Cellular membranes are densely covered by proteins. Steric pressure generated by protein collisions plays a significant role in shaping and curving biological membranes. However, the mechanisms by which proteins generate steric pressure at membrane surfaces remain poorly understood. In particular, many of the proteins involved in membrane shaping are intrinsically disordered. How do these polymer-like domains, which lack a well-defined 3D structure, respond to crowded conditions at membrane surfaces? Here we report that intrinsically disordered domains are capable of generating substantial steric pressure that significantly exceeds the pressure generated by folded protein domains. Our work utilizes a previously developed, FRET-based molecular sensor to measure steric pressure at membrane surfaces. Our studies examined structured and disordered domains of prominent endocytic proteins, AP180 and Epsin1. Our results may appear counter-intuitive, owing to the flexible nature of intrinsically disordered domains. However, it is this same flexibility that gives disordered domains a high degree of conformational entropy, far exceeding that of structured domains. Therefore, when conditions on the membrane become crowded, disordered domains experience a sharp loss of conformational entropy, resulting in a substantial increase in free energy and steric pressure. These results are in agreement with control studies on membrane-tethered polyethylene glycol chains, suggesting the generality of the result. Finally, by placing the FRET sensor within disordered protein domains, we measured their elongation under crowded conditions, confirming the loss of conformational entropy that gives rise to steric pressure. Taken together, these results provide key thermodynamic insights into essential cellular processes that are required for membrane remodeling, further elucidating the biophysical mechanisms that control membrane shape and organization.

Intrinsically disordered proteins generate steric pressure through polymer-like crowding

ACS Spring 2022

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