Computational Prediction of Coiled–Coil Protein Gelation Dynamics and Structure

Abstract

Protein hydrogels represent an important and growing biomaterial for a multitude of applications, including diagnostics and drug delivery. We have previously explored the ability to engineer the thermoresponsive supramolecular assembly of coiled−coil proteins into hydrogels with varying gelation properties, where we have defined important parameters in the coiled−coil hydrogel design. Using Rosetta energy scores and Poisson−Boltzmann electrostatic energies, we iterate a computational design strategy to predict the gelation of coiled−coil proteins while simultaneously exploring five new coiled−coil protein hydrogel sequences. Provided this library, we explore the impact of in silico energies on structure and gelation kinetics, where we also reveal a range of blue autofluorescence that enables hydrogel disassembly and recovery. As a result of this library, we identify the new coiled−coil hydrogel sequence, Q5, capable of gelation within 24 h at 4 °C, a more than 2-fold increase over that of our previous iteration Q2. The fast gelation time of Q5 enables the assessment of structural transition in real time using small-angle X-ray scattering (SAXS) that is correlated to coarse-grained and atomistic molecular dynamics simulations revealing the supramolecular assembling behavior of coiled−coils toward nanofiber assembly and gelation. This work represents the first system of hydrogels with predictable self-assembly, autofluorescent capability, and a molecular model of coiled−coil fiber formation.

Coiled-Coil Protein Hydrogels Engineered with Minimized Fiber Diameters for Sustained Release of Doxorubicin in Triple-Negative Breast Cancer

“…The substantial decrease in surface electrostatic potential energy was also apparent in the electrostatic potential map of Q8 compared to that of Q4 and Q (Figure d). To generate a best pose from an equal starting point to previous Q variant models for comparison, a best scoring Q8 pose of −607 kcal/mol was generated from mutating the original Q input sequence …”

“…Q8 was designed using a modified version of a trimodal Monte Carlo Search developed previously . The Rosetta suite of macromolecular modeling tools (Version 3.5) was used to model protein mutants and calculate Rosetta scores, with lower energy scores indicating higher stability, using the Rosetta Relax protocol with the all-atom energy score function .…”

“…Q8 protein was purified as described previously . First, pellets were lysed in 40 mL of buffer A (50 mM Tris-HCl and 500 mM NaCl; pH 8.0) using a Q500 probe sonicator (QSonica) at 55% amplitude for 2 min with 5 s on and 5 s off pulses on ice.…”

“…The His-tag of the protein was retained following purification, as done with previous Q protein variants. Because the His-tag provides a large contribution to the large N-terminal electrostatic potential, it is considered critical to the supramolecular assembly and is considered in the predictive modeling of ΔEE bcf …”

“…Transmission electron microscopy (TEM) was performed as done previously , to assess hydrogel fiber diameter, morphology, and cross-linking. TEM images were taken with an FEI Talos L120C transmission electron microscope using samples on a Formvar/carbon-coated copper grid.…”