Facile incorporation of non-canonical heme ligands in myoglobin through chemical protein synthesis.

The incorporation of non-canonical amino acids (ncAAs) into the metal coordination environments of proteins has endowed metalloproteins with enhanced properties and novel activities, particularly in hemoproteins. In this work, we disclose a scalable synthetic strategy that enables the production of myoglobin (Mb) variants with non-canonical heme ligands, i.e.

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 -acyl pyrazole can be activated by azole reagents like 3-methylpyrazole or imidazole to facilitate its ligation directly with an N-terminal cysteine peptide.

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 Cu-DPA-modified azurins.

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)). The resulting peptide SAL(-COOH) esters can be activated to afford the reactive peptide SAL(-COOH) esters for subsequent STL/CPL.

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.

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.

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.

Development of Nonheme {FeNO} Complexes Based on the 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, Rd-C9A-{FeNO} and Rd-C42A-{FeNO} (Rd = rubredoxin), by constructing a nonheme {FeNO} center within the redesigned iron-sulfur protein scaffolds.

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.

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

Herein, we developed a bifunctional reagent -2-Br-DMNPA for the late-stage protection of peptide cysteine. Through the identification of its -Bu ester 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.

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.

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.

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.

Elucidation of the heme active site electronic structure affecting the unprecedented nitrite dismutase activity of the ferriheme 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 values over 3.

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.

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.

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.

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.

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.

A caged cyanide.

A photoactivatable caged cyanide, 1-(2-nitrophenyl)ethyl (NPE) cyanide, was synthesized, which upon irradiation in the near UV releases cyanide. It is demonstrated that the compound can be used to induce formation of the Fe(III)-CN(-) complex in the heme protein nitrophorin 4 from Rhodnius prolixus.

Reduction of the lipocalin type heme containing protein nitrophorin -- sensitivity of the fold-stabilizing cysteine disulfides toward routine heme-iron reduction.

The determination of the redox properties of the cofactor in heme proteins provides fundamental insight into the chemical characteristics of this wide-spread class of metalloproteins. For the preparation of the ferroheme state, probably the most widely applied reductant is sodium dithionite, which at neutral pH has a reduction potential well below the reduction potential of most heme centers. In addition to the heme iron, some heme proteins, including the nitrophorins (NPs), contain cysteinecysteine disulfide bonds.

Breaking the proximal Fe(II)-N(His) bond in heme proteins through local structural tension: lessons from the heme b proteins nitrophorin 4, nitrophorin 7, and related site-directed mutant proteins.

The factors leading to the breakage of the proximal iron-histidine bond in the ferroheme protein soluble guanylate cyclase (sGC) are still a matter of debate. This event is a key mechanism in the sensing of NO that leads to the production of the second-messenger molecule cGMP. Surprisingly, in the heme protein nitrophorin 7 (NP7), we noticed by UV-vis absorbance spectroscopy and resonance Raman spectroscopy that heme reduction leads to a loss of the proximal histidine coordination, which is not observed for the other isoproteins (NP1-4).