What can be the possible advantage of mutating Cysteine to Alanine instead of Serine which is the closest replacement for Cysteine? I see many articles where Cysteine has been replaced with Alanine but no body tells why they preferred Alanine. I need some light on this issue.
Serine is not always the closest replacement. It depends on what role the residue is playing in the protein. Cysteine is more hydrophobic than serine, and sometimes an alanine or valine, despite the difference in size, is a better substitute for that hydrophobic character. The other factor is that mutations to alanine are seen as a way to remove the effect of a side chain (the mutation essentially truncates the side chain at the C-beta atom).
Thanks and can u suggest the effect of either mutationothermostability of protein.
Because serine is the closest replacement to cysteine, one may not see any significant changes in the protein's function or cell phenotype. Of course if the cysteine residue is engaged in a disulphide bond formation then a defect may become readily apparent. On the other hand, alanine substitutions are classically done to measure the involvement of side chain interactions. Thus, a cysteine to alanine substitution is more meaningful in deducing whether the replaced SH-group was involved in hydrogen/disulphide bonding.
Dear Kumar, same reply that Jason explained for mutation thermostability assay of protein. Ala scanning is standard approach for such type studies you asked.
Dear Kumar, you must consider the location of the residue in the structure. Since Ala is smaller than Cys, you might introduce a small cavity in the interior of the protein, if it is located there, affecting the stability of the protein. In such case, a Ser replacement will be better than an Ala replacement. Same reasons are important to consider in doing an Ala scanning.
As a graduate student I have used alanine scanning mutational analysis to identify receptor binding region in a protein called interleukin-7. This fine analysis is like using your finger to snuff out a very precise piece of chemistry on the surface of your structure. I have also used cysteine to serine substitution mutational analysis to help define potential disulphide bonds in interleukin-7. My graduate committee argued that we should have performed cysteine to alanine substitutions because there is a chance that two serine side chains could form a hydrogen or salt bridge of some sort. Because I had other types of data to support our disulphide bond assignments the cysteine to serine data was accepted. In the end I believe that each mutational investigation is bound by cost and ability to measure a difference between your control and experiment. Your experiment is defined by your hypothesis. Hence this may help dictate which approach to take.
Hi Arun, I completely agree with all of the above mentioned comments on your question, but nobody here mentioned that introducing serine instead of alanine can create a water mediated hydrogen bonding with the surrounding residues, with which there will be proper H2 bonding distance, b'coz serine being polar residue having -OH grp as its side chain, it is more likely that it will make interaction, but on the other hand cysteine & alanine which has hydrophobic side chain, alanine will have the same effects like in case of cysteine. So most of the time researchers introduce alanine instead of serine in place of cysteine.
Best
Ajay
I also learned so much from your answers, and may helpful for me to design my experiment.Thanks!
I have a protein has two native cysteines that do not form disulfate bond. When I mutated one cysteine to serine, it functioned normally, but another cysteine to serine mutation has the problems with the protein purification and yield. Eventually I mutated it to an alanine, it worked normally. So I figured a serine mutant in one cysteine position probably distablize the protein.
If a cysteine is on the surface of the protein, in contact with water, Ser is usually a well-tolerated substitution, although it may occasionally allow for unexpected post-translational modifications such as phosphorylation or glycosylation. Replacing a Cys in the hydrophobic core of the protein is frequently destabilizing, because the energetic cost of desolvating Ser is higher than for Cys. Therefore it is preferable to replace Cys by Ala. To replace both Cys of a disulfide bond, frequently an Ala-Val pair is least destabilizing option, since due to the large VdW radius of sulfur, replacement by two Ala leaves an underpacked core.
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