Single Molecule Resolution of Immobilized Enzyme Function
Abstract
The objective of this effort is to understand the connectivity between immobilized enzyme heterogeneity, substrate transport, and net reactivity of immobilized enzymes. Model systems that will be investigated are lysozyme (T4 from bacteriophage), lipase (type A from B. subtilis), and diisofluorophosphatase (DFPase). Single-molecule fluorescence resonance energy transfer (FRET) will be used to measure the distribution in folding state of model enzymes upon surface attachment to self-assembled monolayers (SAMs). Distributions in folding states will be measured while varying enzyme orientation and distance from the surface. Constructs of enzyme will be labeled for single-molecule FRET experiments by mutating native residues at specific sites. For site-specific immobilization onto SAMs, a bioorthogonal reactive group (azide) will be added to the enzyme via post-translation modification, varying the insertion of tag on the enzyme. The distance from surface attachment will be varied by varying the PEG linker link. The structural stability of the immobilized enzyme will be measured by following the distribution of folding state over time. The distribution of turnover rates of immobilized enzymed will be measured to to correlate structural stability to activity. For immobilized lysozyme and lipase, fluorescence correlation spectroscopy will be used along with sigle-molecule FRET to measure active site motions. For DFPase, only active site dynamic motions will be measured. By varying the surface chemistry of the immobilization support, surface-mediated substrate diffusion will be correlated to immobilized enzyme activity. Turnover rates will first be measured when fluorescently-labeled substrate mimics (followed by labeled enzymes) are immobilized on modified SAMs of varying hydrophobicity and surface charge. SAMs will be altered with varying mixtures of hexyltriethoxysile and oligoethylene glycol triethoxysilane. Molecular trajectories will be organized by startaing distance between substrate and nearest enzyme, and search efficiencies and time distributions will be measured as a function of starting distance between adsorbing subtrate and immobilized enzyme.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 12, 2017
- Source ID
- W911NF1510141
Entities
People
- Joel L Kaar
Organizations
- Army Contracting Command
- United States Army
- University of Colorado Boulder