Computational Prediction of Coiled-Coil Protein Gelation Dynamics and Structure.

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.

Supramolecular Assembly and Small-Molecule Binding by Protein-Engineered Coiled-Coil Fibers.

The ability to engineer a solvent-exposed surface of self-assembling coiled coils allows one to achieve a higher-order hierarchical assembly such as nano- or microfibers. Currently, these materials are being developed for a range of biomedical applications, including drug delivery systems; however, ways to mechanistically optimize the coiled-coil structure for drug binding are yet to be explored. Our laboratory has previously leveraged the functional properties of the naturally occurring cartilage oligomeric matrix protein coiled coil (C), not only for its favorable motif but also for the presence of a hydrophobic pore to allow for small-molecule binding. This includes the development of Q, a rationally designed pentameric coiled coil derived from C. Here, we present a small library of protein microfibers derived from the parent sequences of C and Q bearing various electrostatic potentials with the aim to investigate the influence of higher-order assembly and encapsulation of candidate small molecule, curcumin. The supramolecular fiber size appears to be well-controlled by sequence-imbued electrostatic surface potential, and protein stability upon curcumin binding is well correlated to relative structure loss, which can be predicted by in silico docking.

Engineered multivalent self-assembled binder protein against SARS-CoV-2 RBD.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic since December 2019, and with it, a push for innovations in rapid testing and neutralizing antibody treatments in an effort to solve the spread and fatality of the disease. One such solution to both of these prevailing issues is targeting the interaction of SARS-CoV-2 spike receptor binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2) receptor protein. Structural studies have shown that the N-terminal alpha-helix comprised of the first 23 residues of ACE2 plays an important role in this interaction. Where it is typical to design a binding domain to fit a target, we have engineered a protein that relies on multivalency rather than the sensitivity of a monomeric ligand to provide avidity to its target by fusing the N-terminal helix of ACE2 to the coiled-coil domain of the cartilage oligomeric matrix protein. The resulting ACE-MAP is able to bind to the SARS-CoV-2 RBD with improved binding affinity, is expressible in E. coli, and is thermally stable and relatively small (62 kDa). These properties suggest ACE-MAP and the MAP scaffold to be a promising route towards developing future diagnostics and therapeutics to SARS-CoV-2.

Protein-based lateral flow assays for COVID-19 detection.

To combat the enduring and dangerous spread of COVID-19, many innovations to rapid diagnostics have been developed based on proteinprotein interactions of the SARS-CoV-2 spike and nucleocapsid proteins to increase testing accessibility. These antigen tests have most prominently been developed using the lateral flow assay (LFA) test platform which has the benefit of administration at point-of-care, delivering quick results, lower cost, and does not require skilled personnel. However, they have gained criticism for an inferior sensitivity. In the last year, much attention has been given to creating a rapid LFA test for detection of COVID-19 antigens that can address its high limit of detection while retaining the advantages of rapid antibodyantigen interaction. In this review, a summary of these proteinprotein interactions as well as the challenges, benefits, and recent improvements to protein based LFA for detection of COVID-19 are discussed.

Effects of Ions and Small Compounds on the Structure of Aß42 Monomers.

Aggregation of amyloid-ß (Aß) proteins in the brain is a hallmark of Alzheimer's disease. This phenomenon can be promoted or inhibited by adding small molecules to the solution where Aß is embedded. These molecules affect the ensemble of conformations sampled by Aß monomers even before aggregation starts. Here, we perform extensive all-atom replica exchange molecular dynamics (REMD) simulations to provide a comparative study of the ensemble of conformations sampled by Aß42 monomers in solutions that promote (i.e., aqueous solution containing NaCl) and inhibit (i.e., aqueous solutions containing scyllo-inositol or 4-aminophenol) aggregation. Simulations performed in pure water are used as our reference. We find that secondary-structure content is only affected in an antagonistic manner by promoters and inhibitors at the C-terminus and the central hydrophilic core. Moreover, the end of the C-terminus binds more favorably to the central hydrophobic core region of Aß42 in NaCl adopting a type of strand-loop-strand structure that is disfavored by inhibitors. Nonpolar residues that form the dry core of larger aggregates of Aß42 (e.g., PDB ID 2BEG) are found at close proximity in these strand-loop-strand structures, suggesting that their formation could play an important role in initiating nucleation. In the presence of inhibitors, the C-terminus binds the central hydrophilic core with a higher probability than in our reference simulation. This sensitivity of the C-terminus, which is affected in an antagonistic manner by inhibitors and promoters, provides evidence for its critical role in accounting for aggregation.

Recent trends in peptide and protein-based hydrogels.

Hydrogels are classic examples of biomaterials that have found its niche in biomedical and allied fields. Here, we describe examples of peptide-based and protein-based hydrogels with a focus on smart gels that respond to various stimuli including temperature, pH, light, and ionic strength. With the recent advancements in computational modeling, it has been possible to predict as well as design peptide and protein sequences that can assemble into hydrogels with unique and improved properties. We briefly discuss coarse grained and atomistic simulations in designing peptides that can form hydrogels. In addition, we highlight the trends that will influence the future design and applications of hydrogels, with emphasis on bioadhesion, exosomes delivery, tissue and organoids engineering, and even intracellular production of gels.

Thermodynamic Stability of Polar and Nonpolar Amyloid Fibrils.

Thermodynamic stabilities of amyloid fibrils remain mostly unknown due to experimental challenges. Here, we combine enhanced sampling methods to simulate all-atom models in explicit water in order to study the stability of nonpolar (Aß16-21) and polar (IAPP28-33) fibrils. We find that the nonpolar fibril becomes more stable with increasing temperature, and its stability is dominated by entropy. In contrast, the polar fibril becomes less stable with increasing temperature, while it is stabilized by enthalpy. Our results show that the nature of side chains in the dry core of amyloid fibrils plays a dominant role in accounting for their thermodynamic stability.

Effects of Trimethylamine-N-oxide on the Conformation of Peptides and its Implications for Proteins.

To provide insights into the stabilizing mechanisms of trimethylamine-N-oxide (TMAO) on protein structures, we perform all-atom molecular dynamics simulations of peptides and the Trp-cage miniprotein. The effects of TMAO on the backbone and charged residues of peptides are found to stabilize compact conformations, whereas effects of TMAO on nonpolar residues lead to peptide swelling. This suggests competing mechanisms of TMAO on proteins, which accounts for hydrophobic swelling, backbone collapse, and stabilization of charge-charge interactions. These mechanisms are observed in Trp cage.

Thermodynamics of Aß16-21 dissociation from a fibril: Enthalpy, entropy, and volumetric properties.

Here, we provide insights into the thermodynamic properties of A ß16-21 dissociation from an amyloid fibril using all-atom molecular dynamics simulations in explicit water. An umbrella sampling protocol is used to compute potentials of mean force (PMF) as a function of the distance ξ between centers-of-mass of the A ß16-21 peptide and the preformed fibril at nine temperatures. Changes in the enthalpy and the entropic energy are determined from the temperature dependence of these PMF(s) and the average volume of the simulation box is computed as a function of ξ. We find that the PMF at 310 K is dominated by enthalpy while the entropic energy does not change significantly during dissociation. The volume of the system decreases during dissociation. Moreover, the magnitude of this volume change also decreases with increasing temperature. By defining dock and lock states using the solvent accessible surface area (SASA), we find that the behavior of the electrostatic energy is different in these two states. It increases (unfavorable) and decreases (favorable) during dissociation in lock and dock states, respectively, while the energy due to Lennard-Jones interactions increases continuously in these states. Our simulations also highlight the importance of hydrophobic interactions in accounting for the stability of A ß16-21.

Role of side-chain interactions on the formation of α-helices in model peptides.

The role played by side-chain interactions on the formation of α-helices is studied using extensive all-atom molecular dynamics simulations of polyalanine-like peptides in explicit TIP4P water. The peptide is described by the OPLS-AA force field except for the Lennard-Jones interaction between Cß-Cß atoms, which is modified systematically. We identify values of the Lennard-Jones parameter that promote α-helix formation. To rationalize these results, potentials of mean force (PMF) between methane-like molecules that mimic side chains in our polyalanine-like peptides are computed. These PMF exhibit a complex distance dependence where global and local minima are separated by an energy barrier. We show that α-helix propensity correlates with values of these PMF at distances corresponding to Cß-Cß of i-i+3 and other nearest neighbors in the α-helix. In particular, the set of Lennard-Jones parameters that promote α-helices is characterized by PMF that exhibit a global minimum at distances corresponding to i-i+3 neighbors in α-helices. Implications of these results are discussed.