8.1 Biochemistry: Extending the Mechanisms of Protein Function to Non-Cellular Environments
Abstract
The objective of the research project is to explore two complementary approaches by which protein function can be stabilized in a non-cellular environment: (1) recapitulating the benign environment inside the cell by optimizing the concentration and composition of osmolytes within a supporting matrix; and (2) covalently appending proteins to a supporting matrix with precise control over intermolecular spacing, ensuring that any molecules that unfold are sufficiently distant from neighboring molecules to prevent aggregation. The research project will encompass two specific aims: 1) Identify the optimal combination of osmolytes required to stabilize water-soluble proteins in non-cellular environments A novel two-dimensional microfluidic gradient generator will be developed and used to screen various osmolytes to determine the optimal combination and concentration needed to stabilize two model proteins, glucose oxidase and glucose dehydrogenase within a hydrogel matrix. Protein stability will be assessed via activity assays performed colorimetrically, fluorometrically or electrochemically under a range of environmental stressors, including high temperatures, strong electric fields, high concentrations of denaturing agents, and multiple freeze/thaw and desiccation cycles. (2) Reproducibly and reliably controlling the intermolecular spacing of proteins to enhance stability Proteins will be covalently attached to a polymer matrix with precise control over intermolecular spacing to prevent molecules that unfold from colliding with other molecules and aggregating. Block copolymer lithography will be used to generate precise nanoscale patterns with long-range order. A block copolymer will be designed to generate a cylindrical phase, and the cylinder-forming domains will contain functional groups to which the proteins will be covalently attached. The cylindrical domain spacing will be modified to assess the impact of protein spacing on protein stability under a variety of environmental conditions. A library of block copolymers with different morphologies will be generated allowing systematic analysis of the effect of protein spacing between 15-60 nm.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Feb 06, 2017
- Source ID
- W911NF1610421
Entities
People
- Sumita Pennathur
Organizations
- Army Contracting Command
- United States Army
- University of California, Santa Barbara