Which mutation is least affecting to the stability of protein if is to be done on surface. If Ala(on surface, with methyl group facing outwards) is mutated to Cys, would it affect the stability? Does the secondary structure of local environment play role?
Some link to respective literature will be much appreciated.
Thanks
In most of the cases, Cys is the best to be replaced with Ala and the other way around. Another alternative is Ser, which, however, is more hydrophylic than Cys.
Mutations on surface residues are less likely to disrupt the fold of a protein but more likely to interfere with the protein interactions (e.g. the solvent). For Cys mutations I'd recommend Ser as substitution.
Replacing Ala with a reactive Cys should be permissible if the environment allows for it, but you need to try...
Hi
As @Stefan mentioned, surface residues generally tolerate replacements - except when they are involved in important interactions. I can give you an example from our work which illustrates each. E. coli glutaredoxin 3 is a thiol-disulfide isomerase and has 2 active site cysteines and a third (whose function is unclear). This third cysteine is exposed and can form unwanted intermolecular disulfides. When mutated to serine it has little effect on function and stability. We have not tried others at this position, but I suppose just about anything would work here. In the active site, the N-terminal cysteine is a thiolate, stabilised by a hydrogen bond network which includes the buried, C-terminal cysteine thiol. We have constructed a stable, active site which can trap the glutathione intermediate by replacing this cysteine with serine. Our structure determination shows this serine replaces completely the hydrogen bonds in the network. We have found that alanine in this position is very un-stabilising.
One can rationalise most things, but in the end you have to try it.
Article Structure, dynamics and electrostatics of the active site of...
Article NMR structure of Escherichia coli glutaredoxin 3-glutathione...
Apparently, there was a discussion in this regard on ResearchGate few years ago: https://www.researchgate.net/post/Mutating_Cysteine_to_Alanine_instead_of_Serine_Why
All said above is reflected there.
Attached are some interesting papers on the properties of Cys hydrogen bonds.
Best.
https://www.researchgate.net/post/Mutating_Cysteine_to_Alanine_instead_of_Serine_Why
As I understand it, you want to introduce a Cystein, presumably to introduce some chemical modification. Therefore, solvent accessibility of the amino acid side chain is the most important parameter, as your protein should not only survive the mutation to Cys, but whatever additional modification you perform on this Cys. Do you have a decent structure of homology model of your protein? In this case, visual inspection and side chain accessibility determination using NACCESS can be used to find the site tat best tolerate the substitution and will lead to a Cys that is accessible to modification. You can also use rosetta ddG prediction and similar methods to verify that the position will tolerate the mutation. If you only have a sequence, you may at least do a secondary structure prediction - hydrophilic loop regions have the best probability to be accessible in the folded protein and are most tolerant to mutations.
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