Question

Are these answers ok?

Fluorescent Intensity Fluorescent Intensity Fluorescent Intensity Wavelength of Emitted Light Wavelength of Emitted Light Wavelength of Emitted Light Figure 1. Cells Expressing X-CFP and Irradiated with 435 nm Light Figure 2. Cells Expressing X-YFP and Irradiated with 435 nm Light Figure 3. Cells Expressing X-YFP and Irradiated with 488 nm Light Fluorescent Intensity Fluorescent Intensity Wavelength of Emitted Light Wavelength of Emitted Light Figure 4. Cells Expressing X-CFP and Y-YFP Irradiated with 435 nm Light Figure 5. Cells Expressing X-CFP and Y-YFP Incubated in Fillipin, a Drug that Removes Cholesterol from the Membrane, and Irradiated with 435 nm Light i. The reason that the curve in Figure 2 is flat is a. there are no fluorescent proteins present in the cells used in the trial b. the fluorescent proteins present are quenched by irradiation at 435 nm c. the fluorescent proteins present have transferred their energy to neighboring fluorescent proteins d. the fluorescent proteins present are not excited by irradiation at 435 nm e. none of the fluorescent proteins are present in membrane microdomain State rational for your answer: The present proteins are not excited by irradiation at 435 nm, therefore they could not transfer energy to the neighboring protein. ii. The reason that there are two peaks of emitted fluorescent light in Figure 4 is a. emission from CFP results in excitation of YFP b. emission from YFP results in excitation of CFP c. excitation of YFP results in excitation of CFP d. excitation of YFP results in quenching of CFP e. excitation of CFP results in quenching of YFP State rational for your answer: Because proteins transfer the energy of irradiation between themselves.

Answer 1

i-d) is correct.

Rationale:
The cells used in Figure 2, express YFP, which absorbs light of wavelength 488 nm. Therefore, cells irradiated with 435 nm light do not show fluorescence as YFP is not excited in these cells.

(There is no energy transfer as there is only one fluorophore present)

ii-a) emission from CFP results in excitation of YFP.

Rationale:
Light absorbed by CFP at 435 nm is emitted at 488 nm, which is in turn absorbed by YFP molecules, resulting in two emission peaks, one of CFP at 488 nm, and another by YFP at a wavelength above 488 nm.

(When two fluorophores are used for FRET experiments, the fluorophore with the lower absorbance wavelength is excited to produce the wavelength absorbed by the fluorophore with the higher absorbance wavelength. Remember, Lower Wavelength = Higher Energy, and the first fluorophore absorbs high energy light, releases light of lower energy, which is absorbed by the second fluorophore to produce light of even lower energy)

Diagrammatically:

High Energy Light --> Absorbed by CFP --> Lower Energy Light Emitted --> Absorbed by YFP --> Even Lower Energy Light Emitted

In terms of Wavelength
435 nm --> CFP absorbs --> CFP emits 488 nm light --> YFP absorbs 488 nm light --> YFP emits light of longer wavelength (~ 530 nm)

iii-c X-CFP and Y-YFP are near neighbors only when membrane microdomains are present

Rationale:
Incubation of cells with Filipin, a drug that releases Cholesterol from the Membrane, causes loss of Energy transfer between CFP linked to X and YFP linked to Y. This suggests that in the absence of Cholesterol, where Membrane microdomains do not form, X and Y are not near neighbors, preventing the Energy transfer from X-CFP to Y-YFP.

(Cholesterol prevents membrane proteins to diffuse away from membrane microdomains)