Dear Silvia, one question to your fluorescence enhancement: Does the emission spectrum change with the increase in fluorescence? Aggregation and precipitation will increase the amount of scattered light reaching your detector; however this effect should be most prominent at the excitation wavelength (elastic scattering) and you should detect this by a change in emission spectrum.

Another possibility could be that the CFP-fused protein coaggregates with the stapled peptides, bringing the fluorescent protein in a hydrophobic environment that may increase its brightness by an influence on the protein structure. This probably will also result in a change of the emission spectrum.

Depending on your detection method (Fluorescence spectrometer, single-molecule detection?), probably it is also just many CFPs coming together in a large aggregate, resulting in a larger brightness of the observed particles.

Dear Franziska,

thanks a lot for your help. In attachment you can see the results of the experiment I performed later, a titration in 96-well plate of the lonely ECFP (which differs from CFP just in two amino acids) with increasing concentrations of stapled peptide. I measured the fluorescence with a spectrometer.

I didn't see any scattering because the emission scan range selected was too high (in terms of wavelengths). But I detect an important increase in the emission, proportional to the concentration of peptides (they start to be insoluble at around 5-15uM).

This is weird to me, since if I should imagine an effect on CFP, I would imagine the opposite, a quenching.

A transfer to a more hydrophobic environment does not automatically have to mean a quenching. Many fluorescent dyes are actually brighter in an organic solvent. Also, for example the ANS dye shows a fluorescence increase when it binds to exposed hydrophobic regions of a protein. However, the fluorophore of your CFP is already well protected from the solvent as it is embedded within the beta-barrel, so a change in the environment will probably not have such a large impact.

The only way you can really increase the brightness of a fluorescent protein is by slightly altering the structure (e.g. making EGFP out of GFP). I have no idea if the stapled proteins are able to do that. If you see an increase in emission even with untagged CFP when the peptides are around, there is probably an unspecific interaction between the two, leading to the fluorescence increase.

The simpler explanation would be that, caused by aggregation, your detector suddenly sees more fluorescent protein in one spot (do you measure from the bottom? Maybe stuff sinks to the bottom of your wells). To rule out this, maybe use another detection method (in a big cuvette on a fluorescence spectrometer, not a plate reader, I didn't quite figure out how you measured the multiwell plate).

To rule out that aggregation in general increases CFP fluorescence, you can try another aggregation-prone protein in your setup. You can also check if increased scattering is the problem by adding various amounts of Ludox colloidal silica solution (or something similar) to your CFP.

On the other hand, if you want to rule out a specific interaction between CFP and the insoluble stapled protein, try another fluorescent protein or even a fluorescent dye together with the stapled protein.

Finally, to rule out that your detection method generically detects an increase in fluorescence of any fluorophore upon any aggregation/scattering, try a different fluorescent protein/dye with a different aggregation-prone protein (probably you can even use Ludox colloidal silica solution to simulate scattering).

I don't know how much you really want to know about why the fluorescence increases,

and if it's not essential for your further work, I would not spend to much time with trying to find out :) However, if you need to figure it out, I would probably start with the last control experiment and work my way up.

Hope that helps, good luck!

I agree with Franziska says, but would also recommend trying the hydrophobic peptides by themselves without the CFP. Many protein aggregates show a fluorescent signal in the approximate range you are detecting (somewhat blueshifted excitation relative to CYP) that is not found in the monomeric protein.

http://pubs.rsc.org/en/Content/ArticleLanding/2013/AN/c3an36798c#!divAbstract

Also I noticed that there is another peak visible at 700 nm. This is odd, as it does not appear to be a second order reflection from the monochrometer (excitation would have to be 350 nm), but the formation of some new species with a very large stokes shift. You can try moving the emission wavelength to check.

Thanks a lot to both of you.

I was puzzled by the fact that the addition of the relatively high concentrations of peptides was triggering both an increase of the CFP emission and a decrease of the NanoLuciferase bioluminescent protein; it's hard to me to figure out the kind of effects that the insoluble peptides can generate on the two proteins.

I don't want to spend too much time looking for an explanation to this problem, actually, but I was wondering if anyone else saw this behavior before.

Thank you again for you suggestions!