We have a GFP mutant that displays a different excitation spectra with emission at 510nm than the WT. However, their emission profiles with excitation at 490nm are the same, and we do not observe the same trend.

We're confused as to what might be happening.

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    $\begingroup$ @AliceD What you said about emission/excitation is wrong. It is actually the opposite, the excitation is always shorter than the emission as shorter wavelengths are more energetic. The question is therefore valid in this regard. I think he is referring to an experiment they have done rather than a paper. Yet more informations would be indeed quite helpful for providing an answer. For example can you post the actual spectra? Also can you please provide the ranges you used for emission/excitation wavelengths in both experiments? $\endgroup$ – cagliari2005 Apr 22 '15 at 4:37
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    $\begingroup$ @cagliari2005 - yep noticed it, and removed comment. Thanks $\endgroup$ – AliceD Apr 22 '15 at 4:41
  • $\begingroup$ emission spectra should not depend on excitation wavelength. Only scales. $\endgroup$ – aaaaaa Apr 22 '15 at 6:03
  • $\begingroup$ @aandreev What? The emission spectrum totally depends on the fixed excitation wavelength... And vice-versa for a fixed emission wavelength. $\endgroup$ – cagliari2005 Apr 22 '15 at 6:10
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    $\begingroup$ Hi, OP here, thank you guys so much for the help. It turns out there was a calibration error, and when we recollected the data, the symmetry in peak height was observed for both Wt and our mutant. Much appreciated either way! $\endgroup$ – student Apr 29 '15 at 5:55

Let me start from short description of fluorescence (since we talk about it here).

Fluorescence is one of the processes through which excited molecule can relax, lose its excessive energy. That is, quantum interaction between atoms in molecule (organic dye or complex GFP) create permitted energy levels. There is "ground state" $S_0$, for example, and excited state $S_1$. You "pump" your molecule with excitation light from $S_0$ to $S_1$. All this can be nicely illustrated as absorption in Jablonski diagram (look at a for now):

enter image description here

via http://micro.magnet.fsu.edu/

"Loss of energy" here is some process via which molecule returns to ground state. In case of GFP energy is released as an emission photon. Nature of fluorophores is that they tend to return to ground state as the state of less energy, and almost all systems tends to go to state with lesser energy.

Now, to your question. Why GFP can has different excitation profile but same emission? Notice, how in (a) molecule get pumped to highest state, then relaxes a bit to lowest of $S_1$ states and then go all the way to $S_0$? That initial relaxation is thermal relaxation. Some energy is lost, essentially, to water molecules around GFP. That is why emission is always "more red", i.e. has lower energy than excitation. Initial relaxation is why your GFP spectra also broad. There are many levels GFP can emit from, as well as be excited to. In crystals of GFP transitions will be very narrow, because much less of excitation energy will go not into fluorescence.

So, my point is following. Nobody prevents one from engineering a mutant molecule of GFP that could be pumped to some even higher level $S_2$, which then relaxes to $S_1$ via non-irradiative process (e.g. gives energy to water, but not into fluorescence!). Only requirement is that for mutant direct pathway from $S_0$ to $S_1$ would be forbidden (see b and c).

Another point is that whether your spectra are different in shape or only intensity. Different shape of spectra means there are differences in those levels and transitions.


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