Tailorable Tetrahelical Bundles as a Toolkit for Redox Studies

Abstract

Oxidoreductases have evolved over millions of years to perform a variety of metabolic tasks crucial for life. Understanding how these tasks are engineered relies on delivering external electron donors or acceptors to initiate electron transfer reactions. This is a challenge. Small-molecule redox reagents can act indiscriminately, poisoning the cell. Natural redox proteins are more selective, but finding the right partner can be difficult due to the limited number of redox potentials and difficulty tuning them. De novo proteins offer an alternative path. They are robust and can withstand mutations that allow for tailorable changes. They are also devoid of evolutionary artifacts and readily bind redox cofactors. However, no reliable set of engineering principles have been developed that allow for these proteins to be fine-tuned so their redox midpoint potential ( E m ) can form donor/acceptor pairs with any natural oxidoreductase. This work dissects protein-cofactor interactions that can be tuned to modulate redox potentials of acceptors and donors using a mutable de novo designed tetrahelical protein platform with iron tetrapyrrole cofactors as a test case. We show a series of engineered heme b -binding de novo proteins and quantify their resulting effect on E m . By focusing on the surface charge and buried charges, as well as cofactor placement, chemical modification, and ligation of cofactors, we are able to achieve a broad range of E m values spanning a range of 330 mV. We anticipate this work will guide the design of proteinaceous tools that can interface with natural oxidoreductases inside and outside the cell while shedding light on how natural proteins modulate E m values of bound cofactors.

Lambda CI Binding to Related Phage Operator Sequences Validates Alignment Algorithm and Highlights the Importance of Overlooked Bonds

“…The method was adapted from Solomon et al [19] and Gao et al [20]. The protein used in this paper was obtained as follows.…”

Protein Dynamics Govern the Oxyferrous State Lifetime of an Artificial Oxygen Transport Protein

“…There is a precedent for protein stabilization by fashioning a single-chain protein from a self-assembling helical bundle – when Degrado and coworkers transformed a nonfunctional homotetrameric diporphyrin protein (17) that they had designed into a single-chain tetraporphyrin protein, the overall structure became so stabilized that a number of destabilizing mutations had to me made in order to make the protein flexible enough that it could bind cofactors under non-denaturing conditions (18). Other single-chain four helix heme-binding bundles have been designed (19,20), including one demonstrated to bind oxygen (21), but a detailed examination of dynamic water penetration and oxyferrous state lifetime has not to date been reported.…”

Protein dynamics govern the oxyferrous state lifetime of an artificial oxygen transport protein

“…There is a precedent for protein stabilization by fashioning a single-chain protein from a self-assembling helical bundlewhen Degrado and coworkers transformed a nonfunctional homotetrameric diporphyrin protein (17) that they had designed into a single-chain tetraporphyrin protein, the overall structure became so stabilized that a number of destabilizing mutations had to me made in order to make the protein flexible enough that it could bind cofactors under non-denaturing conditions (18). Other single-chain four helix heme-binding bundles have been designed (19,20), including one demonstrated to bind oxygen (21), but a detailed examination of dynamic water penetration and oxyferrous state lifetime has not to date been reported.…”