Engineering shape memory and morphing protein hydrogels based on protein unfolding and folding

Engineering shape memory/morphing materials have achieved considerable progress in polymer-based systems with broad potential applications. However, engineering protein-based shape memory/morphing materials remains challenging and under-explored. Here we report the design of a bilayer protein-based shape memory/morphing hydrogel based on protein folding-unfolding mechanism. We fabricate the protein-bilayer structure using two tandem modular elastomeric proteins (GB1)8 and (FL)8. Both protein layers display distinct denaturant-dependent swelling profiles and Young’s moduli. Due to such protein unfolding-folding induced changes in swelling, the bilayer hydrogels display highly tunable and reversible bidirectional bending deformation depending upon the denaturant concentration and layer geometry. Based on these programmable and reversible bending behaviors, we further utilize the protein-bilayer structure as hinge to realize one-dimensional to two-dimensional and two-dimensional to three-dimensional folding transformations of patterned hydrogels. The present work will offer new inspirations for the design and fabrication of novel shape morphing materials.

Monitoring protein unfolding transitions by NMR-spectroscopy

NMR-spectroscopy has certain unique advantages for recording unfolding transitions of proteins compared e.g. to optical methods. It enables per-residue monitoring and separate detection of the folded and unfolded state as well as possible equilibrium intermediates. This allows a detailed view on the state and cooperativity of folding of the protein of interest and the correct interpretation of subsequent experiments. Here we summarize in detail practical and theoretical aspects of such experiments. Certain pitfalls can be avoided, and meaningful simplification can be made during the analysis. Especially a good understanding of the NMR exchange regime and relaxation properties of the system of interest is beneficial. We show by a global analysis of signals of the folded and unfolded state of GB1 how accurate values of unfolding can be extracted and what limits different NMR detection and unfolding methods. E.g. commonly used exchangeable amides can lead to a systematic under determination of the thermodynamic protein stability. We give several perspectives of how to deal with more complex proteins and how the knowledge about protein stability at residue resolution helps to understand protein properties under crowding conditions, during phase separation and under high pressure.

Nano Differential Scanning Fluorimetry-Based Thermal Stability Screening and Optimal Buffer Selection for Immunoglobulin G

Nano differential scanning fluorimetry (nanoDSF) is a high-throughput protein stability screening technique that simultaneously monitors protein unfolding and aggregation properties. The thermal stability of immunoglobulin G (IgG) was investigated in three different buffers (sodium acetate, sodium citrate, and sodium phosphate) ranging from pH 4 to 8. In all three buffers, the midpoint temperature of thermal unfolding (Tm) showed a tendency to increase as the pH increased, but the aggregation propensity was different depending on the buffer species. The best stability against aggregation was obtained in the sodium acetate buffers below pH 4.6. On the other hand, IgG in the sodium citrate buffer had higher aggregation and viscosity than in the sodium acetate buffer at the same pH. Difference of aggregation between acetate and citrate buffers at the same pH could be explained by a protein–protein interaction study, performed with dynamic light scattering, which suggested that intermolecular interaction is attractive in citrate buffer but repulsive in acetate buffer. In conclusion, this study indicates that the sodium acetate buffer at pH 4.6 is suitable for IgG formulation, and the nanoDSF method is a powerful tool for thermal stability screening and optimal buffer selection in antibody formulations.

Missense Mutations of Codon 116 in the SOD1 Gene Cause Rapid Progressive Familial ALS and Predict Short Viability With PMA Phenotype

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease, characterized by a great variety of both clinical presentations and genetic causes. Previous studies had identified two different missense mutations in SOD1 (p.R116C and p.R116G) causing familial ALS. In this study, we report a novel heterozygous missense mutation in the SOD1 gene (p.R116S) in a family with inherited ALS manifested as fast-deteriorating pure lower motor neuron symptoms. The patient displayed similar clinical picture and prognostic value to previous reported cases with different R116 substitution mutations. Modeling of all R116 substitutions in the resolved SOD1 protein structure revealed a shared mechanism with destroyed hydrogen bonds between R116 and other two residues, which might lead to protein unfolding and oligomer formation, ultimately conferring neurotoxicity.

Revealing Topological Barriers against Knot Untying in Thermal and Mechanical Protein Unfolding by Molecular Dynamics Simulations

The knot is one of the most remarkable topological features identified in an increasing number of proteins with important functions. However, little is known about how the knot is formed during protein folding, and untied or maintained in protein unfolding. By means of all-atom molecular dynamics simulation, here we employ methyltransferase YbeA as the knotted protein model to analyze changes of the knotted conformation coupled with protein unfolding under thermal and mechanical denaturing conditions. Our results show that the trefoil knot in YbeA is occasionally untied via knot loosening rather than sliding under enhanced thermal fluctuations. Through correlating protein unfolding with changes in the knot position and size, several aspects of barriers that jointly suppress knot untying are revealed. In particular, protein unfolding is always prior to knot untying and starts preferentially from separation of two α-helices (α1 and α5), which protect the hydrophobic core consisting of β-sheets (β1–β4) from exposure to water. These β-sheets form a loop through which α5 is threaded to form the knot. Hydrophobic and hydrogen bonding interactions inside the core stabilize the loop against loosening. In addition, residues at N-terminal of α5 define a rigid turning to impede α5 from sliding out of the loop. Site mutations are designed to specifically eliminate these barriers, and easier knot untying is achieved under the same denaturing conditions. These results provide new molecular level insights into the folding/unfolding of knotted proteins.

Unfolding is the driving force for mitochondrial import and degradation of Parkinson's disease-related protein DJ-1

Diverse genes associated with familial Parkinson's disease (familial Parkinsonism) have been implicated in mitochondrial quality control. One such gene, PARK7 encodes the protein DJ-1, pathogenic mutations of which trigger its translocation from the cytosol to the mitochondrial matrix. The translocation of steady-state cytosolic proteins like DJ-1 to the mitochondrial matrix by missense mutations is rare and the underlying mechanism remains to be elucidated. Here, we show that the protein unfolding associated with various DJ-1 mutations drives its import into the mitochondrial matrix. Increasing the structural stability of these DJ-1 mutants restores cytosolic localization. Mechanistically, we show that a reduction in the structural stability of DJ-1 exposes a cryptic N-terminal mitochondrial targeting signal (MTS) including Leu10 that promotes DJ-1 import into the mitochondrial matrix for subsequent degradation. Our work describes a novel cellular mechanism for targeting a destabilized cytosolic protein to the mitochondria for degradation.