Assembly Requirements for the Construction of Large-Scale Binary Protein Structures.

The precise assembly of multiple biomacromolecules into well-defined structures and materials is of great importance for various biomedical and nanobiotechnological applications. In this study, we investigate the assembly requirements for two-component materials using charged protein nanocages as building blocks. To achieve this, we designed several variants of ferritin nanocages to determine the surface characteristics necessary for the formation of large-scale binary three-dimensional (3D) assemblies. These nanocage variants were employed in protein crystallization experiments and macromolecular crystallography analyses, complemented by computational methods. Through the screening of nanocage variant combinations at various ionic strengths, we identified three essential features for successful assembly: (1) the presence of a favored crystal contact region, (2) the presence of a charged patch not involved in crystal contacts, and (3) sufficient distinctiveness between the nanocages. Surprisingly, the absence of noncrystal contact mediating patches had a detrimental effect on the assemblies, highlighting their unexpected importance. Intriguingly, we observed the formation of not only binary structures but also both negatively and positively charged unitary structures under previously exclusively binary conditions. Overall, our findings will inform future design strategies by providing some design rules, showcasing the utility of supercharging symmetric building blocks in facilitating the assembly of biomacromolecules into large-scale binary 3D assemblies.

Overcoming Non-Specific Interactions for Efficient Encapsulation of Doxorubicin in Ferritin Nanocages for Targeted Drug Delivery.

Due to its beneficial pharmacological properties, ferritin (Ftn) is considered as an interesting drug delivery vehicle to alleviate the cardiotoxicity of doxorubicin (DOX) in chemotherapy. However, the encapsulation of DOX in Ftn suffers from heavy precipitation and low protein recovery yield which limits its full potential. Here, a new DOX encapsulation strategy by cysteine-maleimide conjugation is proposed. In order to demonstrate that this strategy is more efficient compared to the other approaches, DOX is encapsulated in Ftn variants carrying different surface charges. Furthermore, in contrast to the common belief, this data show that DOX molecules are also found to bind non-specifically to the surface of Ftn. This can be circumvented by the use of Tris(2-carboxyethyl)phosphine (TCEP) during encapsulation or by washing with acidic buffer. The biocompatibility studies of the resulting DOX Ftn variants in MCF-7 and MHS cancer cells shows a complex relationship between the cytotoxicity, the DOX loading and the different surface charges of Ftn. Further investigation on the cell uptake mechanism provides reasonable explanations for the cytotoxicity results and reveals that surface charging of Ftn hinders its transferrin receptor 1 (TfR-1) mediated cellular uptake in MCF-7 cells.

Molecular basis for protein-protein interactions.

This minireview provides an overview on the current knowledge of protein-protein interactions, common characterisation methods to characterise them, and their role in protein complex formation with some examples. A deep understanding of protein-protein interactions and their molecular interactions is important for a number of applications, including drug design. Protein-protein interactions and their discovery are thus an interesting avenue for understanding how protein complexes, which make up the majority of proteins, work.

Higher-Order Structures Based on Molecular Interactions for the Formation of Natural and Artificial Biomaterials.

The assembly of molecular building blocks into highly ordered structures is crucial, both in nature and for the development of novel functional materials. In nature, noncovalent interactions, such as hydrogen bonds or hydrophobic interactions, enable the reversible assembly of biopolymers, such as DNA or proteins. Inspired by these design principles, scientists have created biohybrid materials that employ natural building blocks and their assembly properties. Thus, structures and materials are attainable that cannot be made through other synthetic procedures. Herein, we review current concepts and highlight recent advances.

Metal-Assisted Assembly of Protein Containers Loaded with Inorganic Nanoparticles.

Protein containers are suitable building blocks for bioinorganic materials. Here, we show that high concentrations of magnesium ions induce the formation of a unitary protein scaffold, whereas low magnesium concentration leads to a binary protein scaffold. The molecular interactions in the protein scaffold were characterized with X-ray crystallography to high resolution. We show that the unitary framework can be applied for the assembly of inorganic nanoparticles such as metal oxides into highly ordered bioinorganic structures. Our work emphasizes the structural tunability of protein-container-based materials, important for adjusting emerging properties of such materials.

Free-Standing Metal Oxide Nanoparticle Superlattices Constructed with Engineered Protein Containers Show in Crystallo Catalytic Activity.

The construction of defined nanostructured catalysts is challenging. In previous work, we established a strategy to assemble binary nanoparticle superlattices with oppositely charged protein containers as building blocks. Here, we show that these free-standing nanoparticle superlattices are catalytically active. The metal oxide nanoparticles inside the protein scaffold are accessible for a range of substrates and show oxidase-like and peroxidase-like activity. The stable superlattices can be reused for several reaction cycles. In contrast to bulk nanoparticle-based catalysts, which are prone to aggregation and difficult to characterize, nanoparticle superlattices based on engineered protein containers provide an innovative synthetic route to structurally defined heterogeneous catalysts with control over nanoparticle size and composition.

Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase.

By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.

Binary Protein Crystals for the Assembly of Inorganic Nanoparticle Superlattices.

Biomolecules can act as functional templates for the organization of inorganic particles. Here we use two protein containers, engineered with opposite surface charge, as building blocks for the construction of a new type of biohybrid material. Binary structures with crystalline order were obtained, adopting a tetragonal lattice. Moreover, the cavity of the engineered protein containers can be filled with inorganic nanoparticles. The controlled assembly of these protein-nanoparticle composites yields highly ordered binary nanoparticle superlattices as free-standing crystals, with up to a few hundred micrometers in size. Because the structure and lattice parameters of the protein-nanoparticle crystals are independent of their nanoparticle cargo, the binary protein material may serve as a generally applicable matrix for the assembly of a variety of nanoparticles types.

Solid-state-based laser system as a replacement for Ar+ lasers.

We report on a solid-state-based laser system at 1028 nm. The light is generated by a diode laser seeded ytterbium fiber amplifier. In two build-up cavities, its frequency is doubled and quadrupled to 514 nm and 257 nm, respectively. At 514 nm, the system delivers up to 4.7 W of optical power. In the fourth harmonic, up to 173 mW are available limited by the nonlinear crystal. The frequency of the laser is mode-hop-free tunable by 16 GHz in 10 ms in the UV. Therefore, the system is suitable as a low maintenance, efficient, and tunable narrowband replacement for frequency doubled Ar+ laser systems.

A Promiscuous De†Novo Retro-Aldolase Catalyzes Asymmetric Michael Additions via Schiff Base Intermediates.

Recent advances in computational design have enabled the development of primitive enzymes for a range of mechanistically distinct reactions. Here we show that the rudimentary active sites of these catalysts can give rise to useful chemical promiscuity. Specifically, RA95.5-8, designed and evolved as a retro-aldolase, also promotes asymmetric Michael additions of carbanions to unsaturated ketones with high rates and selectivities. The reactions proceed by amine catalysis, as indicated by mutagenesis and X-ray data. The inherent flexibility and tunability of this catalyst should make it a versatile platform for further optimization and/or mechanistic diversification by directed evolution.

Construction of Matryoshka-type structures from supercharged protein nanocages.

Designing nanoscaled hierarchical structures with increasing levels of complexity is challenging. Here we show that electrostatic interactions between two complementarily supercharged protein nanocages can be effectively utilized to create nested Matryoshka-type structures. Cage-within-cage complexes containing spatially ordered iron oxide nanoparticles spontaneously self-assemble upon mixing positively supercharged ferritin compartments with AaLS-13, a larger shell-forming protein with a negatively supercharged lumen. Exploiting engineered Coulombic interactions and protein dynamics in this way opens up new avenues for creating hierarchically organized supramolecular assemblies for application as delivery vehicles, reaction chambers, and artificial organelles.

Stereochemical determinants of C-terminal specificity in PDZ peptide-binding domains: a novel contribution of the carboxylate-binding loop.

PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for ß-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

SARS-CoV-2 Mpro responds to oxidation by forming disulfide and NOS/SONOS bonds.

The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein's crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.

Structural basis for regulation of human glucokinase by glucokinase regulatory protein.

Glucokinase (GCK) is responsible for maintaining glucose homeostasis in the human body. Dysfunction or misregulation of GCK causes hyperinsulinemia, hypertriglyceridemia, and type 2 diabetes. In the liver, GCK is regulated by interaction with the glucokinase regulatory protein (GKRP), a 68 kDa polypeptide that functions as a competitive inhibitor of glucose binding to GCK. Formation of the mammalian GCK-GKRP complex is stimulated by fructose 6-phosphate and antagonized by fructose 1-phosphate. Here we report the crystal structure of the mammalian GCK-GKRP complex in the presence of fructose 6-phosphate at a resolution of 3.50 Å. The interaction interface, which totals 2060 Å(2) of buried surface area, is characterized by a small number of polar contacts and substantial hydrophobic interactions. The structure of the complex reveals the molecular basis of disease states associated with impaired regulation of GCK by GKRP. It also offers insight into the modulation of complex stability by sugar phosphates. The atomic description of the mammalian GCK-GKRP complex provides a framework for the development of novel diabetes therapeutic agents that disrupt this critical macromolecular regulatory unit.

Neural network enhanced aging time measurements of diary product remaining with infrared spectroscopy.

The determination of the drying degree of food residues on surfaces is an important step before efficient cleaning can be achieved. To accomplish this goal, a rapid evaluation based on a neural network and non-invasive measurement technique is introduced. Two common starch-based products and various yogurts from different manufacturers are used as example contaminants to determine the aging time of dried food residue. Near-infrared spectroscopy serves as a modern and fast measurement technique for investigating food compositions. Two analysis methods were compared for processing the measured near-infrared spectral data. The raw data were analyzed using partial least squares regression in conjunction with necessary preprocessing steps. As an alternative method, three different types of neural networks are employed. The aim of this approach is to compensate for the filtering steps before regression, which are typically necessary for multivariate regression. The challenge is to measure three different types of food and obtain a reliable prediction of moisture content in order to draw conclusions about the drying time. The experiments have shown that simple flat neural networks have similar accuracy compared to conventional regression. The use of a convolutional layer in advance demonstrates a significant improvement in prediction compared to other neural networks and even manages to surpass the accuracy of PLS regression. A network with a convolutional layer can also compensate for the sometimes strong variations between food types.

Crystalline Biohybrid Materials Based on Protein Cages.

Highly ordered superstructures of nanomaterials can be synthesized using protein cages as templates for the assembly of inorganic nanoparticles. Here, we describe in detail the creation of these biohybrid materials. The approach involves computational redesign of ferritin cages, followed by recombinant protein production and purification of the new variants. Metal oxide nanoparticles are synthesized inside the surface-charged variants. The composites are assembled using protein crystallization to yield highly ordered superlattices, which are characterized, for example, with small angle X-ray scattering. This protocol provides a detailed and comprehensive account on our newly established strategy for the synthesis of crystalline biohybrid materials.

Calpeptin is a potent cathepsin inhibitor and drug candidate for SARS-CoV-2 infections.

Several drug screening campaigns identified Calpeptin as a drug candidate against SARS-CoV-2. Initially reported to target the viral main protease (Mpro), its moderate activity in Mpro inhibition assays hints at a second target. Indeed, we show that Calpeptin is an extremely potent cysteine cathepsin inhibitor, a finding additionally supported by X-ray crystallography. Cell infection assays proved Calpeptin's efficacy against SARS-CoV-2. Treatment of SARS-CoV-2-infected Golden Syrian hamsters with sulfonated Calpeptin at a dose of 1 mg/kg body weight reduces the viral load in the trachea. Despite a higher risk of side effects, an intrinsic advantage in targeting host proteins is their mutational stability in contrast to highly mutable viral targets. Here we show that the inhibition of cathepsins, a protein family of the host organism, by calpeptin is a promising approach for the treatment of SARS-CoV-2 and potentially other viral infections.

Encapsulation of Gold Nanoparticles into Redesigned Ferritin Nanocages for the Assembly of Binary Superlattices Composed of Fluorophores and Gold Nanoparticles.

Nanomaterials with a defined composition and structure can be synthesized by exploiting natural templates or biomolecular matrices. In the present work, we use protein nanocages derived from human ferritin as a nanoscale building block for the assembly of gold nanoparticles and fluorescent molecules in the solid state. As a generalizable strategy, we show that prior to material synthesis, the cargo can be encapsulated into the protein nanocages using a dis- and reassembly approach. Toward this end, a new ligand system for gold nanoparticles enables efficient encapsulation of these particles into the nanocages. The gold nanoparticle-loaded protein nanocages are co-assembled with fluorophore-loaded protein nanocages. Binary superlattices are formed because two oppositely charged ferritin nanocages are used as templates for the assembly. The binary crystals show strong exciton-plasmon coupling between the encapsulated fluorophores and gold nanoparticles, which was spatially resolved with fluorescence lifetime imaging. The strategy outlined here offers a modular approach toward binary nanomaterials with highly ordered building blocks.

Assembly of chemically modified protein nanocages into 3D materials for the adsorption of uremic toxins.

Hemodialysis fails to remove protein-bound uremic toxins that are attributed with high cardiovascular risk. Application of adsorption materials is a viable strategy, but suitable biocompatible adsorbents are still not available. Here, we demonstrate that adsorbents based on the bottom-up assembly of the intrinsically biocompatible protein cage ferritin are applicable for toxin adsorption. Due to the size-exclusion effect of its pores, only small molecules such as uremic toxins can enter the protein cage. Protein redesign techniques that target selectively the inner surface were used to introduce anchor sites for chemical modification. Porous crystalline adsorbents were fabricated by bottom-up assembly of the protein cage. Linkage of up to 96 phenylic or aliphatic molecules per container was verified by ESI-MS. Materials based on unmodified ferritin cages can already adsorb the uremic toxins. The adsorption capacity could be increased by about 50% through functionalization with hydrophobic molecules reaching 458 µg g-1 for indoxyl sulfate. The biohybrid materials show no contamination with endotoxins and do not activate blood platelets. These findings demonstrate the great potential of protein-based adsorbents for the clearance of uremic toxins: modifications enhance toxin adsorption without diminishing the biocompatibility of the final protein-based material.