Which of the two is better to study protein unfolding using chemical denaturants?
Hi Manika,
Better in which way? The mechanism of ANS binding to hydrophobic pockets is abundant in the literature, but sypro-orange, as far as I know, is not. I use sypro to obtain the melting temperature of several mutant proteins by thermal denaturation and it works fine. If you want to obtain the denaturing profile of your protein, and get thermodynamic parameters, you could use another signal like CD or intrinsic fluorescence.
Thank you Diego,
I was trying to trap an intermediate state that my protein adopts while unfolding (confirmed it with other techniques) and was curious to find the binding profile of these dyes to that state. Although, they showed similar binding to intermediate state and unfolded protein, ANS shows binding to native protein whereas, sypro orange does not. I guess its because of the small size of ANS that it could penetrate inside the folded protein and bind to hydrophobic patches while the bulky sypro orange could not.
Yes it could be that! If you try to populate the intermediate state, the first question that comes to my mind is the energetic of that state. I mean, if the intermediate is a high-energy intermediate you'll never get that state in solution...otherwise could be a denaturing condition to trap it. You saw the intermediate by kinetic or equilibrium experiments?
Check this paper
https://www.researchgate.net/publication/269411091_The_E_Coli_thioredoxin_folding_mechanism_The_key_role_of_the_C-terminal_helix
Article The E. Coli thioredoxin folding mechanism: The key role of t...
Its an equilibrium intermediate. Not sure about its energetic. it an unfolding intermediate that is formed in intermediate denaturation concentration. Went through your paper, nice work.
Ok, if it is an equilibrium intermediate then the energy in the landscape is between the native and unfolded ensemble and then you should be able to find a condition to trap it. Best!
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