Controlling heme redox properties in peptide amphiphile fibers with sequence and heme loading ratio.

Controlling the reduction midpoint potential of heme B is a key factor in many bioelectrochemical reactions, including long-range electron transport. Currently, there are a number of globular model protein systems to study this biophysical parameter; however, there are none for large polymeric protein model systems (e.g., the OmcS protein from G. sulfurreducens). Peptide amphiphiles, short peptides with a lipid tail that polymerize into fibrous structures, fill this gap. Here, we show a peptide amphiphile model system where one can tune the electrochemical potential of heme B by changing the loading ratio and peptide sequence. Changing the loading ratio resulted in the most significant increase, with values as high as -22 mV down to -224 mV. Circular dichroism spectra of certain sequences show Cotton effects at lower loading ratios that disappear as more heme B is added, indicating an ordered environment that becomes disrupted if heme B is overpacked. These findings can contribute to the design of functional self-assembling biomaterials.

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

Bacteriophage λ's CI repressor protein controls a genetic switch between the virus's lysogenic and lytic lifecycles, in part, by selectively binding to six different DNA sequences within the phage genome-collectively referred to as operator sites. However, the minimal level of information needed for CI to recognize and specifically bind these six unique-but-related sequences is unclear. In a previous study, we introduced an algorithm that extracts the minimal direct readout information needed for λ-CI to recognize and bind its six binding sites. We further revealed direct readout information shared among three evolutionarily related lambdoid phages: λ-phage, Enterobacteria phage VT2-Sakai, and Stx2 converting phage I, suggesting that the λ-CI protein could bind to the operator sites of these other phages. In this study, we show that λ-CI can indeed bind the other two phages' cognate binding sites as predicted using our algorithm, validating the hypotheses from that paper. We go on to demonstrate the importance of specific hydrogen bond donors and acceptors that are maintained despite changes to the nucleobase itself, and another that has an important role in recognition and binding. This in vitro validation of our algorithm supports its use as a tool to predict alternative binding sites for DNA-binding proteins.

Machine learning overcomes human bias in the discovery of self-assembling peptides.

Peptide materials have a wide array of functions, from tissue engineering and surface coatings to catalysis and sensing. Tuning the sequence of amino acids that comprise the peptide modulates peptide functionality, but a small increase in sequence length leads to a dramatic increase in the number of peptide candidates. Traditionally, peptide design is guided by human expertise and intuition and typically yields fewer than ten peptides per study, but these approaches are not easily scalable and are susceptible to human bias. Here we introduce a machine learning workflow-AI-expert-that combines Monte Carlo tree search and random forest with molecular dynamics simulations to develop a fully autonomous computational search engine to discover peptide sequences with high potential for self-assembly. We demonstrate the efficacy of the AI-expert to efficiently search large spaces of tripeptides and pentapeptides. The predictability of AI-expert performs on par or better than our human experts and suggests several non-intuitive sequences with high self-assembly propensity, outlining its potential to overcome human bias and accelerate peptide discovery.

Tailorable Tetrahelical Bundles as a Toolkit for Redox Studies.

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 (Em) 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 Em. 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 Em 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 Em values of bound cofactors.

Alignment of major-groove hydrogen bond arrays uncovers shared information between different DNA sequences that bind the same protein.

Protein-DNA binding is of a great interest due to its importance in many biological processes. Previous studies have presented many factors responsible for the recognition and specificity, but understanding the minimal informational requirements for proteins that bind to multiple DNA-sites is still an understudied area of bioinformatics. Here we focus on the hydrogen bonds displayed by the target DNA in the major groove that take part in protein-binding. We show that analyses focused on the base pair identity may overlook key hydrogen bonds. We have developed an algorithm that converts a nucleotide sequence into an array of hydrogen bond donors and acceptors and methyl groups. It then aligns these non-covalent interaction arrays to identify what information is being maintained among multiple DNA sequences. For three different DNA-binding proteins, Lactose repressor, controller protein and λ-CI repressor, we uncovered the minimal pattern of hydrogen bonds that are common amongst all the binding sequences. Notably in the three proteins, key interacting hydrogen bonds are maintained despite nucleobase mutations in the corresponding binding sites. We believe this work will be useful for developing new DNA binding proteins and shed new light on evolutionary relationships.

Recent Progress Using De Novo Design to Study Protein Structure, Design and Binding Interactions.

De novo protein design is a powerful methodology used to study natural functions in an artificial-protein context. Since its inception, it has been used to reproduce a plethora of reactions and uncover biophysical principles that are often difficult to extract from direct studies of natural proteins. Natural proteins are capable of assuming a variety of different structures and subsequently binding ligands at impressively high levels of both specificity and affinity. Here, we will review recent examples of de novo design studies on binding reactions for small molecules, nucleic acids, and the formation of protein-protein interactions. We will then discuss some new structural advances in the field. Finally, we will discuss some advancements in computational modeling and design approaches and provide an overview of some modern algorithmic tools being used to design these proteins.

Morphological Control of Chromophore Spin State in Zinc Porphyrin-Peptide Assemblies.

Self-assembled peptide micelles and fibers demonstrate unique control over the photophysical properties of the bound, light-activated chromophore, zinc protoporphyrin IX, (PPIX)Zn. Micelles encapsulate either a mixture of uncoordinated and coordinated (PPIX)Zn or all coordinated depending on the ratio of peptide/porphyrin. As the ratio increases toward a 1:1 micelle/porphyrin ratio, providing the chromophore with a discrete coordination environment reminiscent of unstructured proteins, the micelles favor triplet formation. Fibers, however, promote a linear array of porphyrin molecules that dictates exciton hopping and excimer formation at ratios as high as 60:1, peptide/porphyrin. However, even in fibers, the formation of the triplet species increases with increasing peptide/porphyrin ratio due to increased spatial separation between neighboring chromophores facilitating intersystem crossing. Full characterization of the micelles structures and comparison to the fibers lead to the comparison with natural systems and the ability to control the excited populations that have utility in photocatalytic processes. In addition, the incorporation of a second chromophore, heme, yields an electron transfer pathway in both micelles and fibers that highlights the utility of the peptide assemblies when engineering multichromophore arrays as inspired by natural, photosynthetic proteins.

Microenvironment control of porphyrin binding, organization, and function in peptide nanofiber assemblies.

To take peptide materials from predominantly structural to functional assemblies, variations in cofactor binding sites must be engineered and controlled. Here, we have employed the peptide sequence c16-AHX3K3-CO2H where X3 represents the aliphatic structural component of the peptide design that dictates ß-sheet formation and upon self-assembly yields a change in the overall microenvironment surrounding the Zn protoporphyrin IX ((PPIX)Zn) binding site. All peptides studied yield ß-sheet rich nanofibers highlighting the materials' resiliency to amino acid substitution. We highlight that the (PPIX)Zn binding constants correlate strongly with amino acid side chain volume, where X = L or I yields the lowest dissociation constant values (KD). The resulting microenvironment highlights the materials' ability to control interchromophore electronic interactions such that slip-stacked cofacial arrangements are observed via exciton splitting in UV/visible and circular dichroism spectroscopy. Steady state and time-resolved photoluminescence suggests that greater interchromophore packing yields larger excimer populations and corresponding longer excimer association lifetimes (τA) which directly translates to shorter exciton diffusion lengths. In comparison to synthetic porphyrin molecular assemblies, this work demonstrates the ability to employ the peptide assembly to modulate the degree of cofactor arrangement, extent of excimer formation, and the exciton hopping rates all while in a platform amenable for producing polymer-like materials.

Supramolecular control of heme binding and electronic states in multi-heme peptide assemblies.

Nature guides the flow of electrons in biological systems with the assistance of multi-heme proteins called cytochromes. In an effort to understand natures approach to developing electronic systems, three peptides that are compositionally identical but sequentially distinct have been designed to study the impact of morphology and hydrophobicity on heme coordination and function.

Tailorable Exciton Transport in Doped Peptide-Amphiphile Assemblies.

Light-harvesting biomaterials are an attractive target in photovoltaics, photocatalysis, and artificial photosynthesis. Through peptide self-assembly, complex nanostructures can be engineered to study the role of chromophore organization during light absorption and energy transport. To this end, we demonstrate the one-dimensional transport of excitons along naturally occurring, light-harvesting, Zn-protoporphyrin IX chromophores within self-assembled peptide-amphiphile nanofibers. The internal structure of the nanofibers induces packing of the porphyrins into linear chains. We find that this peptide assembly can enable long-range exciton diffusion, yet it also induces the formation of excimers between adjacent molecules, which serve as exciton traps. Electronic coupling between neighboring porphyrin molecules is confirmed by various spectroscopic methods. The exciton diffusion process is then probed through transient photoluminescence and absorption measurements and fit to a model for one-dimensional hopping. Because excimer formation impedes exciton hopping, increasing the interchromophore spacing allows for improved diffusivity, which we control through porphyrin doping levels. We show that diffusion lengths of over 60 nm are possible at low porphyrin doping, representing an order of magnitude improvement over the highest doping fractions.

Control of Heme Coordination and Catalytic Activity by Conformational Changes in Peptide-Amphiphile Assemblies.

Self-assembling peptide materials have gained significant attention, due to well-demonstrated applications, but they are functionally underutilized. To advance their utility, we use noncovalent interactions to incorporate the biological cofactor heme-B for catalysis. Heme-proteins achieve differing functions through structural and coordinative variations. Here, we replicate this phenomenon by highlighting changes in heme reactivity as a function of coordination, sequence, and morphology (micelles versus fibers) in a series of simple peptide amphiphiles with the sequence c16-xyL3K3-CO2H where c16 is a palmitoyl moiety and xy represents the heme binding region: AA, AH, HH, and MH. The morphology of this peptide series is characterized using transmission electron and atomic force microscopies as well as dynamic light scattering. Within this small library of peptide constructs, we show that three spectroscopically (UV/visible and electron paramagnetic resonance) distinct heme environments were generated: noncoordinated/embedded high-spin, five-coordinate high-spin, and six-coordinate low-spin. The resulting material's functional dependence on sequence and supramolecular morphology is highlighted 2-fold. First, the heme active site binds carbon monoxide in both micelles and fibers, demonstrating that the heme active site in both morphologies is accessible to small molecules for catalysis. Second, peroxidase activity was observed in heme-containing micelles yet was significantly reduced in heme-containing fibers. We briefly discuss the implications these findings have in the production of functional, self-assembling peptide materials.

Design and engineering of water-soluble light-harvesting protein maquettes.

Natural selection in photosynthesis has engineered tetrapyrrole based, nanometer scale, light harvesting and energy capture in light-induced charge separation. By designing and creating nanometer scale artificial light harvesting and charge separating proteins, we have the opportunity to reengineer and overcome the limitations of natural selection to extend energy capture to new wavelengths and to tailor efficient systems that better meet human as opposed to cellular energetic needs. While tetrapyrrole cofactor incorporation in natural proteins is complex and often assisted by accessory proteins for cofactor transport and insertion, artificial protein functionalization relies on a practical understanding of the basic physical chemistry of protein and cofactors that drive nanometer scale self-assembly. Patterning and balancing of hydrophobic and hydrophilic tetrapyrrole substituents is critical to avoid natural or synthetic porphyrin and chlorin aggregation in aqueous media and speed cofactor partitioning into the non-polar core of a man-made water soluble protein designed according to elementary first principles of protein folding. This partitioning is followed by site-specific anchoring of tetrapyrroles to histidine ligands strategically placed for design control of rates and efficiencies of light energy and electron transfer while orienting at least one polar group towards the aqueous phase.

Water ordering controls the dynamic equilibrium of micelle-fibre formation in self-assembly of peptide amphiphiles.

Understanding the role of water in governing the kinetics of the self-assembly processes of amphiphilic peptides remains elusive. Here, we use a multistage atomistic-coarse-grained approach, complemented by circular dichroism/infrared spectroscopy and dynamic light scattering experiments to highlight the dual nature of water in driving the self-assembly of peptide amphiphiles (PAs). We show computationally that water cage formation and breakage near the hydrophobic groups control the fusion dynamics and aggregation of PAs in the micellar stage. Simulations also suggest that enhanced structural ordering of vicinal water near the hydrophilic amino acids shifts the equilibrium towards the fibre phase and stimulates structure and order during the PA assembly into nanofibres. Experiments validate our simulation findings; the measured infrared O-H bond stretching frequency is reminiscent of an ice-like bond which suggests that the solvated water becomes increasingly ordered with time in the assembled peptide network, thus shedding light on the role of water in a self-assembly process.

Design and engineering of a man-made diffusive electron-transport protein.

Maquettes are man-made cofactor-binding oxidoreductases designed from first principles with minimal reference to natural protein sequences. Here we focus on water-soluble maquettes designed and engineered to perform diffusive electron transport of the kind typically carried out by cytochromes, ferredoxins and flavodoxins and other small proteins in photosynthetic and respiratory energy conversion and oxido-reductive metabolism. Our designs were tested by analysis of electron transfer between heme maquettes and the well-known natural electron transporter, cytochrome c. Electron-transfer kinetics were measured from seconds to milliseconds by stopped-flow, while sub-millisecond resolution was achieved through laser photolysis of the carbon monoxide maquette heme complex. These measurements demonstrate electron transfer from the maquette to cytochrome c, reproducing the timescales and charge complementarity modulation observed in natural systems. The ionic strength dependence of inter-protein electron transfer from 9.7×10(6) M(-1) s(-1) to 1.2×10(9) M(-1) s(-1) follows a simple Debye-Hückel model for attraction between +8 net charged oxidized cytochrome c and -19 net charged heme maquette, with no indication of significant protein dipole moment steering. Successfully recreating essential components of energy conversion and downstream metabolism in man-made proteins holds promise for in vivo clinical intervention and for the production of fuel or other industrial products. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Engineering the assembly of heme cofactors in man-made proteins.

Timely ligation of one or more chemical cofactors at preselected locations in proteins is a critical preamble for catalysis in many natural enzymes, including the oxidoreductases and allied transport and signaling proteins. Likewise, ligation strategies must be directly addressed when designing oxidoreductase and molecular transport functions in man-made, first-principle protein constructs intended to operate in vitro or in vivo. As one of the most common catalytic cofactors in biology, we have chosen heme B, along with its chemical analogues, to determine the kinetics and barriers to cofactor incorporation and bishistidine ligation in a range of 4-α-helix proteins. We compare five elementary synthetic designs (maquettes) and the natural cytochrome b562 that differ in oligomeric forms, apo- and holo-tertiary structural stability; qualities that we show can either assist or hinder assembly. The cofactor itself also imposes an assembly barrier if amphiphilicity ranges toward too hydrophobic or hydrophilic. With progressive removal of identified barriers, we achieve maquette assembly rates as fast as native cytochrome b562, paving the way to in vivo assembly of man-made hemoprotein maquettes and integration of artificial proteins into enzymatic pathways.

Elementary tetrahelical protein design for diverse oxidoreductase functions.

Emulating functions of natural enzymes in man-made constructs has proven challenging. Here we describe a man-made protein platform that reproduces many of the diverse functions of natural oxidoreductases without importing the complex and obscure interactions common to natural proteins. Our design is founded on an elementary, structurally stable 4-α-helix protein monomer with a minimalist interior malleable enough to accommodate various light- and redox-active cofactors and with an exterior tolerating extensive charge patterning for modulation of redox cofactor potentials and environmental interactions. Despite its modest size, the construct offers several independent domains for functional engineering that targets diverse natural activities, including dioxygen binding and superoxide and peroxide generation, interprotein electron transfer to natural cytochrome c and light-activated intraprotein energy transfer and charge separation approximating the core reactions of photosynthesis, cryptochrome and photolyase. The highly stable, readily expressible and biocompatible characteristics of these open-ended designs promise development of practical in vitro and in vivo applications.

Engineering oxidoreductases: maquette proteins designed from scratch.

The study of natural enzymes is complicated by the fact that only the most recent evolutionary progression can be observed. In particular, natural oxidoreductases stand out as profoundly complex proteins in which the molecular roots of function, structure and biological integration are collectively intertwined and individually obscured. In the present paper, we describe our experimental approach that removes many of these often bewildering complexities to identify in simple terms the necessary and sufficient requirements for oxidoreductase function. Ours is a synthetic biology approach that focuses on from-scratch construction of protein maquettes designed principally to promote or suppress biologically relevant oxidations and reductions. The approach avoids mimicry and divorces the commonly made and almost certainly false ascription of atomistically detailed functionally unique roles to a particular protein primary sequence, to gain a new freedom to explore protein-based enzyme function. Maquette design and construction methods make use of iterative steps, retraceable when necessary, to successfully develop a protein family of sturdy and versatile single-chain three- and four-α-helical structural platforms readily expressible in bacteria. Internally, they prove malleable enough to incorporate in prescribed positions most natural redox cofactors and many more simplified synthetic analogues. External polarity, charge-patterning and chemical linkers direct maquettes to functional assembly in membranes, on nanostructured titania, and to organize on selected planar surfaces and materials. These protein maquettes engage in light harvesting and energy transfer, in photochemical charge separation and electron transfer, in stable dioxygen binding and in simple oxidative chemistry that is the basis of multi-electron oxidative and reductive catalysis.

Design and engineering of an O(2) transport protein.

The principles of natural protein engineering are obscured by overlapping functions and complexity accumulated through natural selection and evolution. Completely artificial proteins offer a clean slate on which to define and test these protein engineering principles, while recreating and extending natural functions. Here we introduce this method with the design of an oxygen transport protein, akin to human neuroglobin. Beginning with a simple and unnatural helix-forming sequence with just three different amino acids, we assembled a four-helix bundle, positioned histidines to bis-histidine ligate haems, and exploited helical rotation and glutamate burial on haem binding to introduce distal histidine strain and facilitate O(2) binding. For stable oxygen binding without haem oxidation, water is excluded by simple packing of the protein interior and loops that reduce helical-interface mobility. O(2) affinities and exchange timescales match natural globins with distal histidines, with the remarkable exception that O(2) binds tighter than CO.