Self‐Assembled Cage‐Like Protein Structures

Proteins and protein‐based assemblies represent the most structurally and functionally diverse molecules found in nature. Protein cages, viruses and bacterial microcompartments are highly organized structures that are composed primarily of protein building blocks and play important roles in molecular ion storage, nucleic acid packaging and catalysis. The outer and inner surface of protein cages can be modified, either chemically or genetically, and the internal cavity can be used to template, store and arrange molecular cargo within a defined space. Owing to their structural, morphological, chemical and thermal diversity, protein cages have been investigated extensively for applications in nanotechnology, nanomedicine and materials science. Here we provide a concise overview of the most common icosahedral viral and nonviral assemblies, their role in nature, and why they are highly attractive scaffolds for the encapsulation of functional materials.     

Conversion of the Native 24‐mer Ferritin Nanocage into Its Non‐Native 16‐mer Analogue by Insertion of Extra Amino Acid Residues

Protein assemblies with high symmetry are widely distributed in nature. Most efforts so far have focused on repurposing these protein assemblies, a strategy that is ultimately limited by the structures available. To overcome this limitation, methods for fabricating novel self‐assembling proteins have received intensive interest. Herein, by reengineering the key subunit interfaces of native 24‐mer protein cage with octahedral symmetry through amino acid residues insertion, we fabricated a 16‐mer lenticular nanocage whose structure is unique among all known protein cages. This newly non‐native protein can be used for encapsulation of bioactive compounds and exhibits high uptake efficiency by cancer cells. More importantly, the above strategy could be applied to other naturally occurring protein assemblies with high symmetry, leading to the generation of new proteins with unexplored functions.     

The Application of Fluorine‐Containing Reagents in Structural Proteomics

Structural proteomics refers to large‐scale mapping of protein structures in order to understand the relationship between protein sequence, structure, and function. Chemical labeling, in combination with mass‐spectrometry (MS) analysis, have emerged as powerful tools to enable a broad range of biological applications in structural proteomics. The key to success is a biocompatible reagent that modifies a protein without affecting its high‐order structure. Fluorine, well‐known to exert profound effects on the physical and chemical properties of reagents, should have an impact on structural proteomics. In this Minireview, we describe several fluorine‐containing reagents that can be applied in structural proteomics. We organize their applications around four MS‐based techniques: a) affinity labeling, b) activity‐based protein profiling (ABPP), c) protein footprinting, and d) protein cross‐linking. Our aim is to provide an overview of the research, development, and application of fluorine‐containing reagents in protein structural studies.     

A Photoactive Carbon‐Monoxide‐Releasing Protein Cage for Dose‐Regulated Delivery in Living Cells

Protein cages can serve as bioinorganic molecular templates for functionalizing metal compounds to regulate cellular signaling. We succeeded in developing a photoactive CO‐releasing system by constructing a composite of ferritin (Fr) containing manganese–carbonyl complexes. When Arg52 adjacent to Cys48 of Fr is replaced with Cys, the Fr mutant stabilizes the retention of 48 Mn–carbonyl moieties, which can release the CO ligands under light irradiation, although wild‐type Fr retains very few Mn moieties. The amount of released CO is regulated by the extent of irradiation. This could reveal an optimized dose for cooperatively activating the nuclear factor κB (NF‐κB) in mammalian cells and the tumor necrosis factor α (TNF‐α). These results suggest that construction of a CO‐releasing protein cage will advance of research in CO biology.     

Site‐directed analysis on protein hydrophobicity

Hydrophobicity of a protein is considered to be one of the major intrinsic factors dictating the protein aggregation propensity. Understanding how protein hydrophobicity is determined is, therefore, of central importance in preventing protein aggregation diseases and in the biotechnological production of human therapeutics. Traditionally, protein hydrophobicity is estimated based on hydrophobicity scales determined for individual free amino acids, assuming that those scales are unaltered when amino acids are embedded in a protein. Here, we investigate how the hydrophobicity of constituent amino acid residues depends on the protein context. To this end, we analyze the hydration free energy—free energy change on hydration quantifying the hydrophobicity—of the wild‐type and 21 mutants of amyloid‐beta protein associated with Alzheimer's disease by performing molecular dynamics simulations and integral‐equation calculations. From detailed analysis of mutation effects on the protein hydrophobicity, we elucidate how the protein global factor such as the total charge as well as underlying protein conformations influence the hydrophobicity of amino acid residues. Our results provide a unique insight into the protein hydrophobicity for rationalizing and predicting the protein aggregation propensity on mutation, and open a new avenue to design aggregation‐resistant proteins as biotherapeutics. © 2014 Wiley Periodicals, Inc.     

Dipole‐Induced Band‐Gap Reduction in an Inorganic Cage

Metal‐doped polyoxotitanium cages are a developing class of inorganic compounds which can be regarded as nano‐ and sub‐nano sized molecular relatives of metal‐doped titania nanoparticles. These species can serve as models for the ways in which dopant metal ions can be incorporated into metal‐doped titania (TiO₂), a technologically important class of photocatalytic materials with broad applications in devices and pollution control. In this study a series of cobalt(II)‐containing cages in the size range ca. 0.7–1.3 nm have been synthesized and structurally characterized, allowing a coherent study of the factors affecting the band gaps in well‐defined metal‐doped model systems. Band structure calculations are consistent with experimental UV/Vis measurements of the Ti_xO_y absorption edges in these species and reveal that molecular dipole moment can have a profound effect on the band gap. The observation of a dipole‐induced band‐gap decrease mechanism provides a potentially general design strategy for the formation of low band‐gap inorganic cages.     

Capture and Recycling of Sortase A through Site‐Specific Labeling with Lithocholic Acid

Enzyme‐mediated protein modification often requires large amounts of biocatalyst, adding significant costs to the process and limiting industrial applications. Herein, we demonstrate a scalable and straightforward strategy for the efficient capture and recycling of enzymes using a small‐molecule affinity tag. A proline variant of an evolved sortase A (SrtA 7M) was N‐terminally labeled with lithocholic acid (LA)—an inexpensive bile acid that exhibits strong binding to β‐cyclodextrin (βCD). Capture and recycling of the LA‐Pro‐SrtA 7M conjugate was achieved using βCD‐modified sepharose resin. The LA‐Pro‐SrtA 7M conjugate retained full enzymatic activity, even after multiple rounds of recycling.     

Rapid sampling of all‐atom peptides using a library‐based polymer‐growth approach

We adapted existing polymer growth strategies for equilibrium sampling of peptides described by modern atomistic forcefields with a simple uniform dielectric solvent. The main novel feature of our approach is the use of precalculated statistical libraries of molecular fragments. A molecule is sampled by combining fragment configurations—of single residues in this study—which are stored in the libraries. Ensembles generated from the independent libraries are reweighted to conform with the Boltzmann‐factor distribution of the forcefield describing the full molecule. In this way, high‐quality equilibrium sampling of small peptides (4–8 residues) typically requires less than one hour of single‐processor wallclock time and can be significantly faster than Langevin simulations. Furthermore, approximate, clash‐free ensembles can be generated for larger peptides (up to 32 residues in this study) in less than a minute of single‐processor computing. We discuss possible applications of our growth procedure to free energy calculation, fragment assembly protein‐structure prediction protocols, and to “multi‐resolution” sampling. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Regio‐ and Enantioselective Photodimerization within the Confined Space of a Homochiral Ruthenium/Palladium Heterometallic Coordination Cage

The photoinduced regio‐ and enantioselective coupling of naphthols and derivatives thereof is achieved in the confined chiral coordination space of a Ru^II metalloligand based cage. The racemic or enantiopure cages encapsulate naphthol guests, which then undergo a regiospecific 1,4‐coupling, rather than the normal 1,1‐coupling, to form 4‐(2‐hydroxy‐1‐naphthyl)‐1,2‐napthoquinones; moderate stereochemical control is achieved with homochiral cages. The photoreactions proceed under both aerobic and anaerobic conditions but through distinct pathways that nevertheless involve the same radical intermediates. This unusual dimerization constitutes a very rare example of asymmetric induction in biaryl coupling by making use of coordination cages with dual functionality—photoredox reactivity and stereoselectivity.     

Protein‐ and homo poly(amino acid)‐based hydrogels with super‐swelling properties

The use of super‐swelling polymers is steadily increasing and the applications in industry are continuing to grow. With the authorization of the superabsorbents in food packaging by the Food and Drug Administration recently, demand may soon take off in the market. However, the increase in prices of petroleum products in recent years may be a drawback for these acrylic‐based materials. Thus, there is now a need to develop natural‐based super‐swelling hydrogels which are more economical and environment friendly. In addition, the super‐swelling gels are promising novel functions in the biomedical and pharmaceutical applications. This review is aimed to highlight research and trends in protein‐ and homo poly(amino acid)‐based super‐swelling hydrogels. Thus, the proteinaceous hydrogels, including chemically modified soy‐, fish‐ and collagen‐based proteins, are discussed. The protein‐polysaccharide, protein‐synthetics, and the inorganic composites are also investigated as hybrid materials. Finally, the super‐swelling hydrogels based on homo polypeptides, i.e. poly(aspartic acid), poly(glutamic acid), and poly(ε‐L‐lysine) are reviewed. Copyright © 2009 John Wiley & Sons, Ltd.     

One‐Pot Assembly of Amino Acid Bridged Hybrid Macromulticyclic Cages through Multiple Multicomponent Macrocyclizations

An important development in the field of macrocyclization strategies towards molecular cages is described. The approach comprises the utilization of a double Ugi four‐component macrocyclization for the assembly of macromulticycles with up to four different tethers, that is, hybrid cages. The innovation of this method rests on setting up the macromulticycle connectivities not through the tethers but through the bridgeheads, which in this case involve N‐substituted amino acids. Both dilution and metal‐template‐driven macrocyclization conditions were implemented with success, enabling the one‐pot formation of cryptands and cages including steroidal, polyether, heterocyclic, peptidic, and aryl tethers. This method demonstrates substantial complexity‐generating character and is suitable for applications in molecular recognition and catalysis.     

A Modular Method for the High‐Yield Synthesis of Site‐Specific Protein–Polymer Therapeutics

A versatile method is described to engineer precisely defined protein/peptide–polymer therapeutics by a modular approach that consists of three steps: 1) fusion of a protein/peptide of interest with an elastin‐like polypeptide that enables facile purification and high yields; 2) installation of a clickable group at the C terminus of the recombinant protein/peptide with almost complete conversion by enzyme‐mediated ligation; and 3) attachment of a polymer by a click reaction with near‐quantitative conversion. We demonstrate that this modular approach is applicable to various protein/peptide drugs and used it to conjugate them to structurally diverse water‐soluble polymers that prolong the plasma circulation duration of these proteins. The protein/peptide–polymer conjugates exhibited significantly improved pharmacokinetics and therapeutic effects over the native protein/peptide upon administration to mice. The studies reported here provide a facile method for the synthesis of protein/peptide–polymer conjugates for therapeutic use and other applications.     

Activation of the hippocampal AC‐cAMP‐PKA‐CREB‐BDNF signaling pathway using WTKYR in depression model rats

Depression, also called “depression disorder,” is characterized by a significant and persistent low mood. It has become a major refractory disease in the 21st century. In recent years, Chinese medicine has shown some important clinical value in the treatment of depression. Among them, the Warming and “Tonifying” Kidney‐Yang Recipe (WTKYR) has been demonstrated to have obvious effects in the clinical treatments of depression; however, the mechanism remains unclear. This study is based on the adenylyl cyclase (AC)—cyclic adenosine monophosphate (cAMP)—protein kinase A (PKA)—cAMP response element‐binding protein (CREB)—brain derived neurotrophic factor (BDNF) signaling pathway, aiming to investigate the mechanism of WTKYR. The results showed that WTKYR can upregulate AC‐cAMP‐PKA‐CREB‐BDNF in the hippocampus of depression model rats and alleviate its depressive symptoms, which may be the mechanism of WTKYR.     

Focused grid‐based resampling for protein docking and mapping

The fast Fourier transform (FFT) sampling algorithm has been used with success in application to protein‐protein docking and for protein mapping, the latter docking a variety of small organic molecules for the identification of binding hot spots on the target protein. Here we explore the local rather than global usage of the FFT sampling approach in docking applications. If the global FFT based search yields a near‐native cluster of docked structures for a protein complex, then focused resampling of the cluster generally leads to a substantial increase in the number of conformations close to the native structure. In protein mapping, focused resampling of the selected hot spot regions generally reveals further hot spots that, while not as strong as the primary hot spots, also contribute to ligand binding. The detection of additional ligand binding regions is shown by the improved overlap between hot spots and bound ligands. © 2016 Wiley Periodicals, Inc.     

Novel drug‐eluting soy‐protein structures for wound healing applications

Naturally derived materials are becoming widely used in the biomedical field. Soy protein has advantages over the various types of natural proteins employed for biomedical applications due to its low price, nonanimal origin, and relatively long storage time and stability. In the current study, novel drug‐eluting soy‐protein films for wound healing applications were developed and studied. The films were prepared using the solvent casting technique. The analgesic drug bupivacaine and two types of wide range antibiotics (gentamicin and clindamycin) were incorporated into the soy‐protein films. The effect of drug incorporation and plasticizers content on the films' mechanical properties, drug release profiles, and cell viability was studied. Drug incorporation had a softening effect of the films, lowering mechanical strength and increasing ductility. Release profiles of bupivacaine and clindamycin exhibited high burst release of 80% to 90% of encapsulated drug within 6 hours, followed by continuous release in a decreasing rate for a period of 2 to 4 days. Gentamicin release was prolonged, probably due to interaction between the gentamicin and the polymer chains. Hybrid soy‐protein/poly (Dl‐lactic‐co‐glycolic acid) (PDLGA) microspheres structure showed potential for long and sustained release of bupivacaine. Films with no drugs and films loaded with gentamicin were found to be noncytotoxic for human fibroblasts, while bupivacaine and clindamycin were found to have some effect on cell growth. In conclusion, our new drug‐loaded soy‐protein films combine good mechanical properties and biocompatibility, with desired drug release profiles, and can therefore be potentially very useful as burn and ulcer dressings.     

A Rhizavidin Monomer with Nearly Multimeric Avidin‐Like Binding Stability Against Biotin Conjugates

Developing a monomeric form of an avidin‐like protein with highly stable biotin binding properties has been a major challenge in biotin‐avidin linking technology. Here we report a monomeric avidin‐like protein—enhanced monoavidin—with off‐rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head‐group‐biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24‐meric avidin probe by fusing eMA to a multimeric cage protein. The 24‐meric avidin and eMA were utilized to demonstrate how artificial clustering of cell‐surface proteins greatly enhances the internalization rates of assembled proteins on live cells.     

Construction of Polycation‐Based Non‐Viral DNA Nanoparticles and Polyanion Multilayers via Layer‐by‐Layer Self‐Assembly

Summary: The multilayers of polycation‐based non‐viral DNA nanoparticles and biodegradable poly(L ‐glutamic acid) (PGA) were constructed by a layer‐by‐layer (LbL) technique. Poly(ethyleneimine) (PEI) was used to condense DNA to develop non‐viral DNA nanoparticles. AFM, UV‐visible spectrometry, and TEM measurements revealed that the PEI‐DNA nanoparticles were successfully incorporated into the multilayers. The well‐structured, easily processed multilayers with the non‐viral DNA nanoparticles may provide a novel approach to precisely control the delivery of DNA, which may have great potential for gene therapy applications in tissue engineering, medical implants, etc.

A TEM image of the cross section of a (PGA/PEI‐DNA nanoparticle)₂₀ multilayer.     

Protein‐Structure‐Directed Metal–Organic Zeolite‐like Networks as Biomacromolecule Carriers

Fabrication of zeolite‐like metal–organic frameworks (ZMOFs) for advanced applications, such as enzyme immobilization, is of great interest but is a great synthetic challenge. Herein, we have developed a new strategy using proteins as structure‐directed agents to direct the formation of new ZMOFs that can act as versatile platforms for the in situ encapsulation of proteins under ambient conditions. Notably, protein incorporation directs the formation of a ZMOF with a sodalite ( sod ) topology instead of a non‐porous diamondoid ( dia ) topology under analogous synthetic conditions. Histidines in proteins play a crucial role in the observed templating effect. Modulating histidine content thereby influenced the resultant MOF product (from dia to dia + sod mixture and, ultimately, to sod MOF). Moreover, the resulting ZMOF‐incorporated proteins preserved their activity even after exposure to high temperatures and organic solvents, demonstrating their potential for biocatalysis and biopharmaceutical applications.     

3D‐Printed Titanium Cage with PVA‐Vancomycin Coating Prevents Surgical Site Infections (SSIs)

Many coating materials have been studied to prevent surgical site infections (SSIs). However, antibacterial coating on surfaces show weak adhesion using the traditional titanium (Ti) cage, resulting in low efficacy for preventing SSIs after spinal surgery. Herein, a 3D‐printed Ti cage combined with a drug‐releasing system is developed for in situ drug release and bacteria killing, leading to prevention of SSIs in vitro and in vivo. First, a 3D‐printed Ti cage is designed and prepared by the Electron Beam Melting (EBM) method. Second, polyvinyl alcohol (PVA) containing hydrophilic vancomycin hydrochloride (VH) is scattered across the surface of 3D‐printed porous Ti (Ti‐VH@PVA) cages. Ti‐VH@PVA cages show an efficient drug‐releasing profile and excellent bactericidal effect for three common bacteria after more than seven days in vitro. In addition, Ti‐VH@PVA cages exhibit reliable inhibition of inflammation associated with Staphylococcus aureus and effective bone regeneration capacity in a rabbit model of SSIs. The results indicate that Ti‐VH@PVA cages have potential advantages for preventing SSIs after spinal surgery.     

Photobodies: Light‐Activatable Single‐Domain Antibody Fragments

Photocaged antibody fragments, termed photobodies, have been developed that are impaired in their antigen‐binding capacity and can be activated by irradiation with UV light (365 nm). This rational design concept builds on the selective photocaging of a single tyrosine in a nanobody (a single‐domain antibody fragment). Tyrosine is a frequently occurring residue in central positions of the paratope region. o‐Nitrobenzyl‐protected tyrosine variants were incorporated into four nanobodies, including examples directed against EGFR and HER2, and photodeprotection restores the native sequence. An anti‐GFP photobody exhibited an at least 10 000‐fold impaired binding affinity before photodeprotection compared with the parent nanobody. A bispecific nanobody–photobody fusion protein was generated to trigger protein heterodimerization by light. Photoactivatable antibodies are expected to become versatile protein reagents and to enable novel approaches in diagnostic and therapeutic applications.