Single Molecule Enzymology Using Femtoliter Arrays.
Abstract: This thesis demonstrates the application of optical fiber based
femtoliter arrays for single molecule enzymology studies. Study of enzymes at single
molecule level provides us with mechanistic insights that cannot be obtained using
traditional bulk studies. Several approaches for isolating and studying single enzyme
molecules have been reported in the literature. These methods are de... read moretailed in Chapter 1
with an emphasis on femtoliter array platforms for single molecule isolation. Chapter 2
describes the application of optical fiber based arrays to study the stochastic binding and
release of the inhibitors to single β-galactosidase molecules. The competition between the
substrate and inhibitor for the enzyme active site is studied at single molecule level. The
effect of inhibition on enzyme activity is also described. In Chapter 3, the
α-complementation reaction of β-galactosidase was studied at single molecule resolution. In
a novel application of the femtoliter arrays, the numbers of active molecules formed in the
complementation reaction were directly counted and the stoichiometry of complementation
reaction was determined. Chapter 4 discusses the effect of temperature on single molecules
of wild type and single surface cysteine variants of β-glucuronidase. The identification of
the thermal switching property of a fraction of the enzyme population in some variants is
reported. Finally, chapter 5 discusses the current progress towards identifying
hetero-oligomers with unnatural backbones that can catalyze notoriously difficult
reactions. The approach of screening large oligomer libraries for the presence of rare
catalysts using the optical fiber femto-well array platform is also
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Chemistry.
Advisor: David Walt.
Committee: Krishna Kumar, Joshua Kritzer, and Jeff Gelles.
Keywords: Chemistry, and Biochemistry.read less