I am performing amino acid substitutions using SDM. Could anyone suggest some sort of guideline in how to decide what amino acid to change the wild-type amino acid to? For instance what would be "safe" to substitute a polar amino acid to, etc.?
What do you want to learn from your experiment?
Identification of interaction sites, influence on stability?
Or do you want to improve a specific functional property of your protein, e.g. solubility?
How much is already known about your protein? Have you looked at an alignment of homologous proteins from different organisms? Highly conserved positions have a high probability of being important for folding and/or function, highly variable positions are normally located on the surface and much more tolerant towards substitution.
As Prem Prasad Lamichhane said, Ala scans are usually performed to see which positions are involved in specific functional interactions, while fairly well tolerated in regard to protein structure.The more conservative substitutions are, the more sensitive your assay has to be to detect an influence of the substitution, but conservative substitutions are less likely to have indirect effects on protein function through perturbing the structure.
hi stephen,
i thing here is article of your interest
1) http://www.medschool.lsuhsc.edu/biochemistry/Courses/Biochemistry201/Haas%20Files/2008/Thermodynamics/Bordo4425.pdf
2) http://biokamikazi.files.wordpress.com/2013/06/aminoacid_and_substitutions.pdf
In general, wild type amino acid is substituted by alanine (called as alanine scanning). Moreover, if you think change in basic or acidic amino acid has any effect on protein function you do that. Otherwise, simply perform alanine scanning.
What do you want to learn from your experiment?
Identification of interaction sites, influence on stability?
Or do you want to improve a specific functional property of your protein, e.g. solubility?
How much is already known about your protein? Have you looked at an alignment of homologous proteins from different organisms? Highly conserved positions have a high probability of being important for folding and/or function, highly variable positions are normally located on the surface and much more tolerant towards substitution.
As Prem Prasad Lamichhane said, Ala scans are usually performed to see which positions are involved in specific functional interactions, while fairly well tolerated in regard to protein structure.The more conservative substitutions are, the more sensitive your assay has to be to detect an influence of the substitution, but conservative substitutions are less likely to have indirect effects on protein function through perturbing the structure.
Thank you all for the answers. The alanine "null" mutations seems to be the way to go since we are trying to determine whether specific amino acids are involved in binding interactions without affecting protein solubility or structure through the substitution.
Ala scanning is indeed a good option. But please consider that it is not always an answer. As an example: Depending on where your site is located and the role of the residue, an exchange of something very bulky (e.g. M, F, W, Y) to Ala MAY cause dramatic changes in local structure.
Please make sure to be careful with your result interpretation when performing Ala scanning. Sometimes a second proof of the working hypothesis is advisable, by e.g. not only mutating to Ala but also to something on the other extreme, e.g. F, or a reverse in polarity etc. (highly depending from where you start)
When analyzing system responses (here binding interactions) it is better not to rely on a single impulse (here Ala mutation) when pertubating a system, better use more and different impulses to see how the system overall reacts. Also the information you gain about your system will be more detailed. If possible consider performing scanning saturation mutagenesis. Alanine scanning limits you (most likely, not in all cases) to the destruction of your properties, whereas saturation mutagenesis will also result in (often, not always) improvements of properties. Therefore the gained insight into your system will be bigger and more detailed.
And, Stephen, after determining what "specific amino acids are involved in binding interactions without affecting protein solubility or structure through the substitution", what are you planning to do? To still improve binding? If so, why don't you simply do a combinatorial scanning mutagenesis, or try it with tyrosine and/or serine. See the following review/papers by Sidhu to have a clearer idea:
Review: http://www.sciencedirect.com/science/article/pii/S1367593107000506
Alanine: http://www.sciencedirect.com/science/article/pii/S0022283605000732
Tyr/Ser: http://www.pnas.org/content/104/16/6632.abstract
Tyrosine: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2829252/
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