Lipid Research Division

What is the best way to study protein-lipid interactions?

I see there is more and more appreciation of lipid-protein interactions being critical for membrane protein activities. Thanks to Cryo-EM technology, many membrane proteins are found to embed/interact with membrane lipids, and it seems there are inevitable effects by the interactions. As Binks pointed out in the early discussion, there would be in general two interacting mechanisms - via specific lipid-protein interactions and via membrane biophysical properties. These interactions may regulate protein functions - transport or enzymatic reactions, oligomeric status, intracellular trafficking, or conformational dynamics. 

But the study seems mostly conducted in in vitro systems, and my question is, how can I capture a native status of lipid-protein interactions and find their biological/biochemical meanings?

I found three ways: one using photoaffinity crosslinking with chemically modified lipids, two using nanodiscs to capture and solubilize membrane proteins and lipids in a 'native' status, and three using MD simulation. 

And I found that photoaffinity crosslinking is doable as a general biochemist, but native mass spectrometry or MD simulations are very difficult for me to access. 

Can anyone suggest another methodology or the best way to study such subjects? I attach one recent review article to bring up the research field. 

Drew, D. & Boudker, O. Ion and lipid orchestration of secondary active transport. 626, 963–974 (2024). DOI: 10.1038/s41586-024-07062-3

Haha Yeongho, it's Neng, from the second floor. I have doubts about photoclickable crosslinking though. Could be a lipid molecule just happen to be nearby in the membrane and then get cross-linked to protein of interest? one can make an argument if proper controls are in position, but still....

What is the best way to study protein-lipid interactions?

I see there is more and more appreciation of lipid-protein interactions being critical for membrane protein activities. Thanks to Cryo-EM technology, many membrane proteins are found to embed/interact with membrane lipids, and it seems there are inevitable effects by the interactions. As Binks pointed out in the early discussion, there would be in general two interacting mechanisms - via specific lipid-protein interactions and via membrane biophysical properties. These interactions may regulate protein functions - transport or enzymatic reactions, oligomeric status, intracellular trafficking, or conformational dynamics. 

But the study seems mostly conducted in in vitro systems, and my question is, how can I capture a native status of lipid-protein interactions and find their biological/biochemical meanings?

I found three ways: one using photoaffinity crosslinking with chemically modified lipids, two using nanodiscs to capture and solubilize membrane proteins and lipids in a 'native' status, and three using MD simulation. 

And I found that photoaffinity crosslinking is doable as a general biochemist, but native mass spectrometry or MD simulations are very difficult for me to access. 

Can anyone suggest another methodology or the best way to study such subjects? I attach one recent review article to bring up the research field. 

Drew, D. & Boudker, O. Ion and lipid orchestration of secondary active transport. 626, 963–974 (2024). DOI: 10.1038/s41586-024-07062-3

What is the best way to study protein-lipid interactions?

I see there is more and more appreciation of lipid-protein interactions being critical for membrane protein activities. Thanks to Cryo-EM technology, many membrane proteins are found to embed/interact with membrane lipids, and it seems there are inevitable effects by the interactions. As Binks pointed out in the early discussion, there would be in general two interacting mechanisms - via specific lipid-protein interactions and via membrane biophysical properties. These interactions may regulate protein functions - transport or enzymatic reactions, oligomeric status, intracellular trafficking, or conformational dynamics. 

But the study seems mostly conducted in in vitro systems, and my question is, how can I capture a native status of lipid-protein interactions and find their biological/biochemical meanings?

I found three ways: one using photoaffinity crosslinking with chemically modified lipids, two using nanodiscs to capture and solubilize membrane proteins and lipids in a 'native' status, and three using MD simulation. 

And I found that photoaffinity crosslinking is doable as a general biochemist, but native mass spectrometry or MD simulations are very difficult for me to access. 

Can anyone suggest another methodology or the best way to study such subjects? I attach one recent review article to bring up the research field. 

Drew, D. & Boudker, O. Ion and lipid orchestration of secondary active transport. 626, 963–974 (2024). DOI: 10.1038/s41586-024-07062-3

Hi 

we have used HTRF assays using biotinylated phospholipids and recombinant protein to study protein-lipid interaction

The assays we developed was a modification of a similar HTRF assay looking for inhibitors of the ceramide binding protein CERT.  We were able to obtain kinetic constants and IC50 valuse for lipid binding competotors...hope this helps

leury, L., Faux, C., Santos, C., Ballereau, S., Genisson, Y., andAusseil, F. (2015) Development of a CERT START Domain-Ceramide HTRF Binding Assay and Application to Pharmacological Studies and Screening J Biomol Screen 20, 779-787 10.1177/1087057115573402

Degorce, F., Card, A., Soh, S., Trinquet, E., Knapik, G. P., andXie, B. (2009) HTRF: A technology tailored for drug discovery - a review of theoretical aspects and recent applications Curr Chem Genomics 3, 22-32 10.2174/1875397300903010022

What is the best way to study protein-lipid interactions?

I see there is more and more appreciation of lipid-protein interactions being critical for membrane protein activities. Thanks to Cryo-EM technology, many membrane proteins are found to embed/interact with membrane lipids, and it seems there are inevitable effects by the interactions. As Binks pointed out in the early discussion, there would be in general two interacting mechanisms - via specific lipid-protein interactions and via membrane biophysical properties. These interactions may regulate protein functions - transport or enzymatic reactions, oligomeric status, intracellular trafficking, or conformational dynamics. 

But the study seems mostly conducted in in vitro systems, and my question is, how can I capture a native status of lipid-protein interactions and find their biological/biochemical meanings?

I found three ways: one using photoaffinity crosslinking with chemically modified lipids, two using nanodiscs to capture and solubilize membrane proteins and lipids in a 'native' status, and three using MD simulation. 

And I found that photoaffinity crosslinking is doable as a general biochemist, but native mass spectrometry or MD simulations are very difficult for me to access. 

Can anyone suggest another methodology or the best way to study such subjects? I attach one recent review article to bring up the research field. 

Drew, D. & Boudker, O. Ion and lipid orchestration of secondary active transport. 626, 963–974 (2024). DOI: 10.1038/s41586-024-07062-3