Interactions at the protein-lipid bilayer interface: quantitative characterization via single-molecule force spectroscopy

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Proteins are important components of cells, which are the building blocks of all living things. Everything that enters and exits a cell (e.g., nutrients and pharmaceuticals) must go through membranes. Yet, little is known about how proteins and membranes interact with each other at the molecular scale. To better understand protein-lipid interactions, we will attach individual proteins to the sharp tip of an atomic force microscope, which is an extremely powerful microscope that is able to reveal interactions at the molecular scale. We will then bring these proteins into the proximity of membranes and use the microscope to record the resulting forces. Measuring these microscopic-level interactions will reveal details that depend on the specific proteins employed. More generally, the work will provide a deeper and quantitative understanding of fundamental activities that take place at the biological membrane. In terms of broader impacts, the project aims to improve STEM education and public science literacy. Several activities are planned to support these goals including developing and implementing a laboratory module for Missouri high school physics students; implementing and refining a new hands-on undergraduate course at the University of Missouri-Columbia targeted to non-science majors; and engaging in direct dialogue with general audiences in mid-Missouri through the Science Café, which is an informal venue that encourages questions from audience members. The overarching goal of this research project is to elucidate interaction strengths, energy landscapes and kinetic pathways of model lipophilic proteins as they negotiate fluid lipid bilayers and thus to provide an improved and quantitative understanding of protein partitioning and folding at the lipid bilayer membrane interface. This will be achieved by combining high precision atomic force microscopy (AFM)-based force spectroscopy methodology with analytical modeling, computer simulations, and biochemistry. The central experimental observable (force) will be measured with state-of-the-art spatial-temporal precision. The project tackles fundamental questions in membrane biophysics such as: How do the protein sequence and the emergence of secondary structure affect the strength of a protein-lipid bilayer interaction? Three model protein systems of differing structural complexity will be employed in conjunction with supported lipid bilayers. Specific research goals include: (i) correlate peptide sequence with peptide-lipid bilayer interaction strength, energy landscape and kinetic pathway; (ii) deconvolve protein secondary structure from primary structure contributions in a model polypeptide-lipid bilayer interaction; and (iii) quantitatively characterize a critical peripheral membrane protein-lipid bilayer interaction underlying protein export activity in E. coli. By directly probing the repeated association and dissociation of model proteins from the lipid bilayer interface, this project is poised to provide an improved understanding of protein partitioning and folding in membranes, a fundamental biophysical process.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.