tag:blogger.com,1999:blog-1136153439451224584.post8963217769059072756..comments2024-03-27T06:45:59.174-07:00Comments on Practical Fragments: The Value of DSFDr. Teddy Zhttp://www.blogger.com/profile/07288045760981372367noreply@blogger.comBlogger5125tag:blogger.com,1999:blog-1136153439451224584.post-54537881886209110752015-10-06T21:22:13.095-07:002015-10-06T21:22:13.095-07:00I Second sgcox above.
Lots of strange things can h...I Second sgcox above.<br />Lots of strange things can happen at high concentrations. <br />When following up hits with DSF dose-response curves, its quite common with bad behavior above 1mM (which i think is the absolute max screening conc for a very soluble Frag library for DSF) and even worse beyond 2mM.ALnoreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-17497414405677126262015-08-07T03:29:53.545-07:002015-08-07T03:29:53.545-07:00In our hands DSF works really well when compound c...In our hands DSF works really well when compound concentration is below 100 uM. Once you go above it, as in the paper with 5 mM (!), you get all sort of artifacts. DSF with environment sensitive dyes can not be run with detergent and compounds aggregation and direct interaction with Sypro orange can simply dominate effect of protein unfolding and data go all other the place. This of course limits the utility of DSF to relatively potent compounds. I am not surprise by all negative experiences when people use it for high concentration screening sgcoxnoreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-78705596064944935712015-08-06T22:03:22.692-07:002015-08-06T22:03:22.692-07:00It is interesting that 5 of the 9 stabilizing frag...It is interesting that 5 of the 9 stabilizing fragments yielded crystal structures, while only 1 of the 12 destabilzing fragments did. This is consistent with anecdotal reports that stabilizers are more likely to yield crystal structures than destabilizers.<br /><br />That said, fairly subtle chemical changes can transform a stabilizer to a destabilizer (F5 vs F5.1, for example), while the affinity, binding mode, and thermodynamic parameters remain roughly the same.<br /><br />One possibility the authors suggest is that the melting point depression might be due to effects other than protein binding, such as reversible bond formation between the primary amine in fragment F5.1 and the PLP cofactor at high temperatures. Alternatively (or additionally), perhaps F5.1 binds preferentially to the unfolded form of the protein, which would also lead to a decrease in Tm.Dan Erlansonhttps://www.blogger.com/profile/07927082337051189270noreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-42916878352888749862015-08-06T04:51:04.227-07:002015-08-06T04:51:04.227-07:00what if a DSF screening only yielded destabilizers...what if a DSF screening only yielded destabilizers without a single stabilizer? can you treat the destabilizers as hits and how to prioritized them?Anonymoushttps://www.blogger.com/profile/16181602851482369507noreply@blogger.comtag:blogger.com,1999:blog-1136153439451224584.post-86877010703895515902015-08-05T23:28:48.989-07:002015-08-05T23:28:48.989-07:00Maybe it just means that DSF (like others methods)...Maybe it just means that DSF (like others methods) must be confirmed by (at least) one orthogonal / independent method(s). Matthieu Dnoreply@blogger.com