NMR-guided directed evolution

Abstract

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...

Second-Shell Residues Contribute to Catalysis by Predominately Preorganizing the Apo State in PafA

“…For example, without a decent starting point for engineering, relying on random saturation mutagenesis and/or directed evolution to improve catalytic properties remains costly and time-consuming; these approaches also require a high-throughput readout or a means to effect a strong selective pressure during evolution. Accordingly, novel experimental and computational approaches have recently emerged to help identify regions that are tightly coupled to activities in the active site. ,, Nevertheless, mutational effects, especially distal ones in the context of exploring new chemical transformations, are still hard to predict. Alternatively, more specific site-directed mutagenesis requires in-depth structural and mechanistic understanding.…”

“…For example, a combination of mutagenesis, kinetic, NMR, and computational studies of the dihydrofolate reductase supported the notion of a coupled network of interactions between residues that are spatially separated in the protein structure, leading to considerable contributions of distal residues to the hydride donor–acceptor distance distributions and therefore hydride transfer kinetics. ,− Moreover, NMR and extensive molecular dynamics (MD) simulations have shown that in many cases, distal mutations lead to a redistribution of pre-existing conformational states that favor a particular catalytic activity ,− or alter the conformational dynamics of essential structural motifs that gate the active site pocket and therefore substrate binding/product dissociation. ,− Building on these mechanistic insights, considerable progress has also been made to identify the location of remote regions that might couple strongly with the active site, which may help reduce the cost of powerful yet time-consuming enzyme engineering approaches such as random saturation mutagenesis and directed evolution , by providing decent starting points. Prominent examples include using chemical shift perturbations to locate sites that respond to binding of inhibitors, using correlation-based analysis of molecular dynamics trajectories to locate residues allosterically coupled to the active site, and using flexibility profiles to identify sites that dictate functionally relevant dynamics of enzymes.…”

Exploring and Learning the Universe of Protein Allostery Using Artificial Intelligence Augmented Biophysical and Computational Approaches

“…These NMR CHESCA adaptations are based on the invariance of core inter-residue correlations to changes in the chemical shifts of the active and inactive conformations interconverting in fast exchange. Integration of NMR spectroscopy and surface plasmon resonance revealed dynamic communication networks of residues linking the ligand-binding site to the activation interface in the glucocorticoid receptor and identified a specific motif acting as a ligand- and coregulator-dependent allosteric switch governing transcriptional activation . A recently introduced NMR-guided directed evolution approach highlighted a new role of NMR in the selection process of mutational libraries as this approach can identify locations of the allosteric hotspots and mutations that can minimize nonessential protein dynamics to achieve high catalytic efficiency without a priori structural information …”

“…Integration of NMR spectroscopy and surface plasmon resonance revealed dynamic communication networks of residues linking the ligand-binding site to the activation interface in the glucocorticoid receptor and identified a specific motif acting as a ligand-and coregulator-dependent allosteric switch governing transcriptional activation. 124 A recently introduced NMR-guided directed evolution approach highlighted a new role of NMR in the selection process of mutational libraries as this approach can identify locations of the allosteric hotspots and mutations that can minimize nonessential protein dynamics to achieve high catalytic efficiency without a priori structural information. 125 Solution NMR experiments and Gaussian-accelerated molecular dynamics (GaMD) simulations examined the structural and dynamic determinants of allosteric signaling within the CRISPR-Cas9 HNH nuclease, advancing our understanding of the allosteric pathway of activation.…”

O2 Carrier Myoglobin Also Exhibits β-Lactamase Activity That Is Regulated by the Heme Coordination State

“…As an ideal model for protein design, Mb has been applied to construct functional enzymes, such as those capable of reduction of O 2 /NO/NO 2 − and the oxidation of organic compounds [ 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. Moreover, Mb has been designed to catalyze carbene transfer reactions and others by modifying the heme center, heme replacement and directed evolution [ 37 , 38 , 39 , 40 , 41 , 42 ]. These achievements provide valuable insights into the relationship between the structure and function of heme proteins.…”