frank at fuerst.de
Thu Mar 18 11:55:46 EST 1999
> Hi all!
> 1) I want to do folding-unfolding studies with my protein.
> Unfortuantely, the single tryptophan is partially exposed. (as from
> homologous xtal strucures and by quenching with KI). The emission maxima
> I obtain is at 346 nm for the native protein with a broad band width and
> for fully unfolded protein(thermal) at 350 nm. Is it possible that most
> of the protein molecles are in an unfolded state?
It is possible. But it also seems that perhaps not all of your protein
is unfolded since lambda max is 350 nm. I don't have experience with
monitoring thermal transitions with fluorescence (and would prefer CD
or absorbance), but protein unfolded using denaturants like urea or
guanidinium chloride usually has a lambda max of about 355 nm. And
I've worked on a protein with a native lambda max of 342 nm,
monitoring (denaturant-induced) unfolding with fluorescence is no
Usually, also the fluorescence intensity changes upon unfolding.
Without intramolecular quenchers (disulfide bridges and perhaps some
cofactors), the fluorescence intensity of a native protein is higher
than that of the unfolded form because of enhanced solvent quenching
of the solvent exposed trp in the unfolded state.
However, if there is/are disulfide bridge(s) in the vicinity of the
trp in the native structure, you should get just the opposite effect:
Enhanced fluorescence in the denatured state.
> How do I check this?
If you don't know the fluorescence and/or CD-spectra of a surely
native state, its really difficult. The most safe thing would be to
test its activity - if you have an assay for that. There are
practically no examples (that means, its generally accepted that, but
there are rare people who claim to have found the opposite) of a
partially unfolded protein showing activity. On the other hand, you'll
have to be carefull: If only 20 % is native, and the specific activity
is 5 times the activity of a homologous protein, you'll get the
Other interesting things with respect to your question are:
- Does the Far-UV-CD-Spectrum show pronounced secondary structure, and
does it look like the spectra of these homologes?
- Does your protein have a tendency to aggregate? This would suggest
that it is partially unfolded. Sometimes aggregates are so small that
you can't see or centrifuge them, but you see light scattering (i.e.
the absorption between 310 and 350 nm is not a horizontal line at
zero, but seems to increase linearly with decreasing wavelength - in
fact its not linear)
> 2) How is the correction for decrease in fluorescence due to increasing
> temperature is done in thermal unfolding?
I don't know, but you don't need that usually. When you denature your
protein with a chemical denaturant, you get the typical form of a
cooperative unfolding transition. The baselines (before and after the
transition) are approximately, but not exactly horizontal. Not
exactly, because flourescence intensity changes not only upon
unfolding, but also with varying solvent conditions. Upon thermal
unfolding, you get the same picture, only that the slopes of these
baselines are steeper.
So there's an other way to judge if your protein is unfolded in native
buffer at RT: If you get a cooperative transition, you know that you
have either native protein or at least a stable intermediate. If you
get something that looks like the second half of a transition, its
probable that your protein is partially denatured (that means, n per
cent of the molecules are denatured). But again, I would recommend
using CD or chemical denaturant. Because, as you don't know the
baselines' slopes, if you don't see any cooperative transition, you
cannot say if this is because there isn't any, of because you cannot
resolve it. This is especially, but not only, a problem if the
influences of temperature and structure compensate each other, that is
if the flourescence is quenched in the native state and increases upon
- If you don't know what I mean by "the typical form of a cooperative
transition", mail to me, I'll look for some graphs in the web. Or, if
you have access to standard journals like Biochemistry or JMB, I could
tell you some articles with graphs of unfolding transitions.
- Using 7 M Guanidinium chloride and incubating some hours,you should
be on the safe side - I don't think any soluble protein would stand
Frank Fuerst, Institut für Biochemie der Uni Potsdam
Im Biotechnologiepark, 14943 Luckenwalde
Tel.: +49-3371-681334; Fax.: +49-3371-681339
ffrank at rz.uni-potsdam.de
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