Analysis and Control of Chain Mobility in Protein Hydrogels

Abstract

Coiled-coil domains can direct the assembly of protein block copolymers into physically crosslinked, viscoelastic hydrogels. Here we describe the use of fluorescence recovery after photobleaching (FRAP) to probe chain mobility in reversible hydrogels assembled from engineered proteins bearing terminal coiled-coil domains. We show that chain mobility can be related to the underlying dynamics of the coiled-coil domains by application of a 3-state "hopping" model of chain migration. We further show that genetic programming allows the effective mobility of network chains to be varied 500-fold through modest changes in protein sequence. Destabilization of the coiled-coil domains by site-directed mutagenesis increases the effective diffusivity of probe chains. Conversely, probe mobility is reduced by expanding the hydrophobic surface area of the coiled-coil domains through introduction of the bulky leucine surrogate homoisoleucine. Predictions from the 3-state model imply asymmetric sequential binding of the terminal domains. Brownian Dynamics simulations suggest that binding asymmetry is a general feature of reversible gels, arising from a loss in entropy as chains transition to a conformationally restricted bridged state.

Mechanisms of Diffusion in Associative Polymer Networks: Evidence for Chain Hopping

“…4 assumes that reversible binding of the chains is fast relative to the time scale of free diffusion during the FRAP experiment, which we validated previously for PEnP gels. 25 For the experimental regime probed here, we estimate that kon * a 2 / D0 ≈ 10 2 -10 3 for the case when the bleach spot radius a = 10 µm, and kon * is the (concentration-dependent) pseudo-first-order association rate constant. Eq.…”

Nucleopore-Inspired Polymer Hydrogels for Selective Biomolecular Transport

“…In contrast, nonspecific, inert biomolecules (I) only undergo Fickian diffusion. Similar binding−diffusion models that have been applied to study the impact of protein binding interactions on transport behavior assume that bound-state diffusivity is negligible on the time-and length-scales of interest; 29,33 however, it has been shown that a mobile bound state is necessary to fully capture diffusive dynamics in experiments and simulations. 20,32 The two-state model is solved analytically as a system of second-order differential equations by considering steady-state operation of a 1D polymer membrane of thickness L (Figure 1b), in the limit of dilute biomolecular solutes in comparison to polymer network recognition sites (constant [S]).…”

“…27,28 Recently, molecular-based transport theories have reconsidered the role of biomolecular binding kinetics on diffusivity in the context of intracellular mobility, 29,30 lateral biomolecular transport at surfaces, 18,19,31 and self-diffusion in associative protein hydrogels. 20,32,33 Aspects of these binding−diffusion models can be applied to understand biomolecular solutes that interact with a polymeric hydrogel and to generate designs for selectively permeable hydrogels.…”

Measuring the effects of α1‐antitrypsin polymerisation on the structure and biophysical properties of the endoplasmic reticulum

“…However, the physical nature of the hydrogel has complex effects on the movement of proteins, since it can both hinder diffusion by acting as a partial barrier, but also speed the movement of large molecules through size exclusion effects. Existing techniques, such as FRAP and fluorescence anisotropy have been used to probe the effects of some physical parameters of hydrogels, such as polymer density and cross‐linking, on rotational and translational diffusion of model proteins, but important questions remain [Bertz et al., ; Rapp et al., ]. To what extent, for example, is protein mobility affected by changes to ER microviscosity rather than through effects on protein binding or crowding?…”