Characterizing the Locus of a Peripheral Membrane Protein–Lipid Bilayer Interaction Underlying Protein Export Activity in E. coli

Abstract

Quantitative characterization of the strength of peripheral membrane protein–lipid bilayer interactions is fundamental in the understanding of many protein targeting pathways. SecA is a peripheral membrane protein that plays a central role in translocating precursor proteins across the inner membrane of E. coli. The membrane binding activity of the extreme N-terminus of SecA is critical for translocase function. Yet, the mechanical strength of the interaction and the kinetic pathways that this segment of SecA experiences when in proximity of an E. coli polar lipid bilayer has not been characterized. We directly measured the N-terminal SecA-lipid bilayer interaction using precision single molecule atomic force microscope (AFM)-based dynamic force spectroscopy. To provide conformational data inaccessible to AFM, we also performed all-atom molecular dynamics simulations and circular dichroism measurements. The N-terminal 10 amino acids of SecA have little secondary structure when bound to zwitterionic lipid head groups, but secondary structure, which rigidifies the lipid-bound protein segment, emerges when negatively charged lipids are present. Analysis of the single molecule protein–lipid dissociation data converged to a well-defined lipid-bound-state lifetime in the absence of force, τ0 lipid = 0.9 s, which is well separated from and longer than the fundamental time scale of the secretion process, defined as the time required to translocate a single amino acid residue (∼50 ms). This value of τ0 lipid is likely to represent a lower limit of the in vivo membrane-bound lifetime due to factors including the minimal system employed here.

High force catch bond mechanism of bacterial adhesion in the human gut

“…2f and Supplementary Figs. 8 and 9 ) showed a crossover regime around 300 pN where the off rate decreased with increasing force, consistent with previously reported multi-pathway systems leading to catch bond behavior 62 , 63 . We note that comparison of the fitted energy landscape parameters between these two models in Table 1 is complicated by the fact that the BE model assumes Δ x ‡ is constant and independent of the force, while the DHS model assumes both Δ x ‡ and Δ G ‡ to be force-dependent.…”

“…(6) to calculate the force-dependent off-rate based on combining the histograms from P1 and P2 rupture events ( Supplementary Fig. 9) 62,63 . The rationale for combining the analysis of P1 and P2 rupture events was motivated by the evidence for dual-binding modes (see below).…”

Bridging Biochemical Activities with Conformational Dynamics Observed in Atomic Force Microscopy

“…Kosztin's group (MU Physics). [31,90,91]. One thing to note is that each barrier is associated with a different weight.…”

“…One thing to note is that each barrier is associated with a different weight. The calculated parameters correspond to the height of the energy barriers (∆G ‡ i ), the distances to the transition states (∆x ‡ i ), and the inverse of the off-rates in the absence of force (τ i ) which were determined for the interaction between the SecA2-11 peptide and a POPC lipid bilayer [31,90,91]. Force histograms obtained using the FIB modified biolever mini cantilever are shown in Figure 42.…”

“…The energy landscape governing the peptide-lipid interaction can be reconstructed and characterized once the force is measured. In previous work, our group used AFM to measure the force associated with the extreme N-terminal 10 amino acids of SecA (SecA2-11) interacting with a model lipid bilayer via force spectroscopy measurement [31,90,91]. Here, we study the same interaction but at higher temporal precision achieved through focused ion beam (FIB) modifications of cantilevers [10,11,15,16].…”