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.

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.

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.

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.

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.

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.

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.

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. The section on combinatorial design describes recent advances in the discovery of catalytic peptides. We present the future prospects of these and other new approaches in a broader context, including implications for functional material design.

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 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. The ability to create new catalysts that can be turned on and off using allosteric interactions would greatly expand the chemical biology toolbox and will allow for detection of environmental pollutants and disease biomarkers and facilitate studies of cellular processes and metal homeostasis. Thus, design of allosterically regulated protein catalysts represents an actively growing area of research. In this paper, we describe various approaches to achieving regulation of catalysis.

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. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

Kinetics and mechanistic analysis of an extremely rapid carbon dioxide fixation reaction.

Carbon dioxide may react with free or metal-bound hydroxide to afford products containing bicarbonate or carbonate, often captured as ligands bridging two or three metal sites. We report the kinetics and probable mechanism of an extremely rapid fixation reaction mediated by a planar nickel complex [Ni(II)(NNN)(OH)](1-) containing a tridentate 2,6-pyridinedicarboxamidate pincer ligand and a terminal hydroxide ligand. The minimal generalized reaction is M-OH + CO(2) → M-OCO(2)H; with variant M, previous rate constants are ≲10(3) M(-1) s(-1) in aqueous solution. For the present bimolecular reaction, the (extrapolated) rate constant is 9.5 × 10(5) M(-1) s(-1) in N,N'-dimethylformamide at 298 K, a value within the range of k(cat)/K(M)≈10(5)-10(8) M(-1) s(-1) for carbonic anhydrase, the most efficient catalyst of CO(2) fixation reactions. The enthalpy profile of the fixation reaction was calculated by density functional theory. The initial event is the formation of a weak precursor complex between the Ni-OH group and CO(2), followed by insertion of a CO(2) oxygen atom into the Ni-OH bond to generate a four center Ni(η(2)-OCO(2)H) transition state similar to that at the zinc site in carbonic anhydrase. Thereafter, the Ni-OH bond detaches to afford the Ni(η(1)-OCO(2)H) fragment, after which the molecule passes through a second, lower energy transition state as the bicarbonate ligand rearranges to a conformation very similar to that in the crystalline product. Theoretical values of metric parameters and activation enthalpy are in good agreement with experimental values [ΔH(‡) = 3.2(5) kcal/mol].

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.

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.

Fast carbon dioxide fixation by 2,6-pyridinedicarboxamidato-nickel(II)-hydroxide complexes: influence of changes in reactive site environment on reaction rates.

The planar complexes [Ni(II)(pyN(2)(R2))(OH)](-), containing a terminal hydroxo group, are readily prepared from N,N'-(2,6-C(6)H(3)R(2))-2,6-pyridinedicarboxamidate(2-) tridentate pincer ligands (R(4)N)(OH), and Ni(OTf)(2). These complexes react cleanly and completely with carbon dioxide in DMF solution in a process of CO(2) fixation with formation of the bicarbonate product complexes [Ni(II)(pyN(2)(R2))(HCO(3))](-) having η(1)-OCO(2)H ligation. Fixation reactions follow second-order kinetics (rate = k(2)'[Ni(II)-OH][CO(2)]) with negative activation entropies (-17 to -28 eu). Reactions were monitored by growth and decay of metal-to-ligand charge-transfer (MLCT) bands at 350-450 nm. The rate order R = Me > macro > Et > Pr(i) > Bu(i) > Ph at 298 K (macro = macrocylic pincer ligand) reflects increasing steric hindrance at the reactive site. The inherent highly reactive nature of these complexes follows from k(2)' ≈ 10(6) M(-1) s(-1) for the R = Me system that is attenuated by only 100-fold in the R = Ph complex. A reaction mechanism is proposed based on computation of the enthalpic reaction profile for the R = Pr(i) system by DFT methods. The R = Et, Pr(i), and Bu(i) systems display biphasic kinetics in which the initial fast process is followed by a slower first order process currently of uncertain origin.

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.

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.

Aromatic hydroxylation at a non-heme iron center: observed intermediates and insights into the nature of the active species.

Mechanism of substrate oxidations with hydrogen peroxide in the presence of a highly reactive, biomimetic, iron aminopyridine complex, [Fe(II)(bpmen)(CH(3)CN)(2)][ClO(4)](2) (1; bpmen=N,N'-dimethyl-N,N'-bis(2-pyridylmethyl)ethane-1,2-diamine), is elucidated. Complex 1 has been shown to be an excellent catalyst for epoxidation and functional-group-directed aromatic hydroxylation using H(2)O(2), although its mechanism of action remains largely unknown. Efficient intermolecular hydroxylation of unfunctionalized benzene and substituted benzenes with H(2)O(2) in the presence of 1 is found in the present work. Detailed mechanistic studies of the formation of iron(III)-phenolate products are reported. We have identified, generated in high yield, and experimentally characterized the key Fe(III)(OOH) intermediate (λ(max)=560 nm, rhombic EPR signal with g=2.21, 2.14, 1.96) formed by 1 and H(2)O(2). Stopped-flow kinetic studies showed that Fe(III)(OOH) does not directly hydroxylate the aromatic rings, but undergoes rate-limiting self-decomposition producing transient reactive oxidant. The formation of the reactive species is facilitated by acid-assisted cleavage of the O-O bond in the iron-hydroperoxide intermediate. Acid-assisted benzene hydroxylation with 1 and a mechanistic probe, 2-Methyl-1-phenyl-2-propyl hydroperoxide (MPPH), correlates with O-O bond heterolysis. Independently generated Fe(IV)=O species, which may originate from O-O bond homolysis in Fe(III)(OOH), proved to be inactive toward aromatic substrates. The reactive oxidant derived from 1 exchanges its oxygen atom with water and electrophilically attacks the aromatic ring (giving rise to an inverse H/D kinetic isotope effect of 0.8). These results have revealed a detailed experimental mechanistic picture of the oxidation reactions catalyzed by 1, based on direct characterization of the intermediates and products, and kinetic analysis of the individual reaction steps. Our detailed understanding of the mechanism of this reaction revealed both similarities and differences between synthetic and enzymatic aromatic hydroxylation reactions.

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.