AW: [RASMB] Fluorescence question

[Titus M. Franzmann]

Perben, 
what you might be observing is that your protein unfolds. In many cases
Trp-residues are quenched in the folded structure. Upon unfolding the Trp
get solvent exposed and the solvent quenching acting on the unfolded
structure can be less than the static or dynamic quenching in the folded
structure. 
To get a first idea about this you can record corrected trp - fluorescence
specta:
Record a buffer / water spectrum: Excite at 295 nm, record emission from 295
- 500 nm at a slit width of 1-5 nm.
Record protein spectrum with the same settings. Typtically 1-5 µM Trp will
give you a good fluorescence signal. Subtract the buffer from the sample. If
the trp residues are is an hydrophobic environment the peak max will be
somewhere between 310-350 nm.
Next denature your protein (same concentration as used above) with 6 M
Guanidinium hydrochloride, incubate for 10 min, or longer. Record a GdmCl
buffer spec, then the denatured sample and subtract the buffer form the
sample. You should see that the peak max has shifted to 355 nm, which is
typical for Trp in aqueous solution.
Use a buffer, such as HEPES, Tri, or NaP to asure that the spectra are
recorded at the same pH. 
>From this you will learn, if the trp-residues are quenched in the folded
structure and if your observation with the increase in signal is due to
unfolding/aggregation.

Another thing could be that your protein is in a non-native conformation or
in an oligomeric state that changes when you dilute the sample into the
cuvette. This you may have to investigate in more detail also recording
spectra at the beginning and the end of the reaction. These you may then
compare to the GdmCl and "native" spectra.

Best Titus

-----Ursprüngliche Nachricht-----
Von: rasmb-bounces at server1.bbri.org [mailto:rasmb-bounces at server1.bbri.org]
Im Auftrag von J. Preben Morth
Gesendet: Donnerstag, 25. Juni 2009 04:46
An: rasmb at server1.bbri.org
Betreff: [RASMB] Fluorescence question

> Hi
We are working on a soluble protein which show a constant increase in  
intrinsic Trp fluorescence changes
by plotting the 295nm/320nm ratio over time (up to 10min), we expect a  
drop. but the change seems to increase linearly? Can someone explain  
this, It might possibly be my lack of knowledge in this particular  
field.
The protein is soluble in water. and measured in water no ligands  
added or anything. When we add SDS the protein denatures and we see  
the expected downward bleaching curve.

cheers
Preben



Jens Preben Morth, Ph.D
Aarhus University
Department of Molecular Biology
Gustav Wieds Vej 10 C
DK - 8000 Aarhus C
Tel. +45 8942 5257, Fax. +45 8612 3178
jpm at mb.au.dk



_______________________________________________
RASMB mailing list
RASMB at rasmb.bbri.org
http://rasmb.bbri.org/mailman/listinfo/rasmb