Hi Padmapriya Kumar

I would agree with Sebastian. If you have an idea of the family of protein and thereafter look at literature of possible domains that interact - site directed mutagenisis may be something to look into to systematically mutate amino acids to evaluate the abolishing of interactions (analyze via Co-IP or SPR). Another method which is used widely is chemical cross linking (after purifying and incubating the target proteins of interest), this is followed by sequential digestion and MS which can give you an idea of the cross linked domains which you can further trap and determine exact sequences that are inter-cross linked.

Structural studies such as CD spectroscopy and ofcourse x-ray crystallography can help in the structural aspects. But ideally you want to have some idea of the proposed domains you believe are interacting and take it from there.

Hi,

I agree with Blake and Sebastian. What I would add/suggest if you have an idea of which domains could be responsible for the interaction is to run Co-IPs only using said domains to confirm this. Alternatively, if you are still looking for these domains you could construct multiple vectors expressing fragments of your interacting partner and run Co-IPs with each, systematically narrowing down the possible interacting site to a small region. Then do site directed mutagenesis on this region to see if you can disrupt the interaction.

I hope this helps,

Max

Thank you for the valuable suggestions!

I am planning to start with Co-IPs of individual domains with intercating partner and then narrow down to specific small regions by site-directed mutagenesis. I could also perform an SPR or similar biophysical analysis to identify the effect of mutagenesis on binding affinity.

Hi Padmapriya,

If you want to know the exact interaction sites, you can use structural biology techniques, including X-ray crystallography, NMR, and cryo-EM to solve this problem. Different techniques have different pros and cons, you need to choose the appropriate approach based on your protein properties. If you don’t have the resources or equipment, you can work together with a reliable partner to solve this problem.

Recommend an article for you, maybe can help you choose experimental method to determine the structure, https://www.creative-biostructure.com/comparison-of-crystallography-nmr-and-em_6.htm

Hope it helps!

Roger Davis Thank you for the response. I would surely consider using structural biology techniques.

Although I have specialization in the past in structural biology, I am considering other options for this project to start with due to lack of resources.

Hi Padmapriya Kumar , If you need accuracy to enterprise how to occur these interactions. I suggest computational approaches of molecular fragmentation as MFCC.

I hope you can help.

Take a look at this article:

Quantum binding energy features of the T3-785 collagen-like triple-helical peptide