Abstract

Membrane proteins play crucial roles in various biological processes, and understanding their folding mechanisms is of paramount importance for elucidating their structure-function relationships and developing therapeutics. However, studying membrane protein folding presents unique challenges due to the hydrophobic nature of the lipid bilayer environment. In this study, we introduce a novel approach, termed Steric Catching Methodology, for targeting the folding of helical membrane proteins. Leveraging the principles of steric hindrance, this methodology employs strategically designed molecular probes to selectively stabilize folding intermediates and trap transient states during the folding process. Through a combination of computational modeling, biophysical experiments, and structural analysis, we demonstrate the utility of the Steric Catching Methodology in dissecting the folding pathways of helical membrane proteins. Our findings provide valuable insights into the structural determinants driving membrane protein folding and pave the way for the development of innovative strategies for membrane protein engineering and drug discovery.