Reduced CFP and YFP fluorescence

[Pablo German]

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Dear List Members,

I am working with 7 trans-membrane domain receptors. These are not
GPCRs. I have made chimeras by inserting CFP and YFP molecules at the
N- and C-terminus and in the intra-cellular loops similarly to what
other people have done previously (Villardaga, 2003) to be able to
detect conformational changes with FRET.

I have noticed that when I insert CFP or YFP in the intra-cellular
loops the fluorescence intensity of both is reduced when compared to
N-terminus or C-terminus. I have shown by Western blot that the
expression levels is similar, therefore the fluorescent proteins have
lower fluorescent intensity.

Does anyone have an idea why this could be happening? Could the
structure of the fluorescent proteins be affected by being held within
two trans-membrane domains? Or is it because the molecule is not free
to rotate?

Any help would be appreciated.

Kind regards,
Pablo

--
Pablo German
PhD Candidate

Plant and Food Research
Private Bag 92169
Auckland Mail Centre
Auckland 1142
New Zealand
DDI: [phone]
Mobile: [phone]

[George McNamara]

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Hi Pablo,

Could be poor folding. That is, at the single molecule level, a
correctly folded FP may produce the same number of counts per second
(photons) for a given excitation level as a free FP. Typically tested
by increasing linker length on both ends of the FP. Using a
Superfolder FP (they are available in several colors), plus longer
linkers, may be useful. Another tool is to add a another FP
(different color, matures well), at a free end - preferably on the
extracellular side (in your case) to avoid FRET.

At a trivial level, since you already have the constructs, growing
your cells at lower temperature for a day or more, for example, 30 C,
may enable better maturation/folding.

Plenty of recent reviews on FP's - see especially:


<http://www.ncbi.nlm.nih.gov/sites//pubmed/20664080>Fluorescent
proteins and their applications in imaging living cells and tissues.

Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA.

Physiol Rev. 2010 Jul;90(3):1103-63. Review.PMID: 20664080

Carl and I also have one:

<http://www.formatex.org/microscopy3/papers.htm>Modern Research and
Educational Topics on Microscopy - Content
G. McNamara and C.A. Boswell.
A Thousand Proteins of Light: 15 Years of Advances in Fluorescent Proteins
www.formatex.org/microscopy3/papers.htm
(article and Excel file is about 1/4 way down ... search the web page
for Boswell)

spectra for most (older) FPs are in the xlsx file inside the ZIP file at
http://sylvester.org/research/shared-resources/laboratory-resources/analytical-imaging-core-facility/analytical-imaging-core-facility-links-forms/pubspectra-data

Speaking of PubSpectra ... Everyone reading this - researchers or
vendors - who have spectra you want to add to PubSpectra, please
contact me directly.

I am especially interested in objective lens transmission curves
(vendors), LED and other novel light sources, filters, filter sets,
internal filters (ex. the filters inside the LSM710/LSM780, SP5,
SP5-II), multiphoton excitation (I recently organized the Xu/Webb
Cornell data but not yet posted online), new FPs, new organic dyes,
new QDots, new other Dots, photonconvertible/switchable/photo-etc
before and after, SHG spectra (see recent PNAS paper from Scott
Fraser's lab), STED depletion spectra. However, I want data, ideally
in 1 nm intervals in Excel (and best of all organized as in
PubSpectra), since un-scanning graphs has gotten old.

I am happy to add other spectra to Pubspectra, and can always put
other Excel files in the ZIP file. One could be Raman spectra. Leica
CW-STED uses a 592 nm laser. The anti-Raman Stokes shifts for H2O
Raman lines are 508 nm for 3600 cm-1 and 502 nm for 3300 cm-1 (I've
never been sure which or both of these to use). I am also curious
what Raman shifts would be useful to have for other mounting media,
such as glycerol, thiodiethanol, Mowiol/polyvinyl alcohol, and
additives such as the STED ROXS reagents (Kasper et al 2010 Small,
PubMed 20521266).

PubSpectra is data - data are facts, facts are not copyrightable (at
least in the USA, which has a much more enlightened laws than the
E.U.). PubSpectra is and always will be free to use. Zeiss already
uses it for "Smart Setup" inside ZEN. I encourage all other vendors
to incorporate PubSpectra into their software to make the spectra
easy to find and graph.

Enjoy,

George




At 10:44 PM 9/26/2010, you wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Dear List Members,
>
>I am working with 7 trans-membrane domain receptors. These are not
>GPCRs. I have made chimeras by inserting CFP and YFP molecules at the
>N- and C-terminus and in the intra-cellular loops similarly to what
>other people have done previously (Villardaga, 2003) to be able to
>detect conformational changes with FRET.
>
>I have noticed that when I insert CFP or YFP in the intra-cellular
>loops the fluorescence intensity of both is reduced when compared to
>N-terminus or C-terminus. I have shown by Western blot that the
>expression levels is similar, therefore the fluorescent proteins have
>lower fluorescent intensity.
>
>Does anyone have an idea why this could be happening? Could the
>structure of the fluorescent proteins be affected by being held within
>two trans-membrane domains? Or is it because the molecule is not free
>to rotate?
>
>Any help would be appreciated.
>
>Kind regards,
>Pablo
>
>--
>Pablo German
>PhD Candidate
>
>Plant and Food Research
>Private Bag 92169
>Auckland Mail Centre
>Auckland 1142
>New Zealand
>DDI: [phone]
>Mobile: [phone]

George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
Miami, FL 33136
[hidden email]
[hidden email]
305-243-8436 office
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+
spectra .xlsx file is in the zip file)
http://home.earthlink.net/~geomcnamara