Elusive Intermediates Research Articles

Watching the RNA Polymerase Transcription by Time-Dependent Soak-Trigger-Freeze X-Ray Crystallography

The challenge for structural biology is to understand atomic-level macromolecular motions during enzymatic reaction. X-ray crystallography can reveal high resolution structures; however, one perceived limitation is that it reveals only static views. Here we use time-dependent soak-trigger-freeze X-ray crystallography, namely, soaking nucleotide and divalent metal into the bacteriophage RNA polymerase (RNAP)-promoter DNA complex crystals to trigger the nucleotidyl transfer reaction and freezing crystals at different time points, to capture real-time intermediates in the pathway of transcription. In each crystal structure, different intensities and shapes of electron density maps corresponding to the nucleotide and metal were revealed at the RNAP active site which allow watching the nucleotide and metal bindings and the phosphodiester bond formation in a time perspective. Our study provides the temporal order of substrate assembly and metal co-factor binding at the active site of enzyme which completes our understanding of the two-metal-ion mechanism and fidelity mechanism in single-subunit RNAPs. The nucleotide-binding metal (MeB) is coordinated at the active site prior to the catalytic metal (MeA). MeA coordination is only temporal, established just before and dissociated immediately after phosphodiester bond formation. We captured these elusive intermediates exploiting the slow enzymatic reaction in crystallo. These results demonstrate that the simple time-dependent soak-trigger-freeze X-ray crystallography offers a direct means for monitoring enzymatic reactions. [Ref: Basu, R.S., and K.S. Murakami (2013). J Biol Chem., 288, 3305-3311]

Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.

Toward Highlighting the Ultrafast Electron Transfer Dynamics at the Optically Dark Sites of Photocatalysts.

Building a detailed understanding of the structure-function relationship is a crucial step in the optimization of molecular photocatalysts employed in water splitting schemes. The optically dark nature of their active sites usually prevents a complete mapping of the photoinduced dynamics. In this work, transient X-ray absorption spectroscopy highlights the electronic and geometric changes that affect such a center in a bimetallic model complex. Upon selective excitation of the ruthenium chromophore, the cobalt moiety is reduced through intramolecular electron transfer and undergoes a spin flip accompanied by an average bond elongation of 0.20 ± 0.03 Å. The analysis is supported by simulations based on density functional theory structures (B3LYP*/TZVP) and FEFF 9.0 multiple scattering calculations. More generally, these results exemplify the large potential of the technique for tracking elusive intermediates that impart unique functionalities in photochemical devices.

Watching the Bacteriophage N4 RNA Polymerase Transcription by Time-dependent Soak-trigger-freeze X-ray Crystallography

The challenge for structural biology is to understand atomic-level macromolecular motions during enzymatic reaction. X-ray crystallography can reveal high resolution structures; however, one perceived limitation is that it reveals only static views. Here we use time-dependent soak-trigger-freeze X-ray crystallography, namely, soaking nucleotide and divalent metal into the bacteriophage RNA polymerase (RNAP)-promoter DNA complex crystals to trigger the nucleotidyl transfer reaction and freezing crystals at different time points, to capture real-time intermediates in the pathway of transcription. In each crystal structure, different intensities and shapes of electron density maps corresponding to the nucleotide and metal were revealed at the RNAP active site which allow watching the nucleotide and metal bindings and the phosphodiester bond formation in a time perspective. Our study provides the temporal order of substrate assembly and metal co-factor binding at the active site of enzyme which completes our understanding of the two-metal-ion mechanism and fidelity mechanism in single-subunit RNAPs. The nucleotide-binding metal (Me(B)) is coordinated at the active site prior to the catalytic metal (Me(A)). Me(A) coordination is only temporal, established just before and dissociated immediately after phosphodiester bond formation. We captured these elusive intermediates exploiting the slow enzymatic reaction in crystallo. These results demonstrate that the simple time-dependent soak-trigger-freeze X-ray crystallography offers a direct means for monitoring enzymatic reactions.

Seeking a Mechanistic Analogue of the Water–Gas Shift Reaction: Carboxamido Ligand Formation and Isocyanate Elimination from Complexes Containing the Tp′PtMe Fragment

A series of stable, isolable Tp'Pt(IV) carboxamido complexes of the type Tp'PtMe(2)(C(O)NHR) (R = Et, (n)Pr, (i)Pr, (t)Bu, Bn, Ph) has been synthesized by addition of amide nucleophiles to the carbonyl ligand in Tp'Pt(Me)(CO) followed by trapping of the Pt(II) intermediate with methyl iodide as the methylating reagent. These compounds mimic elusive intermediates resulting from hydroxide addition to platinum-bound CO in the Water-Gas Shift Reaction (WGSR). Seeking parallels to WGSR chemistry, we find that deprotonation of the carboxamido NH initiates elimination and the isocyanate-derived products form; the resulting platinum fragment can be protonated to reoxidize the metal center and generate Tp'PtMe(2)H, the synthetic precursor to Tp'Pt(Me)(CO). Mechanistic studies on the formation of and elimination from Tp'PtMe(2)(C(O)NHR) suggest a stepwise process with deprotonation from a Pt(IV) species as the key step prompting elimination.

Trapping Intermediates in an [8 + 2] Cycloaddition Reaction with the Help of DFT Calculations

DFT calculations predict that the [8 + 2] cycloaddition reaction between ketenes and 8-azaheptafulvenes occurs stepwise through antiaromatic zwitterionic intermediates. With adequate modifications of both the electronic properties of the ketene and the reaction conditions, these elusive intermediates have been successfully trapped and fully characterized (X-ray), thus confirming the predicted stepwise nature of the transformation.

ChemInform Abstract: Detection of Carbocationic Species in Zeolites: Large Crystals Pave the Way

AbstractReview: 43 refs.

Protein Vivisection Reveals Elusive Intermediates in Folding

Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here, we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu→Glu−) to destabilize and unfold a specific region of the protein. We applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the β5-strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states.

S–S bond reactivity in metal-perthiocarboxylato compounds

While M-percarboxylato species are elusive intermediates, their sulfur containing analogues are known in some cases. The feasibility of isolation of M-perthioacetato compounds allowed, in this work, to obtain new insights into the pathways that follow the reactivity of M-E-ER (E = O, S) fragments. Herein we report on the isolation of two new M-perthioacetato compounds: trans-[Pt(CH(3)CS(2)S)(2)] () and [Ni(CH(3)CSS)(CH(3)CS(2)S)] (), which have been fully characterized, including X-ray structures. Reactivity of these compounds towards PPh(3) has been studied combining UV-vis monitorization and NMR measurements. Overall the accumulated data suggest that the evolution of the perthioacetato ligand in complexes and by reaction with PPh(3) consists of a complex multistep pathway in which the sulfur transfer is preceded by electron transfer. Cyclic voltammetry measurements indicate that the transference of two electrons from the phosphorus to the sulfur atom is not concerted, suggesting that the first step of the reaction with PPh(3) is the monoelectronic electron transfer followed by P-S bond formation. The results presented here show a novel pathway in the field of S-S bond reactivity processes relevant in biological, synthetic systems and in hydrocarbon desulfurization processes.

Direct observation of CuI/CuIII redox steps relevant to Ullmann-type coupling reactions

A series of aryl–copper(III)-halide complexes have been synthesized and characterized by NMR and UV-visible spectroscopy, cyclic voltammetry and X-ray crystallography. These complexes closely resemble elusive intermediates often invoked in catalytic reactions, such as Ullmann–Goldberg cross-coupling reactions, and their preparation has enabled direct observation and preliminary characterization of aryl halide reductive elimination from CuIII and oxidative addition to CuI centers. In situ spectroscopic studies (1H NMR, UV-visible) of a Cu-catalyzed C–N coupling reaction provides definitive evidence for the involvement of an aryl-copper(III)-halide intermediate in the catalytic mechanism. These results provide the first direct observation of the CuI/CuIII redox steps relevant to Ullmann-type coupling reactions.

Structures and Properties of Nonchelated, d0 Alkyl Alkene Complexes of the Type [Cp2ZrMe(alkene)]+: Elusive Intermediates during Ziegler–Natta Polymerizations of Alkenes

Caught in the act: An alkyl alkene Zr(IV) complex (see picture; Cp = C(5)H(5)) has been synthesized and characterized for the first time. The alkene bonding mode is highly asymmetric, and C2 is quite carbocationic. There is also evidence for rotation about the C1-C2 bond. This extremely unusual complex provides an exemplar of previously unknown intermediates in Ziegler-Natta and carbocationic polymerization reactions of alkenes.

Structures and Properties of Nonchelated, d0 Alkyl Alkene Complexes of the Type [Cp2ZrMe(alkene)]+: Elusive Intermediates during Ziegler–Natta Polymerizations of Alkenes

AbstractAuf frischer Tat ertappt: Der abgebildete Alkyl(alken)zirconium(IV)‐Komplex (Cp=C5H5) wurde synthetisiert und charakterisiert. Das Alken ist sehr unsymmetrisch gebunden, sodass C2 einen stark carbokationischen Charakter hat. Ferner war eine Rotation um die C1‐C2‐Bindung nachweisbar. Dieser außergewöhnliche Komplex kann als Modell für bislang unbekannte Zwischenstufen in Ziegler‐Natta‐ und carbokationischer Alkenpolymerisation angesehen werden.magnified image

A CRYSTAL WITH A STORY TO TELL

RETURN TO ISSUEPREVNewsNEXTA CRYSTAL WITH A STORY TO TELLBIOCHEMISTRY: Single crystal traps elusive intermediates in 0 activation by a nonheme iron enzymeCELIA ARNAUDCite this: Chem. Eng. News 2007, 85, 17, 7Publication Date (Print):April 23, 2007Publication History Published online29 October 2010Published inissue 23 April 2007https://doi.org/10.1021/cen-v085n017.p007Copyright © 2007 AMERICAN CHEMICAL SOCIETYArticle Views9Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (565 KB) SUBJECTS:Iron,Peptides and proteins Get e-Alerts

Nanoscale Molecular Cavities for Stabilization of Highly Reactive Species

Several kinds of bowl-type molecules were designed for regulation of the reactivities of the functional groups in their cavities, especially for kinetic stabilization of highly reactive species which have been difficult of access by conventional methods. The “peripheral steric protection” by these molecular cavities effectively prevents the bimolecular decomposition of the reactive species such as dimerization or self-condensation, and enabled the isolation of various species including a sulfenic acid, a selenenic acid, an S-nitrosothiol, and a Se-nitrososelenol, which have been known as important but elusive intermediates in biological reactions. Utilization of the molecular bowls as the ligands for transition metal complexes is also described.

Mechanistic aspects of C–C bond formation involving allylpalladium complexes: the role of computational studies

Computational studies have proven to be a useful tool for the study of mechanistic aspects of reactions involving allylpalladium complexes as intermediates. Previous work on the mechanisms of reactions involving such intermediates is discussed, as well as our contributions in this field. Thus, we report on the study of the mechanism of the insertion of alkenes into allylpalladium intermediates involved in the Oppolzer carbocyclization, the possible reaction pathways for the allylstannylation of alkynes, and the carbon-carbon reductive elimination from bis(allyl)palladium complexes. Structures for elusive intermediates, transition states, and even a novel mechanism for the reductive elimination in allyl-allyl cross-coupling reactions have been proposed.

Synthetic active site analogues of heme–thiolate proteins: Characterization and identification of intermediates of the catalytic cycles of cytochrome P450 cam and chloroperoxidase

In view of recent results from different sources, the reaction mechanisms of two heme–thiolate proteins, cytochrome P450 cam and chloroperoxidase (CPO), are discussed. In this context a mechanism of CPO is proposed which includes H 2O 2 cleavage, subsequent formation of compound I and the identification of two elusive intermediates. The HOCl adduct of the iron(III)porpyhrin is the catalytically competent Cl + donor chlorinating activated C–H bonds of substrates bound to the enzyme. Pulse-EPR characterization of an enzyme model of the resting state of P450 cam suggests a role of the electric field of the protein for stabilizing the low-spin state of the cofactor of the enzyme. It is further suggested that the same effect of the protein may trigger the reactivity of compound I such that both concerted and two-step reactions are feasible within the concept of a Two-State-Reactivity. This review emphasizes the value of synthetic enzyme models complementing investigations of the native proteins.

Analyzing protein functions in four dimensions.

Time-resolved structural studies on biomolecular function are coming of age. Focus has shifted from studies on 'systems of opportunities' to a more problem-oriented approach, addressing significant questions in biology and chemistry. An important step in this direction has been the use of physical and chemical trapping methods to capture and then freeze reaction intermediates in crystals. Subsequent monochromatic data collection at cryogenic temperatures can produce high resolution structures of otherwise elusive intermediates. The combination of diffraction methods with spectroscopic techniques provides a means to directly correlate electronic transitions with structural transitions in the sample, eliminating much of the guesswork from experiments. Studies on cytochrome P450, isopenicillin N synthase, cytochrome cd1 nitrite reductase, copper amine oxidase and bacteriorhodopsin were selected as examples, and the results are discussed.

The Formation of 1,3-Dithiolanes from Aromatic Thioketones and Diazomethane − The Mechanism of the Schönberg Reaction

Reactions of diaryl thioketones with diazomethane at room temperature afford 4,4,5,5-tetraaryl-1,3-dithiolanes; the scope of this surprising 2:1 interaction has been studied for decades (Schonberg Reaction). The clue to the mechanism was our observation that the stoichiometry is 1:1 at −78 °C, and 2,5-dihydro-2,2-diaryl-1,3,4-thiadiazoles are formed as primary [2+3] cycloadducts. They lose N2 at −45 °C in first-order reactions generating diaryl thioketone S-methylides which can be intercepted by thioketones (1,3-dithiolanes), multiple CC bonds, or acids HX. In the absence of trapping reagents, the elusive intermediates either dimerize furnishing 2,2,3,3-tetraaryl-1,4-dithianes or give rise to 2,2-diarylthiiranes by electrocyclization. Beyond thiobenzophenone and diazomethane, our main model reaction, the studies involve fluorene-9-thione, 4,4-dimethoxy- and 4,4-dichlorothiobenzophenone. The ring of 2,5-dihydro-2,2-diphenyl-1,3,4-thiadiazole (8) is opened by LDA at −78 °C and derivatives of anion 12 are obtained. − In summa: The Schonberg reaction consists of two 1,3-dipolar cycloadditions, linked by a 1,3-dipolar cycloreversion.

Towards a new class of synthetic antibacterials acting on lipopolysaccharide biosynthesis

Increasing antibiotic resistance in microorganisms and newly emerging pathogens has become a major problem in present society. The recent confluence of powerful drug discovery techniques, including rational design of mechanism-based inhibitors and combinatorial chemistry accompanied with high-throughput screening methods, will expedite the search for new antimicrobials. This review article describes recent attempts in the design of selective inhibitors of lipopolysaccharide biosynthesis, which are the target for the development of new antibiotics. The results introduced here illustrate the value of organic synthesis to characterize enzymatic reaction. Mechanistic postulates have pointed the way to the design of potent inhibitors, and these in turn have provided insight into details of the reaction, as well as useful structural models for elusive intermediates. Drug Dev. Res. 50:416–424, 2000. © 2000 Wiley-Liss, Inc.

ChemInform Abstract: Structure and Reactivity of Thiophanium Salts, the Elusive Intermediates in ArS+‐Mediated One‐Pot Sequence of Three AdE Reactions.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.