I am studying the binding of a bacterial protein without a crystal structure to a small molecule. Through predicted structure and molecular docking, I have found several amino acid sites with significant changes in the binding ability of small molecules, but the reviewers suspect that some sites are The contribution is to maintain the protein structure, not the direct binding site. What experiment should I do to respond? What I think of is to use some experiments to detect whether some parameters of the WT and point mutant proteins are the same. Is something like circular dichroism feasible? Or is there a better way.
Dear Jacky Liang
i tihnk that you have to show to the reviewers that this mutation induce just local changes in the protein folding and do not induce general protein unfolding.
I tihnk that for this purpose you can use DSF (differenzial scanning fluorimetry) or DSC (differential scanning calorimetry) by monitoring the effect of the mutation in the protein thermal stability or you can acquire a 1D NMR spectra (peack dispertion in NMR provide an indication of folding)
Circular dichroism could be also possible but it is able to detect only very strong changes.
if you are interested to know some more about DSF, you can give a look to the following video present on my blog, proteoCool
https://www.blogger.com/video.g?token=AD6v5dzr9KqZslTx9NeEjXx1XyPti8dU15eqYoE9TT8OEDaZQcNINuDKwzCAzVXkFWZA0btipKTr81doABh-VjpprwCcRukVcLhAXE_T5BzPxYbeQQA7VMFmc4DdpjS4XbOfxkwAhIWu
good luck
Manuele
Is there any other function of the protein that you can assess as a surrogate for normal folded structure, such as enzymatic activity or binding to some other protein? Otherwise Manuele Martinelli gave you good advice regarding biophysical approaches
There are several techniques that can be used to determine if point mutations in a protein alter its proper folding:
It is worth noting that the choice of technique will depend on the specific requirements of your study and the availability of suitable reagents and equipment. Some techniques may also be more suitable for certain types of proteins or mutations. Additionally, multiple techniques may need to be used to validate the results and provide a comprehensive understanding of the effect of the point mutations on protein folding.
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