Temperature-Induced Collapse of Elastin-like Peptides Studied by 2DIR Spectroscopy.

Elastin-like peptides are hydrophobic biopolymers that exhibit a reversible coacervation transition when the temperature is raised above a critical point. Here, we use a combination of linear infrared spectroscopy, two-dimensional infrared spectroscopy, and molecular dynamics simulations to study the structural dynamics of two elastin-like peptides. Specifically, we investigate the effect of the solvent environment and temperature on the structural dynamics of a short (5-residue) elastin-like peptide and of a long (450-residue) elastin-like peptide. We identify two vibrational energy transfer processes that take place within the amide I' band of both peptides. We observe that the rate constant of one of the exchange processes is strongly dependent on the solvent environment and argue that the coacervation transition is accompanied by a desolvation of the peptide backbone where up to 75% of the water molecules are displaced. We also study the spectral diffusion dynamics of the valine(1) residue that is present in both peptides. We find that these dynamics are relatively slow and indicative of an amide group that is shielded from the solvent. We conclude that the coacervation transition of elastin-like peptides is probably not associated with a conformational change involving this residue.

Metal Ion-Induced Self-Assembly and Packaging of CCMV Nanocapsules.

The controlled self-assembly of protein cages is vital for the use of these nanocompartments in biomedical and nanotechnological applications. Recently, we showed that by combining different structural peptide elements, it is possible to assemble viral capsid proteins in distinct well-defined morphologies. In this chapter, a triblock copolypeptide is discussed, consisting of a metal ion-coordinating hexahistidine tag, a stimulus-responsive elastin-like polypeptide and a pH-responsive self-assembling viral capsid protein. This protein is able to form two different types of capsids, depending on the assembly pathway that is followed. Here, we focus on the metal ion-induced assembly process and describe the relevant experimental procedures to induce and utilize this assembly behavior.

Residue-Specific Incorporation of Noncanonical Amino Acids for Protein Engineering.

The incorporation of noncanonical amino acids has given protein chemists access to an expanded repertoire of amino acids. This methodology has significantly broadened the scope of protein engineering allowing introduction of amino acids with non-native functionalities, such as bioorthogonal reactive handles (azides and alkynes) and hydrophobic fluorinated side chains. Here, we describe the efficient residue-specific replacement of methionine by azidonorleucine in an engineered green fluorescent protein using a bacterial expression system to introduce a single reactive site for the strain-promoted azide-alkyne cycloaddition.

Modular, Bioorthogonal Strategy for the Controlled Loading of Cargo into a Protein Nanocage.

Virus capsids, i.e., viruses devoid of their genetic material, are suitable nanocarriers for biomedical applications such as drug delivery and diagnostic imaging. For this purpose, the reliable encapsulation of cargo in such a protein nanocage is crucial, which can be accomplished by the covalent attachment of the compounds of interest to the protein domains positioned at the interior of the cage. This approach is particularly valid for the capsid proteins of the cowpea chlorotic mottle virus (CCMV), which have their N-termini located at the inside of the capsid structure. Here, we examined several site-selective modification methods for covalent attachment and encapsulation of cargo at the N-terminus of the CCMV protein. Initially, we explored approaches to introduce an N-terminal azide functionality, which would allow the subsequent bioorthogonal modification with a strained alkyne to attach the desired cargo. As these methods showed compatibility issues with the CCMV capsid proteins, a strategy based on 2-pyridinecarboxaldehydes for site-specific N-terminal protein modification was employed. This method allowed the successful modification of the proteins, and was applied for the introduction of a bioorthogonal vinylboronic acid moiety. In a subsequent reaction, the proteins could be modified further with a fluorophore using the tetrazine ligation. The application of capsid assembly conditions on the functionalized proteins led to successful particle formation, showing the potential of this covalent encapsulation strategy.

Selective Proteomic Analysis of Antibiotic-Tolerant Cellular Subpopulations in Pseudomonas aeruginosa Biofilms.

Biofilm infections exhibit high tolerance against antibiotic treatment. The study of biofilms is complicated by phenotypic heterogeneity; biofilm subpopulations differ in their metabolic activities and their responses to antibiotics. Here, we describe the use of the bio-orthogonal noncanonical amino acid tagging (BONCAT) method to enable selective proteomic analysis of a Pseudomonas aeruginosa biofilm subpopulation. Through controlled expression of a mutant methionyl-tRNA synthetase, we targeted BONCAT labeling to cells in the regions of biofilm microcolonies that showed increased tolerance to antibiotics. We enriched and identified proteins synthesized by cells in these regions. Compared to the entire biofilm proteome, the labeled subpopulation was characterized by a lower abundance of ribosomal proteins and was enriched in proteins of unknown function. We performed a pulse-labeling experiment to determine the dynamic proteomic response of the tolerant subpopulation to supra-MIC treatment with the fluoroquinolone antibiotic ciprofloxacin. The adaptive response included the upregulation of proteins required for sensing and repairing DNA damage and substantial changes in the expression of enzymes involved in central carbon metabolism. We differentiated the immediate proteomic response, characterized by an increase in flagellar motility, from the long-term adaptive strategy, which included the upregulation of purine synthesis. This targeted, selective analysis of a bacterial subpopulation demonstrates how the study of proteome dynamics can enhance our understanding of biofilm heterogeneity and antibiotic tolerance.IMPORTANCE Bacterial growth is frequently characterized by behavioral heterogeneity at the single-cell level. Heterogeneity is especially evident in the physiology of biofilms, in which distinct cellular subpopulations can respond differently to stresses, including subpopulations of pathogenic biofilms that are more tolerant to antibiotics. Global proteomic analysis affords insights into cellular physiology but cannot identify proteins expressed in a particular subpopulation of interest. Here, we report a chemical biology method to selectively label, enrich, and identify proteins expressed by cells within distinct regions of biofilm microcolonies. We used this approach to study changes in protein synthesis by the subpopulation of antibiotic-tolerant cells throughout a course of treatment. We found substantial differences between the initial response and the long-term adaptive strategy that biofilm cells use to cope with antibiotic stress. The method we describe is readily applicable to investigations of bacterial heterogeneity in diverse contexts.

Metal Ion-Induced Self-Assembly of a Multi-Responsive Block Copolypeptide into Well-Defined Nanocapsules.

Protein cages are an interesting class of biomaterials with potential applications in bionanotechnology. Therefore, substantial effort is spent on the development of capsule-forming designer polypeptides with a tailor-made assembly profile. The expanded assembly profile of a triblock copolypeptide consisting of a metal ion chelating hexahistidine-tag, a stimulus-responsive elastin-like polypeptide block, and a pH-responsive morphology-controlling viral capsid protein is presented. The self-assembly of this multi-responsive protein-based block copolymer is triggered by the addition of divalent metal ions. This assembly process yields monodisperse nanocapsules with a 20 nm diameter composed of 60 polypeptides. The well-defined nanoparticles are the result of the emergent properties of all the blocks of the polypeptide. These results demonstrate the feasibility of hexahistidine-tags to function as supramolecular cross-linkers. Furthermore, their potential for the metal ion-mediated encapsulation of hexahistidine-tagged proteins is shown.

Natural supramolecular protein assemblies.

Supramolecular protein assemblies are an emerging area within the chemical sciences, which combine the topological structures of the field of supramolecular chemistry and the state-of-the-art chemical biology approaches to unravel the formation and function of protein assemblies. Recent chemical and biological studies on natural multimeric protein structures, including fibers, rings, tubes, catenanes, knots, and cages, have shown that the quaternary structures of proteins are a prerequisite for their highly specific biological functions. In this review, we illustrate that a striking structural diversity of protein assemblies is present in nature. Furthermore, we describe structure-function relationship studies for selected classes of protein architectures, and we highlight the techniques that enable the characterisation of supramolecular protein structures.

Elastin-like polypeptide based nanoparticles: design rationale toward nanomedicine.

Elastin-like polypeptides (ELPs) are characterized by a high sequence control, temperature responsiveness and biocompatibility, which make them highly interesting as smart materials for application in nanomedicine. In particular the construction of ELP-based nanoparticles has recently become a focal point of attention in materials research. This review will give an overview of the ELP-based nanoparticles that have been developed until now and their underlying design principles. First a short introduction on ELPs and their stimulus-responsive behavior will be given. This characteristic has been applied for the development of ELP-based block copolymers that can self-assemble into nanoparticles. Both the fully ELP-based as well as several ELP hybrid materials that have been reported to form nanoparticles will be discussed, which is followed by a concise description of the promising biomedical applications reported for this class of materials.

Synthesis and self-assembly of well-defined elastin-like polypeptide-poly(ethylene glycol) conjugates.

A series of stimulus-responsive elastin-like polypeptide-poly(ethylene glycol) (ELP-PEG) block copolymers was synthesized. The polymeric building blocks were conjugated via the efficient and specific strain-promoted alkyne-azide cycloaddition (SPAAC). For this purpose, ELP and PEG blocks were functionalized with azide and cyclooctyne moieties, respectively. Azides were introduced by applying a recently developed pH-controlled diazotransfer reaction on the primary amines present in ELP (N-terminus and lysine side chains). By varying pH, ELP-blocks with one or two azides were obtained, which subsequently allowed us to synthesize both ELP-PEG diblock copolymers and miktoarm star polymers. Triggering the phase transition of the ELP-block resulted in the formation of an amphiphilic block copolymer, which self-assembled into micelles. This is the first example of an ELP-containing hybrid block copolymer in which PEG as the hydrophilic corona-forming domain is combined with a stimulus-responsive ELP-block. The encapsulation of a hydrophobic fluorescent dye was shown to exemplify the potential of the micelles to serve as nanocarriers for hydrophobic drugs, with the PEG corona providing stealth and steric protection of encapsulated materials.

Thermodynamic investigation of Z33-antibody interaction leads to selective purification of human antibodies.

Antibodies, such as IgGs, are widely applied as detection probes, purification ligands and targeting moieties in research and medicine. Protein A from Staphylococcus aureus is capable of selectively binding to antibodies. Z33, a 33 amino acid peptide sequence derived from Protein A, is a minimized binding domain with comparable interaction potential. This peptide was fused to two different proteins without perturbing the properties of both the protein and the Z33-domain. The thermodynamic parameters for the interaction of the fusion proteins with antibodies from various species were determined by isothermal titration calorimetry. This showed that binding was enthalpically driven and entropically unfavorable. A difference in Z33 binding affinity of several orders of magnitude was observed between human and bovine antibodies. This selectivity toward human IgGs was utilized for the efficient and selective purification of human IgGs from mixtures containing bovine IgGs and other proteins by affinity precipitation employing a fusion protein of Z33 and a stimulus-responsive elastin-like polypeptide.

General strategy for ordered noncovalent protein assembly on well-defined nanoscaffolds.

Here we develop a novel approach allowing the noncovalent assembly of proteins on well-defined nanoscaffolds such as virus particles. The antibody-binding peptide Z33 was genetically fused to the monomeric yellow fluorescent protein and 4-coumarate:CoA-ligase 2. This Z33 "tag" allowed their patterning on the surface of zucchini yellow mosaic virus by means of specific antibodies directed against the coat protein of the virus. The approach was validated by affinity assays and correlative microscopy. The coverage efficiency was ≈ 87%. Fluorescence and enzymatic activity were fully retained after assembly. The principle of using the combination of a scaffold-specific antibody and Z33-fusion proteins can be extended to a wide variety of proteins/enzymes and antigenic scaffolds to support coupling for creating functional "biochips" with optical or catalytic properties.

Evidence that the catenane form of CS2 hydrolase is not an artefact.

CS2 hydrolase, a zinc-dependent enzyme that converts carbon disulfide to carbon dioxide and hydrogen sulfide, exists as a mixture of octameric ring and hexadecameric catenane forms in solution. A combination of size exclusion chromatography, multi-angle laser light scattering, and mass spectrometric analyses revealed that the unusual catenane structure is not an artefact, but a naturally occurring structure.

Site-specific peptide and protein immobilization on surface plasmon resonance chips via strain-promoted cycloaddition.

Surface plasmon resonance (SPR) is a powerful label-free diagnostic tool to study biomolecular interactions. However, one of the drawbacks of SPR is the lack of controlled immobilization of ligands on the sensor surface. We have developed a modular platform for the fast, reagent-free and site-specific immobilization of azide-containing ligands by strain-promoted cycloaddition onto a cyclooctyne-modified SPR sensor surface. The usefulness of the concept was shown in a study with a papain model system, and up to 150 experiments were performed without loss of surface quality. Furthermore, azide-containing green fluorescent protein (GFP) was also effectively immobilized. Taken together, cyclooctyne-modified SPR chips enable smooth and site-selective immobilization of ligands and prove to be more robust than traditionally functionalized systems.

N-Terminal dual protein functionalization by strain-promoted alkyne-nitrone cycloaddition.

Strain-promoted alkyne-nitrone cycloadditon (SPANC) was optimized as a versatile strategy for dual functionalization of peptides and proteins. The usefulness of the dual labeling protocol is first exemplified by the simultaneous introduction of a chloroquine and a stearyl moiety, two endosomal escape-improving functional groups, into the cell-penetrating peptide hLF (human lactoferrin). Additionally, we demonstrate that dual labeling of proteins is feasible by combining metal-free and copper-catalyzed click chemistry. First, SPANC is applied to enhanced green fluorescent protein to introduce both biotin and a terminal alkyne. The terminal acetylene then serves as a convenient anchor point for the CuAAC reaction with azido-containing fluorescein, thereby demonstrating the potential of combined SPANC and CuAAC for the straightforward, dual functionalization of proteins.

Designing two self-assembly mechanisms into one viral capsid protein.

ELP-CP, a structural fusion protein of the thermally responsive elastin-like polypeptide (ELP) and a viral capsid protein (CP), was designed, and its assembly properties were investigated. Interestingly, this protein-based block copolymer could be self-assembled via two mechanisms into two different, well-defined nanocapsules: (1) pH-induced assembly yielded 28 nm virus-like particles, and (2) ELP-induced assembly yielded 18 nm virus-like particles. The latter were a result of the emergent properties of the fusion protein. This work shows the feasibility of creating a self-assembly system with new properties by combining two structural protein elements.

Elastomeric polypeptides.

Elastomeric polypeptides are very interesting biopolymers and are characterized by rubber-like elasticity, large extensibility before rupture, reversible deformation without loss of energy, and high resilience upon stretching. Their useful properties have motivated their use in a wide variety of materials and biological applications. This chapter focuses on elastin and resilin - two elastomeric biopolymers - and the recombinant polypeptides derived from them (elastin-like polypeptides and resilin-like polypeptides). This chapter also discusses the applications of these recombinant polypeptides in the fields of purification, drug delivery, and tissue engineering.

Staudinger ligation as a method for bioconjugation.

In 1919 the German chemist Hermann Staudinger was the first to describe the reaction between an azide and a phosphine. It was not until recently, however, that Bertozzi and co-workers recognized the potential of this reaction as a method for bioconjugation and transformed it into the so-called Staudinger ligation. The bio-orthogonal character of both the azide and the phosphine functions has resulted in the Staudinger ligation finding numerous applications in various complex biological systems. For example, the Staudinger ligation has been utilized to label glycans, lipids, DNA, and proteins. Moreover, the Staudinger ligation has been used as a synthetic method to construct glycopeptides, microarrays, and functional biopolymers. In the emerging field of bio-orthogonal ligation strategies, the Staudinger ligation has set a high standard to which most of the new techniques are often compared. This Review summarizes recent developments and new applications of the Staudinger ligation.

Site-specific modification of Candida antarctica lipase B via residue-specific incorporation of a non-canonical amino acid.

In order to modify proteins in a controlled way, new functionalities need to be introduced in a defined manner. One way to accomplish this is by the incorporation of a non-natural amino acid of which the side chain can selectively be reacted to other molecules. We have investigated whether the relatively simple method of residue-specific replacement of methionine by azidohomoalanine can be used to achieve monofunctionalization of the model enzyme Candida antarctica lipase B. A protein variant was engineered with one additional methionine residue. Due to the high hydrophobicity and low abundance of methionine, this was the only residue out of five that was exposed to the solvent. The use of the Cu (I)-catalyzed [3 + 2] cycloaddition under native conditions resulted in a monofunctionalized enzyme which retained hydrolytic activity. The strategy can be considered a convenient tool to modify proteins at a single position as long as one solvent-exposed methionine is available.