Hello Zirui, generally speaking (in my experience) folks tend to mutate residues to alanine. If you want to make a more informed decision, you can do the following. Use DeepMSA2 to generate a sequence logo of your protein of interest: https://zhanggroup.org/DeepMSA/. Now you know what other residues (if any) are found at particular amino acid positions. If, for example, you generate a sequence logo and you see that alanine occurs as frequently at that amino acid position as glycine, then alanine may not be the best choice. A better and more comprehensive approach would be to use a deep mutational scanning tool such as ESM-Scan (see below). Those suggestions assume that the protein is evolved to bind the ligand (i.e. residue conservation reflects importance in ligand binding). Alternatively, if you have a structure of the protein bound to the ligand then you can take a more rational design approach. For example, if the residue(s) involved in binding the ligand is polar or charged, then you can mutate it to something nonpolar. Good luck!

Article ESM‐scan—A tool to guide amino acid substitutions

Thank you for your helpful and detailed answer! Jamison Drew Law

Zirui, just a quick follow up. If an experimentally determined structure of your protein + ligand is not available, you can predict one using AlphaFold3, Boltz-1 (recommended), Chai-1, and ProteinX. Alternatively, you can use online docking tools. Just exercise some caution with these predictive programs. Try looking at the following website to start: https://neurosnap.ai/services?gad_source=1&gclid=CjwKCAiAnpy9BhAkEiwA-P8N4gvge-aGv8TirKWRwlkvNvxevRvYxfv3aqVHlB7aZPcg6tecglZWexoCu6UQAvD_BwE. Good luck!

Thanks for the additional message. Actually, I already get the normal ligand + proetin binding site through online docking tools. So the following step is experimentally test this binding site. And I am now stuck with what kind of amino acid to mutate to. As you kindly mentioned before, normally it should be ala. But I just wondered why(hh)? So I ask this question trying to conduct this mutate more logically! After looking into your instruction, my strategy now is to mutate according to the charge and polar. Meanwhile , I wonder if it is necessary to test that the mutation does not change the original structure of the protein? (so the "not-binding" after the mutation is purely caused by the binding site, not the structure change caused by the mutation?) Jamison Drew Law

Zirui, whether the mutation(s) change the structure or not will need to be determined empirically. There are online tools that can help predict the effects of mutations, see DynaMut tool: https://biosig.lab.uq.edu.au/dynamut/. You can use circular dichroism to assess if there are changes in secondary structure for the mutants. A caveat to that strategy is that if the mutation is in an unstructured region of the protein then CD will not be of help. Good luck!

Zirui, there are many other webservers that you can try using for assessing the impact of mutations on protein stability. You just neex to dig around Google and PubMed a bit. MutationExplorer is a recent example: https://academic.oup.com/nar/article/52/W1/W132/7655781. Good luck!