Benchmarking of Cph1 Mutants and DrBphP for Light‐Responsive Phytochrome‐Based Hydrogels with Reversibly Adjustable Mechanical Properties

Abstract

In the rapidly expanding field of molecular optogenetics, the performance of the engineered systems relies on the switching properties of the underlying genetically encoded photoreceptors. In this study, the bacterial phytochromes Cph1 and DrBphP are engineered, recombinantly produced in Escherichia coli, and characterized regarding their switching properties in order to synthesize biohybrid hydrogels with increased light‐responsive stiffness modulations. The R472A mutant of the cyanobacterial phytochrome 1 (Cph1) is identified to confer the phytochrome‐based hydrogels with an increased dynamic range for the storage modulus but a different light‐response for the loss modulus compared to the original Cph1‐based hydrogel. Stiffness measurements of human atrial fibroblasts grown on these hydrogels suggest that differences in the loss modulus at comparable changes in the storage modulus affect cell stiffness and thus underline the importance of matrix viscoelasticity on cellular mechanotransduction. The hydrogels presented here are of interest for analyzing how mammalian cells respond to dynamic viscoelastic cues. Moreover, the Cph1‐R472A mutant, as well as the benchmarking of the other phytochrome variants, are expected to foster the development and performance of future optogenetic systems.

Extracellular Optogenetics at the Interface of Synthetic Biology and Materials Science

“…Spatial ( Wu et al (2018) ; Hörner et al (2019) ; Liu L. et al (2018) ) and temporal ( Hörner et al (2019) ; Wu et al (2018) ; Liu L. et al (2018) ; Narayan et al (2021) ; Jiang et al (2020) ; Xiang et al (2020) ; Wang et al (2017) ; Hopkins et al (2021) ; Duan et al (2021) ; Yang et al (2022) ; Emig et al (2022) ) control of OptoGel stiffness has been demonstrated using atomic force microscopy (AFM) and/or rheology ( Figure 3A,B ). Two commonly reported rheological quantities are storage modulus ( G ′) and loss modulus ( G ″ ) describing elastic and viscous responses of a material, respectively.…”

“…Reversible mechanical and rheological properties offer additional dimensions of light-programmable control. Reversibility with negligible hysteresis has been reported for many OptoGels, though the number of ON/OFF cycles tested has ranged from less than five to over 100 ( Zhang et al (2015) ; Wu et al (2018) ; Liu L. et al (2018) ; Hörner et al (2019) ; Hopkins et al (2021) ; Duan et al (2021) ; Emig et al (2022) ). Reversibility has also been observed in absorbance measurements with spectrophotometry ( Figure 3E ).…”

“…There are three main building blocks in what we call an OptoGel: a photoswitchable protein, a conjugation system, and a polymer network, with a few exceptions. Optoproteins have been incorporated into networks with synthetic polymers ( Hörner et al (2019) ; Wu et al (2018) ; Liu L. et al (2018) ; Xiang et al (2020) ; Hammer et al (2020) ; Emig et al (2022) ), natural polymers ( Zhang et al (2015) ; Hopkins et al (2021) ; Narayan et al (2021) ), and optoprotein-based OptoGels without additional polymer networks ( Duan et al (2021) ; Wang et al (2017) ; Lyu et al (2017) ; Jiang et al (2020) ; Yang et al (2022) ). Conjugation chemistries that do not disrupt optoprotein function make combinatorial assembly efficient.…”

“…Multiple other conjugation chemistries have been reported in the literature. Cph1-based OptoGels used cysteine thiols to bind vinyl-sulfone (VS.) groups on functionalized PEG ( Hörner et al (2019) ; Emig et al (2022) ). UVR8-based OptoGels used hexa-peptides to bind tax-interacting proteins on supramolecular nanofibers ( Zhang et al (2015) ).…”

“…Other studies have begun to apply OptoGels to understand the role of dynamical mechanical cues on cell migration and differentiation, taking advantage of the OptoGels’ spatiotemporal control. Nanoindentations of cells grown on Cph1-based OptoGels under different illumination conditions showed that both storage and loss moduli of the underlaying gel influence cell stiffening ( Emig et al (2022) ). Dronpa-based OptoGels have been used to dynamically control cell migration rates by tuning substrate stiffness; increasing ECM crosslink density may speed up wound healing ( Wu et al (2018) ).…”