Directed evolution can rapidly achieve dramatic improvements in the properties of a protein or bestow entirely new functions on it. We have discovered a strong correlation between the probability of nding a productive mutation at a particular position of a protein and a chemical shift perturbation in Nuclear Magnetic Resonance spectra upon addition of an inhibitor for the chemical reaction it promotes. In a proof-of-concept study we converted myoglobin, a non-enzymatic protein, into the most active Kemp eliminase reported to date using only three mutations. The observed levels of catalytic e ciency are on par with the levels shown by natural enzymes. This simple approach, that requires no a priori structural or bioinformatic knowledge, is widely applicable and will unleash the full potential of directed evolution. Full TextDirected evolution is a powerful tool for improving existing properties and imparting completely new functionalities onto proteins. [1][2][3][4] Nonetheless, even in small proteins its potential 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 20 100 combinations, which is currently beyond any existing experimental approach. Fortunately, in practice, selective modi cation of relatively few residues is su cient for e cient improvement, functional enhancement and repurposing of existing proteins. 5 Moreover, computational methods have been developed to predict the location, and, in certain cases, identities of potentially productive mutations. [6][7][8][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 limited ability to identify bene cial mutations far from the active site, even though such changes may dramatically improve the catalytic properties of an enzyme. 10 Here we show that mutagenic hot spots in enzymes can be identi ed using Nuclear Magnetic Resonance (NMR) spectroscopy. In a proof-of-concept study we converted myoglobin, a non-enzymatic oxygen storage protein, into a highly e cient Kemp eliminase using only three mutations. The observed levels of catalytic e ciency (k cat /K M of 2.8 x 10 6 M -1 s -1 and k cat /k uncat > 10 8 ) are the highest reported for any designed protein and are on par with the levels shown by natural enzymes for the reactions they are evolved to catalyze. 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 unleash the full potential of directed enzyme evolution.Recent paradigm shifting advances in understanding the fundamental principles that drive enzyme evolution point to a major role of global conformational selection for productive arrangements of functional groups to perfect transition state stabilization, as well as steric and electrostatic interactio...

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.

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.

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.

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