Structure-Property Relationships of Elastin-like Polypeptides: A Review of Experimental and Computational Studies.

Elastin is a structural protein with outstanding mechanical properties (e.g., elasticity and resilience) and biologically relevant functions (e.g., triggering responses like cell adhesion or chemotaxis). It is formed from its precursor tropoelastin, a 60-72 kDa water-soluble and temperature-responsive protein that coacervates at physiological temperature, undergoing a phenomenon termed lower critical solution temperature (LCST). Inspired by this behavior, many scientists and engineers are developing recombinantly produced elastin-inspired biopolymers, usually termed elastin-like polypeptides (ELPs). These ELPs are generally comprised of repetitive motifs with the sequence VPGXG, which corresponds to repeats of a small part of the tropoelastin sequence, X being any amino acid except proline. ELPs display LCST and mechanical properties similar to tropoelastin, which renders them promising candidates for the development of elastic and stimuli-responsive protein-based materials. Unveiling the structure-property relationships of ELPs can aid in the development of these materials by establishing the connections between the ELP amino acid sequence and the macroscopic properties of the materials. Here we present a review of the structure-property relationships of ELPs and ELP-based materials, with a focus on LCST and mechanical properties and how experimental and computational studies have aided in their understanding.

Post-production modification of industrial enzymes.

Industry has an increasing interest in the use of enzymes as environmentally friendly, highly efficient, and specific bio-catalysts. Enzymes have primarily evolved to function in aqueous environments at ambient temperature and pressure. These conditions however do not always correspond with industrial processes or applications, and only a small portion of all known enzymes are therefore suitable for industrial use. Protein engineering can sometimes be applied to convey more desirable properties to enzymes, such as increased stability, but is limited to the 20 naturally occurring amino acids or homologs thereof. Using post-production modification, which has the potential to combine desirable properties from the enzyme and the conjugated compounds, enzymes can be modified with both natural and synthetic molecules. This offers access to a myriad of possibilities for tuning the properties of enzymes. At this moment, however, the effects of post-production modification cannot yet be reliably predicted. The increasing number of applications will improve this so that the potential of this technology can be fully exploited. This review will focus on post-production modification of enzymes and its use and opportunities in industry.

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.

Constrained and UV-activatable cell-penetrating peptides for intracellular delivery of liposomes.

Herein we report on the development of a novel method of constraining a cell-penetrating peptide, which can be used to trigger transport of liposomes into cells upon in this case radiation with UV-light. A cell-penetrating peptide, which was modified on both termini with an alkyl chain, was anchored to the liposomal surface in a constrained and deactivated form. Since one of the two alkyl chains was connected to the peptide via a UV-cleavable linker, disconnection of this alkyl chain upon irradiation led to the exposure of the cell-penetrating peptide, and mediated the transport of the entire liposome particle into cells.

Reactions inside nanoscale protein cages.

Chemical reactions are traditionally carried out in bulk solution, but in nature confined spaces, like cell organelles, are used to obtain control in time and space of conversion. One way of studying these reactions in confinement is the development and use of small reaction vessels dispersed in solution, such as vesicles and micelles. The utilization of protein cages as reaction vessels is a relatively new field and very promising as these capsules are inherently monodisperse, in that way providing uniform reaction conditions, and are readily accessible to both chemical and genetic modifications. In this review, we aim to give an overview of the different kinds of nanoscale protein cages that have been employed as confined reaction spaces.

Metal-ion-induced formation and stabilization of protein cages based on the cowpea chlorotic mottle virus.

The cowpea chlorotic mottle virus (CCMV) is a versatile building block for the construction of nanoreactors and functional materials. Upon RNA removal, the capsid can be reversibly assembled and disassembed by adjusting the pH. At pH 5.0 the capsid is in the native assembled conformation, while at pH 7.5 it disassembles into 90 capsid protein dimers. This special property enables the encapsulation of various molecules, such as protein and enzymes, but only at low pH. It is possible to stabilize the capsid at pH 7.5 by addition of negatively charged polyelectrolytes or negatively charged particles, but these methods all fill the interior of the capsid, leaving little or no space for other cargo molecules. This pH restriction therefore severely limits the range of enzymes that can be encapsulated, and hampers the investigation of the CCMV capsid as a nanoreactor for the study of enzymes in confined spaces. Herein, the interaction of N-terminal histidine-tag-modified capsid proteins with several metal ions is reported. Depending on the conditions used, nanometer-sized protein particles or capsidlike architectures are formed that are stable at pH 7.5. This metal-mediated stabilization methodology is employed to form stable capsids containing multiple proteins at pH 7.5, thereby greatly expanding the scope of the CCMV capsid as a nanoreactor.

A modular and noncovalent transduction system for leucine-zipper-tagged proteins.

Modes of transport: A leucine-zipper-tagged GFP was transported into cells by "zipping" it (red) to it's complementary leucine zipper (blue) functionalized with a cell-penetrating peptide (CPP). This transport system has an inherent modularity as the CPP is "clicked" to the leucine zipper, and then noncovalently bound to the protein, thus making it system particularly useful for targeting studies.

Virus-like particles templated by DNA micelles: a general method for loading virus nanocarriers.

DNA amphiphile particles template formation of virus capsids and enable their loading.

Complex assembly behavior during the encapsulation of green fluorescent protein analogs in virus derived protein capsules.

Enzymes encapsulated in nanocontainers are a better model of the conditions inside a living cell than free enzymes in solution. In a first step toward the encapsulation of multiple enzymes inside the cowpea chlorotic mottle virus (CCMV) capsid, enhanced green fluorescent protein (EGFP) was attached to CCMV capsid proteins. The capsid protein-EGFP complex was then co-assembled with wild-type capsid protein (wt CP) in various ratios. At higher complex to wt CP ratios, the number of EGFP per capsid decreased instead of leveling off. We propose that this unexpected behavior is caused by pH-induced disassembly of the capsid protein-EGFP complex as well as by concentration and ratio dependent dimerization of the complex, making it partially unavailable for incorporation into the capsid.

Controlled encapsulation of multiple proteins in virus capsids.

Multiple proteins can be bound within the Cowpea Chlorotic Mottle Virus capsid shell in an efficient and controlled manner by using heterodimeric coiled-coil peptide oligomers. Through genetic modification, these oligomers can be attached to the capsid protein and an enhanced green fluorescent protein (EGFP). In this way, the capsid proteins can be noncovalently bound to EGFP prior to the induction of the capsid assembly. Up to 15 EGFP proteins can be encapsulated per capsid in a controlled and efficient manner.

CCMV capsid formation induced by a functional negatively charged polymer.

A functional negatively charged polyelectrolyte, polyferrocenylsilane (PFS) was encapsulated in cowpea chlorotic mottle virus (CCMV) capsid proteins, yielding monodisperse particles of 18 nm in size with altered redox properties compared to the parent materials.

Controlled disassembly of peptide amphiphile fibres.

In this paper, the introduction of both a methionine residue and a nitrobenzyl derivative as a labile linker between the peptide part and the hydrophobic alkyl chain of a peptide amphiphile are presented. These modifications are shown not to inhibit the formation of structured assemblies that analogous peptide amphiphiles lacking the linkers are able to form. Moreover, the introduction of either labile linker allows removal of the peptide amphiphile's stabilizing hydrophobic moieties to initiate a controlled disassembly of fibre aggregates. This is achieved by either treatment with CNBr or UV irradiation, respectively. These disassembly mechanisms could be the starting point for methodology that allows further manipulation of self-assembled peptide amphiphile architectures.

Disassembling peptide-based fibres by switching the hydrophobic-hydrophilic balance.

Amyloid-like model peptides, modified on the N-terminus with an alkyl tail and on the C-terminus with a PEG chain, yielded fibres that were susceptible to triggered disassembly by removal of the alkyl chain, which affected the hydrophobic-hydrophilic balance.