Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis.

Native chemical ligation (NCL) has been playing an increasingly important role in chemical protein synthesis (CPS). More efficient ligation methods that circumvent the requirement of a peptidyl thioester and thiol additive-which allow the following desulfurization or refolding in one pot-are urgently needed for the synthesis of more complex protein targets and in large quantities. Herein, we discover that the weak acyl donor peptidyl N-acyl pyrazole can be activated by azole reagents like 3-methylpyrazole or imidazole to facilitate its ligation directly with an N-terminal cysteine peptide. As it requires no thioester or thiol additive, this ligation strategy can be conveniently combined with metal-free desulfurization (MFD) or oxidative protein folding to allow various one-pot protocols. The utility and generality of the strategy are showcased by the total synthesis of ubiquitin via an N-to-C sequential ligation-MFD strategy, the semi-synthesis of the copper protein azurin, and the efficient assembly of a sulfated hirudin variant and the cyclotide kalata B1, all in a one-pot fashion.

Chemical protein synthesis enabled engineering of saccharide oxidative cleavage activity in artificial metalloenzymes.

Chemical protein (semi-)synthesis is a powerful technique allowing the incorporation of unnatural functionalities at any desired protein site. Herein we describe a facile one-pot semi-synthetic strategy for the construction of a type 2 copper center in the active site of azurin, which is achieved by substitution of Met121 with unnatural amino acid residues bearing a strong ligand N,N-bis(pyridylmethyl)amine (DPA) to mimic the function of typical histidine brace-bearing copper monooxygenases, such as lytic polysaccharide monooxygenases (LPMOs) involved in polysaccharide breakdown. The semi-synthetic proteins were routinely obtained in over 10-mg scales to allow for spectroscopic measurements (UV-Vis, CD, and EPR), which provides structural evidences for the CuII-DPA-modified azurins. 4-nitrophenyl-ß-D-glucopyranoside (PNPG) was used as a model substrate for the H2O2-driven oxidative cleavage reaction facilitated by semi-synthetic azurins, and the CuII-6 complex showed a highest activity (TTN 253). Interestingly, our semi-synthetic azurins were able to tolerate high H2O2 concentrations (up to 4000-fold of the enzyme), making them promising for practical applications. Collectively, we establish that chemical protein synthesis can be exploited as a reliable technology in affording large quantities of artificial metalloproteins to facilitate the transformation of challenging chemical reactions.

Oxidation and Phenolysis of Peptide/Protein C-Terminal Hydrazides Afford Salicylaldehyde Ester Surrogates for Chemical Protein Synthesis.

With the growing popularity of serine/threonine ligation (STL) and cysteine/penicillamine ligation (CPL) in chemical protein synthesis, facile and general approaches for the preparation of peptide salicylaldehyde (SAL) esters are urgently needed, especially those viable for obtaining expressed protein SAL esters. Herein, we report the access of SAL ester surrogates from peptide hydrazides (obtained either synthetically or recombinantly) via nitrite oxidation and phenolysis by 3-(1,3-dithian-2-yl)-4-hydroxybenzoic acid (SAL(-COOH)PDT). The resulting peptide SAL(-COOH)PDT esters can be activated to afford the reactive peptide SAL(-COOH) esters for subsequent STL/CPL. While being operationally simple for both synthetic peptides and expressed proteins, the current strategy facilitates convergent protein synthesis and combined application of STL with NCL. The generality of the strategy is showcased by the N-terminal ubiquitination of the growth arrest and DNA damage-inducible protein (Gadd45a), the efficient synthesis of ubiquitin-like protein 5 (UBL-5) via a combined N-to-C NCL-STL strategy, and the C-to-N semisynthesis of a myoglobin (Mb) variant.

Functional expression of a thrombin exosite I inhibitor triabin in Escherichia coli and elucidation of the role of key residues in its inhibitory activity.

Triabin, a lipocalin-like thrombin inhibitor from the saliva of the blood-sucking triatomine bug Triatoma pallidipennis, exhibits effective inhibition comparable to hirudin despite binding exclusively at exosite I. Interestingly, it was reported that higher triabin doses would not inhibit thrombin completely, which makes it a promising antithrombotic candidate agent with a larger therapeutic window. However, few structural and functional studies about triabin have been reported in the past three decades, mostly due to the lack of a reliable and practicable recombinant expression technology for this seemingly small protein. In this work, we have adopted the SUMO fusion technology for the expression of triabin in E. coli cells-with facile refolding and purification procedures-and the bioactive triabin was produced in ∼12 mg/L culture medium. Subsequently, the structure-function studies through extensive site-directed mutagenesis reveal that triabin's Phe-106 involved in the hydrophobic contacts plays a surprisingly important role in the thrombin inhibition, in contrast to the negatively charged residues Asp-135 or Glu-128 involved in the salt-bridge interaction. As such, this study complements our understanding of the interaction mechanism of natural thrombin inhibitors, which should facilitate the development of anticoagulant drugs with a novel mode of action against thrombin.

Chemical synthesis of human selenoprotein F and elucidation of its thiol-disulfide oxidoreductase activity.

Selenoprotein F (SelF) is an endoplasmic reticulum-residing eukaryotic protein that contains a selenocysteine (Sec) residue. It has been suggested to be involved in a number of physiological processes by acting as a thiol-disulfide oxidoreductase, but the exact role has remained unclear due to the lack of a reliable production method. We document herein a robust synthesis of the human SelF through a three-segment two-ligation semisynthesis strategy. Highlighted in this synthetic route are the use of a mild desulfurization process to protect the side-chain of the Sec residue from being affected and the simultaneous removal of acetamidomethyl and p-methoxybenzyl protection groups by PdCl2, thus facilitating the synthesis of multi-milligrams of homogenous SelF. The reduction potential of SelF was determined and the thiol-disulfide oxidoreductase activity was further supported by its ability to catalyze the reduction and isomerization of disulfide bonds.

Protein Ligation and Labeling Enabled by a C-Terminal Tetracysteine Tag.

The hydrazinolysis of S-cyanylated peptide provides an alternative way to afford protein α-hydrazide, a key reagent used in native chemical ligation (NCL), without the aid of any inteins or enzymes. The currently used non-selective S-cyanylation, however, allows no other cysteine in the protein besides the one at the cleavage site. Herein, we report a regioselective S-cyanylation and hydrazinolysis strategy achieved via the fusion of a tetracysteine tag to the C-terminal of the protein of interest. We term it tetracysteine enabled protein ligation (TCEPL). While highly selective, the strategy is applicable for proteins expressed as inclusion bodies, and this was showcased by the efficient semi-synthesis of an iron-sulfur protein rubredoxin and the catalytic and hinge domains of matrix metalloprotease-14 (MMP-14) containing 207 amino acid residues. Furthermore, the TCEPL strategy was exploited for protein C-terminal labeling with amino reagents bearing a variety of functional groups, demonstrating its versatility and generality.

Development of Nonheme {FeNO}7 Complexes Based on the Pyrococcus furiosus Rubredoxin for Red-Light-Controllable Nitric Oxide Release.

Nitric oxide (NO) is an essential biological messenger, contributing a significant role in a diverse range of physiological processes. The light-controllable NO releasers are of great interest because of their potential as agents for NO-related research and therapeutics. Herein, we developed a pair of red-light-controllable NO releasers, pfRd-C9A-{FeNO}7 and pfRd-C42A-{FeNO}7 (pfRd = Pyrococcus furiosus rubredoxin), by constructing a nonheme {FeNO}7 center within the redesigned iron-sulfur protein scaffolds. While shown to be both air and thermally stable, these complexes are highly sensitive to red-light irradiation with temporal precision, which was confirmed by electron paramagnetic resonance spin trapping and Griess assay. The temporally controlled NO release from these complexes was also demonstrated in DNA cleavage assay. Overall, this study demonstrates that such a protein-based nonheme iron nitrosyl system could be a viable chemical tool for precise NO administration.

Chemical Synthesis of the Sec-To-Cys Homologue of Human Selenoprotein F and Elucidation of Its Disulfide-pairing Mode.

Herein, we document a highly optimized synthesis of the Sec-to-Cys homologue of the human selenoprotein F (SelF) through a three-segment two-ligation semisynthesis strategy. Highlighted in this synthetic route are two one-pot manipulations, i.e. the first ligation followed by a desulfurization and the second ligation followed by the protein refolding. This way multi-milligrams of the folded synthetic protein was obtained, which set the stage for the synthesis of the natural selenoprotein. Moreover, the disulfide pairing mode of the SelF was elucidated through a combination of site-directed mutagenesis and LC-MS study. It provides not only a criterion to judge the viability of the synthetic protein, and more importantly, useful structural insights into the previously unresolved UGGT-binding domain of SelF.

On Demand Attachment and Detachment of rac-2-Br-DMNPA Tailoring to Facilitate Chemical Protein Synthesis.

Herein, we developed a bifunctional reagent rac-2-Br-DMNPA 2 for the late-stage protection of peptide cysteine. Through the identification of its t-Bu ester 1 as a more competent form under ligation conditions, facile N-terminal and side-chain caging for the model peptide and protein were accomplished. Building upon this, a one-pot ligation and photolysis strategy was applied in the synthesis of the mini-protein chlorotoxin. More importantly, we extended the utility of 2 as a bifunctional linker for traceless solid-phase chemical ligation.

Solid-phase synthesis of peptide Mn(i)-carbonyl bioconjugates and their CO release upon visible light activation.

A one-pot synthetic route has been developed for the assembly of peptide Mn(i)-carbonyl bioconjugates. It allows the installation of a variety of chelating agents at the late stage, and after just one purification step the TAT-MnCO complexes can be obtained. The resulting bioconjugates showed different and tunable CO releasing kinetics upon visible light activation.

Streamlined purification and characterization of Pyrococcus furiosus rubredoxins with different N-terminal modifications by reversed-phase HPLC.

Rubredoxins (Rds), like those from Pyrococcus furious (Pf), have largely been found to be expressed in Escherichia coli (E. coli) as a mixture of different N-terminal forms, which may affect the properties of the protein. The typical procedures for the purification of Rds are cumbersome and usually with low yield. We present herein a streamlined purification strategy based on the reversed-phase high performance liquid chromatography (RP-HPLC), which offers high yield and high resolution after simply one-step purification following pre-treatment. We also show that RP-HPLC can be a valuable tool to gain information related to the thermal decomposition pathway of Pf-Rds.

Facile synthesis of sulfotyrosine-containing α-conotoxins.

α-Conotoxins (Ctx) can selectively target distinct subtypes of nicotinic acetylcholine receptors (nAChRs), which are closely related to a number of neurological diseases, and they have been considered as ideal probes and model peptide drugs. Sulfotyrosine (sY) is an important post-translational modification and believed to modulate certain key protein-protein interactions. Although sY modification has been indicated in several α-Ctx, its biological consequence has largely remained unexplored, mostly because of the difficulties in both its extraction from biological samples and chemical synthesis. Herein, we report a facile synthesis and folding strategy for obtaining the sY modified α-Ctx. This strategy is based on the development of a simple and controlled deprotection of the neopentyl protecting group of the sulfate ester as well as its compatibility with a step-wise oxidative folding of the two disulfide bonds. Eight sY modified α-Ctx peptides were successfully synthesized in good yield and with high purity, and their serum stabilities were almost comparable with non-modified peptides.

Functional expression and purification of the untagged C-terminal domain of MMP-2 from Escherichia coli inclusion bodies.

The C-terminal domain (CTD) of MMP-2, which includes a hemopexin-like domain, has been increasingly studied as an alternative target in developing selective intervention strategies towards MMP-2. Moreover, The CTD itself has been implicated in a growing number of biological events, either MMP-dependent or -independent. The production of CTD, however, has been mostly based on the uncontrolled lysis of the latent ProMMP-2 or fusion protein expression that leaves a fusion tag. In this work we present a facile production of the untagged CTD in E. coli. The target protein was expressed as inclusion bodies, and we established an efficient wash and refolding strategy that allows us to obtain the target protein in extremely high purity. The yield was established at ~6 mg/L of the culture medium, which would greatly facilitate the production and hence the biological study of CTD. The method described herein might also prove useful for related (domain) proteins in MMP family and beyond.

Elucidation of the heme active site electronic structure affecting the unprecedented nitrite dismutase activity of the ferriheme b proteins, the nitrophorins.

Nitrophorins (NPs) catalyze the nitrite dismutation reaction that is unprecedented in ferriheme proteins. Despite progress in studying the reaction mechanism, fundamental issues regarding the correlation of the structural features with the nitrite dismutase activity of NPs remain elusive. On the other hand, it has been shown that the nitrite complexes of NPs are unique among those of the ferriheme proteins since some of their electron paramagnetic resonance (EPR) spectra show significant highly anisotropic low spin (HALS) signals with large gmax values over 3.2. The origin of HALS signals in ferriheme proteins or models is not well understood, especially in cases where axial ligands other than histidine are present. In this study several mutations were introduced in NP4. The related nitrite coordination and dismutation reaction were investigated. As a result, the EPR spectra of the NP-nitrite complexes were found to be tightly correlated with the extent of heme ruffling and protonation state of the proximal His ligand-dictated by an extended H-bonding network at the heme active site. Furthermore, it is established that the two factors are essential in determining the nitrite dismutase activity of NPs. These results may provide a valuable guide for identifying or designing novel heme proteins with similar activity.

Chemical Synthesis of the 20†kDa Heme Protein Nitrophorin†4 by α-Ketoacid-Hydroxylamine (KAHA) Ligation.

The chemical synthesis of the 184-residue ferric heme-binding protein nitrophorin 4 was accomplished by sequential couplings of five unprotected peptide segments using α-ketoacid-hydroxylamine (KAHA) ligation reactions. The fully assembled protein was folded to its native structure and coordinated to the ferric heme b cofactor. The synthetic holoprotein, despite four homoserine residues at the ligation sites, showed identical properties to the wild-type protein in nitric oxide binding and nitrite dismutase reactivity. This work establishes the KAHA ligation as a valuable and viable approach for the chemical synthesis of proteins up to 20 kDa and demonstrates that it is well-suited for the preparation of hydrophobic protein targets.

Nitrite dismutase reaction mechanism: kinetic and spectroscopic investigation of the interaction between nitrophorin and nitrite.

Nitrite is an important metabolite in the physiological pathways of NO and other nitrogen oxides in both enzymatic and nonenzymatic reactions. The ferric heme b protein nitrophorin 4 (NP4) is capable of catalyzing nitrite disproportionation at neutral pH, producing NO. Here we attempt to resolve its disproportionation mechanism. Isothermal titration calorimetry of a gallium(III) derivative of NP4 demonstrates that the heme iron coordinates the first substrate nitrite. Contrary to previous low-temperature EPR measurements, which assigned the NP4-nitrite complex electronic configuration solely to a low-spin (S = 1/2) species, electronic absorption and resonance Raman spectroscopy presented here demonstrate that the NP4-NO2(-) cofactor exists in a high-spin/low-spin equilibrium of 7:3 which is in fast exchange in solution. Spin-state interchange is taken as evidence for dynamic NO2(-) coordination, with the high-spin configuration (S = 5/2) representing the reactive species. Subsequent kinetic measurements reveal that the dismutation reaction proceeds in two discrete steps and identify an {FeNO}(7) intermediate species. The first reaction step, generating the {FeNO}(7) intermediate, represents an oxygen atom transfer from the iron bound nitrite to a second nitrite molecule in the protein pocket. In the second step this intermediate reduces a third nitrite substrate yielding two NO molecules. A nearby aspartic acid residue side-chain transiently stores protons required for the reaction, which is crucial for NPs' function as nitrite dismutase.

Complexes of ferriheme nitrophorin 4 with low-molecular weight thiol(ate)s occurring in blood plasma.

Nitrophorins are proteins occurring in the saliva of the blood-sucking insect Rhodnius prolixus to carry NO as a vasodilator and blood-coagulation inhibitor into the victim's tissue. It was suggested that the rate of NO release can be enhanced by the blood-plasma component L-cysteine [J.M.C.Ribeiro, Insect Biochem. Mol. Biol. 26 (1996) 899-905]. However, the mechanism of the reaction is not clear. In the attempt to exploit the reaction in detail, complexes of nitrophorin 4 (NP4) with the thiols 2-mercaptoethanol, L-cysteine, and L-homocysteine and with HS(-) were formed and characterized under anaerobic conditions using absorption spectroscopy, X-ray crystallography, and EPR spectroscopy. In contrast to met-myoglobin, which is reduced by L-cysteine, all four compounds form low-spin Fe(III) complexes with NP4. The weak equilibration constants (167-5200 M(-1)) neither support significant complexation nor the simple displacement of NO in vivo. Both amino acid based thiols form additional H-bonds with side chains of the heme pocket entry. Glutathione and L-methionine did not form a complex, indicating the specificity of the complexes with L-cysteine and L-homocysteine. Continuous wave EPR spectroscopy reveals the simultaneous existence of three low-spin systems in each case that are attributed to various protonation and/or conformational stages in the heme pocket. Electron nuclear double resonance (ENDOR) spectroscopy demonstrates that the thiol sulfurs are, at least in part, protonated. Overall, the results not only demonstrate the good accessibility of the NP4 heme center by biologically relevant thiols, but also represent the first structural characterization of a ferriheme protein in complex with L-cysteine L-homocysteine.

Insertion of an H-bonding residue into the distal pocket of the ferriheme protein nitrophorin 4: effect on nitrite-iron coordination and nitrite disproportionation.

Heme proteins are important entities for the metabolism of nitrite. Inspection of the structural features of the reported hemoprotein-nitrite crystal structures reveals that, except for nitrophorin 4 (NP4), H-bonding to the nitrite ligand is accomplished via histidine or arginine residues. These H-bonds probably play an important role for the nitrite coordination and/or reactivities. In nitrophorins, which catalyze the nitrite disproportionation reaction, such a residue is missing. Here, we report on the L130R mutant of the NP isoprotein NP4 that provides the Arg130 residue as part of the flexible G-H loop as a potential H-bonding residue in the distal heme pocket. Similar to the wild-type protein, nitrite remains N-bonded in the crystal structure of NP4(L130R). However, spectroscopic investigations show that, in solution, a second ligand-rotational orientation exists, which is in fast-exchange equilibrium with the normal, parallel ligand orientation. Moreover, the nitrite disproportionation is inhibited in NP4(L130R). Comparison with another, also less active mutant NP4(D30N) suggests that the displacement of H(2)O molecules from the heme cavity prevents the proton donation pathway through Asp30.

Heterogeneous kinetics of the carbon monoxide association and dissociation reaction to nitrophorin 4 and 7 coincide with structural heterogeneity of the gate-loop.

NO is an important signaling molecule in human tissue. However, the mechanisms by which this molecule is controlled and directed are currently little understood. Nitrophorins (NPs) comprise a group of ferriheme proteins originating from blood-sucking insects that are tailored to protect and deliver NO via coordination to and release from the heme iron. Therefore, the kinetics of the association and dissociation reactions were studied in this work using the ferroheme-CO complexes of NP4, NP4(D30N), and NP7 as isoelectronic models for the ferriheme-NO complexes. The kinetic measurements performed by nanosecond laser-flash-photolysis and stopped-flow are accompanied by resonance Raman and FT-IR spectroscopy to characterize the carbonyl species. Careful analysis of the CO rebinding kinetics reveals that in NP4 and, to a larger extent, NP7 internal gas binding cavities are located, which temporarily trap photodissociated ligands. Moreover, changes in the free energy barriers throughout the rebinding and release pathway upon increase of the pH are surprisingly small in case of NP4. Also in case of NP4, a heterogeneous kinetic trace is obtained at pH 7.5, which corresponds to the presence of two carbonyl species in the heme cavity that are seen in vibrational spectroscopy and that are due to the change of the distal heme pocket polarity. Quantification of the two species from FT-IR spectra allowed the fitting of the kinetic traces as two processes, corresponding to the previously reported open and closed conformation of the A-B and G-H loops. With the use of the A-B loop mutant NP4(D30N), it was confirmed that the kinetic heterogeneity is controlled by pH through the disruption of the H-bond between the Asp30 side chain and the Leu130 backbone carbonyl. Overall, this first study on the slow phase of the dynamics of diatomic gas molecule interaction with NPs comprises an important experimental contribution for the understanding of the dynamics involved in the binding/release processes of NO/CO in NPs.