RESUMEN
Multicomponent bioluminescence imaging in vivo requires an expanded collection of tissue-penetrant probes. Toward this end, we generated a new class of near-infrared (NIR) emitting coumarin luciferin analogues (CouLuc-3s). The scaffolds were easily accessed from commercially available dyes. Complementary mutant luciferases for the CouLuc-3 analogues were also identified. The brightest probes enabled sensitive imaging in vivo. The CouLuc-3 scaffolds are also orthogonal to popular bioluminescent reporters and can be used for multicomponent imaging applications. Collectively, this work showcases a new set of bioluminescent tools that can be readily implemented for multiplexed imaging in a variety of biological settings.
Asunto(s)
Luciferina de Luciérnaga , Luciferinas , Mediciones Luminiscentes/métodos , Luciferasas , CumarinasRESUMEN
Engineered luciferase-luciferin pairs have expanded the number of cellular targets that can be visualized in tandem. While light production relies on selective processing of synthetic luciferins by mutant luciferases, little is known about the origin of selectivity. The development of new and improved pairs requires a better understanding of the structure-function relationship of bioluminescent probes. In this work, we report a biochemical approach to assessing and optimizing two popular bioluminescent pairs: Cashew/d-luc and Pecan/4'-BrLuc. Single mutants derived from Cashew and Pecan revealed key residues for selectivity and thermal stability. Stability was further improved through a rational addition of beneficial residues. In addition to providing increased stability, the known stabilizing mutations surprisingly also improved selectivity. The resultant improved pair of luciferases are >100-fold selective for their respective substrates and highly thermally stable. Collectively, this work highlights the importance of mechanistic insight for improving bioluminescent pairs and provides significantly improved Cashew and Pecan enzymes which should be immediately suitable for multicomponent imaging applications.
Asunto(s)
Luciferina de Luciérnaga , Mediciones Luminiscentes , Luciferina de Luciérnaga/química , Mediciones Luminiscentes/métodos , Luciferasas/genética , Luciferasas/química , Luciferinas , MutaciónRESUMEN
Bioluminescence imaging enables the sensitive tracking of cell populations and the visualization of biological processes in living systems. Bioluminescent luciferase/luciferin pairs with far-red and near-infrared emission benefit from the reduced competitive absorption by blood and tissue while also facilitating multiplexing strategies. Luciferins with extended π-systems, such as AkaLumine and recently reported CouLuc-1 and -3, can be used for bioluminescence imaging in this long wavelength regime. Existing synthetic routes to AkaLumine and similar π-extended compounds require a multistep sequence to install the thiazoline heterocycle. Here we detail the development of a two-step strategy for accessing these molecules via a Horner-Wadsworth-Emmons reaction and cysteine condensation sequence from readily available aldehyde starting materials. We detail an improved synthesis of AkaLumine, as well as the corresponding two-carbon homologues, Tri- and Tetra-AkaLumine. We then extended this approach to prepare coumarin- and naphthalene-derived luciferins. These putative luciferins were tested against a panel of luciferases to identify capable emitters. Of these, an easily prepared naphthalene derivative exhibits photon emission on par with that of the broadly used Akaluc/AkaLumine pair with similar emission maxima. Overall, this chemistry provides efficient access to several bioluminescent probes for a variety of imaging applications.
RESUMEN
Fluorescent noncanonical amino acids (fNCAAs) could serve as starting points for the rational design of protein-based fluorescent sensors of biological activity. However, efforts toward this goal are likely hampered by a lack of atomic-level characterization of fNCAAs within proteins. Here, we describe the spectroscopic and structural characterization of five streptavidin mutants that contain the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) at sites proximal to the binding site of its substrate, biotin. Many of the mutants exhibited altered fluorescence spectra in response to biotin binding, which included both increases and decreases in fluorescence intensity as well as red- or blue-shifted emission maxima. Structural data were also obtained for three of the five mutants. The crystal structures shed light on interactions between 7-HCAA and functional groups, contributed either by the protein or by the substrate, that may be responsible for the observed changes in the 7-HCAA spectra. These data could be used in future studies aimed at the rational design of fluorescent, protein-based sensors of small molecule binding or dissociation.
Asunto(s)
Aminoácidos/química , Biotina/química , Proteínas Recombinantes/química , Estreptavidina/química , Sitios de Unión , Fenómenos Biofísicos , Cristalografía por Rayos X/métodos , Fluorescencia , Ligandos , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in ß-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A ß-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on ß-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of ß-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes.
Asunto(s)
Proteínas Mutantes/genética , Conformación Proteica , Ingeniería de Proteínas , beta-Lactamasas/genética , Aminoácidos/genética , Bacterias/enzimología , Sitios de Unión/genética , Dominio Catalítico/genética , Biología Computacional , Estabilidad de Enzimas/genética , Escherichia coli/enzimología , Modelos Moleculares , Simulación de Dinámica Molecular , Proteínas Mutantes/ultraestructura , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , beta-Lactamasas/ultraestructuraRESUMEN
Multi-component bioluminescence imaging requires an expanded collection of luciferase-luciferin pairs that emit far-red or near-infrared light. Toward this end, we prepared a new class of luciferins based on a red-shifted coumarin scaffold. These probes (CouLuc-1s) were accessed in a two-step sequence via direct modification of commercial dyes. The bioluminescent properties of the CouLuc-1 analogs were also characterized, and complementary luciferase enzymes were identified using a two-pronged screening strategy. The optimized enzyme-substrate pairs displayed robust photon outputs and emitted a significant portion of near-infrared light. The CouLuc-1 scaffolds are also structurally distinct from existing probes, enabling rapid multi-component imaging. Collectively, this work provides novel bioluminescent tools along with a blueprint for crafting additional fluorophore-derived probes for multiplexed imaging.