ligand/receptor analysis question

[chef]

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what experiments are best suited to study where on the receptor ligand binds? both the ligand & receptor are cloned and characterized.. alanine scanning mutagenesis, covalent crosslinking of ligand, mutagenesis studies,, etc?

 

[structurelab]

what experiments are best suited to study where on the receptor ligand binds? both the ligand & receptor are cloned and characterized.. alanine scanning mutagenesis, covalent crosslinking of ligand, mutagenesis studies,, etc?

I would start with a homology model of the protein. In other words, see where ligand binds the receptor on other similar receptors. You can then try mutagenesis studies to see if you can knock out ligand responsiveness. However, BEWARE. Just because the receptor is no longer responsive to ligand does not mean that ligand is not binding. The protein may be rendered in a conformation that is not susceptible to change upon ligand binding. On the other hand, the protein may not bind ligand due to other reasons. For example, the mutant protein may have local structural changes that prevents ligand from binding. Also, there is a point that is often overlooked. The protein binds ligand to reach a lower energy state. If the mutation results in a more stable (ie lower energy state) protein conformation, the ligand may not bind due to energetic reasons. Alanine scanning is often used, but runs into problems all in its own. Alanine shifts the probablility of a portion of the protein becoming more alpha helical. This in itself can change the dynamics of the protein. So, in short, I would use homology modeling and mutagenesis to setup activity and ligand binding assays. If your lab is not geared toward structural biology, you may want to collaborate with a structural lab that will try to crystallize and determine the structure of the protein. If it works, it will take alot of the guess work out of your experiments.

 

[xanthines]

The above sounds like good advice. If you're not into mol bio and don't know any structural biologists/crystallographers....

Depending on the size of your molecules (and the size of your wallet!), you could try synthesizing your stuff with the right isotopes for NMR analysis. This would give you atomic resolution.

For molecular resolution, you could try putting your stuff on EM grids and visualizing them that way. If you want more details about this, I can post it if you're interested. It's not nearly as clear but it could be better than nothing, especially if your proteins don't crystallize and NMR is out of the question.

Also, you don't have to use alanines for mutagenesis studies. It's just the AA that everyone typically defaults to. Depending on the microenvironment and the sequences of your molecules and the structure (if known), F's could be a better substitution.

Just my two cents.

-X

what experiments are best suited to study where on the receptor ligand binds? both the ligand & receptor are cloned and characterized.. alanine scanning mutagenesis, covalent crosslinking of ligand, mutagenesis studies,, etc?