We have found that mutation of firefly luciferase can dramatically improve aminoluciferin substrate utilization and selectivity, and allow the use of these substrates to monitor luciferase expression in cells and cell lysates. For example, the F247L mutant improves light output from 6’-aminoluciferin by almost five-fold and can be recommended for improved imaging of this substrate (; Figure S1
). This has particular significance for bioluminescence assays of protease activity that rely on detection of this substrate (Monsees et al., 1994
; Shah et al., 2005
; Fan et al., 2007
; Dragulescu-Andrasi et al., 2009
; Hickson et al., 2010
; Scabini et al., 2011
). Light output from all aminoluciferins was greatly increased by the R218K mutant (e.g., 14-fold for CycLuc1, 20-fold for CycLuc2), suggesting this mutant as a starting point for measuring light emission from these and other novel alkylated aminoluciferins. Moreover, mutation of serine 347 to threonine or alanine gives similarly improved light emission from CycLuc1 but also exhibits strong selectivity for CycLuc1 over D-luciferin both in vitro
and in live cells (, , S2, S4
). Combination of mutations such as the ones described here is expected to allow further enhancement of aminoluciferin light output and selectivity. For instance, we have found that the S347A/L286M double mutant gives twice the light output of S347A with CycLuc1, and further discriminates against D-luciferin (Figure S2B
The rate at which light is generated by luciferase is dependent on several factors, including the rate of adenylation and oxidation to afford the oxyluciferin excited state, the quantum yield of light emission, and the rate of product release. To gain insight into the underlying mechanisms by which light emission from aminoluciferins is improved with these luciferase mutants, we monitored the rate of light output over the first 50 seconds following substrate addition (; Figure S3
). For CycLuc1 and 6’-MeNH-LH2
, all mutants result in a rapid initial burst that is similar to that observed for the wild-type protein (Reddy et al., 2010
), suggesting that there are no substantive improvements in the ability to form the respective luciferyl-AMP, its subsequent oxidation to afford the excited-state oxyluciferin, or the quantum yield of light emission. Rather, higher light output was observed after the initial burst (; Figure S3
), implicating a reduction in product inhibition as the primarily factor responsible for the observed improvement in sustained light emission. Even with D-luciferin, the molecular basis for product inhibition is still unresolved, potentially including contributions from both dehydroluciferyl-AMP (L-AMP) and oxyluciferin (Fraga, 2008
). Lowered product inhibition could result from a simple reduction in affinity for aminoluciferin substrates and their corresponding products. Alternatively, it is possible that some mutants function in part by reducing the formation of the corresponding L-AMP analog. A better molecular understanding of the nature of the product inhibition with these substrates may help guide future optimization efforts.
Notably, both the burst and sustained light emission from CycLuc1 exceeds that of 6’-MeNH-LH2
with all of the luciferases we have characterized, and is consistent with a role for cyclization in optimizing aminoluciferin light output (Reddy et al., 2010
). Like CycLuc1, light emission from CycLuc2 is superior to its acyclic counterpart 6’-Me2
for every luciferase we have tested. In contrast to CycLuc1, we find that the burst emission with CycLuc2 is substantially more rapid and intense with the mutant luciferase R218K than with wild-type (; Figure S3
), indicating an improvement in one or more of the enzymatic steps required to form this excited-state oxyluciferin (e.g., adenylation and/or oxidation). We postulate that the bulk and rigidity of CycLuc2 enforces a sub-optimal alignment for the production of the excited-state oxyluciferin in the wild-type luciferase. Enlarging the luciferin binding pocket with the R218K mutation improves the alignment of CycLuc2 within the active site, allowing more rapid production of an efficient light-emitting oxyluciferin excited state (). By comparison, the more flexible substrate 6’-Me2
gives a rapid but weak burst for all mutants (; Figure S3
). We hypothesize that the free bond rotation about the aryl amine bond of 6’-Me2
can facilitate the alignment necessary for the rapid formation of the oxyluciferin, but also limits the efficiency of light output from this excited-state molecule ().
For light emission from live cells, the cell-permeability and Km
of the substrate are important for access to the intracellular luciferase and the efficiency of light output under sub-saturating concentrations. CycLuc2 has a lower Km
than CycLuc1 (Table S2
) and is predicted to be more cell-permeable than CycLuc1 because of the replacement of a polar amine proton with a methyl group (cLogP of 2.5 versus 2.0). These differences are therefore likely to explain the better relative performance of CycLuc2 in live versus lysed cells. Moreover, this suggests that optimization of cell permeability and light output at low substrate concentrations – rather than just maximal light output – may be particularly important for imaging in live cells and organisms, because insufficient substrate is delivered into the live cell to achieve maximal light emission.
The bioluminescence emission wavelength of D-luciferin from different beetle luciferases (firefly, click beetle, railroad worm) and their mutants ranges from ~540-622 nm (Viviani et al., 1999
; Branchini et al., 2010
). Beyond simple changes in the polarity of the environment of the light emitter (Morton et al., 1969
), explanations for this sensitivity include the rigidity of the active site (Nakatsu et al., 2006
) and perturbations in the ionic interactions of the phenolate of D-luciferin (Hirano et al., 2009
). The emission behavior of aminoluciferins can lend insight into this question because they lack an ionizable phenolate (). With the mutant R218K, all luciferins exhibit a modest red-shift in emission wavelength (Table S3
). Because oxyluciferins are asymmetric charge-transfer molecules, some shared solvatochromatic sensitivity to the polarity of the luciferin binding site is expected (Naumov et al., 2009
). However, D-luciferin exhibits anomalous emission behavior with several mutants: the L286M mutation causes a red-shift in the emission of D-luciferin but a blue-shift in the emission of all aminoluciferins, and D-luciferin uniquely gives bimodal emission with S347A (Table S3
). These differences are consistent with a role for the ionization state and ionic interactions of the phenol in determining the bioluminescence emission wavelength when D-luciferin is the substrate.
The efficient chemical generation of light by firefly luciferase has been widely used as a sensitive reporter system for gene expression (de Wet et al., 1987
; Prescher et al., 2010
). However, the application of bioluminescence detection as a general optical reporter of cellular status has lagged behind that of fluorescence. Chemical modification of the luciferin substrate can red-shift the emission wavelength of bioluminescence beyond that of D-luciferin (Reddy et al., 2010
). Moreover, the combination of synthetic luciferins and mutant luciferases described here not only extends the wavelength of bioluminescence emission and broadens the scope of substrates that can be used for bioluminescent assays both in vitro
and in mammalian cells, but also suggests that orthogonal luciferases could be developed to allow multiplexing of bioluminescent signals. Further chemical modification of luciferin substrates and corresponding mutation of firefly and other beetle luciferases is therefore anticipated to be a fruitful avenue for expanding the power and scope of bioluminescence assays and imaging.
Aminoluciferin, in which the 6’-hydroxyl group of D-luciferin is replaced by a 6’-amino group, is a firefly luciferase substrate that is widely used for bioluminescent protease assays. Recently we and others have discovered that a wide variety of modifications of the 6’-amino group retain bioluminescent light emission, including alkylation of the 6’-amino group and 5’,6’-fused ring structures. This family of synthetic aminoluciferin substrates exhibits desirable properties such as light emission at longer wavelengths than D-luciferin and increased affinity for luciferase, but is limited by low sustained light output after an initial flash of light. Here we have described the creation of mutant luciferases that yield improved sustained light emission with aminoluciferins, both in vitro and in lysed and live mammalian cells. Light output from 6’-aminoluciferin is greatly increased with the luciferase mutant F247L, which has implications for the use of this substrate in bioluminescent protease assays. The luciferase mutant R218K gives broadly improved light output with a wide variety of aminoluciferins, including monoalkylated, dialkylated, and cyclic aminoluciferin substrates. Significant discrimination between luciferin substrates was observed for many mutants, suggesting that orthogonal luciferases could be developed to allow multiplexing of bioluminescent signals. For example, the cyclic alkylaminoluciferin CycLuc1 is a better substrate for S347A luciferase than D-luciferin. Aminoluciferins possess unique physical and photophysical properties that have yet to be fully exploited in bioluminescent assays and imaging, and these mutant luciferases broaden the scope and utility of their application.