blocks. Such control is of value in designing hydrogels with predetermined physical properties and makes these biosynthetic triblock copolymer systems attractive candidates for use in molecular and cellular encapsulation and in controlled reagent delivery

Cleavable Biotin Probes for Labeling of Biomolecules via Azide−Alkyne Cycloaddition

The azide−alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide−alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na_2S_2O_4, 2% HOCH_2CH_2SH, 10% HCO_2H, 95% CF_3CO_2H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO_2H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies

Evolution of a fluorinated green fluorescent protein

The fluorescence of bacterial cells expressing a variant (GFPm) of the green fluorescent protein (GFP) was reduced to background levels by global replacement of the leucine residues of GFPm by 5,5,5-trifluoroleucine. Eleven rounds of random mutagenesis and screening via fluorescence-activated cell sorting yielded a GFP mutant containing 20 amino acid substitutions. The mutant protein in fluorinated form showed improved folding efficiency both in vivo and in vitro, and the median fluorescence of cells expressing the fluorinated protein was improved {approx}650-fold in comparison to that of cells expressing fluorinated GFPm. The success of this approach demonstrates the feasibility of engineering functional proteins containing many copies of abiological amino acid constituents

Yielding Behavior in Injectable Hydrogels from Telechelic Proteins

Injectable hydrogels show substantial promise for use in minimally invasive tissue engineering and drug delivery procedures. A new injectable hydrogel material, developed from recombinant telechelic proteins expressed in E. coli, demonstrates shear thinning by 3 orders of magnitude at large strains. Large-amplitude oscillatory shear illustrates that shear thinning is due to yielding within the bulk of the gel, and the rheological response and flow profiles are consistent with a shear-banding mechanism for yielding. The sharp yielding transition and large magnitude of the apparent shear thinning allow gels to be injected through narrow gauge needles with only gentle hand pressure. After injection the gels reset to full elastic strength in seconds due to rapid re-formation of the physical network junctions, allowing self-supporting structures to be formed. The shear thinning and recovery behavior is largely independent of the midblock length, enabling genetic engineering to be used to control the equilibrium modulus of the gel without loss of the characteristic yielding behavior. The shear-banding mechanism localizes deformation during flow into narrow regions of the gels, allowing more than 95% of seeded cells to survive the injection process

Genetic Synthesis of Periodic Protein Materials

Genetic engineering offers a novel approach to the development of advanced polymeric materials, in particular protein-based materials. Biological synthesis provides levels of control of polymer chain architecture that cannot yet be attained by current methods of chemical synthesis. In addition to employing naturally occurring genetic templates artificial genes can be designed to encode completely new materials with customized properties. In the present paper we: 1) review the concepts and technology of creating protein-based materials by genetic engineering, 2) discuss the merits of producing crystalline lamellar proteins by this approach, and 3) review progress made by our group in generating such materials by genetic strategies. Full descriptions appear elsewhere about the parameters to be considered in designing artificial protein genes of this type, the effectiveness of different gene construction and expression strategies utilized by us thus far and, the specific properties of the various materials derived from these efforts (1,2). Progress made by other groups involved in developing periodic proteins by molecular biological strategies are described in refs. 3-8. The latter studies include genetic engineering of artificial silk-like proteins (3,4), poly-aspartylphenylalanine (5), an α/β barrel domain (octarellin; 6), the collagen tripeptide GlyProPro (7) and human tropoelastin (8). Advances with the silk-like proteins (SLP) have been particularly impressive. In addition to producing multi-gram quantities of pure SLP homopolymers, this group has successfully generated block copolymers of SLP interspersed with core peptides of mammalian elastin and the human fibronectin cell attachment element. While publications are still lacking it appears that a numiber of groups are striving to create genetically engineered variants of the repetitive bioadhesive proteins produced by mussels and barnacles (9)

A BODIPY-Cyclooctyne for Protein Imaging in Live Cells

Cellular proteins that bear reactive azides can be imaged by fluorescence microscopy following strain-promoted ligation to cyclooctyne dyes. Here we describe BODIPY-cyclooctyne (BDPY), a membrane-permeant fluorophore that can be used to label intracellular proteins in live mammalian cells. Flow cytometry reveals fluorescence signals more than 25-fold above background after labeling of azide-tagged cells with BDPY