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.

Protein cage self-assembly enables encapsulation and sequestration of small molecules, macromolecules, and nanomaterials for many applications in bionanotechnology. Notably, wild-type thermophilic ferritin from Archaeoglobus fulgidus (AfFtn) exists as a stable dimer of four-helix bundle proteins at low ionic strength, and the protein forms a hollow assembly of 24 protomers at high ionic strength (∼800 mM NaCl). This assembly process can also be initiated by highly charged gold nanoparticles (AuNPs) in solution, leading to encapsulation. These data suggest that salt solutions or charged AuNPs can shield unfavorable electrostatic interactions at AfFtn dimer-dimer interfaces, but specific “hot-spot” residues controlling assembly have not been identified. To investigate this further, we computationally designed three AfFtn mutants (E65R, D138K, A127R) that introduce a single positive charge at sites along the dimer-dimer interface. These proteins exhibited different assembly kinetics and thermodynamics, which were ranked in order of increasing 24mer propensity: A127R < WT < D138K ≪ E65R. E65R assembled to the 24mer across a wide range of ionic strengths (0 – 800 mM NaCl), and the dissociation temperature for the 24mer was 98 °C. X-ray crystal structure analysis of the E65R mutant identified a more compact, closed-pore cage geometry. A127R and D138K mutants exhibited wild-type ability to encapsulate and stabilize 5-nm AuNPs, whereas E65R gained ability to remain assembled in apo-form. This work illustrates designed protein cages with distinct assembly and encapsulation properties.

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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.

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...