Investigating How Membrane Mechanical Properties Affect the Expression, Folding, and Function of a Model Mechanosensitive Channel Protein

Jacobs, Miranda

doi:https://doi.org/10.21985/n2-dt1a-a092

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Investigating How Membrane Mechanical Properties Affect the Expression, Folding, and Function of a Model Mechanosensitive Channel Protein

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Mechanosensation is an essential behavior for cellular health that depends on the interactions between the plasma membrane and stretch-activated ion channels. To date, a few studies have measured the effect of membrane composition on protein behaviors from folding to function. However, the relationship between membrane mechanical properties and these behaviors remains poorly understood. The objective of this thesis is to uncover the relationship between membrane properties and protein behavior. My central hypothesis is that by decreasing membrane elastic moduli, protein folding efficiency may be improved and the activation sensitivity of a model mechanosensitive channel may be decreased, and that this property can be modulated with membrane amphiphiles. To investigate this relationship, we turned to the mechanosensitive channel of large conductance (MscL). As MscL is activated by membrane tension, MscL activity is expected to be altered by membrane mechanical properties that alter tension propagation. First, we determined the relationship between membrane elasticity and membrane protein folding using cell-free protein expression systems, and synthetic vesicles constructed from non-natural amphiphiles to modify membrane properties. Through this study, we found that by decreasing the area expansion modulus of the membrane, MscL folding was improved. We then wondered how certain natural amphiphiles modulate membrane mechanical properties. Using micropipette aspiration techniques, we determined that polyunsaturated fatty acids decrease the area expansion modulus of pure phospholipid membranes. However, in the presence of high amounts of cholesterol, this relationship depends on fatty acid identity. Finally, we wondered how membrane mechanical properties impact MscL behavior. Using patch-clamp electrophysiology with an integrated pressure-transducer, we determined that MscL sensitivity was decreased in the presence of amphiphiles that decrease membrane area expansion modulus. The results of these studies contribute to a critical unmet need in the field of membrane mechanobiology and highlight the role of membrane area expansion modulus on membrane protein folding and function. Together, the studies in this thesis demonstrate an important role of membrane amphiphiles in modulating membrane biophysical properties through affecting membrane protein folding and function.

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