INSPIRE: Concentrated Dispersions of Equilibrium Protein Nanoclusters that Reversibly Dissociate into Active Monomers

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This INSPIRE award to University of Texas at Austin by the Interfacial Processes and Thermodynamics program in the Division of Chemical, Bioengineering, Environmental and Transport (CBET) in the Directorate for Engineering (ENG) is cofunded by the Particulate and Multiphase Processes in the CBET-ENG; and the Biomaterials program in the Division of Materials Research in the Directorate for the Mathematical and Physical Sciences. Protein-based drugs represent some of the most promising therapies for a wide range of diseases, including cancer. Subcutaneous injection is the preferred method of delivery, but its usefulness is currently limited by unwanted outcomes such as protein aggregation and gelation that occur for high doses. Previous attempts to address these problems by modifying the amino-acid sequence of potential therapeutics have been expensive and often unsuccessful. The investigators have recently reported a new method for creating highly concentrated, low-viscosity dispersions of stable protein nanoclusters that are not only of great fundamental interest but also could provide a basis for an unconventional means for solving major challenges in the protein-based therapeutics. However, at present, the answers to basic questions about the origins of the nanoclusters are lacking. Furthermore, the relationships between specific nanocluster characteristics and physical properties of the resulting dispersions are currently unknown.Intellectual MeritThe aqueous protein nanocluster dispersions to be studied in this proposal represent an entirely new form of soft condensed matter. The goal of the proposal is to explore and develop a fundamental understanding of how the protein nanoclusters form, why they stabilize the folded state of the proteins, and the impact of the clusters on the physical properties of dispersions. The investigators will use experiments, statistical mechanical theory, and computer simulations to test the hypothesis that clusters spontaneously form due to an equilibrium, self-assembly process where the addition of a small molecule ?crowder? molecule induces attractions between proteins that are balanced by weak electrostatic repulsions near the protein?s isoelectric point. In doing so, they will also address key open questions about the pathway dependence of nanocluster dispersions, the role and design of novel molecular crowders in nanocluster assembly, and the structure and dynamics of the nanoclusters. Since protein nanocluster dispersions are inherently multiscale (clusters, proteins, crowders, and solvent each introduce characteristic length scales), they are challenging to characterize experimentally. The PIs will determine the extent to which light-scattering, cryo-SEM and TEM, neutron scattering, and x-ray scattering can be used provide insights into the structure, dynamics, and stability of self-crowded proteins in the nanoclusters. They will also investigate appropriate multiscale strategies for modelingthese systems.Broader ImpactsIf successful, the proposed research will provide resolutions to important fundamental questions about the possibility of forming aqueous dispersions of equilibrium nanoclusters with tunable size that dissociate to monomers upon dilution. Anticipated outcomes include a general method for creating (and tuning the properties of) aqueous nanocluster dispersions of interest for technologies that range fromdrug delivery to biofuel production, as well as a theoretical understanding for why previous attempts using alternative strategies were only able to produce nanoclusters that were small, dilute, and short lived. In this grant, both undergraduate and graduate students will have the opportunity to work on important fundamental research with unusually strong interdisciplinary and technological components. In addition to other outreach efforts, the PIs propose to integrate the science on protein stability and crowding from the project into an undergraduate course on biological physics through a series of interactive, simulation- and theory-based modules.