Probing membrane protein dynamics and interactions with lipids at the single molecule level
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Membrane proteins are difficult to characterize via traditional techniques, yet they constitute [approx]30 percent of proteins expressed in cells and perform critical functions. It is also essential to study the interactions with these proteins and membrane interfaces, as they can help shed light on protein stability and folding processes. The challenge in studying these interactions resides in not only the complexity of the biochemical system in question, but the length and time scales these dynamics/interactions take place in. At the single molecule level, atomic force microscopy (AFM) has become increasingly useful for direct imaging of membrane proteins in near-native conditions. Though the past decades have seen significant enhancements in scanning speed, AFM remains a serial imaging process in which a single tip is physically raster-scanned in x and y over a region of interest. AFM-based single molecule force spectroscopy (SMFS) can also be applied to studying the interactions between certain peptide sequences and supported lipid bilayers, which help to determine the energy landscapes for these peptides. However, this approach can lead to low throughput of high-quality data, as mechanical limitations of AFM cantilevers can reduce the amount of detectable interactions above a certain noise level. A long-standing question is,"How can one improve the spatial and/or temporal resolution without sacrificing precision?" This work probes this question for both AFM and force spectroscopy by studying two different systems, respectively: (1) protein translocation in bacteria, and (2) dissociation pathways of oligopeptide-lipid interactions. Translocation of polypeptide chains across membranes is an essential activity of all life forms. The general secretory (Sec) system is a primary method of exporting unfolded proteins from the cytosol of E. coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances the polypeptide secretion process that is driven by the Sec translocase, consisting of translocon SecYEG and peripheral ATPase SecA. Working models have been developed to describe the structure and functions of SecDF, but important mechanistic questions remain unanswered. Here, we acquired AFM images of SecDF protrusions in supported lipid bilayers, and compared the resulting height distriubtion to simulated images based on reported crystal structures. Orientational assignment was bolstered via imaging certain mutations of SecDF lacking critical domains. As a complement, we acquire kymographs ([greater than] 100 ms resolution) to probe the conformational dynamics of SecDF across time. Conformational shifts and increased transition rates between states were observed upon interaction with SecYEG. The method of kymograph analysis is further analyzed using simulated images of SecDF, studying the effect of certain imaging artifacts such as instrument drift. Taken together, the data provides a novel vista of SecDF dynamics in physiologically relevant conditions. Protein-lipid interfaces are a fundamental component of biology. A multitude of membraneactive peptides are known to partition into membranes and develop higher order structures. The ability to quantitatively characterize the interaction strength between peptides and lipids would be beneficial in studying the folding and stability of certain membrane proteins. When studying these interactions via bulk biochemical assays, one can extract salient information about the peptide(s) being studied, such as their free energy of transfer (bilayer to solution). However, asynchronous activity resulting from these ensembles prevent us from probing more mechanical details about the interactions. In an attempt the complement these results, we applied AFM-based force spectroscopy to two classes of peptides, both of which stem from the hydrophobicity scale developed by Wimley and White: (1) Polyleucines W-Ln and (2) Host-guest Pentapeptides W - L - L - L - X (X is a guest residue). By studying the rupture force distributions P(F) and dissociation rate k(F), we can study the peptide-lipid interactions' dependence on both peptide length and amino acid sequence. These results were coupled with coarse-grained (CG) MD simulations, allowing us to construct the free energy profile for certain peptide-lipid systems. The proposed methods can extract from the AFM data the P(F) corresponding to a single peptide, regardless of the number of peptides attached to the AFM tip. We also compare manual vs. automated methods of analyzing rupture force data in order to reduce potential user bias. These investigations will help shed light on the kinetics underlying interactions between membranes and smaller peptides, paving the way for future experiments with more complex amino acid sequences.
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Ph. D.