Understanding Intracellular Organization Using Aqueous Phase Model Systems

Open Access
Author:
Aumiller, William Michael
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
June 11, 2015
Committee Members:
  • Christine Dolan Keating, Dissertation Advisor
  • Christine Dolan Keating, Committee Chair
  • Carsten Krebs, Committee Member
  • Scott A Showalter, Committee Member
  • Antonios Armaou, Committee Member
Keywords:
  • aqueous phase separation
  • macromolecular crowding
  • phosphorylation
  • coacervate
  • sequential enzymes
  • compartmentalization
Abstract:
The intracellular milieu is a heterogeneous environment composed of many different organelles and compartments that perform specific functions simultaneously in spatially confined areas. This environment is incredibly complex, which can make studying enzyme reactions inside cells challenging. The aqueous phase model systems described here aim to provide insight as to the mechanisms used by cells to control enzyme activity, compartment formation, and compartmentalization of biomolecules. Chapter 1 provides a general background of intracellular organization in biological cells, followed by a discussion of fundamental principles governing biomolecule activity within a nonideal solution. The types of aqueous phase separation that appear in later chapters are discussed, as well as a review of related aqueous phase-separated model systems. Chapter 2 describes complex coacervation of a long poly(uridylic acid) RNA (poly U, a polyanion) and short cationic peptide (RRASLRRASL) that undergo reversible phase separation in response to the phosphorylation state (and therefore charge state) of the peptide. Phosphorylation was controlled enzymatically by a kinase and phosphatase pair. This demonstrates that phosphorylation could be a viable means to control compartment formation and dissolution in vivo. Additionally, the poly U/RRASLRRASL coacervates were capable of concentrating solutes up to 1150×. In Chapter 3, sequential enzyme activity is studied in a polyethylene glycol (PEG)/sodium citrate biphasic system. The enzymes glucose oxidase (GOX) and horseradish peroxidase (HRP) and substrate Amplex Red partitioned to opposite phases and required mass transport across an interface for product formation. A mathematical model was developed to describe the complex kinetics in the system, and the model was validated when it accurately described other experimental conditions. Chapter 4 looks at the reaction of HRP with two different substrates that differed in their relative hydrophobicity. Catalysis by HRP was highly dependent on solution composition because attractive interactions between the more hydrophobic substrate and more hydrophobic crowders and cosolutes (different molecular weights of PEG) caused a larger decrease in enzyme activity. This was confirmed by diffusion nuclear magnetic resonance (NMR) measurements. This is significant because it is the first systematic study of substrate-crowder interactions within the macromolecular crowding community. In Chapter 5, enzymes and substrates of the purine biosynthesis pathway were partitioned to the dextran-rich phase of a PEG/dextran biphasic system. The mathematical model developed in Chapter 3 was used in this phase system to describe the kinetics and mass transport under conditions that were difficult to study experimentally. The results suggest that significant advantages of enzyme colocalization may only be observed if essentially all of the enzyme is restricted to compartments, rather than only being weakly partitioned. Finally, Chapter 6 offers some general conclusions from this work, as well as some future outlook on using aqueous phase systems as model cells.