College of Science

Seminar: Krishna Sigdel [Univ. Missouri-Columbia]

[BIOPHYSICS SEMINAR] Insights into protein-membrane interactions and membrane protein structure via atomic force microscopy

Jan 30, 2018 11:00 AM to Jan 30, 2018 12:00 PM at Building 8, Room 241

Understanding the interactions of proteins with the complex environment of cellular membranes is a longstanding and fundamental problem in biophysics. These interactions are important because they determine the three-dimensional structure and ultimately the function of membrane proteins, a large class of pharmaceutically relevant macromolecules that reside within cellular membranes. Conventional biochemical methods have been used to study protein-membrane interactions, but elucidating mechanistic details from such assays has proven to be challenging. In this talk I will discuss a single-molecule approach to measure the interaction between a protein and a membrane. To achieve this, a small protein (i.e., a peptide) of interest was affixed to the tip of an atomic force microscope (AFM). Protein binding was measured by lowering the tip onto the membrane, while dissociation was measured by raising it again. The mechanical nature of the method allowed binding/unbinding data to be collected in physiological buffer solution. Distinct interaction patterns were observed between peptides that differed only by a single monomer unit. These measurements, together with analytical modeling and molecular dynamics simulations, provide a basis for improved understanding of the structural energetics of membrane proteins and their partitioning into the membrane. I will also discuss a project focused on the cancer-related membrane protein P-glycoprotein (P-gp) that plays a major role in human disease and drug disposition because of its ability to pump a chemically diverse range of drugs out of the cell. Deciphering the drug-induced conformational changes is key to understanding the molecular basis of transport and to developing novel pharmaceuticals. We directly imaged individual P-gp transporters reconstituted into a membrane and studied the protein structure and structural dynamics in physiological buffer solution. Analysis of the AFM images revealed discrete populations with differences in P-gp protrusion volume, consistent with the large cytoplasmic domain and the smaller extracellular domain of the transporter. The domains were then positively identified by volume changes in the presence of domain specific antibodies. Experimentally measured AFM images of the P-gp protrusions above the membrane were in good agreement with simulated AFM images. This work provides a foundation for future studies such as imaging the structural consequences of cancer drug binding to P-gp in near-native conditions.

10:50 am Refreshments

11:00 am Seminar

Building 8, Room 241