Stopped-flow measurement of CO 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.

A Simulation Independent Analysis of Single- and Multi-Component cw ESR Spectra.

The accurate analysis of continuous-wave electron spin resonance (cw ESR) spectra of biological or organic free-radicals and paramagnetic metal complexes is key to understanding their structure-function relationships and electrochemical properties. The current methods of analysis based on simulations often fail to extract the spectral information accurately. In addition, such analyses are highly sensitive to spectral resolution and artifacts, users' defined input parameters and spectral complexity.

Antibacterial and Cytocompatible pH-Responsive Peptide Hydrogel.

A short peptide, FHHF-11, was designed to change stiffness as a function of pH due to changing degree of protonation of histidines. As pH changes in the physiologically relevant range, G' was measured at 0 Pa (pH 6) and 50,000 Pa (pH 8). This peptide-based hydrogel is antimicrobial and cytocompatible with skin cells (fibroblasts).

NMR-guided directed evolution.

Directed evolution is a powerful tool for improving existing properties and imparting completely new functionalities to proteins. Nonetheless, its potential in even small proteins is inherently limited by the astronomical number of possible amino acid sequences. Sampling the complete sequence space of a 100-residue protein would require testing of 20 combinations, which is beyond any existing experimental approach.

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.

Mechanistic studies of the cofactor assembly in class Ib ribonucleotide reductases and protein affinity for MnII and FeII.

Ribonucleotide reductase (RNR) is an essential enzyme found in all organisms. The function of RNR is to catalyze the conversion of nucleotides to deoxynucleotides. RNRs rely on metallocofactors to oxidize a conserved cysteine in the active site of the enzyme into a thiyl radical, which then initiates nucleotide reduction.

Beneficial Impacts of Incorporating the Non-Natural Amino Acid Azulenyl-Alanine into the Trp-Rich Antimicrobial Peptide buCATHL4B.

Antimicrobial peptides (AMPs) present a promising scaffold for the development of potent antimicrobial agents. Substitution of tryptophan by non-natural amino acid Azulenyl-Alanine (AzAla) would allow studying the mechanism of action of AMPs by using unique properties of this amino acid, such as ability to be excited separately from tryptophan in a multi-Trp AMPs and environmental insensitivity. In this work, we investigate the effect of Trp→AzAla substitution in antimicrobial peptide buCATHL4B (contains three Trp side chains).

Characteristics and therapeutic applications of antimicrobial peptides.

The demand for novel antimicrobial compounds is rapidly growing due to the phenomenon of antibiotic resistance in bacteria. In response, numerous alternative approaches are being taken including use of polymers, metals, combinatorial approaches, and antimicrobial peptides (AMPs). AMPs are a naturally occurring part of the immune system of all higher organisms and display remarkable broad-spectrum activity and high selectivity for bacterial cells over host cells.

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 β-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.

Contributions of primary coordination ligands and importance of outer sphere interactions in UFsc, a de novo designed protein with high affinity for metal ions.

Metalloproteins constitute nearly half of all proteins and catalyze some of the most complex chemical reactions. Recently, we reported a design of 4G-UFsc (Uno Ferro single chain), a single chain four-helical bundle with extraordinarily high (30 pM) affinity for zinc. We evaluated the contribution of different side chains to binding of Co(II), Ni(II), Zn(II) and Mn(II) using systematic mutagenesis of the amino acids that constitute the primary metal coordination and outer spheres.

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.

Kemp Eliminases of the AlleyCat Family Possess High Substrate Promiscuity.

Minimalist enzymes designed to catalyze model reactions provide useful starting points for creating catalysts for practically important chemical transformations. We have shown that Kemp eliminases of the AlleyCat family facilitate conversion of leflunomide (an immunosupressor pro-drug) to its active form teriflunomide with outstanding rate enhancement (nearly four orders of magnitude) and catalytic proficiency (more than seven orders of magnitude) without any additional optimization. This remarkable activity is achieved by properly positioning the substrate in close proximity to the catalytic glutamate with very high pK.

Uno Ferro, a de novo Designed Protein, Binds Transition Metals with High Affinity and Stabilizes Semiquinone Radical Anion.

Metalloenzymes often utilize radicals in order to facilitate chemical reactions. Recently, DeGrado and co-workers have discovered that model proteins can efficiently stabilize semiquinone radical anion produced by oxidation of 3,5-di-tert-butylcatechol (DTBC) in the presence of two zinc ions. Here, we show that the number and the nature of metal ions have relatively minor effect on semiquinone stabilization in model proteins, with a single metal ion being sufficient for radical stabilization.

Copper-Containing Catalytic Amyloids Promote Phosphoester Hydrolysis and Tandem Reactions.

Self-assembly of short de novo designed peptides gives rise to catalytic amyloids capable of facilitating multiple chemical transformations. We show that catalytic amyloids can efficiently hydrolyze paraoxon, a widely used, highly toxic organophosphate pesticide. Moreover, these robust and inexpensive metal-containing materials can be easily deposited on various surfaces producing catalytic flow devices.

A Designed Enzyme Promotes Selective Post-translational Acylation.

A computationally designed, allosterically regulated catalyst (CaM M144H) produced by substituting a single residue in calmodulin, a non-enzymatic protein, is capable of efficient and site selective post-translational acylation of lysines in peptides with highly diverse sequences. Calmodulin's binding partners are involved in regulating a large number of cellular processes; this new chemical-biology tool will help to identify them and provide structural insight into their interactions with calmodulin.

Zinc-binding structure of a catalytic amyloid from solid-state NMR.

Throughout biology, amyloids are key structures in both functional proteins and the end product of pathologic protein misfolding. Amyloids might also represent an early precursor in the evolution of life because of their small molecular size and their ability to self-purify and catalyze chemical reactions. They also provide attractive backbones for advanced materials.

Functional tuning of the catalytic residue pK in a de novo designed esterase.

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p-nitrophenyl acetate (pNPA) and 4-nitrophenyl-(2-phenyl)-propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single-histidine residue (His ) into a hydrophobic pocket of calmodulin.

Catalytic Amyloid Fibrils That Bind Copper to Activate Oxygen.

Amyloid-like fibrils assembled from de novo designed peptides lock ligands in a conformation optimal for metal binding and catalysis in a manner similar to how metalloenzymes provide proper coordination environment through fold. These supramolecular assemblies efficiently catalyze p-nitrophenyl ester hydrolysis in the presence of zinc and phenol oxidation by dioxygen in the presence of copper. The resulting heterogeneous catalysts are inherently switchable, as addition and removal of the metal ions turns the catalytic activity on and off, respectively.

Secretion of functional formate dehydrogenase in Pichia pastoris.

Biofuels are an important tool for the reduction of carbon dioxide and other greenhouse emissions. NAD+-dependent formate dehydrogenase has been previously shown to be capable of the electrochemical reduction of carbon dioxide into formate, which can be ultimately converted to methanol. We established that a functional enzyme, tagged for immobilization, could be continuously secreted by Pichia pastoris.

Short Self-Assembling Peptides Are Able to Bind to Copper and Activate Oxygen.

We have shown that de novo designed peptides self-assemble in the presence of copper to create supramolecular assemblies capable of carrying out the oxidation of dimethoxyphenol in the presence of dioxygen. Formation of the supramolecular assembly, which is akin to a protein fold, is critical for productive catalysis since peptides possessing the same functional groups but lacking the ability to self-assemble do not catalyze substrate oxidation. The ease with which we have discovered robust and productive oxygen activation catalysts suggests that these prion-like assemblies might have served as intermediates in the evolution of enzymatic function and opens the path for the development of new catalyst nanomaterials.

Design of Catalytic Peptides and Proteins Through Rational and Combinatorial Approaches.

This review focuses on recent progress in noncomputational methods to introduce catalytic function into proteins, peptides, and peptide assemblies. We discuss various approaches to creating catalytic activity and classification of noncomputational methods into rational and combinatorial classes. The section on rational design covers recent progress in the development of short peptides and oligomeric peptide assemblies for various natural and unnatural reactions.

New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities.

Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases.

Design of allosterically regulated protein catalysts.

Activity of allosteric protein catalysts is regulated by an external stimulus, such as protein or small molecule binding, light activation, pH change, etc., at a location away from the active site of the enzyme. Since its original introduction in 1961, the concept of allosteric regulation has undergone substantial expansion, and many, if not most, enzymes have been shown to possess some degree of allosteric regulation.