Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases

Abstract

Directed evolution has proven to be an invaluable tool for protein engineering; however, there is still a need for developing new approaches to continue to improve the efficiency and efficacy of these methods. Here, we demonstrate a new method for library design that applies a previously developed bioinformatic method, Statistical Coupling Analysis (SCA). SCA uses homologous enzymes to identify amino acid positions that are mutable and functionally important and engage in synergistic interactions between amino acids. We use SCA to guide a library of the protein luciferase and demonstrate that, in a single round of selection, we can identify luciferase mutants with several valuable properties. Specifically, we identify luciferase mutants that possess both red-shifted emission spectra and improved stability relative to those of the wild-type enzyme. We also identify luciferase mutants that possess a >50-fold change in specificity for modified luciferins. To understand the mutational origin of these improved mutants, we demonstrate the role of mutations at N229, S239, and G246 in altered function. These studies show that SCA can be used to guide library design and rapidly identify synergistic amino acid mutations from a small library.

Orthogonal Bioluminescent Probes from Disubstituted Luciferins

“…Potential sites of steric clashing were observed, but such interactions would likely be advantageous for orthogonal luciferase development. Enzyme engineering can be used to overcome steric penalties and provide more substrate-specific luciferases. ,, …”

“…Enzyme engineering can be used to overcome steric penalties and provide more substrate-specific luciferases. 20,34,35 The disubstituted analogues were prepared using a general method previously reported by our laboratory. 36,37 This route features functionalized anilines and Appel's salt (blue, Scheme 1) to access key cyanobenzothiazole intermediates.…”

Advances in bioluminescence imaging: new probes from old recipes

“…SCA was used to analyze amino acid positions that were mutable and functionally important, along with networks of potentially synergistic interactions. [39*] In a single round of selection, mutants with desirable emission spectra and improved thermostability were identified. Mutants with >50-fold changes in specificity for modified luciferins were also found.…”

Systematic Comparison of Beetle Luciferase-Luciferin Pairs as Sources of Near-Infrared Light for In Vitro and In Vivo Applications

“…Longer wavelength photon production has been accomplished without the assistance of energy transfer processes by, in most instances, extending the π conjugation of the natural substrate in several distinct structural designs [ 10 , 11 , 12 , 13 , 14 ], including the incorporation of a naphthalene ring (NH 2 -NpLH2 and OH-NpLH2) [ 14 ] ( Figure 1 ). For substrate analogs AkaLumine-HCl (Aka) [ 15 ], infraluciferin (iLH 2 ) [ 16 , 17 ], 4′-BrLuc [ 18 , 19 ], NH 2 -NpLH2 [ 14 ] and OH-NpLH2 [ 14 ], the initial BL properties determined with Fluc, a mammalian codon optimized version of P. pyralis Luc (Luc2), or click beetle red Luc (CBR) were optimized for noninvasive in vivo bioluminescence imaging (BLI) applications by mutagenesis strategies including directed evolution.…”

“…Recognizing that enzyme/substrate pairs that produce nIR light are of great importance to the continued development and improvement of in vivo BLI methods, we focused on combinations that, with the exception of the quinoline analogs NH 2 -QLH 2 and OH-QLH 2 , had been successfully employed in BLI studies [ 14 , 15 , 16 , 17 , 18 , 19 , 30 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ]. PLR3 was matched with the quinoline-containing analogs based on the complete in vitro and live cell testing results with the seven Lucs ( Table S1 ).…”