Life is far from thermodynamic equilibrium. Hence, life must extract energy from the environment. On Earth, that energy is driven by networks of metabolic reactions in all cells which ultimately move electrons and protons (i.e. hydrogen atoms) across the planet. The origin of metabolism required the emergence and evolution of proteins. Proteins are nanometre-scale chemical machines-i.e. literal nanomachines which physically move. These nanomachines enable living systems to perform essential biochemical tasks from replication to metabolism; the latter being the engines of life. In all extant life on Earth, a small set of these nanomachines, called oxidoreductases, couple chemical energy from the environment with core redox reactions including photosynthesis, respiration and nitrogen fixation. The origins and emergence of complex life have been intimately tied with evolution of oxidoreductases. Here, using structure-based analyses, we describe the evolution of the protein catalysts in three biological epochs. First, thermodynamically driven polymerization reactions generated simple metal-binding peptides with specific sequences that catalysed core metabolic reactions. Second, these catalysts were incorporated in small structural 'folds'. In the third epoch, these folds served as building blocks for extant, complex nanomachines.This article is part of the discussion meeting issue 'Chance and purpose in the evolution of biospheres'.

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, are a resource for photosynthetic, photonic and fluorescence labeling elements. They cover an exceptionally broad spectral range, but the complex superstructure and assembly have been an obstacle. By replacing in Synechocystis sp. PCC 6803 the biliverdin reductases, we studied the role of chromophores in the assembly of the phycobilisome core. Introduction of the green-absorbing phycoerythrobilin instead of the red-absorbing phycocyanobilin inhibited aggregation. A novel, trimeric allophycocyanin (Dic-APC) was obtained. In the small (110 kDa) unit, the two chromophores, phycoerythrobilin and phytochromobilin, cover a wide spectral range (550 to 660 nm). Due to efficient energy transfer, it provides an efficient artificial light-harvesting element. Dic-APC was generated in vitro by using the contained core-linker, L-C, for template-assisted purification and assembly. Labeling the linker provides a method for targeting Dic-APC.

Photosystem II reaction centers extract electrons from water, providing the basis of oxygenic life on earth. Among the light-sensitive pigments of the reaction center, a central chlorophyll a dimer, known as the special pair, so far has escaped a complete theoretical characterization of its excited state properties. The close proximity of the special pair pigments gives rise to short-range effects that comprise a coupling between local and charge transfer (CT) excited states as well as other intermolecular quantum effects. Using a multiscale simulation and a diabatization technique, we show that the coupling to CT states is responsible for 45% of the excitonic coupling in the special pair. The other short-range effects cause a nonconservative nature of the circular dichroism spectrum of the reaction center by effectively rotating the electric transition dipole moments of the special pair pigments inverting and strongly enhancing their intrinsic rotational strength.

The molecular structure of mBDFP, a far-red fluorescent protein (FPs) derived from an allophycocyanin homolog was resolved to 2.52 angstrom. Its biliverdin chromophore was found to be attached to the protein in an unusual way that was never observed in natural phycobiliproteins, and only once in a sub-population of artificial bacteriophytochrome-derived FPs. One of the biliverdin's vinyl groups had two cysteine residues covalently bound to its two carbon atoms. This reduces the conjugation length of the biliverdin pi-electron system, which shifts the absorption and emission spectra by about 40 nm, from the near-infrared to the far-red region of the spectrum. By spectrally characterizing a set of mBDFP mutants, we show that such spectral shifts can be induced by modifying a single residue in either one of two critical positions in the vicinity of the binding cysteines. This changes the reactivity of biliverdin and the cysteine's thiols towards forming one, or two thioether bonds to the vinyl group. The ability to control the spectral properties of BDFP by specific point mutations opens many possibilities for rational design of far-red and near-infrared FPs that are of great interest to the development of fluorescence markers for bioimaging since most biological tissues are transparent in this spectral window.

Iron-sulfur proteins are ubiquitous among all living organisms and are indispensable for almost all metabolic pathways ranging from photosynthesis, respiration, nitrogen, and carbon dioxide cycles. The iron-sulfur clusters primarily serve as electron acceptors and donors and transfer electrons to active sites of various enzymes, thus driving the energy metabolism. Prokaryotes like E. coli have ISC and SUF pathways that help in the assembly and maturation of iron-sulfur proteins. These iron-sulfur proteins, especially with [4Fe-4S] clusters, are highly sensitive to molecular oxygen, and it would be advantageous if the de novo proteins and native proteins having iron-sulfur binding sites are expressed and isolated under anaerobic conditions. Bacterially assembled iron-sulfur proteins, when isolated and purified anaerobically, exhibit improved biochemical and biophysical stabilities in comparison to the counterparts expressed and purified aerobically and reconstituted under anaerobic conditions. This protocol outlines the expression and purification of the artificial protein, Coiled-Coil Iron-Sulfur (CCIS). It may be deployed to both natural and artificial [4Fe-4S] proteins when heterologously expressed in E. coli.

Iron-sulfur proteins are primordial catalysts and biological electron carriers that today drive major metabolic pathways across all forms of life. They can access a diversity of oxidation states and can mediate electron transfer over an extended range of reduction potentials spanning more than 1 V. Depending on the protein micro-environment and geometry of ligand, co-ordination the iron-sulfur clusters can occur in different forms [2Fe-2S], [3Fe-4S], HiPIP [4Fe-4S], and [4Fe-4S]. There are several spectroscopic methods available to characterize the composition and electronic configuration of the iron-sulfur clusters, such as optical methods and electron paramagnetic resonance. This paper presents the protocols used to characterize the metal center of Coiled-Coil Iron-Sulfur (CCIS), an artificial metalloprotein containing one [4Fe-4S] cluster. It is expected that these protocols will be of general utility for other iron-sulfur proteins.

Water-soluble chlorophyll-binding proteins (WSCPs) from Brassicaceae constitute a small family of non-photosynthetic proteins that may provide a useful benchmark and model system for studying molecular aspects of chlorophyll-protein interactions such as the tuning of absorption and emission spectra, and binding selectivity. WSCP apo-proteins are readily expressed by recombinant DNA techniques and can be assembled in vitro with natural and synthetic chlorophyll derivatives. The complexes with native chlorophylls are exceptionally stable toward thermal dissociation and protein denaturation due to hydrophobic interactions with the chlorophyll's phytyl chains that stabilize the core of the WSCP tetrameric complexes. However, assembly requires the use of detergents or water-in-oil emulsions to introduce the hydrophobic pigments into the water-soluble apo-proteins. Here, we explore the direct assembly of recombinant WSCPs with the water-soluble phytyl-free chlorophyll analogue chlorophyllide a in aqueous solutions. We show that the complexes formed by mixing chlorophyllide and WSCP apo-proteins are exclusively tetrameric, and while they lack the extreme thermostability of the respective chlorophyll complexes, they are still thermostable up to around 60°C. Their absorption and CD spectra are very similar to the chlorophyll complexes albeit slight peak shifts and broadening of the bands indicate variations in pigment and protein conformations, and less rigid structures. Simplifying the assembly process of WSCPs opens new possibilities for their use in modelling natural chlorophyll-protein complexes, and as templates for designing novel artificial protein-pigment complexes.

The phycobilisomes (PBSs) of cyanobacteria and red-algae are unique megadaltons light-harvesting protein-pigment complexes that utilize bilin derivatives for light absorption and energy transfer. Recently, the high-resolution molecular structures of red-algal PBSs revealed how the multi-domain core-membrane linker (LCM ) specifically organizes the allophycocyanin subunits in the PBS's core. But, the topology of LCM in these structures was different than that suggested for cyanobacterial PBSs based on lower-resolution structures. Particularly, the model for cyanobacteria assumed that the Arm2 domain of LCM connects the two basal allophycocyanin cylinders, whereas the red-algal PBS structures revealed that Arm2 is partly buried in the core of one basal cylinder and connects it to the top cylinder. Here, we show by biochemical analysis of mutations in the apcE gene that encodes LCM , that the cyanobacterial and red-algal LCM topologies are actually the same. We found that removing the top cylinder linker domain in LCM splits the PBS core longitudinally into two separate basal cylinders. Deleting either all or part of the helix-loop-helix domain at the N-terminal end of Arm2, disassembled the basal cylinders and resulted in degradation of the part containing the terminal emitter, ApcD. Deleting the following 30 amino-acids loop severely affected the assembly of the basal cylinders, but further deletion of the amino-acids at the C-terminal half of Arm2 had only minor effects on this assembly. Altogether, the biochemical data are consistent with the red-algal LCM topology, suggesting that the PBS cores in cyanobacteria and red-algae assemble in the same way.

Significance We determined the crystal structure of the full-length amphibian antimicrobial peptide (AMP) uperin 3.5 and showed fibrillation into helical “cross-α” amyloid fibril, correlated with its antibacterial activity. This provides a molecular basis for the link between AMPs which are largely helical in nature, and amyloid formation. Uperin 3.5 is a cross-α amyloid discovered in eukaryotes, following a previously reported cross-α amyloid fibril for Staphylococcus aureus PSMα3 cytotoxin, hence demonstrating the existence of the cross-α amyloid architecture across kingdoms of life, with potential functional roles in early evolution. Furthermore, the findings revealed a chameleon cross-α/cross-β secondary structure switch of uperin 3.5 fibrils, likely related to regulation of its activity.

In photosynthetic complexes, tuning of chlorophyll light-absorption spectra by the protein environment is crucial to their efficiency and robustness. Water Soluble Chlorophyll-binding Proteins from Brassicaceae (WSCPs) are useful for studying spectral tuning mechanisms due to their symmetric homotetramer structure, the ability to rigorously modify the chlorophyll’s protein surroundings, and the availability of crystal structures. Here, we present a rigorous analysis based on hybrid Quantum Mechanics and Molecular Mechanics simulations with conformational sampling to quantify the relative contributions of steric and electrostatic factors to the absorption spectra of WSCP-chlorophyll complexes. We show that when considering conformational dynamics, chlorophyll ring deformation accounts for about one-third of the spectral shift, whereas protein electrostatics accounts for the remaining two-thirds. From a practical perspective, protein electrostatics is easier to manipulate than chlorophyll conformations, thus, it may be more readily implemented in designing artificial protein-chlorophyll complexes with desired spectral shift.

Molecular oxygen (O2) is a highly reactive oxidizing agent and is harmful to many biological and industrial systems. Although O2 often interacts via metals or reducing agents, a binding mechanism involving an organic supramolecular structure has not been described to date. In this work, the prominent dipeptide hydrogelator fluorenylmethyloxycarbonyl-diphenylalanine is shown to encage O2 and significantly limit its diffusion and penetration through the hydrogel. Molecular dynamics simulations suggested that the O2 binding mechanism is governed by pockets formed between the aromatic rings in the supramolecular structure of the gel, which bind O2 through hydrophobic interactions. This phenomenon is harnessed to maintain the activity of the O2-hypersensitive enzyme [FeFe]-hydrogenase, which holds promising potential for utilizing hydrogen gas for sustainable energy applications. Hydrogenase encapsulation within the gel allows hydrogen production following exposure to ambient O2. This phenomenon may lead to utilization of this low molecular weight gelator in a wide range of O2-sensitive applications.

Type‐II water‐soluble chlorophyll (Chl) proteins (WSCPs) of Brassicaceae are promising models for understanding how protein sequence and structure affect Chl binding and spectral tuning in photosynthetic Chl–protein complexes. However, to date, their use has been limited by the small number of known WSCPs, which also limited understanding their physiological roles. To overcome these limitations, we performed a phylogenetic analysis to compile a more comprehensive and complete set of natural type‐II WSCP homologues. The identified homologues were heterologously expressed in Escherichia coli, purified, tested for assembly with chlorophylls, and spectroscopically characterized. The analyses led to the discovery of previously unrecognized type‐IIa and IIb subclass WSCPs, as well as of a new subclass that did not bind chlorophylls. Further analysis by ancestral sequence reconstruction yielded sequences of putative ancestors of the three subclasses, which were subsequently recombinantly expressed in E. coli, purified and characterized. Combining the phylogenetic and spectroscopic data with molecular structural information revealed distinct Chl‐binding motifs, and identified residues critically impacting spectral tuning. The distinct Chl‐binding properties of the WSCP archetypes suggest that the non‐Chl‐binding subclass evolved from a Chl‐binding ancestor that most likely lost its Chl‐binding capacity upon localization in the plant tissues with low Chl content. This dual evolutionary trajectory is consistent with WSCPs association with the Kunitz‐type protease inhibitors superfamily, and indications of their inhibitory activity in response to various forms of stress in plants. These findings suggest new directions for exploring the physiological roles of WSCPs and the correlation, if any, between Chl‐binding and protease inhibition functionality.

In vivo expression of metalloproteins requires specific metal trafficking and incorporation machinery inside the cell. Synthetic designed metalloproteins are typically purified without the target metal, which is subsequently introduced through in vitro reconstitution. The extra step complicates protein optimization by high-throughput library screening or laboratory evolution. We demonstrate that a designed coiled-coil iron-sulfur protein (CCIS) assembles robustly with [4Fe-4S] clusters in vivo. While in vitro reconstitution produces a mixture of oligomers that depends on solution conditions, in vivo production generates a stable homotrimer coordinating a single, diamagnetic [4Fe-4S]2+ cluster. The multinuclear cluster of in vivo assembled CCIS is more resistant to degradation by molecular oxygen. Only one of the two metal coordinating half-sites is required in vivo, indicating specificity of molecular recognition in recruitment of the metal cluster. CCIS, unbiased by evolution, is a unique platform to examine iron-sulfur protein biogenesis and develop synthetic multinuclear oxidoreductases.

Simple optical spectroscopy measurements, namely circular dichroism (CD) and absorption spectra of interacting pigments, can be used for deriving details of their molecular geometry. Unlike X-ray crystallography and NMR that provide highly detailed structural information but consume time and resources, optical spectroscopic measurements that provide valuable information on local interactions are fast, and easy to perform. Unfortunately, structural information may be extracted from optical spectra only upon solving an ill-defined inverse-problem (spectrum → structure). In this paper, we present a computational approach for addressing this problem, relying on simulation-based optimization. We introduce quantum theoretical simulations of both CD and absorption spectra of interacting chlorophylls, integrated with an effective graphical user interface to facilitate an expert's estimation of the chlorophyll geometry. The inverse-problem is then efficiently solved by decomposing it into two approximately independent subproblems and employing two different derandomized Evolution Strategies, while relying on the expert's initial search-point. Our approach is implemented for deriving the geometry of interacting chlorophylls incorporated within natural proteins. It is then demonstrated to retrieve chlorophyll geometries with low errors when compared to the respective geometries determined to near-atomic resolution by X-ray crystallography. The observations are reported and investigated in the light of non-uniqueness and uncertainty.

The ability to tune the light-absorption properties of chlorophylls by their protein environment is the key to the robustness and high efficiency of photosynthetic light-harvesting proteins. Unfortunately, the intricacy of the natural complexes makes it very difficult to identify and isolate specific protein-pigment interactions that underlie the spectral-tuning mechanisms. Herein we identify and demonstrate the tuning mechanism of chlorophyll spectra in type II water-soluble chlorophyll binding proteins from Brassicaceae (WSCPs). By comparing the molecular structures of two natural WSCPs we correlate a shift in the chlorophyll red absorption band with deformation of its tetrapyrrole macrocycle that is induced by changing the position of a nearby tryptophan residue. We show by a set of reciprocal point mutations that this change accounts for up to 2/3 of the observed spectral shift between the two natural variants.

This study describes new recombinant water‐soluble chlorophyll (Chl)‐binding proteins ( WSCP ) from Lepidium virginicum (Lv WSCP ). This complex binds four Chls ( i.e . two dimers of Chls) per protein tetramer. We show that absorption, emission, hole‐burned ( HB ) spectra and the shape of the zero‐phonon hole ( ZPH ) action spectrum are consistent with the presence of uncorrelated excitation energy transfer between two Chl dimers. Thus, there is no need to include slow protein relaxation within the lowest excited state (as suggested in a previous analysis of cauliflower WSCP [Schmitt, F.‐J. et al . (2008) J. Phys. Chem. B , 112, 13951; Pieper, J. et al . (2011) J. Phys. Chem. B , 115, 4053]) in order to explain the large shift observed between the maxima of the ZPH action and emission spectra. Experimental evidence is provided which shows that electron exchange between lowest energy Chls and the protein may occur, i.e . electrons can be trapped at low temperature by nearby aromatic amino acids. The latter explains the shape of nonresonant HB spectra ( i.e . the absence of antihole), demonstrating that the hole‐burning process in Lv WSCP is largely photochemical in nature, though a small contribution from nonphotochemical hole burning (in resonant holes) is also observed.

Graphical Abstract Die Deformation aromatischer Ringe wurde lange als eine Möglichkeit diskutiert, die Absorptionsspektren von Chlorophyllen in ihrer natürlichen Proteinumgebung zu beeinflussen. In der Zuschrift auf S. 7015 ff. demonstrieren D. Noy et al. erstmals direkt einen solchen Mechanismus am WSCP (wasserlösliches Chlorophyll-Protein) von Brassicaceae. Änderungen in der Wasserstoffbrückenbindung an einen Tryptophanrest positionieren es in der Weise neu, dass die Planarität des Chlorophyllmakrocyclus gestört wird, was signifikante Rotverschiebungen der Absorption bewirkt.

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport.Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes.In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics.One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins.However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult.Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments.Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls.The new method was validated by assembling recombinant versions of the watersoluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a.We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

Iron-sulfur centers in metalloproteins can access multiple oxidation states over a broad range of potentials, allowing them to participate in a variety of electron transfer reactions and serving as catalysts for high-energy redox processes. The nitrogenase FeMoCO cluster converts di-nitrogen to ammonia in an eight-electron transfer step. The 2(Fe4S4) containing bacterial ferredoxin is an evolutionarily ancient metalloprotein fold and is thought to be a primordial progenitor of extant oxidoreductases. Controlling chemical transformations mediated by iron-sulfur centers such as nitrogen fixation, hydrogen production as well as electron transfer reactions involved in photosynthesis are of tremendous importance for sustainable chemistry and energy production initiatives. As such, there is significant interest in the design of iron-sulfur proteins as minimal models to gain fundamental understanding of complex natural systems and as lead-molecules for industrial and energy applications. Herein, we discuss salient structural characteristics of natural iron-sulfur proteins and how they guide principles for design. Model structures of past designs are analyzed in the context of these principles and potential directions for enhanced designs are presented, and new areas of iron-sulfur protein design are proposed. This article is part of a Special issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, protein networks, edited by Ronald L. Koder and J.L Ross Anderson.