Cyclic AMP receptor protein-aequorin molecular switch for cyclic AMP. 2011

Daniel Scott, and Krystal Teasley Hamorsky, and C Mark Ensor, and Kimberly W Anderson, and Sylvia Daunert
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506, USA.

Molecular switches are designer molecules that combine the functionality of two individual proteins into one, capable of manifesting an "on/off" signal in response to a stimulus. These switches have unique properties and functionalities and thus, can be employed as nanosensors in a variety of applications. To that end, we have developed a bioluminescent molecular switch for cyclic AMP. Bioluminescence offers many advantages over fluorescence and other detection methods including the fact that there is essentially zero background signal in physiological fluids, allowing for more sensitive detection and monitoring. The switch was created by combining the properties of the cyclic AMP receptor protein (CRP), a transcriptional regulatory protein from E. Coli that binds selectively to cAMP with those of aequorin, a bioluminescent photoprotein native of the jellyfish Aequorea victoria . Genetic manipulation to split the genetic coding sequence of aequorin in two and genetically attach the fragments to the N and C termini of CRP resulted in a hybrid protein molecular switch. The conformational change experienced by CRP upon the binding of cyclic AMP is suspected to result in the observed loss of the bioluminescent signal from aequorin. The "on/off" bioluminescence can be modulated by cyclic AMP over a range of several orders of magnitude in a linear fashion in addition to the capacity to detect changes in cellular cyclic AMP of intact cells exposed to different external stimuli without the need to lyse the cells. We envision that the molecular switch could find applications in vitro as well as In Vivo cyclic AMP detection and/or imaging.

UI MeSH Term Description Entries
D008163 Luminescent Measurements Techniques used for determining the values of photometric parameters of light resulting from LUMINESCENCE. Bioluminescence Measurements,Bioluminescent Assays,Bioluminescent Measurements,Chemiluminescence Measurements,Chemiluminescent Assays,Chemiluminescent Measurements,Chemoluminescence Measurements,Luminescence Measurements,Luminescent Assays,Luminescent Techniques,Phosphorescence Measurements,Phosphorescent Assays,Phosphorescent Measurements,Assay, Bioluminescent,Assay, Chemiluminescent,Assay, Luminescent,Assay, Phosphorescent,Assays, Bioluminescent,Assays, Chemiluminescent,Assays, Luminescent,Assays, Phosphorescent,Bioluminescence Measurement,Bioluminescent Assay,Bioluminescent Measurement,Chemiluminescence Measurement,Chemiluminescent Assay,Chemiluminescent Measurement,Chemoluminescence Measurement,Luminescence Measurement,Luminescent Assay,Luminescent Measurement,Luminescent Technique,Measurement, Bioluminescence,Measurement, Bioluminescent,Measurement, Chemiluminescence,Measurement, Chemiluminescent,Measurement, Chemoluminescence,Measurement, Luminescence,Measurement, Luminescent,Measurement, Phosphorescence,Measurement, Phosphorescent,Measurements, Bioluminescence,Measurements, Bioluminescent,Measurements, Chemiluminescence,Measurements, Chemiluminescent,Measurements, Chemoluminescence,Measurements, Luminescence,Measurements, Luminescent,Measurements, Phosphorescence,Measurements, Phosphorescent,Phosphorescence Measurement,Phosphorescent Assay,Phosphorescent Measurement,Technique, Luminescent,Techniques, Luminescent
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D011487 Protein Conformation The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain). Conformation, Protein,Conformations, Protein,Protein Conformations
D011953 Receptors, Cyclic AMP Cell surface proteins that bind cyclic AMP with high affinity and trigger intracellular changes which influence the behavior of cells. The best characterized cyclic AMP receptors are those of the slime mold Dictyostelium discoideum. The transcription regulator CYCLIC AMP RECEPTOR PROTEIN of prokaryotes is not included nor are the eukaryotic cytoplasmic cyclic AMP receptor proteins which are the regulatory subunits of CYCLIC AMP-DEPENDENT PROTEIN KINASES. Cyclic AMP Receptors,cAMP Receptors,Cyclic AMP Receptor,Receptors, cAMP,cAMP Receptor,Receptor, Cyclic AMP,Receptor, cAMP
D011993 Recombinant Fusion Proteins Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes. Fusion Proteins, Recombinant,Recombinant Chimeric Protein,Recombinant Fusion Protein,Recombinant Hybrid Protein,Chimeric Proteins, Recombinant,Hybrid Proteins, Recombinant,Recombinant Chimeric Proteins,Recombinant Hybrid Proteins,Chimeric Protein, Recombinant,Fusion Protein, Recombinant,Hybrid Protein, Recombinant,Protein, Recombinant Chimeric,Protein, Recombinant Fusion,Protein, Recombinant Hybrid,Proteins, Recombinant Chimeric,Proteins, Recombinant Fusion,Proteins, Recombinant Hybrid
D004305 Dose-Response Relationship, Drug The relationship between the dose of an administered drug and the response of the organism to the drug. Dose Response Relationship, Drug,Dose-Response Relationships, Drug,Drug Dose-Response Relationship,Drug Dose-Response Relationships,Relationship, Drug Dose-Response,Relationships, Drug Dose-Response
D000242 Cyclic AMP An adenine nucleotide containing one phosphate group which is esterified to both the 3'- and 5'-positions of the sugar moiety. It is a second messenger and a key intracellular regulator, functioning as a mediator of activity for a number of hormones, including epinephrine, glucagon, and ACTH. Adenosine Cyclic 3',5'-Monophosphate,Adenosine Cyclic 3,5 Monophosphate,Adenosine Cyclic Monophosphate,Adenosine Cyclic-3',5'-Monophosphate,Cyclic AMP, (R)-Isomer,Cyclic AMP, Disodium Salt,Cyclic AMP, Monoammonium Salt,Cyclic AMP, Monopotassium Salt,Cyclic AMP, Monosodium Salt,Cyclic AMP, Sodium Salt,3',5'-Monophosphate, Adenosine Cyclic,AMP, Cyclic,Adenosine Cyclic 3',5' Monophosphate,Cyclic 3',5'-Monophosphate, Adenosine,Cyclic Monophosphate, Adenosine,Cyclic-3',5'-Monophosphate, Adenosine,Monophosphate, Adenosine Cyclic
D000331 Aequorin A photoprotein isolated from the bioluminescent jellyfish Aequorea. It emits visible light by an intramolecular reaction when a trace amount of calcium ion is added. The light-emitting moiety in the bioluminescence reaction is believed to be 2-amino-3-benzyl-5-(p-hydroxyphenyl)pyrazine (AF-350). Aequorine
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D015202 Protein Engineering Procedures by which protein structure and function are changed or created in vitro by altering existing or synthesizing new structural genes that direct the synthesis of proteins with sought-after properties. Such procedures may include the design of MOLECULAR MODELS of proteins using COMPUTER GRAPHICS or other molecular modeling techniques; site-specific mutagenesis (MUTAGENESIS, SITE-SPECIFIC) of existing genes; and DIRECTED MOLECULAR EVOLUTION techniques to create new genes. Genetic Engineering of Proteins,Genetic Engineering, Protein,Proteins, Genetic Engineering,Engineering, Protein,Engineering, Protein Genetic,Protein Genetic Engineering

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