(4cw) Biomimetic and Microfluidic Approaches to Biomolecular Function and Application

Living cells have evolved sophisticated intracellular organization strategies that are challenging to reproduce synthetically. Biomolecular function depends on both the structure of the molecule itself and the properties of the surrounding medium. The ability to correlate structure to function at biologically relevant timescales, simulate the in vivo environment, and isolate biological networks for study in an artificial milieu without sacrificing the crowding, structure, and compartmentalization of a cellular environment, represent engineering challenges with tremendous potential to impact both biological studies and biomedical applications.

Efforts to understand biomolecular structure-function relationships have relied heavily on static structural depictions that do not fully describe the dynamic nature of reaction processes. This limitation is the result of difficulties in applying dynamic or time-resolved crystallographic methods to a majority of biomolecular targets. These challenges are associated with radiation damage and/or the need to simultaneously and repetitively trigger the biomolecular reaction within a crystal. X-ray transparent microfluidic platforms for protein crystallography can be used to address these challenges, enabling the serial analysis of a large number of crystals so as to avoid both radiation damage and the necessity of repeatedly cycling a reaction.  The fine control over compositions on-chip enables both the easy growth of a large number of isomorphous crystals for analysis and reproducible chemical triggering of reactions (i.e. ligand addition), challenges that are difficult to address using traditional, single-crystal methods.  Such chips can also enable high throughput structural analysis with respect to many variables including pH and ionic strength to better understand biomolecule function. 

The challenge in designing synthetic organelles and in vivo microenvironments is maintaining both crowding and compartmentalization while controlling the available intermolecular interactions.  Emerging experience has shown that complex coacervates (liquid-liquid phase separation) utilizing biomolecules produces an effective biomimetic microenvironment.  Initial efforts are focused on understanding how functionalities, introduced through sequence-specific motifs, affect the emergent properties of these materials on both the bulk and molecular scale, and thus alter biomolecule sequestration and function.  Molecular design enables the use of structure and microphase separation as additional design parameters. Using these strategies, I propose the development of artificial organelles for applications in biochemistry, bioenergetics, biocatalysis, and biomedicine. The understanding gained from the development of these systems may provide insight into the function of analogous membraneless organelles such as carboxysomes, p-bodies, and p-granules as well as elucidation of potential pathways for the evolution of prebiotic life.