The figure below shows the fluorescence emission spectra of fluorescein (FI) and

Question

The figure below shows the fluorescence emission spectra of fluorescein (FI) and Texas-Red (TR) when covalently attached to the catalytic (C) or regulatory (R) subunit ofd cAMP-dependent protein kinase (PKI). When the subunits are associated, resonance energy transfer (RET) occurs from FL to TR. The associated from is C2R2. Read the figure legend and provide a possible explanation for the spectral data. Figure 4: Effect of cAMP and PKI on the steady-state emission spectra of the heterochromatic holoenzyme. Carboxyfluorcsccin-labeled catalytic (^CF C)(donor) and Texas Red-labeled regulatory (^TR R_II) (acceptor) subunits were reassociated to form as described in Materials and Methods. The sample was diluted with 50 mM MES. pH 6.8, 5 mM beta-mercaptoethanol to a final concentration of 20nM in 0.35 mL. Emission spectrum of the donor-acceptor pair (dotted line) is compared with the spectrum generated after the addition of cAMP (20 mu M) (dot/dash line) and then again after the addition of PKI (300 nm) (solid line). Samples were excited at 470 nm.

Explanation / Answer

Consider, the first peak is raised with respect to fluorescein and second peak with respect to Texas-red. According to the given data and graph, the binding of fluorescein to the catalytic site is high in presence of both cAMP and protein kinase and least in absence of both or only existence of catalytic site. In contrast, Texas-red binding to the catalytic site is high in absence of both or only existence of catalytic site and least in presence of both cAMP and protein kinase.

The order of fluorescence in decreasing order with respect to fluorescein is as follows:

In presence of both cAMP and protein kinase

in presence of cAMP

Only catalytic site.

The order of fluorescence in decreasing order with respect to Texas-red is as follows:

Only catalytic site

in presence of cAMP

In presence of both cAMP and protein kinase.

In all the conditions, samples were excited at same nm.

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