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.

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. We introduce a simulation-independent spectral analysis approach that enables broader application of ESR. We use a wavelet packet transform-based method for extracting g values and hyperfine (A) constants directly from cw ESR spectra. We show that our method overcomes the challenges associated with simulation-based methods for analyzing poorly/partially resolved and unresolved spectra, which is common in most cases. The accuracy and consistency of the method are demonstrated on a series of experimental spectra of organic radicals and copper-nitrogen complexes. We showed that for a two-component system, the method identifies their individual spectral features even at a relative concentration of 5% for the minor component.

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). It was demonstrated that the incorporation of unnatural AzAla tryptophan analog residue improves the antimicrobial properties of the hydrogel. The material developed can have a practical application and be a paradigm shift in the approach to wound treatment, and it will improve healing outcomes for millions of patients each year.

NMR-guided directed evolution.

Directed evolution is a powerful tool for improving existing properties and imparting completely new functionalities to proteins1-4. 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 20100 combinations, which is beyond any existing experimental approach. In practice, selective modification of relatively few residues is sufficient for efficient improvement, functional enhancement and repurposing of existing proteins5. Moreover, computational methods have been developed to predict the locations and, in certain cases, identities of potentially productive mutations6-9. Importantly, all current approaches for prediction of hot spots and productive mutations rely heavily on structural information and/or bioinformatics, which is not always available for proteins of interest. Moreover, they offer a limited ability to identify beneficial mutations far from the active site, even though such changes may markedly improve the catalytic properties of an enzyme10. Machine learning methods have recently showed promise in predicting productive mutations11, but they frequently require large, high-quality training datasets, which are difficult to obtain in directed evolution experiments. Here we show that mutagenic hot spots in enzymes can be identified using NMR spectroscopy. In a proof-of-concept study, we converted myoglobin, a non-enzymatic oxygen storage protein, into a highly efficient Kemp eliminase using only three mutations. The observed levels of catalytic efficiency exceed those of proteins designed using current approaches and are similar with those of natural enzymes for the reactions that they are evolved to catalyse. Given the simplicity of this experimental approach, which requires no a priori structural or bioinformatic knowledge, we expect it to be widely applicable and to enable the full potential of directed enzyme evolution.

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.

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. The proteins required for MnIII2-Y• cluster formation in class Ib RNRs are NrdF (ß-subunit) and NrdI (flavodoxin). An oxidant is channeled from the FMN cofactor in NrdI to the dimanganese center in NrdF, where it oxidizes the dimanganese center and a tyrosyl radical (Y•) is formed. Both Streptococcus sanguinis and Escherichia coli MnII2-NrdF structures have a constriction in the channel immediately above the metal site. In E. coli, the constriction is formed by the side chain of S159, whereas in the S. sanguinis system it involves T158. This serine-to-threonine substitution was investigated using S. sanguinis and Streptococcus pneumoniae class Ib RNRs but it is also present in other pathogenic streptococci. Using stopped-flow kinetics, we investigate the role of this substitution in the mechanism of MnIII2-Y• cluster formation. In addition to different kinetics observed in the studied streptococci, we found that affinity constants of NrdF for MnII and FeII are about 1 µM and the previously reported preference for MnII could not be explained by affinity only.

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). We found that antimicrobial and bactericidal activity of the original peptide was preserved, while cytocompatibility with human cells and proteolytic stability was improved. We envision that AzAla will find applications as a tool for studies of the mechanism of action of AMPs. In addition, incorporation of this non-natural amino acid into AMP sequences could enhance their application properties.

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.

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. However, despite good activity and safety profiles, AMPs have struggled to find success in the clinic. In this review, we outline the fundamental properties of AMPs that make them effective antimicrobials and extend this into three main approaches being used to help AMPs become viable clinical options. These three approaches are the incorporation of non-natural amino acids into the AMP sequence to impart better pharmacological properties, the incorporation of AMPs in hydrogels, and the chemical modification of surfaces with AMPs for device applications. These approaches are being developed to enhance the biocompatibility, stability, and/or bioavailability of AMPs as clinical options.

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. The binding affinity of proteins for metals was then measured using isothermal titration calorimetry. Our results show that both primary metal coordination environment and side chains in the outer sphere of UFsc are highly sensitive to even slight changes and can be adapted to binding different 3d metals, including hard-to-tightly bind metal ions such as Mn(II). The studies on the origins of tight metal binding will guide future metalloprotein design efforts.

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.

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 pKa.

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. The radical is stabilized by both metal ion, hydrophobic sequestration, and interactions with the hydrophilic residues in the protein interior resulting in a remarkable, nearly 500 mV change in the redox potential of the SQ. - /catechol couple compared to bulk aqueous solution. Moreover, we have created 4G-UFsc, a single metal ion-binding protein with pm affinity for zinc that is higher than any other reported model systems and is on par with many natural zinc-containing proteins. We expect that the robust and easy-to-modify DFsc/UFsc family of proteins will become a versatile tool for mechanistic model studies of metalloenzymes.

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. Finally, functional promiscuity of catalytic amyloids promotes tandem hydrolysis/oxidation reactions. High efficiency discovered in a very small library of peptides suggests an enormous potential for further improvement of catalytic properties both in terms of catalytic efficiency and substrate scope.

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. When ß-strands of an amyloid are arranged parallel and in register, side chains from the same position of each chain align, facilitating metal chelation when the residues are good ligands such as histidine. High-resolution structures of metalloamyloids are needed to understand the molecular bases of metal-amyloid interactions. Here we combine solid-state NMR and structural bioinformatics to determine the structure of a zinc-bound metalloamyloid that catalyzes ester hydrolysis. The peptide forms amphiphilic parallel ß-sheets that assemble into stacked bilayers with alternating hydrophobic and polar interfaces. The hydrophobic interface is stabilized by apolar side chains from adjacent sheets, whereas the hydrated polar interface houses the Zn2+-binding histidines with binding geometries unusual in proteins. Each Zn2+ has two bis-coordinated histidine ligands, which bridge adjacent strands to form an infinite metal-ligand chain along the fibril axis. A third histidine completes the protein ligand environment, leaving a free site on the Zn2+ for water activation. This structure defines a class of materials, which we call metal-peptide frameworks. The structure reveals a delicate interplay through which metal ions stabilize the amyloid structure, which in turn shapes the ligand geometry and catalytic reactivity of Zn2.

Functional tuning of the catalytic residue pKa 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 (His144 ) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pKa value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pKa calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pKa value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pKa value of this residue in water. We determined that a short-range favorable interaction with Glu127 contributes to the elevated pKa of His144 . We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pKa of its active nucleophile, His144 , by 0.7 units. As a direct result of the decrease in the His144 pKa value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine-promoted catalysis. Proteins 2017; 85:1656-1665. © 2017 Wiley Periodicals, Inc.

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. The ease of peptide preparation and self-assembly makes amyloid-like fibrils an attractive platform for developing catalysts for a broad range of chemical reactions. Here, we present a detailed protocol for the preparation of copper-containing fibrils and for kinetic characterization of their abilities to oxidize phenols.

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. The protein can be easily separated from the growth media and its activity remains constant over an extended period of time. This is an important first step in creating a self-sustaining system capable of producing biofuels with minimal resources and space required.