Sheehan, Molly Marie

Email Address
Research Projects
Organizational Units
Research Interests

Search Results

Now showing 1 - 1 of 1
  • Publication
    Engineering Oxygen Reactivity in Heme-Protein Maquettes
    (2014-01-01) Sheehan, Molly Marie
    Fundamental questions of protein cofactor oxygen reactivity are left unanswered even after years of research due to the limitations imposed by the complexity of natural systems. Natural electron transport proteins are often large with many spectrally overlapping cofactors and fragile to mutation due to multiple roles of each amino acid. The study of the products oxygen reduction is further made difficult by inadequate available methods for kinetic resolution with quantitative yields of different reactive oxygen species (ROS). To first examine how proteins can avoid oxygen reduction for oxygen transport, as in natural globins, an oxygen transporter is designed from first principles of protein folding with each intermediate characterized. This is the first example of a transparently-crafted protein design functioning at natural rates. The ease of this design process, only three steps, and the minimalism of the design, only 8 different amino acids, calls into question many assumptions of what is necessary for a functional protein. This work suggests that functional success is easier reached by modification of gross properties of proteins rather than attempting structural exactness. This work may also aid future designs without necessitating a return to first principles because it not only produces a functional design but a toolbox from which to engineer. Oxygen reactivity was further studied through the development of a system for differentiating the rates and yields of superoxide, hydrogen peroxide and hydroxyl radicals. A series of man-made proteins were tested for oxygen binding, each aforementioned ROS production, and ligand exchange rates to uncover differences in ROS species production and mechanisms. It was found that it takes a single repetitive mutation to design from stable oxygen binder to a rapid outer-sphere superoxide generator. Further, another small set of changes confer preferential hydrogen peroxide generation without a superoxide intermediate through an inner-sphere mechanism. The ability to elucidate mechanistic details of highly homologous proteins and resolve each species of ROS allows us to learn about protein oxygen chemistry and apply this knowledge to the interpretation of data from natural systems, as well as use this knowledge to engineer forward towards industrially- and medically-applicable designer proteins.