A Thermostable Protein Matrix for Spectroscopic Analysis of Organic Semiconductors.

Advances in protein design and engineering have yielded peptide assemblies with enhanced and non-native functionalities. Here, various molecular organic semiconductors (OSCs), with known excitonic up- and down-conversion properties, are attached to a de novo-designed protein, conferring entirely novel functions on the peptide scaffolds. The protein-OSC complexes form similarly sized, stable, water-soluble nanoparticles that are robust to cryogenic freezing and processing into the solid-state. The peptide matrix enables the formation of protein-OSC-trehalose glasses that fix the proteins in their folded states under oxygen-limited conditions. The encapsulation dramatically enhances the stability of protein-OSC complexes to photodamage, increasing the lifetime of the chromophores from several hours to more than 10 weeks under constant illumination. Comparison of the photophysical properties of astaxanthin aggregates in mixed-solvent systems and proteins shows that the peptide environment does not alter the underlying electronic processes of the incorporated materials, exemplified here by singlet exciton fission followed by separation into weakly bound, localized triplets. This adaptable protein-based approach lays the foundation for spectroscopic assessment of a broad range of molecular OSCs in aqueous solutions and the solid-state, circumventing the laborious procedure of identifying the experimental conditions necessary for aggregate generation or film formation. The non-native protein functions also raise the prospect of future biocompatible devices where peptide assemblies could complex with native and non-native systems to generate novel functional materials.

Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo-designed heme protein.

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.

Ultrafast flavin/tryptophan radical pair kinetics in a magnetically sensitive artificial protein.

Radical pair formation and decay are implicated in a wide range of biological processes including avian magnetoreception. However, studying such biological radical pairs is complicated by both the complexity and relative fragility of natural systems. To resolve open questions about how natural flavin-amino acid radical pair systems are engineered, and to create new systems with novel properties, we developed a stable and highly adaptable de novo artificial protein system. These protein maquettes are designed with intentional simplicity and transparency to tolerate aggressive manipulations that are impractical or impossible in natural proteins. Here we characterize the ultrafast dynamics of a series of maquettes with differing electron-transfer distance between a covalently ligated flavin and a tryptophan in an environment free of other potential radical centers. We resolve the spectral signatures of the cysteine-ligated flavin singlet and triplet states and reveal the picosecond formation and recombination of singlet-born radical pairs. Magnetic field-sensitive triplet-born radical pair formation and recombination occurs at longer timescales. These results suggest that both triplet- and singlet-born radical pairs could be exploited as biological magnetic sensors.

Rational Construction of Compact de Novo-Designed Biliverdin-Binding Proteins.

We report the rational construction of de novo-designed biliverdin-binding proteins by first principles of protein design, informed by energy minimization modeling in Rosetta. The self-assembling tetrahelical bundles bind biliverdin IXa (BV) cofactor autocatalytically in vitro, like photosensory proteins that bind BV (and related bilins or linear tetrapyrroles) despite lacking sequence and structural homology to the natural counterparts. Upon identification of a suitable site for ligation of the cofactor to the protein scaffold, stepwise placement of residues stabilized BV within the hydrophobic core. Rosetta modeling was used in the absence of a high-resolution structure to inform the structure-function relationships of the cofactor binding pocket. Holoprotein formation stabilized BV, resulting in increased far-red BV fluorescence. Via removal of segments extraneous to cofactor stabilization or bundle stability, the initial 15 kDa de novo-designed fluorescence-activating protein was truncated without any change to its optical properties, down to a miniature 10 kDa "mini", in which the protein scaffold extends only a half-heptad repeat beyond the hypothetical position of the bilin D-ring. This work demonstrates how highly compact holoprotein fluorochromes can be rationally constructed using de novo protein design technology and natural cofactors.

Magnetically Sensitive Radical Photochemistry of Non-natural Flavoproteins.

It is a remarkable fact that ∼50 µT magnetic fields can alter the rates and yields of certain free-radical reactions and that such effects might be the basis of the light-dependent ability of migratory birds to sense the direction of the Earth's magnetic field. The most likely sensory molecule at the heart of this chemical compass is cryptochrome, a flavin-containing protein that undergoes intramolecular, blue-light-induced electron transfer to produce magnetically sensitive radical pairs. To learn more about the factors that control the magnetic sensitivity of cryptochromes, we have used a set of de novo designed protein maquettes that self-assemble as four-α-helical proteins incorporating a single tryptophan residue as an electron donor placed approximately 0.6, 1.1, or 1.7 nm away from a covalently attached riboflavin as chromophore and electron acceptor. Using a specifically developed form of cavity ring-down spectroscopy, we have characterized the photochemistry of these designed flavoprotein maquettes to determine the identities and kinetics of the transient radicals responsible for the magnetic field effects. Given the gross structural and dynamic differences from the natural proteins, it is remarkable that the maquettes show magnetic field effects that are so similar to those observed for cryptochromes.

Single arm two-stage studies: Improved designs for molecularly targeted agents.

Mechanistic understanding of cancers and their potential interactions with molecularly targeted agents is driving the need for stratified medicine to ensure each participant receives the best possible care. This understanding, backed by scientific research, should be used to guide the design of clinical trials for these agents. The mechanism of action of a molecularly targeted agent often suggests that a biomarker can be used as a predictor of activity of the agent on the targeted disease. A biomarker driven trial is needed to confirm that the molecularly targeted agent stratifies the participant population with disease into high and low responder groups. We assume that the biomarker of interest can be dichotomised and propose a balanced parallel two-stage single-arm phase II trial that builds on existing two-stage single-arm designs. A single-arm trial cannot distinguish between a marker being predictive in the population as a whole and the agent causing an increased response in the marker positive group, but it is a first step. We compare this approach to the existing single-arm approaches, sequential enrichment, tandem two-stage, and parallel two-stage designs, and discuss the advantages and disadvantages of each design. We show that our design compares favourably to existing designs in the Bayesian framework, making a more efficient use of collected data. We recommend using the parallel two-stage balanced or sequential enrichment designs when randomisation is not practical in a phase II trial.

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.

First principles design of a core bioenergetic transmembrane electron-transfer protein.

Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. 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.

Strong Coupling of Localized Surface Plasmons to Excitons in Light-Harvesting Complexes.

Gold nanostructure arrays exhibit surface plasmon resonances that split after attaching light harvesting complexes 1 and 2 (LH1 and LH2) from purple bacteria. The splitting is attributed to strong coupling between the localized surface plasmon resonances and excitons in the light-harvesting complexes. Wild-type and mutant LH1 and LH2 from Rhodobacter sphaeroides containing different carotenoids yield different splitting energies, demonstrating that the coupling mechanism is sensitive to the electronic states in the light harvesting complexes. Plasmon-exciton coupling models reveal different coupling strengths depending on the molecular organization and the protein coverage, consistent with strong coupling. Strong coupling was also observed for self-assembling polypeptide maquettes that contain only chlorins. However, it is not observed for monolayers of bacteriochlorophyll, indicating that strong plasmon-exciton coupling is sensitive to the specific presentation of the pigment molecules.

Engineering an Artificial Flavoprotein Magnetosensor.

Migratory birds use the Earth's magnetic field as a source of navigational information. This light-dependent magnetic compass is thought to be mediated by cryptochrome proteins in the retina. Upon light activation, electron transfer between the flavin adenine dinucleotide cofactor and tryptophan residues leads to the formation of a spin-correlated radical pair, whose subsequent fate is sensitive to external magnetic fields. To learn more about the functional requirements of this complex chemical compass, we have created a family of simplified, adaptable proteins-maquettes-that contain a single tryptophan residue at different distances from a covalently bound flavin. Despite the complete absence of structural resemblance to the native cryptochrome fold or sequence, the maquettes exhibit a strong magnetic field effect that rivals those observed in the natural proteins in vitro. These novel maquette designs offer unprecedented flexibility to explore the basic requirements for magnetic sensing in a protein environment.

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.

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.

Designing Light-Activated Charge-Separating Proteins with a Naphthoquinone Amino Acid.

The first principles design of manmade redox-protein maquettes is used to clarify the physical/chemical engineering supporting the mechanisms of natural enzymes with a view to recapitulate and surpass natural performance. Herein, we use intein-based protein semisynthesis to pair a synthetic naphthoquinone amino acid (Naq) with histidine-ligated photoactive metal-tetrapyrrole cofactors, creating a 100 µs photochemical charge separation unit akin to photosynthetic reaction centers. By using propargyl groups to protect the redox-active para-quinone during synthesis and assembly while permitting selective activation, we gain the ability to employ the quinone amino acid redox cofactor with the full set of natural amino acids in protein design. Direct anchoring of quinone to the protein backbone permits secure and adaptable control of intraprotein electron-tunneling distances and rates.

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.

Impact of HER2 copy number in IHC2+/FISH-amplified breast cancer on outcome of adjuvant trastuzumab treatment in a large UK cancer network.

BACKGROUND: Adjuvant trastuzumab with chemotherapy is standard treatment for HER2-positive breast cancer, defined as either HER2 IHC3+ or IHC2+ and FISH amplified. The aim of this study was to investigate the degree to which HER2 amplification in terms of HER2 gene copy numbers in HER2+IHC2+ cancers affected the outcome in a community setting. METHODS: Case records of 311 consecutive patients with early breast cancer presenting between 1st January 2005 and 31st December 2008 were reviewed. Progression-free survival and overall survival were calculated with the Kaplan-Meier method using STATA 13. RESULTS: Among 3+ cases (n=230) 163 received T vs 67 no-T. Among 2+ cases (n=81) 59 received T vs 22 no-T. Among 59 IHC2+-treated cases n=28 had an average of >12, n=13 had >6 to <12, and n=18 had >2 to <6 HER2 gene copies, respectively. The time of progression and overall survival of high and low copy number patients was similar and better than the intermediate copy number and the untreated cohorts. CONCLUSIONS: High HER2 copy number (>12) appears to be associated with consistently better response compared with patients with intermediate HER2 copy numbers (6-12). In light of emerging data of patients showing insensivity to trastuzumab therapy, we propose that the HER2 gene copy number value should be included as an additional indicator for stratifying both the management and the follow-up of breast cancer patients.

The efficacy of selective arterial embolization in the management of colonic bleeding.

BACKGROUND: The aim of the present study was to determine the efficacy of mesenteric embolization in the management of acute haemorrhage from the colon. METHODS: A retrospective review was performed of a consecutive series of patients who underwent selective arterial embolization between 2002 and 2010 at two Australian institutions. An analysis was performed of each patient's present and past medical history, procedural details and subsequent post-procedural recovery. RESULTS: Seventy-one patients were reviewed in the study. Sixty-one patients (86 %) had immediate cessation of bleeding following embolization. In total, 20 % had some form of morbidity due to mesenteric embolization being performed, the three most common being worsening renal function, groin haematoma and contrast allergy (11, 9 and 7 %, respectively). Only one patient developed superficial bowel ischaemia. Overall, 11 patients (18 %) had recurrent bleeding. Of these patients, five had repeat embolization. Of the patients who underwent re-embolization, three stopped bleeding. Surgery was required in 5 patients 2 of whom died postoperatively of systemic complications. CONCLUSIONS: Colonic bleeding can be treated successfully in most patients by embolization, without causing ischaemia. Eighteen per cent of patients rebleed during the first hospital admission, and 20 % patients experienced a procedure-related complication. In those patients that proceed to surgery, the morbidity, mortality and length of hospital stay increase dramatically.

Acute myocardial infarction in pregnancy following unlicensed use of methylenedioxymethamphetamine ('Ecstasy').

Acute myocardial infarction is rare in pregnancy; however, the emerging trend towards advanced maternal age and the rising prevalence of obesity and diabetes suggest that more cases of myocardial infarction are likely to be encountered in pregnancy. However, there is scanty evidence on the risk of myocardial infarction with regard to socially misused substances in pregnancy. We describe the management of a case of acute myocardial infarction following unlicensed use of 3,4-methylenedioxymethamphetamine ('Ecstasy') in pregnancy. The case highlights a rare but serious risk associated with substance misuse in pregnancy.

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.

The aprotic electrochemistry of quinones.

Quinones play important roles in biological electron transfer reactions in almost all organisms, with specific roles in many physiological processes and chemotherapy. Quinones participate in two-electron, two-proton reactions in aqueous solution at equilibrium near neutral pH, but protons often lag behind the electron transfers. The relevant reactions in proteins are often sequential one electron redox processes without involving protons. Here we report the aprotic electrochemistry of the two half-couples, Q/Q.- and Q.-/Q=, of 11 parent quinones and 118 substituted 1,4-benzoquinones, 91 1,4-naphthoquinones, and 107 9,10-anthraquinones. The measured redox potentials are fit quite well with the Hammett para sigma (σpara) parameter. Occasional exceptions can involve important groups, such as methoxy substituents in ubiquinone and hydroxy substituents in therapeutics. These can generally be explained by reasonable conjectures involving steric clashes and internal hydrogen bonds. We also provide data for 25 other quinones, 2 double quinones and 15 non-quinones, all measured under similar conditions.

Rational design of photosynthetic reaction center protein maquettes.

New technologies for efficient solar-to-fuel energy conversion will help facilitate a global shift from dependence on fossil fuels to renewable energy. Nature uses photosynthetic reaction centers to convert photon energy into a cascade of electron-transfer reactions that eventually produce chemical fuel. The design of new reaction centers de novo deepens our understanding of photosynthetic charge separation and may one day allow production of biofuels with higher thermodynamic efficiency than natural photosystems. Recently, we described the multi-step electron-transfer activity of a designed reaction center maquette protein (the RC maquette), which can assemble metal ions, tyrosine, a Zn tetrapyrrole, and heme into an electron-transport chain. Here, we detail our modular strategy for rational protein design and show that the intended RC maquette design agrees with crystal structures in various states of assembly. A flexible, dynamic apo-state collapses by design into a more ordered holo-state upon cofactor binding. Crystal structures illustrate the structural transitions upon binding of different cofactors. Spectroscopic assays demonstrate that the RC maquette binds various electron donors, pigments, and electron acceptors with high affinity. We close with a critique of the present RC maquette design and use electron-tunneling theory to envision a path toward a designed RC with a substantially higher thermodynamic efficiency than natural photosystems.