Proximity of the Nucleotide Binding Domains of the P-glycoprotein Multidrug Transporter to the Membrane Surface:  A Resonance Energy Transfer Study†

Biochemistry ◽  
1998 ◽  
Vol 37 (18) ◽  
pp. 6503-6512 ◽  
Author(s):  
Ronghua Liu ◽  
Frances J. Sharom
Keyword(s):  
Energy Transfer ◽  
Membrane Surface ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nucleotide Binding ◽  
Multidrug Transporter ◽  
Binding Domains ◽  
P Glycoprotein ◽  
Transfer Study

Rubredoxin mutant A51C unfolding dynamics: A Förster Resonance Energy Transfer study

2010 ◽  
Vol 148 (1-3) ◽  
pp. 131-137 ◽  
Author(s):  
Andrea Santos ◽  
Américo G. Duarte ◽  
Alexander Fedorov ◽  
José M.G. Martinho ◽  
Isabel Moura
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Transfer Study ◽  
Förster Resonance

scholarly journals Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

2007 ◽  
Vol 407 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Michael Russwurm ◽  
Florian Mullershausen ◽  
Andreas Friebe ◽  
Ronald Jäger ◽  
Corina Russwurm ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Systematic Approach ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Binding Domains ◽  
Fluorescence Resonance

The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 μM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.

scholarly journals Association/Dissociation of the Nucleotide-binding Domains of the ATP-binding Cassette Protein MsbA Measured during Continuous Hydrolysis

2013 ◽  
Vol 288 (29) ◽  
pp. 20785-20796 ◽  
Author(s):  
Rebecca S. Cooper ◽  
Guillermo A. Altenberg
Keyword(s):  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Binding Domains ◽  
Partial Opening ◽  
Contact Models ◽  
Bacterial Lipid ◽  
Atp Binding Cassette

In ATP-binding cassette proteins, the two nucleotide-binding domains (NBDs) work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. It is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or partial opening of the dimers such that the NBDs remain in contact during the hydrolysis cycle. We studied the bacterial lipid flippase MsbA by luminescence resonance energy transfer (LRET). The LRET signal between optical probes reacted with single-cysteine mutants was employed to follow NBD association/dissociation in real time. The intermonomer distances calculated from LRET data indicate that the NBDs separate completely following ATP hydrolysis, even in the presence of mm MgATP, and that the dissociation occurs following each hydrolysis cycle. The results support association/dissociation, as opposed to constant contact models, for the mode of operation of ATP-binding cassette proteins.

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