I used RSM for optimization refolding process of an enzyme. Results from ANOVA shows that pH has the strongest impact on refolding, compared to L-Arginine and Redox agent. It seems that this outcome provokes some doubt. I would appreciate if anyone could help me through this problem. Is there any work which has a focus on pH effect on refolding? What is the impact of L-arginine and redox agents at different pH levels on protein structure during refolding process?
I presume the redox reagent is used for the formation of disulfide bonds by reshuffling of the thiols from SH to S-S or vice versa within proteins. At different pH, the rate of reshuffling is varied due to the reactivity of thiols. In general, the refolding rate is slower at low pH. Indeed, some proteins contain multiple disulphide bonds require slower reaction rate to perform refolding in order to form the correct SS bond.
For the L-arginine, most people believe it acts as an alternative to guanidine as you can see the functional group of arginine is actually called guanidino group. So it somehow act as a denature agent to interfere the H-bond with in proteins.
Hi, Oveis,
which kind or redox agents have you used?
It is very true that pH have a strong impact on protein folding, in many cases even stronger that redox agents: Arginine gropup were altered these charges under different pH. Good luck!
Dear Clarence,
Thanks for your respond. Would you please introduce some papers on effect of pH on protein structure? In fact, my protein has 9 disulfide bridges. my current results shows that pH effect is "much higher" than L-arginine and GSSG. Is that make sense?
Dear Taras,
Thanks for your comment on pH effect, it will help me a lot. I use GSSG/GSH for S-S reshuffling. In fact, I am trying to find a rational relationship between redox and L-arginine level. Now, results show that pH has the most important effect. But in current papers I don't see much things about pH effect. Researchers prefer to show effect of L-arginine and redox, rather than pH. what tests and analyzes I should do for investigation true effect of pH on protein? I am thinking that pH has a "non-linear" effect on protein structure!
I do not have any papers in hand about the pH on protein folding. This is because most of the proteins are folded in physiological pH, otherwise it is considered "denature" . Unless there are some very unusual proteins that present in abnormal environment. As a result, most studies are performed in standard pH. But of course change pH affects many thing. Notably, your high concentration of Arg is indeed turning the folding buffer into highly basic.
I do not find it surprising that pH has such a large effect on the refolding of your protein. Changes in pH directly effect the charge of amino acid. Depending on the pH you can gain or loose charge charges around the protein, this disrupts salt bridge formation and can also create charged residues in buried hydrophobic regions, making them more hydrophilic. You should look at the structure of your protein, if it is known to look for likely amino acids that would disrupt folding by a change in charge. If the 3d structure is not known you can still get pretty good 2nd structure prediction and where alpha helices and beta strands are being disrupted.
As for the effects of redox regents, i would guess that they are having less of an effect because a protein first needs to fold into the right position before correct disulfide bridge is formed. This is why it is generally good practice to refold protein under reducing conditions and then allow them to oxidize and form disulfide bridges. If the protein is reduce the it is like still folding into the right shape, but the lack of disulfide bridges would reduce the stability.
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