PhD Thesis colloquium: Avijeet Kulshrestha

Talk: Computational study of membrane driven secondary structural changes in proteins

Conformational changes in proteins, the most abundant biomolecule found in all living organisms, are ubiquitous and triggered by several factors. Protein conformational changes typically occur on time scales of tens of microseconds to milliseconds, lying well outside the sampling regime of conventional molecular dynamics (MD) simulations. In this thesis, we present a finite temperature string method path based approach to obtain the free energy of protein conformational changes utilizing path collective variables. We rigorously test and validate our approach and demonstrate its application to capture the α-helix to β-sheet transformation in the mini G-protein in a reduced two-dimensional collective variable space. We apply the method to study phospholipid membrane driven protein conformational changes associated with the assembly of bacterial pore forming toxins (PFTs) and antimicrobial peptides (AMPs).

The mammalian cell membrane contains cholesterol and several proteins of PFT family require cholesterol recognition for lytic activity. However, the role of cholesterol for Cytolysin A (ClyA), an α-PFT, expressed by E. coli remains elusive. Using our string method approach, we unravel the critical role played by cholesterol in assisting pore formation by stabilizing an unfolded on-pathway intermediate of the membrane inserted β-tongue motif. Specifically, a tyrosine residue was critical in catalyzing unfolding. Using extensive thermal unfolding MD studies on point mutations of the protein, we concluded that loss of flexibility in key membrane binding domains is detrimental to pore formation consistent with experimental observations.

We next applied the string method approach to study the insertion free energy and mechanism of insertion of the AMP ‘CM15’ in the inner bacterial membrane. Our free energy analysis showed that a membrane-bound single peptide unfolded state is more stable than a membrane-inserted folded state, with the insertion mechanism triggered by the N-terminus interactions with the cardiolipin lipid molecules of the bacterial membrane. Cardiolipin has not been considered in the previous studies, and our study points to the vital role for this four tail lipid in AMP-membrane interactions. We also report strong interactions of water molecules with one side of the membrane-inserted amphiphilic peptide, which can potentially be responsible for bacterial cell lysis.

In summary, the string method based approach developed in this work can be applied to a wide variety of protein conformational changes and can be used to study complex membrane driven protein unfolding and refolding phenomena.