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

Luciferases catalyze light-emitting reactions that produce a rainbow of colors from their substrates (luciferins), molecular oxygen, and often additional cofactors. These bioluminescence (BL) systems have afforded an incredible variety of basic research and medical applications. Driven by the importance of BL-based non-invasive animal imaging (BLI) applications, especially in support of cancer research, new BL systems have been developed by engineering beetle luciferase (Luc) variants and synthetic substrate combinations to produce red to near-infrared (nIR) light to improve imaging sensitivity and resolution. To stimulate the application of BLI research and advance the development of improved reagents for BLI, we undertook a systematic comparison of the spectroscopic and BL properties of seven beetle Lucs with LH2 and nine substrates, which included two new quinoline ring-containing analogs. The results of these experiments with purified Luc enzymes in vitro and in live HEK293T cells transfected with luc genes have enabled us to identify Luc/analog combinations with improved properties compared to those previously reported and to provide live cell BL data that may be relevant to in vivo imaging applications. Additionally, we found strong candidate enzyme/substrate pairs for in vitro biomarker applications requiring nIR sources with minimal visible light components. Notably, one of our new substrates paired with a previously developed Luc variant was demonstrated to be an excellent in vitro source of nIR and a potentially useful BL system for improved resolution in BLI.

Mutagenesis and Structural Studies Reveal the Basis for the Activity and Stability Properties That Distinguish the Photinus Luciferases scintillans and pyralis.

The dazzling yellow-green light emission of the common North American firefly Photinus pyralis and other bioluminescent organisms has provided a wide variety of prominent research applications like reporter gene assays and in vivo imaging methods. While the P. pyralis enzyme has been extensively studied, only recently has a second Photinus luciferase been cloned from the species scintillans. Even though the enzymes share very high sequence identity (89.8%), the color of the light they emit, their specific activity and their stability to heat, pH, and chemical denaturation are quite different with the scintillans luciferase being generally more resistant. Through the construction and evaluation of the properties of chimeric domain swapped, single point, and various combined variants, we have determined that only six amino acid changes are necessary to confer all of the properties of the scintillans enzyme to wild-type P. pyralis luciferase. Altered stability properties were attributed to four of the amino acid changes (T214N/S276T/H332N/E354N), and single mutations each predominantly changed emission color (Y255F) and specific activity (A222C). Results of a crystallographic study of the P. pyralis enzyme containing the six changes (Pps6) provide some insight into the structural basis for some of the documented property differences.

Metal-citrate complex transport in Kineococcus radiotolerans.

The growth of an organism is highly dependent on the acquisition of carbon and metals, and availability of these nutrients in the environment affects its survival. Organisms can obtain both nutrients simultaneously through proteins of the CitMHS superfamily. Bioinformatic studies suggested a CitMHS gene (Accession number ABS03965.1) in Kineococcus radiotolerans. Radio flux assays following 14-C radiolabelled citrate, either free or complexed to a variety of metal ions, in K. radiotolerans demonstrated internalization of the citrate when bound to select metal ions only, primarily in the form of calcium-citrate. A pH response was also observed, consistent with a permease (ATP independent) mechanism as noted for other CitMHS family members, with greater uptake at pH 7 compared to pH 10. These results confirm the ability of K. radiotolerans to transport complexed citrate.

The Lysosomal Protein Saposin†B Binds Chloroquine.

Chloroquine (CQ) has been widely used in the treatment of malaria since the 1950s, though toxicity and resistance is increasingly limiting its use in the clinic. More recently, CQ is also becoming recognized as an important therapeutic compound for the treatment of autoimmune disorders and has shown activity as an anticancer agent. However, the full extent of CQ pharmacology in humans is still unclear. Herein, we demonstrate that the lysosomal protein saposin B (sapB), critical for select lipid degradation, binds CQ with implications for both CQ function and toxicity. Using isothermal titration calorimetry (ITC) and fluorescence quenching experiments, CQ was shown to bind to the dimeric form of sapB at both pH 5.5 and pH 7.4 with an average binding affinity of 2.3×10(4) m(-1). X-ray crystallography confirmed this, and the first complete crystal structure of sapB with a bound small molecule (CQ) is reported. The results suggest that sapB might play a role in mitigating CQ-based toxicity and that sapB might itself be overwhelmed by CQ causing impaired lipid degradation.