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.

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.

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.

Design of an allosterically regulated retroaldolase.

We employed a minimalist approach for design of an allosterically controlled retroaldolase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000-fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatR's activity is allosterically regulated by Ca(2+) ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (∼2000-fold) and the change in the apparent pKa of the active lysine residue.