Professor Ron Koder
City University of New York
Hosted by Professor Sagar Khare
Tuesday December 4, 2018
11:00AM, CCB* Auditorium
*New Chemistry and Chemical Biology Building
"The Importance of Dynamics in Designed Enzymes"
The evolution of enzymatic function is believed to begin with a dynamic, promiscuous enzyme which then evolves to be more rigid and specific. By contrast, the field of enzyme design has historically targeted highly stable and inflexible protein scaffolds. Here we show that a dynamic single chain, single heme variant of a designed four-helix oxygen transport protein, created using simple physical-chemical principles as opposed to computational optimization, catalyzes the detoxification of the neuronal signaling molecule nitric oxide at a rate superior to that of any natural hemoprotein nitric oxide dioxygenase (NOD). The enzyme is functional in in vivo complementation assays of E. coli knockouts of the nitric oxide defense enzyme flavohemoglobin: the first demonstration of a fully artificial protein being capable of supporting life in vivo without selection-based optimization. Importantly, increasing the structural specificity of the enzyme results in an enzyme with significantly lower activity. We further report the rational redesign of DF2, an extremely rigid, computationally designed homodimeric diiron protein, into the functional enzyme CDM13, a single-chain dimetal-containing four-helix bundle, by increasing the dynamic motion of the protein via the reversal of the amino acid sequence of two of the helices, greatly reducing the packing complementarity in the protein core. The diiron form of CDM13 activates oxygen, forming µ-oxo-bridged diferric clusters at a rate far exceeding that of the initial tightly-folded designed single-chain diiron protein. This diiron protein hydroxylates methane and other hydrocarbons at rates comparable to that of natural methane monooxygenase enzymes. We further demonstrate a new design method – protein surface supercharging – that introduces a 'dynamics dial' that allows us to manipulate the degree of disorder in the protein by changing the counterion concentration. This manipulation enables to examine at a new level of detail the effect that dynamic disorder has on fundamental protein enzyme functions such as ligand binding and catalytic action. Together these results demonstrate that even for enzymes with complex multi-step reaction pathways the activity achieved upon binding cofactors with the correct ligation into a dynamic protein is enough to create protein catalysts that are comparable in activity to those found in Nature and underscores the need to move beyond the paradigm of structure in protein design to encompass a more realistic view that encompasses protein and enzyme dynamics.