DMREF: Collaborative Research: Helical Protein Assemblies by Design

Emory University, Atlanta GA

Investigators

Abstract

NON-TECHNICAL SUMMARY Molecular self-assembly is a fundamental principle of life, with cells having mastered this process to encode incredible diversity of function. Helical protein assemblies organize much of the intracellular and extracellular structure, and direct all movement. The ability to emulate such functions by designing synthetic protein assemblies would transform modern molecular science, with far-reaching applications including locomotion, controlled release, directional transport, dynamic switching, and shape-selective catalysis. However, structurally ordered supramolecular materials on the nanometer length-scale are the most challenging to rationally construct and the most difficult to structurally analyze. The size and structural complexity of these extended protein assemblies present a significant challenge to current computational design methods. The rules that govern protein-protein interactions are more complex and difficult to reliably predict than for DNA. In this project, a novel intellectual framework for the targeted design of synthetic protein assemblies at atomic-level accuracy will be established, validated, and made available to the research community. Enabled by the combined expertise of the three investigators involved, this approach will merge significant advances in modeling and computational design with never-before-possible experimental techniques for structural determination of protein assemblies at the atomic level. On the way to developing this framework, fundamental questions of acute significance to biology, chemistry, and materials science will be addressed: from development of an understanding of the functional roles of native biological assemblies to construction of synthetic assemblies for technological applications. Students (graduate and undergraduate), postdoctorals, and faculty involved in this project will gain experience in a variety of computational, synthetic, and analytical methods in research areas of fundamental technological interest that will prepare them well for future scientific careers. An exchange program between the three academic institutions (Emory University, University of Virginia, and Dartmouth College) will be established that will permit students and postdoctorals to become involved in the different aspects of this research project. TECHNICAL SUMMARY Helical protein assemblies in biological systems exhibit a rich portfolio of structure and function; capturing these within simpler and more tractable synthetic materials would amount to a major leap in molecular science. Recent technological advances in genome sequencing, bioinformatic analysis, near-atomic resolution cryo-EM structural determination, and computational protein design, in combination with extant synthetic and analytical methods, present an unprecedented opportunity to engineer novel protein assemblies that emulate and improve upon their native counter-parts. This project will employ protein designability, estimated on the basis of native structural representation, as a mechanism to promote and control association between folded protein motifs with an aim to create protein-based materials of defined structure and function. Designability in the context of protein engineering refers to robustness of a protein fold in sequence space. A proxy for designability is the frequency of occurrence of a structural motif within the Protein Data Bank (PDB). This approach will be employed to search for designable interfaces between protomers within the protein structural databank. The ultimate objective of the proposed research will be to define sequences based on simple secondary or tertiary structural elements that are competent for self-assembly into nano-scale materials with extended helical symmetry. Computational methods will be employed to interrogate the protein structural databank to identify designable interfaces within robust structural motifs. Suitable candidate sequences will be computationally optimized and synthesized. Proven biophysical methods will be employed initially to identify sequences with promising self-assembly behavior. State-of-the-art high-resolution structural analyses will be performed on these assemblies using Iterative Helical Real-Space Reconstruction (IHRSR) from cryo-EM images. Dramatic improvements in imaging hardware, reconstruction algorithms, and computational methods of structural refinement have provided rapid access to near-atomic resolution structures of native and synthetic helical assemblies. These analyses will inform future rounds of computational modeling and design, thus establishing a dynamic feedback loop between theory, synthesis, and advanced methods of structural analysis.