Hello,
we had an interesting case in the lab many years ago... Using the
shift-assay, a student managed to identify conditions that markedly
stabilized the protein of interest. To cut a long story short, in the
end it turned out conditions were identified that gave monomers while
the biological active unit is clearly multimeric! Actually, the monomer
never crystallized while the multimer did...
Keep in mind that a compound may induce a structural change and that the
resulting structure may indeed be less stable. An increase in ΔTm would
certainly "desirable" but this alone is not to be used as a measure. If
you are interested in the complex, a more important parameter will be
the affinity of the compound for the protein. Example, let's assume the
affinity is about 50 µM, then you will need at least 10 times that value
in the crystallization droplet to achieve about 90% occupation. Problem
may be with concentrations > 1 mM, you can not achieve that high
concentration in the solute and will have to add for example DMSO which
may in turn have negative efefcts on your protein of interest.
Furthermore, strange scenarios could be, the compound itself interferes
in protein-protein contact formation at higher concentrations or, could
promote protein-protein contact formation (work as a glue) and not show
up in the active site at all...
Question in the end will be not about ΔTm (which is one useful tool for
identifying buffers or compounds worth using/testing), but whether the
complex can in fact be co-crystallized (the ultimate "test"!). So yes,
go ahead and test it.
Best,
Jeroen
--
*Dr.math. et dis. nat.Jeroen R. Mesters*
Deputy, Lecturer, Program Coordinator /Infection Biology
/
<http://www.uni-luebeck.de/studium/studiengaenge/infection-biology/introduction.html>Visiting
Professorship (South Bohemian University) in Biophysics
*University of Lübeck*
Center for Structural and Cell Biology in Medicine
*Institute of Biochemistry*
Tel +49 451 3101 3105 (secretariate 3101)
Fax +49 451 3101 3104
jeroen.mest...@uni-luebeck.de <mailto:jeroen.mest...@uni-luebeck.de>
www.biochem.uni-luebeck.de <http://www.biochem.uni-luebeck.de>
*Ratzeburger Allee 160
23538 Lübeck, Schleswig-Holstein
Germany*
Am 25.02.21 um 15:55 schrieb Saif Mohd:
Hello everyone,
1) How much change in Tm (ΔTm) in a thermal shift assay is considered
to be significant ?
2) A negative ΔTm infers that the compound is making the protein
unstable. In such a case, will the co-crystallization be difficult or
just impossible or on the contrary it shouldn't matter much?
Thanks and best regards,
Saif
------------------------------------------------------------------------
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
<https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1>
**
########################################################################
To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list
hosted by www.jiscmail.ac.uk, terms & conditions are available at
https://www.jiscmail.ac.uk/policyandsecurity/
