In article <tliang-0703961306260001 at bmb155.med.uth.tmc.edu>, tliang at utmmg.med.uth.tmc.edu (T. Chyau Liang) writes:
|> In article <4hmg4j$n7l at sun4.bham.ac.uk>, bcm4jah2 at bham.ac.uk (Jim) wrote:
|>|> > Can anybody please tell me the best way to look at the conformational changes
|> > in proteins as they are bound by substrates, inhibitors, etc.
|> > Thanks very much James Harper.
Exactly what do you want to know about these conformational changes?
Just that something happened? Or do you need precise information about
the changes in atomic positions?
|>|> Hi Jim,
|>|> You have several possibilities:
|>|> (1) Fluorescence spectroscopy: very sentitive and can tell you whether
|> there is conformational change or not. However, it does not tell what
|> kind of conformational changes. Often, you can monitor the intrinsic
|> fluorophore on the protein or the substrate/inhibitor.
Or it could seem to indicate a conformational change when none occurs.
For example, imagine a protein that has a hydrophobic pocket. Imagine that
fluorophore can bind into it without causing any changes in its conformation
or that of the protein. Simply because of the lack of solvent quenching,
the fluorophore's fluorescence will change dramatically. I'd call this
a false-positive indication of conformational change upon ligand binding.
|>|> (2) Circular dichroism (CD): sensitivity comparable to UV/VIS spectroscopy
|> and can tell you more info on conformational changes (e.g., extent of
|> alpha-helix or beta sheet changes).
This isn't a bad suggestion if the original poster just wants to know that
something happened upon ligand binding, but CD can't really be interpreted
beyond gross changes in structure.
|>|> (3) NMR: a very insensitive method, but can potentially provide more info.
|> The same can be said about X-ray.
NMR can give atomic-level information about the changes in conformation
and dynamics that occur in proteins upon ligand binding. Its sensitivity
is lower than fluorescence (you need ~0.5 ml of ~1 mM protein solution to
get decent spectra, and if you want to do a structure, you'll probably
have to make a sample that's labeled with 13C and 15N), but it usually
produces vastly more information about protein structure and dynamics
than fluorescence or CD.
|>|> These are just a few mthods come to my mind. Others may be able to provide
|> you alternate methods.
Analytical ultracentrifugation can tell you whether ligand binding
induces gross changes in the protein's conformation or
Boy, this sounds like an answer to the final in my methods in physical
biochemistry class. 8-)
|>|> hope this helps.
|>|> T. Chyau Liang, Ph.D.
|> U. Texas-Houston
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"Biophysics has driven me to an attitude of apocalyptic doom"