A Switch Controlling Biomolecular Reconfiguration: "Tunably Controlled, Calibrated Assembly"
Abstract
In our current ARO-supported research, we discovered the mechanism by which chargeneutralization precisely regulates the size of assembly of the reflectin protein, and how this assembly governs the osmotically tunable color and brightness of light reflected from intracellular Bragg reflectors in specialized cells in squid skin (1-17). Most recently we discovered that (a) this charge-regulated assembly of reflectin proceeds through a liquid nano-droplet phase, and (b) it is likely that the physics of this liquid state governs the size of the assemblies, and thus, the precise calibration between the excitatory neuronal signal and the output color. We call this process and its underlying mechanism ÒTunably Controlled, Calibrated Assembly.Ó We first discovered that reflectin s assembly is triggered (as many neuronally and hormonally activated processes are) by the neurotransmitter-activated, signal transduction cascade-mediated phosphorylation of the protein, which we realized serves to neutralize the Coulombic repulsion of the positively charged, initially disordered protein. Based on this finding, we then developed several "surrogates" for this neutralizing phosphorylation that we can use conveniently to progressively and reversibly drive charge neutralization and thus fine-tune the assembly of the purified recombinant protein in vitro; these include pH-titration, genetic engineering, ionic charge screening, and low voltage (< 1V) electrochemical bias. Based on these discoveries, we now propose to extend our research to other proteins, both to determine the extent of generality of the mechanisms we have discovered, and to enable us to harness and control the form and properties of other proteins that do not normally assemble, This proposal requests a 1-year continuation of our current project, to serve as a bridge to extend our latest discoveries to a suitable second protein, both to test and extend our conclusions made with the tunable reflectin from squids, and then to use this second protein as a tunably controlled, calibrated assembly- directing carrier for other proteins. As the first candidate we propose to explore the use of amylin, a small (37- amino acid) protein that offers several advantages for our proposed study: like reflectin, amylin is cationic, initially disordered, and capable of assembling via extended beta structure stacking. As a fall-back if our efforts with amylin do not succeed, we propose to genetically modify the initially non-tunable reflectin from octopus, converting it to one that can be harnessed as carrier for the precisely calibrated, finely tunable assembly of other non-assembling proteins. If we are successful in our proposed identification of the structural modifications and conditions necessary to precisely and predictively control the size and extent of amylin (or octopus reflectin) assembly and its reversibility, follow-on work beyond this one-year bridging effort will then aim to develop the engineered protein as a robust platform for bringing non-assembling proteins under finely tunable, controlled assembly, enabling us to harness their functions in fiber, thin-film or other physical forms useful for Army applications.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jul 09, 2020
- Source ID
- W911NF2010257
Entities
People
- Daniel E Morse
Organizations
- Army Contracting Command
- United States Army
- University of California, Santa Barbara