RESUMO
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.
Assuntos
Luciferina de Vaga-Lumes , Luciferinas , Medições Luminescentes/métodos , Luciferases , CumarínicosRESUMO
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.
Assuntos
Luciferina de Vaga-Lumes , Medições Luminescentes , Luciferina de Vaga-Lumes/química , Medições Luminescentes/métodos , Luciferases/genética , Luciferases/química , Luciferinas , MutaçãoRESUMO
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.
RESUMO
The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore-environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore-environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions.
Assuntos
Análise Espectral Raman , Análise Espectral Raman/métodos , Proteínas de Fluorescência Verde/metabolismo , Domínio CatalíticoRESUMO
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.
Assuntos
Aminoácidos/química , Biotina/química , Proteínas Recombinantes/química , Estreptavidina/química , Sítios de Ligação , Fenômenos Biofísicos , Cristalografia por Raios X/métodos , Fluorescência , Ligantes , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Proteínas Mutantes/genética , Conformação Proteica , Engenharia de Proteínas , beta-Lactamases/genética , Aminoácidos/genética , Bactérias/enzimologia , Sítios de Ligação/genética , Domínio Catalítico/genética , Biologia Computacional , Estabilidade Enzimática/genética , Escherichia coli/enzimologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/ultraestrutura , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , beta-Lactamases/ultraestruturaRESUMO
Genetically encoded fluorescent noncanonical amino acids (fNCAAs) could be used to develop novel fluorescent sensors of protein function. Previous efforts toward this goal have been limited by the lack of extensive physicochemical and structural characterizations of protein-based sensors containing fNCAAs. Here, we report the steady-state spectroscopic properties and first structural analyses of an fNCAA-containing Fab fragment of the 5c8 antibody, which binds human CD40L. A previously reported 5c8 variant in which the light chain residue IleL98 is replaced with the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) exhibits a 1.7-fold increase in fluorescence upon antigen binding. Determination and comparison of the apparent pKas of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in pKa. Crystal structures of the fNCAA-containing Fab in the apo and bound forms reveal interactions between the 7-HCAA side chain and surrounding residues that are disrupted upon antigen binding. This structural characterization not only provides insight into the manner in which protein environments can modulate the fluorescence properties of 7-HCAA but also could serve as a starting point for the rational design of new fluorescent protein-based reporters of protein function.
Assuntos
Aminoácidos/química , Sítios de Ligação de Anticorpos , Ligante de CD40/química , Fluorescência , Corantes Fluorescentes/química , Fragmentos Fab das Imunoglobulinas/química , Aminoácidos/metabolismo , Ligante de CD40/metabolismo , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Conformação ProteicaRESUMO
Bioluminescence imaging with luciferase-luciferin pairs is commonly used for monitoring biological processes in cells and whole organisms. Traditional bioluminescent probes are limited in scope, though, as they cannot be easily distinguished in biological environments, precluding efforts to visualize multicellular processes. Additionally, many luciferase-luciferin pairs emit light that is poorly tissue penetrant, hindering efforts to visualize targets in deep tissues. To address these issues, we synthesized a set of π-extended luciferins that were predicted to be red-shifted luminophores. The scaffolds were designed to be rotationally labile such that they produced light only when paired with luciferases capable of enforcing planarity. A luciferin comprising an intramolecular "lock" was identified as a viable light-emitting probe. Native luciferases were unable to efficiently process the analog, but a complementary luciferase was identified via Rosetta-guided enzyme design. The unique enzyme-substrate pair is red-shifted compared to well-known bioluminescent tools. The probe set is also orthogonal to other luciferase-luciferin probes and can be used for multicomponent imaging. Four substrate-resolved luciferases were imaged in a single session. Collectively, this work provides the first example of Rosetta-guided design in engineering bioluminescent tools and expands the scope of orthogonal imaging probes.
Assuntos
Luciferina de Vaga-Lumes/química , Luciferases/química , Substâncias Luminescentes/química , Medições Luminescentes , Luciferina de Vaga-Lumes/síntese química , Luciferases/metabolismo , Luminescência , Substâncias Luminescentes/síntese química , Estrutura MolecularRESUMO
We describe an approach for the development of fluorescent sensors of metabolite binding in which a genetically encoded fluorescent non-canonical amino acid (fNCAA) containing a 7-hydroxycoumarin moiety (7-HCAA) forms a FRET pair with native tryptophan residues. Although previous studies demonstrated the potential for using 7-HCAA as an acceptor for tryptophan, this approach has not yet been explored within a single protein containing multiple tryptophan residues. A structure-based analysis of a hexokinase enzyme with multiple native tryptophan residues identified glutamate 50 as a potential site of 7-HCAA incorporation; Glu50 moves closer to the native tryptophans upon substrate binding. Substitution of 7-HCAA at residue 50 led to an increase in FRET efficiency in the presence of the substrate; this effect was not observed in a control protein where no change in distance between 7-HCAA and the native tryptophans occurs on substrate binding. This system was then used to directly observe differences in binding affinity of the hexokinase that occur at a number of pH values. Our approach builds on previous research in that it eliminates the need for the incorporation of multiple fNCAAs or fluorescent labels within a target protein and can be used to study substrate binding with native ligands. As such, it serves to expand the versatility of FRET-based techniques.
Assuntos
Aminoácidos/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Hexoquinase/química , Umbeliferonas/química , Corantes Fluorescentes/síntese química , Hexoquinase/metabolismo , Ligantes , Modelos Moleculares , Conformação MolecularRESUMO
Synthetic auxotrophy in which bacterial viability depends on the presence of a synthetic amino acid provides a robust strategy for the containment of genetically modified organisms and the development of safe, live vaccines. However, a simple, general strategy to evolve essential proteins to be dependent on synthetic amino acids is lacking. Using a temperature-sensitive selection system, we evolved an Escherichia coli (E. coli) sliding clamp variant with an orthogonal protein-protein interface, which contains a Leu273 to p-benzoylphenyl alanine (pBzF) mutation. The E. coli strain with this variant DNA clamp has a very low escape frequency (<10-10), and its growth is strictly dependent on the presence of pBzF. This selection strategy can be generally applied to create ncAA dependence of other organisms with DNA clamp homologues.
Assuntos
Aminoácidos/classificação , Aminoácidos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Engenharia Genética , Viabilidade Microbiana , Engenharia de ProteínasRESUMO
Metal-chelating heteroaryl small molecules have found widespread use as building blocks for coordination-driven, self-assembling nanostructures. The metal-chelating noncanonical amino acid (2,2'-bipyridin-5yl)alanine (Bpy-ala) could, in principle, be used to nucleate specific metalloprotein assemblies if introduced into proteins such that one assembly had much lower free energy than all alternatives. Here we describe the use of the Rosetta computational methodology to design a self-assembling homotrimeric protein with [Fe(Bpy-ala)3]2+ complexes at the interface between monomers. X-ray crystallographic analysis of the homotrimer showed that the design process had near-atomic-level accuracy: The all-atom rmsd between the design model and crystal structure for the residues at the protein interface is â¼1.4 Å. These results demonstrate that computational protein design together with genetically encoded noncanonical amino acids can be used to drive formation of precisely specified metal-mediated protein assemblies that could find use in a wide range of photophysical applications.
Assuntos
Metaloproteínas/química , Engenharia de Proteínas/métodos , Piridinas/química , Aminoácidos/química , Clonagem Molecular , Biologia Computacional/métodos , Simulação por Computador , Cristalografia por Raios X , Metais/química , Modelos Moleculares , Conformação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , SoftwareRESUMO
The Violence Risk Appraisal Guide-Revised (VRAG-R) was developed to replace the original VRAG based on an updated and larger sample with an extended follow-up period. Using a sample of 120 adult male correctional offenders, the current study examined the interrater reliability and predictive and comparative validity of the VRAG-R to the VRAG, the Psychopathy Checklist-Revised, the Statistical Information on Recidivism-Revised, and the Two-Tiered Violence Risk Estimate over a follow-up period of up to 22 years postrelease. The VRAG-R achieved moderate levels of predictive validity for both general and violent recidivism that was sustained over time as evidenced by time-dependent area under the curve (AUC) analysis. Further, moderate predictive validity was evident when the Antisociality item was both removed and then subsequently replaced with a substitute measure of antisociality. Results of the individual item analyses for the VRAG and VRAG-R revealed that only a small number of items are significant predictors of violent recidivism. The results of this study have implications for the application of the VRAG-R to the assessment of violent recidivism among correctional offenders. (PsycINFO Database Record