Daniel W. Watkins

Constructing a man-made c-type cytochrome maquette in vivo: electron transfer, oxygen transport and conversion to a photoactive light harvesting maquette.

The successful use of man-made proteins to advance synthetic biology requires both the fabrication of functional artificial proteins in a living environment, and the ability of these proteins to interact productively with other proteins and substrates in that environment. Proteins made by the maquette method integrate sophisticated oxidoreductase function into evolutionarily naive, non-computationally designed protein constructs with sequences that are entirely unrelated to any natural protein. Nevertheless, we show here that we can efficiently interface with the natural cellular machinery that covalently incorporates heme into natural cytochromes c to produce in vivo an artificial c-type cytochrome maquette. Furthermore, this c-type cytochrome maquette is designed with a displaceable histidine heme ligand that opens to allow functional oxygen binding, the primary event in more sophisticated functions ranging from oxygen storage and transport to catalytic hydroxylation. To exploit the range of functions that comes from the freedom to bind a variety of redox cofactors within a single maquette framework, this c-type cytochrome maquette is designed with a second, non-heme C, tetrapyrrole binding site, enabling the construction of an elementary electron transport chain, and when the heme C iron is replaced with zinc to create a Zn porphyrin, a light-activatable artificial redox protein. The work we describe here represents a major advance in de novo protein design, offering a robust platform for new c-type heme based oxidoreductase designs and an equally important proof-of-principle that cofactor-equipped man-made proteins can be expressed in living cells, paving the way for constructing functionally useful man-made proteins in vivo.

De novo protein components for oxidoreductase assembly and biological integration

Manmade protein design is founded on the concept that a protein with minimal evolutionary complexity is a viable scaffold for incorporating simple engineering elements responsible for function in natural proteins and enzymes. There has been significant, recent success both in fabricating manmade protein components that exhibit functional elements inspired by natural oxidoreductases, and the functional integration of this componentry with natural proteins and biochemical pathways. Here we discuss the state of the art in de novo oxidoreductase construction, focusing on the diverse manmade componentry available and how their functions might be interfaced and integrated within living organisms.

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2. The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.Catalytic mechanisms of enzymes are well understood, but achieving diverse reaction chemistries in re-engineered proteins can be difficult. Here the authors show a highly efficient and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H2O2.

A suite of de novo c-type cytochromes for functional oxidoreductase engineering ☆

Central to the design of an efficient de novo enzyme is a robust yet mutable protein scaffold. The maquette approach to protein design offers precisely this, employing simple four-α-helix bundle scaffolds devoid of evolutionary complexity and with proven tolerance towards iterative protein engineering. We recently described the design of C2, a de novo designed c-type cytochrome maquette that undergoes post-translational modification in E. coli to covalently graft heme onto the protein backbone in vivo. This de novo cytochrome is capable of reversible oxygen binding, an obligate step in the catalytic cycle of many oxygen-activating oxidoreductases. Here we demonstrate the flexibility of both the maquette platform and the post-translational machinery of E. coli by creating a suite of functional de novo designed c-type cytochromes. We explore the engineering tolerances of the maquette by selecting alternative binding sites for heme C attachment and creating di-heme maquettes either by appending an additional heme C binding motif to the maquette scaffold or by binding heme B through simple bis-histidine ligation to a second binding site. The new designs retain the essential properties of the parent design but with significant improvements in structural stability. Molecular dynamics simulations aid the rationalization of these functional improvements while providing insight into the rules for engineering heme C binding sites in future iterations. This versatile, functional suite of de novo c-type cytochromes shows significant promise in providing robust platforms for the future engineering of de novo oxygen-activating oxidoreductases. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electron transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Dynamic action of the Sec machinery during initiation, protein translocation and termination

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.

Dynamic action of the Sec machinery during initiation, protein translocation and termination revealed by single molecule fluorescence

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. This is mediated, for the majority of proteins, by the highly conserved Sec machinery. The bacterial translocon – SecYMKEG – resides in the plasma membrane, where translocation is driven through rounds of ATP hydrolysis by the cytoplasmic SecA ATPase, and the proton motive force (PMF). We have used single molecule Förster resonance energy transfer (FRET) alongside a combination of confocal and total internal reflection microscopy to gain access to SecY pore dynamics and translocation kinetics on timescales spanning milliseconds to minutes. This allows us to dissect and characterise the translocation process in unprecedented detail. We show that SecA, signal sequence, pre-protein and ATP hydrolysis each have important and specific roles in unlocking and opening the Sec channel, priming it for transport. After channel opening, translocation proceeds in two phases: an initiation phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~ 40 amino acids per second for the model pre-protein substrate proOmpA. The initiation and translocation phases are both coupled to ATP hydrolysis while termination is ATP-independent. Distributions of translocation rates reflect the stochastic nature of the translocation process and are consistent with the recently proposed Brownian ratchet model [Allen et al. doi: 10.7554/eLife.15598]. The results allow us unparalleled access to the kinetics of the complex reaction and provide a framework for understanding the molecular mechanism of protein secretion.

Specific cardiolipin–SecY interactions are required for proton-motive force stimulation of protein secretion

Significance Many proteins are located in lipid membranes surrounding cells and subcellular organelles. The membrane can impart important structural and functional effects on the protein, making understanding of this interaction critical. Here, we apply computational simulation to the identification of conserved lipid binding sites on an important highly conserved bacterial membrane protein, the Sec translocase (SecA-SecYEG), which uses ATP and the proton-motive force (PMF) to secrete proteins across the bacterial plasma membrane. We experimentally validate these binding sites and use functional analyses to investigate the biological significance of the interactions. We demonstrate that these interactions are specific, transient, and critical for both ATP- and PMF-driven protein secretion. The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of preproteins across the plasma membrane, powered by ATP hydrolysis and the transmembrane proton-motive force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly cardiolipin (CL), a specialized phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific CL binding sites on the Thermotoga maritima SecA–SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry, biochemical analysis, and fluorescence spectroscopy of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional CL binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for CL in the conferral of PMF stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery, and thereby stimulate protein transport, by a hitherto unexplored mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, toward investigation of both the nature and functional implications of protein–lipid interactions.