Calmodulin conformational changes in the activation of protein kinases

2002 ◽  
Vol 30 (2) ◽  
pp. 55-61 ◽  
Author(s):  
K. Török
Keyword(s):  
Energy Transfer ◽  
Protein Kinases ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Activation Process ◽  
Dependent Protein ◽  
Peptide Complexes ◽  
Muscle Myosin ◽  
Kinetics Of

The conformation of Ca2+/calmodulin changes from extended when free in solution to compact when bound in peptide complexes. The extent and kinetics of calmodulin compaction in association with Ca2+/calmodulin-dependent protein kinases (CaMKs), as well as target peptides, were investigated by fluorescence, resonance energy transfer and stopped-flow kinetics. Compaction of Ca2+/ calmodulin labelled with resonance energy-transfer probes in association with target peptides was rapid (>350 s−1). With the target enzymes smooth-muscle myosin light-chain kinase, CaMKIV and αcCaMKII, the rates of calmodulin compaction were one-two orders of magnitude lower compared with those of the peptides and in the case of αCaMKII, ATP binding and Thr286 auto-phosphorylation were required for calmodulin compaction. In the absence of nucleotides, Ca2+/calmodulin bound to αCaMKII in extended conformations, initially probably attached by one lobe only. Kinetic data suggest that in the activation process of Ca2+/ calmodulin-dependent protein kinases, productive as well as unproductive complexes are formed. The formation of productive complexes with Ca2+/calmodulin thus may determine the rate of activation.

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.

Fluorescent resonance energy transfer -based biosensor for detecting conformational changes of Pin1

2018 ◽  
Vol 505 (2) ◽  
pp. 399-404 ◽  
Author(s):  
Masafumi Hidaka ◽  
Emiko Okabe ◽  
Kodai Hatakeyama ◽  
Heather Zook ◽  
Chiyoko Uchida ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescent Resonance Energy Transfer
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