A Closer Look at Type I Left-Handed ß-Helices Provides a Better Understanding in Their Sequence-Structure Relationship: Toward Their Rational Design.

Understanding the sequence-structure relationship in protein is of fundamental interest, but has practical applications such as the rational design of peptides and proteins. This relationship in the Type I left-handed ß-helix containing proteins is updated and revisited in this study. Analyzing the available experimental structures in the Protein Data Bank, we could describe, further in detail, the structural features that are important for the stability of this fold, as well as its nucleation and termination. This study is meant to complete previous work, as it provides a separate analysis of the N-terminal and C-terminal rungs of the helix. Particular sequence motifs of these rungs are described along with the structural element they form.

Quest for a stable Cu-ligand complex with a high catalytic activity to produce reactive oxygen species.

Metal ion-catalyzed overproduction of reactive oxygen species (ROS) is believed to contribute significantly to oxidative stress and be involved in several biological processes, from immune defense to development of diseases. Among the essential metal ions, copper is one of the most efficient catalysts in ROS production in the presence of O2 and a physiological reducing agent such as ascorbate. To control this chemistry, Cu ions are tightly coordinated to biomolecules. Free or loosely bound Cu ions are generally avoided to prevent their toxicity. In the present report, we aim to find stable Cu-ligand complexes (Cu-L) that can efficiently catalyze the production of ROS in the presence of ascorbate under aerobic conditions. Thermodynamic stability would be needed to avoid dissociation in the biological environment, and high ROS catalysis is of interest for applications as antimicrobial or anticancer agents. A series of Cu complexes with the well-known tripodal and tetradentate ligands containing a central amine linked to three pyridyl-alkyl arms of different lengths were investigated. Two of them with mixed arm length showed a higher catalytic activity in the oxidation of ascorbate and subsequent ROS production than Cu salts in buffer, which is an unprecedented result. Despite these high catalytic activities, no increased antimicrobial activity toward Escherichia coli or cytotoxicity against eukaryotic AGS cells in culture related to Cu-L-based ROS production could be observed. The potential reasons for discrepancy between in vitro and in cell data are discussed.

Revisiting the pro-oxidant activity of copper: interplay of ascorbate, cysteine, and glutathione.

Copper (Cu) is essential for most organisms, but it can be poisonous in excess, through mechanisms such as protein aggregation, trans-metallation, and oxidative stress. The latter could implicate the formation of potentially harmful reactive oxygen species (O2•-, H2O2, and HO•) via the redox cycling between Cu(II)/Cu(I) states in the presence of dioxygen and physiological reducing agents such as ascorbate (AscH), cysteine (Cys), and the tripeptide glutathione (GSH). Although the reactivity of Cu with these reductants has been previously investigated, the reactions taking place in a more physiologically relevant mixture of these biomolecules are not known. Hence, we report here on the reactivity of Cu with binary and ternary mixtures of AscH, Cys, and GSH. By measuring AscH and thiol oxidation, as well as HO• formation, we show that Cu reacts preferentially with GSH and Cys, halting AscH oxidation and also HO• release. This could be explained by the formation of Cu-thiolate clusters with both GSH and, as we first demonstrate here, Cys. Moreover, we observed a remarkable acceleration of Cu-catalyzed GSH oxidation in the presence of Cys. We provide evidence that both thiol-disulfide exchange and the generated H2O2 contribute to this effect. Based on these findings, we speculate that Cu-induced oxidative stress may be mainly driven by GSH depletion and/or protein disulfide formation rather than by HO• and envision a synergistic effect of Cys on Cu toxicity.

How trimerization of CTR1 N-terminal model peptides tunes Cu-binding and redox-chemistry.

Employing peptide-based models of copper transporter 1 (CTR1), we show that the trimeric arrangement of its N-terminus tunes its reactivity with Cu, promoting Cu(ii) reduction and stabilizing Cu(i). Hence, the employed multimeric models of CTR1 provide an important contribution to studies on early steps of Cu uptake by cells.

Copper-binding motifs Xxx-His or Xxx-Zzz-His (ATCUN) linked to an antimicrobial peptide: Cu-binding, antimicrobial activity and ROS production.

Depending on the coordination, copper ions can have a very high activity in catalyzing the production of reactive oxygen species. Thus interest arose in increasing the activity of antimicrobial peptides (AMPs) by equipping them with a Cu-binding unit. Several examples, native and engineered, have been investigated with the motif Xxx-Zzz-His, called Amino Terminal Cu(II)- and Ni(II)-binding (ATCUN) motif. Here we investigate a short AMP that was equipped either with Xxx-Zzz-His or Xxx-His. Xxx-His is a shorter motif and yields a more redox active copper complex. The control AMP, Xxx-His-AMP and Xxx-Zzz-His-AMP were investigated toward Cu-binding, Reactive Oxygen Species (ROS) production and antimicrobial activity in E. coli. The data indicate that these Cu-binding motifs have very limited impact on antimicrobial activity and low ROS production capability.

Reversible turn-on fluorescent Cu(ii) sensors: rather dream than reality?

Reversible turn-on fluorescent sensors of Cu(ii) are of high interest for biological studies. We re-investigate a reported sensor, showing that turn-on occurs via irreversible Cu(ii)-induced sensor oxidation only in the presence of acetonitrile. This prevents its application in biological studies and highlights the challenge of establishing a reversible Cu(ii) turn-on sensor.

Model peptide for anti-sigma factor domain HHCC zinc fingers: high reactivity toward 1O2 leads to domain unfolding.

All organisms have to cope with the deleterious effects of reactive oxygen species. Some of them are able to mount a transcriptional response to various oxidative stresses, which involves sensor proteins capable of assessing the redox status of the cell or to detect reactive oxygen species. In this article, we describe the design, synthesis and characterization of Zn·LASD(HHCC), a model for the Zn(Cys)2(His)2 zinc finger site of ChrR, a sensor protein involved in the bacterial defence against singlet oxygen that belongs to the family of zinc-binding anti-sigma factors possessing a characteristic H/C-X24/25-H-X3-C-X2-C motif. The 46-amino acid model peptide LASD(HHCC) was synthetized by solid phase peptide synthesis and its Zn2+-binding properties were investigated using electronic absorption, circular dichroism and NMR. LASD(HHCC) forms a 1 : 1 complex with Zn2+, namely Zn·LASD(HHCC), that adopts a well-defined conformation with the Zn2+ ion capping a 3-helix core that reproduces almost perfectly the fold of the ChrR in the vicinity of its zinc site. H2O2 reacts with Zn·LASD(HHCC) to yield a disulfide with a second order rate constant of 0.030 ± 0.002 M-1 s-1. Zn·LASD(HHCC) reacts rapidly with singlet oxygen to yield sulfinates and sulfonates. A lower limit of the chemical reaction rate constant between Zn·LASD(HHCC) and 1O2 was determined to be 3.9 × 106 M-1 s-1. Therefore, the Zn(Cys)2(His)2 site of Zn·LASD(HHCC) appears to be at least 5 times more reactive toward these two oxidants than that of a classical ßßα zinc finger. Consequences for the activation mechanism of ChrR are discussed.

Artificial Metalloenzymes: Reaction Scope and Optimization Strategies.

The incorporation of a synthetic, catalytically competent metallocofactor into a protein scaffold to generate an artificial metalloenzyme (ArM) has been explored since the late 1970's. Progress in the ensuing years was limited by the tools available for both organometallic synthesis and protein engineering. Advances in both of these areas, combined with increased appreciation of the potential benefits of combining attractive features of both homogeneous catalysis and enzymatic catalysis, led to a resurgence of interest in ArMs starting in the early 2000's. Perhaps the most intriguing of potential ArM properties is their ability to endow homogeneous catalysts with a genetic memory. Indeed, incorporating a homogeneous catalyst into a genetically encoded scaffold offers the opportunity to improve ArM performance by directed evolution. This capability could, in turn, lead to improvements in ArM efficiency similar to those obtained for natural enzymes, providing systems suitable for practical applications and greater insight into the role of second coordination sphere interactions in organometallic catalysis. Since its renaissance in the early 2000's, different aspects of artificial metalloenzymes have been extensively reviewed and highlighted. Our intent is to provide a comprehensive overview of all work in the field up to December 2016, organized according to reaction class. Because of the wide range of non-natural reactions catalyzed by ArMs, this was done using a functional-group transformation classification. The review begins with a summary of the proteins and the anchoring strategies used to date for the creation of ArMs, followed by a historical perspective. Then follows a summary of the reactions catalyzed by ArMs and a concluding critical outlook. This analysis allows for comparison of similar reactions catalyzed by ArMs constructed using different metallocofactor anchoring strategies, cofactors, protein scaffolds, and mutagenesis strategies. These data will be used to construct a searchable Web site on ArMs that will be updated regularly by the authors.

Towards Cellular Activation of an Artificial Metalloenzyme: Development of an Artificial Zymogen.

Regulation of enzymatic activity is essential for living organisms. Nowadays, with the emergence of synthetic biology, there is a rising interest in placing the activity of synthetic catalysts under the control of a cell. This short review aims at summarizing the regulation strategies developed to date, and at presenting the development of an artificial zymogen, which is upregulated by a natural protease. In our view, this constitutes a first step towards the cellular control of the activity of an artificial metalloenzyme.

Upregulation of an Artificial Zymogen by Proteolysis.

Regulation of enzymatic activity is vital to living organisms. Here, we report the development and the genetic optimization of an artificial zymogen requiring the action of a natural protease to upregulate its latent asymmetric transfer hydrogenase activity.

Anion-π Enzymes.

In this report, we introduce artificial enzymes that operate with anion-π interactions, an interaction that is essentially new to nature. The possibility to stabilize anionic intermediates and transition states on an π-acidic surface has been recently demonstrated, using the addition of malonate half thioesters to enolate acceptors as a biologically relevant example. The best chiral anion-π catalysts operate with an addition/decarboxylation ratio of 4:1, but without any stereoselectivity. To catalyze this important but intrinsically disfavored reaction stereoselectively, a series of anion-π catalysts was equipped with biotin and screened against a collection of streptavidin mutants. With the best hit, the S112Y mutant, the reaction occurred with 95% ee and complete suppression of the intrinsically favored side product from decarboxylation. This performance of anion-π enzymes rivals, if not exceeds, that of the best conventional organocatalysts. Inhibition of the S112Y mutant by nitrate but not by bulky anions supports that contributions from anion-π interactions exist and matter, also within proteins. In agreement with docking results, K121 is shown to be essential, presumably to lower the pK a of the tertiary amine catalyst to operate at the optimum pH around 3, that is below the pK a of the substrate. Most importantly, increasing enantioselectivity with different mutants always coincides with increasing rates and conversion, i.e., selective transition-state stabilization.

Interfacing Functional Systems.

The objective of molecular systems engineering is to move beyond functional components and primary systems, towards cumulate emergent properties in interfaced higher-order systems of unprecedented multifunctionality and sophistication.

Near diffusion-controlled reaction of a Zn(Cys)4 zinc finger with hypochlorous acid.

Hypochlorous acid (HOCl) is one of the strongest oxidants produced in mammals to kill invading microorganisms. The bacterial response to HOCl involves proteins that are able to sense HOCl using methionine, free cysteines or zinc-bound cysteines of zinc finger sites. Although the reactivity of methionine or free cysteine with HOCl is well documented at the molecular level, this is not the case for zinc-bound cysteines. We present here a study that aims at filling this gap. Using a model peptide of the Zn(Cys)4 zinc finger site of the chaperone Hsp33, a protein involved in the defence against HOCl in bacteria, we show that HOCl oxidation of this model leads to the formation of two disulfides. A detailed mechanistic and kinetic study of this reaction, relying on stopped-flow measurements and competitive oxidation with methionine, reveals very high rate constants: the absolute second-order rate constants for the reaction of the model zinc finger with HOCl and its conjugated base ClO- are (9.3 ± 0.8) × 108 M-1 s-1 and (1.2 ± 0.2) × 104 M-1 s-1, the former approaching the diffusion limit. Revised values of the second-order rate constants for the reaction of methionine with HOCl and ClO- were also determined to be (5.5 ± 0.8) × 108 M-1 s-1 and (7 ± 5) × 102 M-1 s-1, respectively. At physiological pH, the zinc finger site reacts faster with HOCl than methionine and glutathione or cysteine. This study demonstrates that zinc fingers are potent targets for HOCl and confirms that they may serve as HOCl sensors as proposed for Hsp33.

Reactivity of a Zn(Cys)2(His)2 Zinc Finger with Singlet Oxygen: Oxidation Directed toward Cysteines but not Histidines.

Singlet oxygen ((1)O2) is an important reactive oxygen species in biology that has deleterious effects. Proteins constitute the main target of (1)O2 in cells. Several organisms are able to mount a transcriptional defense against (1)O2. ChrR and MBS are two proteins with Zn(Cys)2(His)2 zinc finger sites that are involved in the regulation of the defense against (1)O2. In this article, we investigate the reactivity of Zn⋅CPF, a Zn(Cys)2(His)2 classical ßßα zinc finger, with (1)O2. We show that Zn⋅CPF interacts with (1)O2 mainly by physical quenching using a combination of (1)O2 luminescence quenching and kinetic competition experiments. The chemical reaction, which accounts for 5% of the interaction, leads to oxidation of cysteines but not histidines. Primary photooxidation products, identified by HPLC and mass spectrometry, are sulfinate (75±5%) and disulfides (25±5%). The peptides that have a single cysteine thiolate oxidized into a sulfinate are still able to bind one equivalent Zn(2+) but with a dramatic reduction of the binding constant compared to Zn⋅CPF despite the preservation of the ßßα fold, as shown by NMR and CD titrations. Finally, Zn⋅CPF is compared to Zn⋅LTC, a treble clef Zn(Cys)4 zinc finger, to gain further insight into the behavior of zinc fingers toward (1)O2.

Efficient oxidation and destabilization of Zn(Cys)4 zinc fingers by singlet oxygen.

Singlet oxygen ((1)O2) plays an important role in oxidative stress in all types of organisms, most of them being able to mount a defense against this oxidant. Recently, zinc finger proteins have been proposed to be involved in its cellular detection but the molecular basis of this process still remains unknown. We have studied the reactivity of a Zn(Cys)4 zinc finger with (1)O2 by combinations of spectroscopic and analytical techniques, focusing on the products formed and the kinetics of the reaction. We report that the cysteines of this zinc finger are oxidized to sulfinates by (1)O2. The reaction of the ZnS4 core with (1)O2 is very fast and efficient with almost no physical quenching of (1)O2. A drastic (ca. five orders of magnitude) decrease of the Zn(2+) binding constant was observed upon oxidation. This suggests that the Zn(Cys)4 zinc finger proteins would release their Zn(2+) ion and unfold upon reaction with (1)O2 under cellular conditions and that zinc finger sites are likely targets for (1)O2.

Catalytic zinc complexes for phosphate diester hydrolysis.

Creating efficient artificial catalysts that can compete with biocatalysis has been an enduring challenge which has yet to be met. Reported herein is the synthesis and characterization of a series of zinc complexes designed to catalyze the hydrolysis of phosphate diesters. By introducing a hydrated aldehyde into the ligand we achieve turnover for DNA-like substrates which, combined with ligand methylation, increases reactivity by two orders of magnitude. In contrast to current orthodoxy and mechanistic explanations, we propose a mechanism where the nucleophile is not coordinated to the metal ion, but involves a tautomer with a more effective Lewis acid and more reactive nucleophile. This data suggests a new strategy for creating more efficient metal ion based catalysts, and highlights a possible mode of action for metalloenzymes.

On the design of zinc-finger models with cyclic peptides bearing a linear tail.

Cyclic peptides with a linear tail (CPLT) have been successfully used to model two zinc fingers (ZFs) adopting the treble-clef- and loosened zinc-ribbon folds. In this article, we examine the factors that may influence the design of such ZF models: mutations in the sequence, size of the cycle, and size of the tail. For this purpose, several peptides derived from the CPLT-based models of the treble-clef- and loosened zinc-ribbon ZF were synthesized and studied. CPLT-based models appear to be robust toward mutations, accommodate various cycle sizes, and are sensible to the size of the linking region of the tail located between the cycle and the coordinating amino acids. Based on these criteria, we describe the design of a new CPLT-based model for the zinc-ribbon ZFs, LZR , and compare it to a linear analogue, LZR(lin) . The model complex Zn⋅LZR is able to fold correctly around the metal ion contrary to Zn⋅LZR(lin) , suggesting that CPLT-based models are more likely to yield structurally meaningful models of ZF sites than linear peptide models. Finally, we draw some rules that could allow the design of new CPLT-based metallopeptides with a controlled fold.