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.

Figure 1.

SCA-identified amino acids used to encode library. The crystal structure of the homologous Luciola cruciata luciferase (PDB 2D1S) is shown with D-luciferin shown in goldenrod with surface. Amino acid positions included in the library (dark blue), amino acid positions removed due to lack of natural diversity in luciferases (light blue) or removed due to difficulty in coding amino acids (gray) are highlighted in the structure.

Figure 2.

Identified mutant luciferases with red-shifted emission spectra. (A) 620:528 emission ratio of wildtype and isolated mutants (n=3). For each mutant, individual lysates were incubated with D-luc for two minutes and luminescence recorded using either a 528nm filter or a 620nm filter and 1s integrations (B) Genotype of identified active mutants. Only amino acid positions mutated in the library are shown (mutations highlighted in red); no mutations outside of those encoded by the library were observed. (C) Thermal stability of Fluc wildtype and isolated mutants (n=3). For each replicate, a single lysate was split and portions were incubated at varying temperatures for 2 minutes and immediately assayed for luminescence using D-luc. (D) Emission spectrum of COL2. (E) Emission spectrum of COL4.

Figure 3.

Selective luciferases identified in this study. (A) Relative luminescence units for emission in the presence of 4’Br-Luc and 7’Pyr-Luc. A single lysate was split and incubated with either modified luciferin and unfiltered emission was recorded for 10s. (B) Genotypes of isolated mutants at library-encoded positions. Only amino acid positions mutated in the library are shown (mutations highlighted in red); no mutations outside of those encoded by the library were observed.

Figure 4.

620:528 ratio for mutants isolated in (A) frequently occurring mutants and (B) alanine mutants corresponding to SCA library positions. For each mutant, individual lysates were incubated with D-luc for two minutes and luminescence recorded using either a 528nm filter or a 620nm filter and 1s integrations. Each mutant was assayed in triplicate.

Figure 5.

T₅₀ values for frequently observed mutations. For each replicate, a single lysate was split and portions were incubated at varying temperatures for 2 minutes and immediately assayed for luminescence using D-luc. Each luciferase was assayed in triplicate.

Figure 6.

Selectivity of frequently occurring mutants. For each replicate, a single lysate was split and incubated with either modified luciferin and unfiltered emission was recorded for 10s. Enzymes indicated with an asterisk indicate no 7’Pyr-Luc activity was observed; thus, the observed ratio is a lower limit on selectivity. All mutants were assayed in triplicate.

Figure 7.

Positions of frequently observed mutations. Active site of homologous enzyme L. cruciata (PDB ID: 2D1S) with luciferin analog shown in goldenrod with surface, key mutated amino acids shown in red, and other SCA-identified positions shown in blue. Although T251 is most proximal to the 4’ position of luciferin, it is not observed to be mutated in our studies while other amino acid positions (G246, F250, N229T) are observed to be mutated in all recovered mutants.