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1.
Curr Opin Chem Biol ; 35: 150-158, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27768949

RESUMO

Metabolic engineering offers the potential to renewably produce important classes of chemicals, particularly biofuels, at an industrial scale. DNA synthesis and editing techniques can generate large pathway libraries, yet identifying the best variants is slow and cumbersome. Traditionally, analytical methods like chromatography and mass spectrometry have been used to evaluate pathway variants, but such techniques cannot be performed with high throughput. Biosensors - genetically encoded components that actuate a cellular output in response to a change in metabolite concentration - are therefore a promising tool for rapid and high-throughput evaluation of candidate pathway variants. Applying biosensors can also dynamically tune pathways in response to metabolic changes, improving balance and productivity. Here, we describe the major classes of biosensors and briefly highlight recent progress in applying them to biofuel-related metabolic pathway engineering.


Assuntos
Biocombustíveis , Técnicas Biossensoriais , Engenharia Metabólica/métodos , Transferência Ressonante de Energia de Fluorescência , RNA/metabolismo
2.
Nat Commun ; 7: 12266, 2016 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-27470466

RESUMO

Single-fluorescent protein biosensors (SFPBs) are an important class of probes that enable the single-cell quantification of analytes in vivo. Despite advantages over other detection technologies, their use has been limited by the inherent challenges of their construction. Specifically, the rational design of green fluorescent protein (GFP) insertion into a ligand-binding domain, generating the requisite allosteric coupling, remains a rate-limiting step. Here, we describe an unbiased approach, termed domain-insertion profiling with DNA sequencing (DIP-seq), that combines the rapid creation of diverse libraries of potential SFPBs and high-throughput activity assays to identify functional biosensors. As a proof of concept, we construct an SFPB for the important regulatory sugar trehalose. DIP-seq analysis of a trehalose-binding-protein reveals allosteric hotspots for GFP insertion and results in high-dynamic range biosensors that function robustly in vivo. Taken together, DIP-seq simultaneously accelerates metabolite biosensor construction and provides a novel tool for interrogating protein allostery.


Assuntos
Técnicas Biossensoriais , Proteínas de Fluorescência Verde , Sondas Moleculares , Elementos de DNA Transponíveis , Escherichia coli , Proteínas Ligantes de Maltose , Técnicas de Sonda Molecular , Thermococcus , Trealose/análise
3.
Biochemistry ; 50(7): 1226-37, 2011 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-21214273

RESUMO

Photocontrolled transcription factors could be powerful tools for probing the roles of transcriptional processes in a variety of settings. Previously, we designed a photocontrolled DNA-binding protein based on a fusion between the bZIP region of GCN4 and photoactive yellow protein from Halorhodospira halophila [Morgan, S. A., et al. (2010) J. Mol. Biol. 399, 94-112]. Here we report a structure-based attempt to improve the degree of photoswitching observed with this chimeric protein. Using computational design tools PoPMuSiC 2.0, Rosetta, Eris, and bCIPA, we identified a series of single- and multiple-point mutations that were expected to stabilize the folded dark state of the protein and thereby enhance the degree of photoswitching. While a number of these mutations, particularly those that introduced a hydrophobic residue at position 143, did significantly enhance dark-state protein stability as judged by urea denaturation studies, dark-state stability did not correlate directly with the degree of photoswitching. Instead, the influence of mutations on the degree of photoswitching was found to be related to their effects on the degree to which DNA binding slowed the pB to pG transition in the PYP photocycle. One mutant, K143F, caused an ∼10-fold slowing of the photocycle and also showed the largest difference in the apparent K(d) for DNA binding, 3.5-fold lower, upon irradiation. This change in the apparent K(d) causes a 12-fold enhancement in the fraction bound DNA upon irradiation due to the cooperativity of DNA binding by this family of proteins. The results highlight the strengths and weaknesses of current approaches to a practical problem in protein design and suggest strategies for further improvement of designed photocontrolled transcription factors.


Assuntos
Proteínas de Ligação a DNA/síntese química , Luz , Proteínas Luminescentes/síntese química , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Cristalografia por Raios X , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Halorhodospira halophila/genética , Halorhodospira halophila/metabolismo , Proteínas Luminescentes/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/métodos , Processos Fotoquímicos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína/genética , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/genética
4.
Photochem Photobiol Sci ; 9(10): 1320-6, 2010 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-20835493

RESUMO

Photo-controlled DNA-binding proteins promise to be useful tools for probing complex spatiotemporal patterns of gene expression in living organisms. Here we report a novel photoswitchable DNA-binding protein, GCN4(S)Δ25PYP, based on a truncated GCN4-photoactive yellow protein chimera. In contrast to previously reported designed photoswitchable proteins where DNA binding affinity is enhanced upon irradiation, GCN4(S)Δ25PYP dissociates from DNA when irradiated with blue light. In addition, the rate of thermal relaxation to the ground state, part of the PYP photocycle, is enhanced by DNA binding whereas in previous reported constructs it is slowed. The origins of this reversed photoactivity are analyzed in structural terms.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Fotorreceptores Microbianos/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Dicroísmo Circular , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Luz , Dados de Sequência Molecular , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrofotometria Ultravioleta
5.
J Mol Biol ; 399(1): 94-112, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20363227

RESUMO

Photocontrolled transcription factors could be powerful tools for probing the role of transcriptional processes in settings that are spatially or temporally complex. We report the structure-based design of a photocontrolled bZIP-type DNA binding protein that is a hybrid of the prototypical homodimeric bZIP protein GCN4 and photoactive yellow protein (PYP), a blue-light-sensitive protein from Halorhodospira halophila. A fusion of the C-terminal zipper region of GCN4-bZIP with the N-terminal cap of PYP was designed based on examination of available crystal structure data, analysis of amino acid preference rules for leucine zippers, and mutational and amino acid conservation data for PYP, together with Rosetta-guided structural modeling. The designed fusion protein GCN4Delta25PYP-v2 is monomeric in the dark; fluorescence, circular dichroism, NMR, and analytical ultracentrifugation data indicate that the zipper domain is hidden. DNA binding in the dark causes substantial structural reorganization of GCN4Delta25PYP-v2 with concomitant slowing of the photocycle, consistent with conformational coupling of the DNA binding domain and the light-sensitive domain of the protein. Consistent with this finding, blue-light irradiation causes a 2-fold increase in specific DNA binding affinity that reverses in the dark. The structure-based approach suggests strategies for enhancing this activity and for producing a family of related photocontrolled proteins for manipulating bZIP activity.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Halorhodospira halophila/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Raios Ultravioleta
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