Investigation of the mechanism of coupling between two domains of T4 lysozyme: the role of an alpha helix in coupling distant regions

As the molecular biology and metabolic engineering fields move towards more thorough exploration and design of biological systems, it is imperative that there is a wealth of knowledge available about the biological macromolecules that comprise these systems. Proteins are important members of these biological systems and understanding the biophysical complexities of these macromolecules is the central focus of the protein-folding field. Protein folding researchers use a wide array of techniques to elucidate the forces that allow polypeptides to fold into the three-dimensional structures that allow them to function. The work presented in this thesis both unveils more about a hallmark of protein folding and utilizes knowledge of protein folding to alleviate an enzymatic bottleneck in an important recombinant metabolic pathway. In the second chapter of this thesis, I use a number of techniques to probe how cooperativity, a common characteristic of proteins, is conferred in the model protein T4 lysozyme (T4L*). Cooperativity is a property that allows most proteins to exist as either fully folded or fully unfolded without significant population of a partially-unfolded intermediate under equilibrium conditions. The molecular mechanisms that allow for this two-state behavior are not completely understood. T4L* is a single domain protein comprised of two-subdomains that fold cooperatively. In this chapter I uncover an allosteric mechanism allowing for cooperativity between the two subdomains via a central helix. In the third chapter, I utilize what is known about the interplay between protein misfolding and heterologous protein expression to optimize an engineered metabolic pathway. The recombinant glucaric acid pathway utilizes E. coli machinery to express three heterologous enzymes, one of these, myo-inositol oxygenase (MIOX), has been identified as the bottleneck of this pathway. Expression of recombinant MIOX is dependent on its substrate myo-inositol. In this chapter, I demonstrate that myo-inositol acts as a chemical chaperone and its action as a protein folding aid can be replaced by the action of the chaperonin GroEL/ES