Fluorescence resonance energy transfer within a heterochromatic cAMP-dependent protein kinase holoenzyme under equilibrium conditions: New insights into the conformational changes that result in cAMP-dependent activation

Biochemistry ◽  
1993 ◽  
Vol 32 (25) ◽  
pp. 6402-6410 ◽  
Author(s):  
David A. Johnson ◽  
Valerie L. Leathers ◽  
Anna M. Martinez ◽  
Donal A. Walsh ◽  
William H. Fletcher
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Protein Kinase ◽  
Equilibrium Conditions ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals A cGMP-Dependent Protein Kinase Assay for High Throughput Screening Based on Time-Resolved Fluorescence Resonance Energy Transfer

2001 ◽  
Vol 6 (4) ◽  
pp. 255-264 ◽  
Author(s):  
Benjamin Bader ◽  
Elke Butt ◽  
Alois Palmetshofer ◽  
Ulrich Walter ◽  
Thomas Jarchau ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
Cyclic Gmp ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Protein Kinase ◽  
Time Resolved ◽  
Dependent Protein

Activation of cyclic GMP-dependent protein kinase (cGK) is an important event in the regulation of blood pressure and platelet function. Upstream signals are the generation of nitric oxide (NO) by NO syntheses and the subsequent rise in cyclic GMP levels mediated by NO-dependent guanylyl cyclases (GCs). The identification of new cGK activators by high throughput sreening (HTS) may lead to the development of a novel class of therapeutics for the treatment of cardiovascular diseases. Therefore, a homogeneous, nonradioactive assay for cGK activity was developed using a biotinylated peptide derived from vasodilator-stimulated phosphoprotein (VASP), a well-characterized natural cGK substrate. The phosphorylated peptide could be detected by a VASP-specific monoclonal phosphoserine antibody and a fluorescent detection system consisting of a europium-labeled secondary antibody and allophycocyanin (APC)-labeled streptavidin. Fluorescence resonance energy transfer (FRET) from europium to APC was detected in a time-resolved fashion (TR-FRET). Activation and inhibition constants for known substances determined by this new fluorescence-based assay correlated well with published results obtained by conventional radioactive cGK activity assays. The assay proved to be sensitive, robust, highly specific for cGK, and suitable for HTS in 96- and 384-well formats. This assay is applicable to purified enzymes as well as to complex samples such as human platelet extracts.

scholarly journals Fluorescence Resonance Energy Transfer in Quantum Dot-Protein Kinase Assemblies

2007 ◽  
Vol 2007 ◽  
pp. 1-5 ◽  
Author(s):  
Ibrahim Yildiz ◽  
Xinxin Gao ◽  
Thomas K. Harris ◽  
Françisco M. Raymo
Keyword(s):  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinase Domain ◽  
Binding Domain ◽  
Dependent Protein ◽  
Fluorescence Resonance

In search of viable strategies to identify selective inhibitors of protein kinases, we have designed a binding assay to probe the interactions of human phosphoinositide-dependent protein kinase-1 (PDK1) with potential ligands. Our protocol is based on fluorescence resonance energy transfer (FRET) between semiconductor quantum dots (QDs) and organic dyes. Specifically, we have expressed and purified the catalytic kinase domain of PDK1 with an N-terminal histidine tag [His6-PDK1(ΔPH)]. We have conjugated this construct to CdSe-ZnS core-shell QDs coated with dihydrolipoic acid (DHLA) and tested the response of the resulting assembly to a molecular dyad incorporating an ATP ligand and a BODIPY chromophore. The supramolecular association of the BODIPY-ATP dyad with theHis6-PDK1(ΔPH)-QD assembly encourages the transfer of energy from the QDs to the BODIPY dyes upon excitation. The addition of ATP results in the displacement of BODIPY-ATP from the binding domain of theHis6-PDK1(ΔPH) conjugated to the nanoparticles. The competitive binding, however, does not prevent the energy transfer process. A control experiment with QDs, lacking theHis6-PDK1(ΔPH), indicates that the BODIPY-ATP dyad adsorbs nonspecifically on the surface of the nanoparticles, promoting the transfer of energy from the CdSe core to the adsorbed BODIPY dyes. Thus, the implementation of FRET-based assays to probe the binding domain of PDK1 with luminescent QDs requires the identification of energy acceptors unable to interact nonspecifically with the surface of the nanoparticles.

Structural rearrangements of the motor protein prestin revealed by fluorescence resonance energy transfer

2009 ◽  
Vol 297 (2) ◽  
pp. C290-C298 ◽  
Author(s):  
Kristin Rule Gleitsman ◽  
Michihiro Tateyama ◽  
Yoshihiro Kubo
Keyword(s):  
Energy Transfer ◽  
Membrane Potential ◽  
Fluorescence Resonance Energy Transfer ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Motor Protein ◽  
Resonance Energy ◽  
Outer Hair Cells ◽  
Nonlinear Capacitance ◽  
Fluorescence Resonance

Prestin is a membrane protein expressed in the outer hair cells (OHCs) in the cochlea that is essential for hearing. This unique motor protein transduces a change in membrane potential into a considerable mechanical force, which leads to a cell length change in the OHC. The nonlinear capacitance in cells expressing prestin is recognized to reflect the voltage-dependent conformational change of prestin, of which its precise nature remains unknown. In the present work, we aimed to detect the conformational changes of prestin by a fluorescence resonance energy transfer (FRET)-based technique. We heterologously expressed prestin labeled with fluorophores at the COOH- or NH2-terminus in human embryonic kidney-293T cells, and monitored FRET changes on depolarization-inducing high KCl application. We detected a significant decrease in intersubunit FRET both between the COOH-termini and between the COOH- and NH2-termini. A similar FRET decrease was observed when membrane potential was directly and precisely controlled by simultaneous patch clamp. Changes in FRET were suppressed by either of two treatments known to abolish nonlinear capacitance, V499G/Y501H mutation and sodium salicylate. Our results are consistent with significant movements in the COOH-terminal domain of prestin upon change in membrane potential, providing the first dynamic information on its molecular rearrangements.

scholarly journals Phospholemman Phosphorylation Alters Its Fluorescence Resonance Energy Transfer with the Na/K-ATPase Pump

2006 ◽  
Vol 281 (43) ◽  
pp. 32765-32773 ◽  
Author(s):  
Julie Bossuyt ◽  
Sanda Despa ◽  
Jody L. Martin ◽  
Donald M. Bers
Keyword(s):  
Protein Kinase C ◽  
Energy Transfer ◽  
Protein Kinase ◽  
Fluorescence Resonance Energy Transfer ◽  
Cardiac Myocyte ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Hek293 Cells ◽  
Kinase C ◽  
Fluorescence Resonance

Phospholemman (PLM) or FXYD1 is a major cardiac myocyte phosphorylation target upon adrenergic stimulation. Prior immunoprecipitation and functional studies suggest that phospholemman associates with the Na/K-pump (NKA) and mediates adrenergic Na/K-pump regulation. Here, we tested whether the NKA-PLM interaction is close enough to allow fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent (CFP/YFP) fusion proteins of Na/K pump and phospholemman and whether phospholemman phosphorylation alters such FRET. Co-expressed NKA-CFP and PLM-YFP in HEK293 cells co-localized in the plasma membrane and exhibited robust FRET. Selective acceptor photobleach increased donor fluorescence (FCFP) by 21.5 ± 4.1% (n = 13), an effect nearly abolished when co-expressing excess phospholemman lacking YFP. Activation of protein kinase C or A progressively and reversibly decreased FRET assessed by either the fluorescence ratio (FYFP/FCFP) or the enhancement of donor fluorescence after acceptor bleach. After protein kinase C activation, forskolin did not further reduce FRET, but after forskolin pretreatment, protein kinase C could still reduce FRET. This agreed with phospholemman phosphorylation measurements: by protein kinase C at both Ser-63 and Ser-68, but by protein kinase A only at Ser-68. Expression of PLM-YFP and PLM-CFP resulted in even stronger FRET than for NKA-PLM (FCFP increased by 37 ± 1% upon YFP photobleach), and this FRET was enhanced by phospholemman phosphorylation, consistent with phospholemman multimerization. Co-expressed PLM-CFP and Na/Ca exchange-YFP were highly membrane co-localized, but FRET was undetectable. We conclude that phospholemman and Na/K-pump are in very close proximity (FRET occurs) and that phospholemman phosphorylation alters the interaction of Na/K-pump and phospholemman.

Sign in / Sign up

Export Citation Format

Share Document