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Biophysics - Coggle Diagram
Biophysics
FRET
Only occurs between 1-10nm
Greater than 10nm effects usually ignored by the theory of FRET become more prominent
At distances less than 1nm complex formation is more likely and the Ideal Dipole Approximation (IDA), that FRET is based on, breaks down
Non-radiative energy transfer
The emission and absorption spectra of the donor and acceptor chromophore must overlap
Depends on the angle between the two molecules
Theory shows that the FRET efficiency is proportional to the sixth power of the distance between the two molecules
R0 is the distance at which the FRET efficiency is 50% and ranges between 2-7nm
Used to measure protein-protein interactions
ATPsynthase :
Attached fluorescent dyes to ATP synthase molecule, via sulphide bonds, on the gamma domain and the b2 domain
Reconstituted into liposome
Single liposome with single ATP synthase
Liposome moves slowly (several 100ms), so spends longer in the detection volume of a laser confocal microscope.
Bursts of emission detected
In hydrolysis conditions (adding ATP) ATP synthase turned in one direction (1>2>3>1) - measured ratio of donor fluorescence over acceptor fluorescence - produced three distinct levels of FRET efficiency
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ATP synthesis conditions () protein turned in the opposite direction
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Cannot use fluorescent proteins because they are too big
Distance between the donor dye and acceptor dye will vary as the protein rotates during ATP synthesis
Should produce 3 distinct FRET efficiency values
bc1 complex
Bifurcated mechanism
Oxidation of
quinone derivative
(QH2) at Q0 site
First electron transferred to Fe-S and then cytochrome c2
second electron transferred to BL then BH and then to the Q1 site where it reduces a ubiquinone (Q) to form a semi-quinone radical (SQ)
Process repeated to produce QH2 at the Q1 site
Two consecutive turnovers of the enzyme
2H+ released on one side of the membrane per e- for every QH2 oxidised
Not understood why the second electron does not also follow the energetically stable pathway
However, this must happen or no charge is translocated across the membrane so electric potential is formed which would be energetically disastrous
Many mechanisms proposed over the years
'Double occupancy' model
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'Proton-gated charge transfer' model
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cytochrome c2 complex
four transmembrane cytochrome b
BH (high potential) b-type haem
BL (low potential) b-type haem
Rieske domain 2Fe2S