Hi all,

In order to see conformational change upon two proteins binding I am very interested in applying protein fluorescence methods.

I wonder if there is any web page or similar to take knowledge about this method.

Thanks in advance

Celvas

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This is interesting....I've not heard of this technique, but I'd be interested if anyone knows anything about it. Out of curiosity, how would you visualize conformational change with flourescence? Do you have some antibody that is specific for an epitope generated by only one of the conformations? or is lost when the proteins bind?

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hm ... could you describe your idea in more detail? there are some well established techniques to study protein<->protein interaction, but I don't know any that has fluorescence involved

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Ok, as I could read in some papers changes in structure and conformation of proteins can be measured as shifts of intrinsic fluorescence. This intrinsic fluorescence is caused by tryptophans side chains and "quenching" is expressed as the ratio F/Fo where Fo represents the intrinsic protein fluorescence and F the protein fluorescence upon structural change.

But this is all I can tell you. We have find a new two proteins interaction and I would want to know if there is possible to check for conformational change upon binding using fluorescnece spectroscopy technique

Thanks a lot in advance

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hi i have used fluorescence to study protein conformation ( but it wasn't protein protein interactions). for that you can look up Forster resonance energy transfer (FRET). you can use that to study protein-protein interactions and adapt that to study the conformational change. you can also do quenching of either intrinsic or extrinsic fluorescence of the protein on binding another protein or ligand.
I actually used this book called principles of fluorescence spectroscopy by joseph lackowicz to help me plan how to use fluorescence to study protein folding. however i recently saw this paper on FRET which might be of some use to you in planning your experiments;hope it works out. if you need more info I'll look it up.

1 Calleja, V., Ameer-Beg, S.M., Vojnovic, B., Woscholski, R., Downward, J. and Larijani, B. (2003) Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy. Biochem. J. 372, 33-40

and a Commentary called
Protein conformation: through a lens, darkly
by Robert INSALL1

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hi to all
certainly there are scuh methods by which one can visulaize in vivo protein folding.
i have attached that paper with this message.
Hope it will work for you.
Best wishes
amit

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Prof. Lakowicz group, Center for Fluorescence Spectroscopy is good:

http://cfs.umbi.umd.edu/

also a ref from:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?DB=pubmed

9: Hibbs RE, Talley TT, Taylor P. Related Articles, Links
Free Full Text Acrylodan-conjugated cysteine side chains reveal conformational state and ligand site locations of the acetylcholine-binding protein.
J Biol Chem. 2004 Jul 2;279(27):28483-91. Epub 2004 Apr 26.
PMID: 15117947 [PubMed - indexed for MEDLINE]

-Larry

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What about fluorescence anisotropy? I haven't done it myself but have read quite a bit about it as I considering using it for my project.

Basically, you have each of your proteins attach to a fluorophore, each protein to a difference colored fluorophore (ie. YFP and CFP). You measure the fluorescence anisotropy of the each of the proteins in solution where there's only one species of the protein then mix the proteins and measure the fluorescence anisotropy again. If they interact, it'll change the anisotropy of the fluorophore.

What you are measuring is actually the polarization of the fluorescence emitted from the fluorophore. The polarization changes when the fluorophore interacts with different proteins.

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