Avoiding common pitfalls in designing kinetic protocols for catalytic amyloid studies.

Kinetic characterization of catalytic amyloids arguably presents a most challenging type of kinetic experiment where careful consideration of many factors is required. Here we outline common pitfalls in devising kinetic studies in such systems. Unlike the more specific protocols for various applications described in this volume, this chapter deals with general issues in setting up kinetic experiments that are incredibly important but often go without explicit mention in the specialized literature. The kinetic fundamentals described here can be also be of use to the enzymologists working with more traditional catalysts.

Screening of oxidative behavior in catalytic amyloid assemblies.

Once considered a thermodynamic minimum of the protein fold or as simply by-products of a misfolding process, amyloids are increasingly showing remarkable potential for promoting enzyme-like catalysis. Recent studies have demonstrated a diverse range of catalytic behaviors that amyloids can promote way beyond the hydrolytic behaviors originally reported. We and others have demonstrated the strong propensity of catalytic amyloids to facilitate redox reactions both in the presence and in the absence of metal cofactors. Here, we present a detailed protocol for measuring the oxidative ability of supramolecular peptide assemblies.

Stopped-flow measurement of CO2 hydration activity by catalytic amyloids.

With the ever-increasing rates of catalysis shown by catalytic amyloids, the use of faster characterization techniques is required for proper kinetic studies. The same is true for inherently fast chemical reactions. Carbon dioxide hydration is of significant interest to the field of enzyme design, given both carbonic anhydrases' status as a "perfect enzyme" and the central role carbonic anhydrase plays in the respiration and existence of all carbon-based life. Carbon dioxide is an underexplored hydrolysis substrate within the literature, and a lack of a direct spectroscopic marker for reaction monitoring can make studies more complex and require specialist equipment. Within this article we present a method for measuring the carbon dioxide hydration activity of amyloid fibrils.

Fishing for Catalysis: Experimental Approaches to Narrowing Search Space in Directed Evolution of Enzymes.

Directed evolution has transformed protein engineering offering a path to rapid improvement of protein properties. Yet, in practice it is limited by the hyper-astronomic protein sequence search space, and approaches to identify mutagenic hot spots, i.e., locations where mutations are most likely to have a productive impact, are needed. In this perspective, we categorize and discuss recent progress in the experimental approaches (broadly defined as structural, bioinformatic, and dynamic) to hot spot identification. Recent successes in harnessing protein dynamics and machine learning approaches provide new opportunities for the field and will undoubtedly help directed evolution reach its full potential.

Peptide hydrogel with self-healing and redox-responsive properties.

We have rationally designed a peptide that assembles into a redox-responsive, antimicrobial metallohydrogel. The resulting self-healing material can be rapidly reduced by ascorbate under physiological conditions and demonstrates a remarkable 160-fold change in hydrogel stiffness upon reduction. We provide a computational model of the hydrogel, explaining why position of nitrogen in non-natural amino acid pyridyl-alanine results in drastically different gelation properties of peptides with metal ions. Given its antimicrobial and rheological properties, the newly designed hydrogel can be used for removable wound dressing application, addressing a major unmet need in clinical care.

Catalytic amyloids: Is misfolding folding?

Originally regarded as a disease symptom, amyloids have shown a rich diversity of functions, including biologically beneficial ones. As such, the traditional view of polypeptide aggregation into amyloid-like structures being 'misfolding' should rather be viewed as 'alternative folding.' Various amyloid folds have been recently used to create highly efficient catalysts with specific catalytic efficiencies rivaling those of enzymes. Here we summarize recent developments and applications of catalytic amyloids, derived from both de novo and bioinspired designs, and discuss how progress in the last 2 years reflects on the field as a whole.

Self-Assembling Catalytic Peptide Nanomaterials Capable of Highly Efficient Peroxidase Activity.

The self-assembly of short peptides gives rise to versatile nanomaterials capable of promoting efficient catalysis. We have shown that short, seven-residue peptides bind hemin to produce functional catalytic materials which display highly efficient peroxidation activity, reaching a catalytic efficiency of 3×105 m-1 s-1 . Self-assembly is essential for catalysis as non-assembling controls show no activity. We have also observed peroxidase activity even in the absence of hemin, suggesting the potential to alter redox properties of substrates upon association with the assemblies. These results demonstrate the practical utility of self-assembled peptides in various catalytic applications and further support the evolutionary link between amyloids and modern-day enzymes.

Covalent Linkage and Macrocylization Preserve and Enhance Synergistic Interactions in Catalytic Amyloids.

The self-assembly of short peptides into catalytic amyloid-like nanomaterials has proven to be a powerful tool in both understanding the evolution of early proteins and identifying new catalysts for practically useful chemical reactions. Here we demonstrate that both parallel and antiparallel arrangements of ß-sheets can accommodate metal ions in catalytically productive coordination environments. Moreover, synergistic relationships, identified in catalytic amyloid mixtures, can be captured in macrocyclic and sheet-loop-sheet species, that offer faster rates of assembly and provide more complex asymmetric arrangements of functional groups, thus paving the way for future designs of amyloid-like catalytic proteins. Our findings show how initial catalytic activity in amyloid assemblies can be propagated and improved in more-complex molecules, providing another link in a complex evolutionary chain between short, potentially abiotically produced peptides and modern-day enzymes.

Synergistic Interactions Are Prevalent in Catalytic Amyloids.

Interactions between multiple functional groups are key to catalysis. Previously, we reported synergistic interactions in catalytic amyloids formed by mixtures of heptameric peptides that lead to significant improvements in esterase activity. Herein, we describe the in-depth investigation of synergistic interactions within a family of amyloid fibrils, exploring the results of functional group interactions, the effects of chirality and the use of mixed enantiomers within fibrils. Remarkably, we find that synergistic interactions (either positive or negative) are found in the vast majority of binary mixtures of catalytic amyloid-forming peptides. The productive arrangements of functionalities rapidly identified by mixing different peptides will undoubtedly lead to the development of more active catalysts for a variety of different transformations.

Nine-Residue Peptide Self-Assembles in the Presence of Silver to Produce a Self-Healing, Cytocompatible, Antimicrobial Hydrogel.

Silver compounds have been used extensively for wound healing because of their antimicrobial properties, but high concentrations of silver are toxic to mammalian cells. We designed a peptide that binds silver and releases only small amounts of this ion over time, therefore overcoming the problem of silver toxicity. Silver binding was achieved through incorporation of an unnatural amino acid, 3'-pyridyl alanine (3'-PyA), into the peptide sequence. Upon the addition of silver ions, the peptide adopts a beta-sheet secondary structure and self-assembles into a strong hydrogel as characterized by rheology, circular dichroism, and transmission electron microscopy. We show that the resulting hydrogel kills Escherichia coli and Staphylococcus aureus but is not toxic to fibroblasts and could be used for wound healing. The amount of Ag(I) released by hydrogels into the solution is less than 4% and this low amount of Ag(I) does not change in the pH range 6-8. These studies provide an initial indication for use of the designed hydrogel as injectable, antimicrobial wound dressing.

Minimalist de novo Design of Protein Catalysts.

The field of protein design has grown enormously in the past few decades. In this review we discuss the minimalist approach to design of artificial enzymes, in which protein sequences are created with the minimum number of elements for folding and function. This method relies on identifying starting points in catalytically inert scaffolds for active site installation. The progress of the field from the original helical assemblies of the 1980s to the more complex structures of the present day is discussed, highlighting the variety of catalytic reactions which have been achieved using these methods. We outline the strengths and weaknesses of the minimalist approaches, describe representative design cases and put it in the general context of the de novo design of proteins.