Question

In a FRET experiment, the donor GFP (GFP absorbs at 487 nm, emits at 510 nm) is attached to protein A, and the acceptor YFP (YFP absorbs at 514 nm, emits at 535 nm) is attached to protein B. The value of ro is 10Å. a. If protein A does not bind to protein B, fluorescence will be observed at what wavelength b. At pH 6, the distance between the proteins is 25 Å. What is the efficiency? c. Increasing the pH from 6 to 12 causes the efficiency to increase to 0.9. Calculate the separation distance at pH 12.

Answer 1

PART A

In the FRET experiment, energy depends inverse sixth=distance between donor and acceptor

using E= R^{6}/( R^{6} +r^{6} )

Where R = Forster distance at which half the distance is tranmsferred, here it is given as 535/2 = 267.5nm

r = Actual distance between donor and acceptor , here it is given as 10 A0 or 100nm

Putting all values in the above equation, E = 0.9972

Hence binding done is 99.72%

PART B

Using the same equation by putting R = 500 /2= 250nm

Value of E = 0.9959

Hence binding done is 99.59%

PARTC

Thiscan be understand by the concept of denaturation of protein.

Whenever a protein is subjected to any physical or chemical change such as change in temperature, change in pH or here in this case, change in the intensity of fluorescense, the native structure of protein is destroyed. In other words we can say that the some of the globular protein must have converted to fibrous protein.Thus due to change in fluorescense, ph changes from 8 to 8 resulting in the decrease in efficiency to 0.1