Computational Analysis of Spectroscopical Properties and Binding Affinities of Oxyluciferin Analogs in Firefly Luciferase Protein

Abstract

Design of novel luciferin substrates as near-infrared bioluminescence probes to improve deep-tissue imaging is an active field of research. Several effective analogs of oxyluciferin, such as AkaLumine (bioluminescence wavelength = 677nm), cyclic alkylamino luciferin (CycLuc1) (bioluminescence wavelength = 604 nm) have indeed resulted in longer bioluminescence wavelengths as compared to standard D-luciferin (bioluminescence wavelength = 562nm). Additionally, lower substrate concentration, bioavailability of substrate in the protein binding pocket and higher quantum yield of bioluminescence are also important criterions for the design of ideal luciferin substrate. In order to investigate the effect of substitutions on the oxyluciferin, we have designed a series of luciferin substrates with various functional groups on both 6-hydroxy-benzothiazole and 4(5H)-thiazolone heterocyclic rings. Through performing TDDFT calculations in both gas phase and condensed-phase (using implicit solvent model), we identified several promising combinations of electron donating groups and electron withdrawing groups, as emitted light from these analogs is predicted to be significantly red-shifted compared to standard oxyluciferin. In order to explain shifts in the computed emission wavelengths of oxyluciferin analogs, energy decomposition analysis was performed to study the interaction between molecular orbitals from both oxyluciferin and substituents. In order to design effective bioluminescent probes, effect of microenvironment will be considered along with substrate modifications. QM/MM calculations on luciferin analogs and various mutant luciferase systems can provide useful insights to explain substrate-protein interactions responsible for modulation of bioluminescence wavelength. Herein, we also report relative binding free energies of newly designed luciferin analogs with native firefly luciferase.