Conformational Flexibility in Beclin 1, an Evolutionarily-conserved, Key Autophagy Protein

North Dakota State University Fargo, Fargo ND

Investigators

Abstract

Project title: Conformational flexibility in Beclin 1, an evolutionarily-conserved, key autophagy protein Autophagy is a critical cell survival pathway responsible for nutrient recycling due to degradation of unwanted, damaged or harmful cellular components. Autophagy plays an important role in several key organismal processes including embryonic development, tissue differentiation, cell-growth, as well as surviving environmental stress and internal stressors like damaged organelles, mutant proteins, free radicals and intracellular pathogens. Beclin1, a protein essential for autophagy, has been directly implicated in these processes. This research will focus on understanding the atomic-resolution structure and flexibility of Beclin1 to provide a better understanding of its critical role in autophagy and cellular maintenance. This project will provide graduate and undergraduate students in North Dakota an unprecedented opportunity to train in classical and cutting edge methods of biophysical and biochemical research, and also have a substantial impact outside the laboratory by educating high-school teachers about biomacromolecular structure and function. Over the period of this project, 40 high-school science teachers will participate in a summer professional development course to help them instruct their students in understanding the structures of biomacromolecules such as proteins and DNA. Thus, the educational activities outlined in this project are designed to educate and mentor students at all levels: high school, undergraduate and graduate; and will have a broader impact far beyond the grant funding period. Beclin1 appears to be a protein interaction hub for autophagy as it binds to over twenty diverse cellular proteins. The research goal of this project is to better understand how Beclin 1 interacts with so many different partners by establishing its structure and conformational flexibility and identifying residues that are key for function. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy will be used to assess secondary structure content, backbone flexibility and dynamic conformational changes. High-resolution structures will be determined by X-ray crystallography or NMR. Small-angle X-ray scattering will be used to obtain low-resolution structural information such as size, oligomerization and molecular envelopes. Combined information from these methods will be used to derive a pseudo-atomic model of full-length Beclin1. The role of motifs and conserved residues in binding will be examined using isothermal titration calorimetry to quantify the thermodynamics of interaction and by cellular assays to assess the impact of mutations on cellular autophagy levels and binding of selected key autophagy proteins. Last, but not least, this research will provide substantial information about poorly understood intrinsically disordered regions and help establish methodology essential for investigating such regions.