NMR-guided directed evolution

Abstract

Directed evolution is a powerful tool for improving existing properties and imparting completely new functionalities to proteins^1-4. 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 mutations^6-9. 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.

Extended Data Fig. 1 |. Selection of negative controls for screening.

Left. We have sorted all residues in myoglobin in bins based on their distance to the docked inhibitor (black) in FerrElCat. The residues in the van der Waals contact with the docked inhibitor were placed in bin 1 (red), the residues in direct contact with the residues in bin 1 were placed in bin 2 (yellow), etc. A total of five bins were devised: red, yellow, orange, green and blue. Right. The list of the residues sorted in the five bins. Residues showing large backbone CSP and their immediate neighbors are highlighted in red and yellow, respectively. Unassigned positions and residues immediately next to unassigned stretches are shown in dark grey and light grey, respectively. Prolines are highlighted in blue. Residues showing small CSP that were selected as controls are either highlighted in green or labeled in green font (when located next to unassigned residues).

Extended Data Fig. 2 |. Substrate turnover by reduced Mb-L29I/H64G/V68A (FerrElCat).

Reaction was monitored using stopped-flow at pH 8.0 for 30 s at 25 °C with 140 μM of 5-NBI and 5 nM of FerrElCat.

Extended Data Fig. 3 |. Catalytic parameters of Kemp eliminases evolved using directed evolution:

k_cat/K_M and k_cat values for the evolved enzymes (left) and improvement in k_cat/K_M and k_cat achieved by directed evolution (right). In cases where only k_cat/K_M was reported, we used 5 mM for K_M, to obtain low estimate of the k_cat.

Extended Data Fig. 4 |. Spectroelectrochemical determination of redox potentials of selected myoglobin mutants.

The proteins were analyzed in 20 mM TRIS-HCl (pH 8.0) at 20 °C in presence of the mediator (100 μM phenazine sulfate). The redox potentials (vs Ag/AgCl) are summarized in Supplementary Table 5.

Extended Data Fig. 5 |. Kemp elimination catalyzed by AlleyCat10 in presence of Ca²⁺ (black) and in absence of Ca²⁺ (red).

The activity of 0.1 μM AlleyCat10 was tested with 0.12–0.96 mM substrate in 20 mM HEPES, 100 mM NaCl (pH 7.0) at 22°C with 10 mM CaCl₂ (black) or 100 μM EDTA (red).

Extended Data Fig. 6 |. Absorbance spectra of Mb-H64V (left) and Mb-L29I/H64G/V68A (FerrElCat) (right) in the oxidized (black) and reduced (red) forms.

Extended Data Fig. 7 |. Michaelis-Menten plots of Kemp elimination catalyzed by reduced (unless otherwise stated) myoglobin mutants and by AlleyCat proteins.

Final reaction mixtures for myoglobin mutant analyses contained 1 mM L-ascorbic acid, 0.1 μM SOD, 20 nM catalase, 140–840 μM substrate, 1.5% acetonitrile in 20 mM TRIS (pH 8.0). The protein concentration was 1 μM for Mb-H64V, 0.1 or 0.25 μM for Mb-H64V-based double variants, 5 nM for Mb-H64G or Mb-H64G-based double or triple variants. AlleyCat reaction mixtures contained 0.1 μM proteins with 0.12–0.96 mM substrate in 1.5% acetonitrile, 20 mM TRIS (pH 8.0), 10 mM CaCl₂, 100 mM NaCl. Kinetic parameters are summarized in Table 1 and Extended Data Table 1.

Extended Data Fig. 7 |. Michaelis-Menten plots of Kemp elimination catalyzed by reduced (unless otherwise stated) myoglobin mutants and by AlleyCat proteins.

Final reaction mixtures for myoglobin mutant analyses contained 1 mM L-ascorbic acid, 0.1 μM SOD, 20 nM catalase, 140–840 μM substrate, 1.5% acetonitrile in 20 mM TRIS (pH 8.0). The protein concentration was 1 μM for Mb-H64V, 0.1 or 0.25 μM for Mb-H64V-based double variants, 5 nM for Mb-H64G or Mb-H64G-based double or triple variants. AlleyCat reaction mixtures contained 0.1 μM proteins with 0.12–0.96 mM substrate in 1.5% acetonitrile, 20 mM TRIS (pH 8.0), 10 mM CaCl₂, 100 mM NaCl. Kinetic parameters are summarized in Table 1 and Extended Data Table 1.

Extended Data Fig. 7 |. Michaelis-Menten plots of Kemp elimination catalyzed by reduced (unless otherwise stated) myoglobin mutants and by AlleyCat proteins.

Final reaction mixtures for myoglobin mutant analyses contained 1 mM L-ascorbic acid, 0.1 μM SOD, 20 nM catalase, 140–840 μM substrate, 1.5% acetonitrile in 20 mM TRIS (pH 8.0). The protein concentration was 1 μM for Mb-H64V, 0.1 or 0.25 μM for Mb-H64V-based double variants, 5 nM for Mb-H64G or Mb-H64G-based double or triple variants. AlleyCat reaction mixtures contained 0.1 μM proteins with 0.12–0.96 mM substrate in 1.5% acetonitrile, 20 mM TRIS (pH 8.0), 10 mM CaCl₂, 100 mM NaCl. Kinetic parameters are summarized in Table 1 and Extended Data Table 1.

Fig. 1 |. Kemp elimination promoted by acid-base or redox mechanisms.

Fig. 2 |. NMR-guided evolution of myoglobin.

a, Backbone amide CSP of Mb-H64V upon addition of 2 molar equivalents of 6-NBT. The red bars indicate the protein regions experiencing large chemical shift perturbation (Z≳1). No bars are provided where no backbone resonance assignment could be made. The positions where productive mutations were found are marked by red asterisks (along with the corresponding increase in k_cat/K_M relative to Mb-H64V, top). Positions where screening identified no productive mutations are marked by blue asterisks. The corresponding representative ¹H-¹⁵N HSQC spectral regions are shown in panel b. c, Michaelis-Menten plots for representative proteins. d, NMR CSP data mapped on the X-ray crystal structure of Mb-H64V (PDB 6cf0) showing the residues with prominent changes (Z≳1) as yellow sticks. The spheres show backbone nitrogen atoms of the residues with identified productive mutations (red) or those for which no productive mutations could be found (blue). e, Overlay of the crystal structures of Mb-H64V (yellow) and FerrElCat with the docked inhibitor (cyan). The newly introduced mutations are shown in red.

Fig. 3 |. NMR-guided evolution of calmodulin.

a-c, Backbone amide CSP of cCaM/AlleyCat (a), AlleyCat7 (b) and AlleyCat8 (c) upon addition of 2 molar equivalents of 6-NBT. The red bars indicate the protein regions experiencing large chemical shift perturbation (Z≳1). The open red and grey bars identify EF hand residues. The catalytic F92E mutation is shown with a solid black bar. The positions where productive mutations were found are marked by red asterisks in b and c (along with the corresponding increase in k_cat/K_M relative to the previous round of design, top). Positions where screening identified no productive mutations are marked by blue asterisks in b. The solid grey bars in b and c refer to residues already mutated in previous rounds. The difference in Z-score of crystallographic B-factors (Cα) for the inhibitor bound and free AlleyCat9 is mapped onto the CSP data on AlleyCat8 (ΔZ_B trace in c). d Michaelis-Menten plots for representative proteins. e Overlay of the crystal structures of C-terminal domain of calmodulin (magenta), AlleyCat9 with the inhibitor (cyan) and AlleyCat10 with the inhibitor (yellow). The residues identified in CSP analysis are shown in red. f Overlay of the crystal structures of apo (cyan) and the inhibitor bound (yellow) in AlleyCat10.