Short-lived Complex Research Articles

Chemical Tools for Probing the Ub/Ubl Conjugation Cascades.

Conjugation of ubiquitin (Ub) and structurally related ubiquitin-like proteins (Ubls), essential for many cellular processes, employs multi-step reactions orchestrated by specific E1, E2 and E3 enzymes. The E1 enzyme activates the Ub/Ubl C-terminus in an ATP-dependent process that results in the formation of a thioester linkage with the E1 active site cysteine. The thioester-activated Ub/Ubl is transferred to the active site of an E2 enzyme which then interacts with an E3 enzyme to promote conjugation to the target substrate. The E1-E2-E3 enzymatic cascades utilize labile intermediates, extensive conformational changes, and vast combinatorial diversity of short-lived protein-protein complexes to conjugate Ub/Ubl to various substrates in a regulated manner. In this review, we discuss various chemical tools and methods used to study the consecutive steps of Ub/Ubl activation and conjugation, which are often too elusive for direct studies. We focus on methods developed to probe enzymatic activities and capture and characterize stable mimics of the transient intermediates and transition states, thereby providing insights into fundamental mechanisms in the Ub/Ubl conjugation pathways.

2.6-Å resolution cryo-EM structure of a class Ia ribonucleotide reductase trapped with mechanism-based inhibitor N3CDP

Ribonucleotide reductases (RNRs) reduce ribonucleotides to deoxyribonucleotides using radical-based chemistry. For class Ia RNRs, the radical species is stored in a separate subunit (β2) from the subunit housing the active site (α2), requiring the formation of a short-lived α2β2 complex and long-range radical transfer (RT). RT occurs via proton-coupled electron transfer (PCET) over a long distance (~32-Å) and involves the formation and decay of multiple amino acid radical species. Here, we use cryogenic electron microscopy and a mechanism-based inhibitor 2'-azido-2'-deoxycytidine-5'-diphosphate (N3CDP) to trap a wild-type α2β2 complex of Escherichia coli class Ia RNR. We find that one α subunit has turned over and that the other is trapped, bound to β in a midturnover state. Instead of N3CDP in the active site, forward RT has resulted in N2 loss, migration of the third nitrogen from the ribose C2' to C3' positions, and attachment of this nitrogen to the sulfur of cysteine-225. In this study, an inhibitor has been visualized as an adduct to an RNR. Additionally, this structure reveals the positions of PCET residues following forward RT, complementing the previous structure that depicted a preturnover PCET pathway and suggesting how PCET is gated at the α-β interface. This N3CDP-trapped structure is also of sufficient resolution (2.6 Å) to visualize water molecules, allowing us to evaluate the proposal that water molecules are proton acceptors and donors as part of the PCET process.

Plastocyanin and Cytochrome f Complex Structures Obtained by NMR, Molecular Dynamics, and AlphaFold 3 Methods Compared to Cryo-EM Data.

Plastocyanin is a small mobile protein that facilitates electron transfer through the formation of short-lived protein-protein complexes with cytochrome bf and photosystem 1. Due to the transient nature of plastocyanin-cytochrome f complex, the lack of a long-lived tight complex makes it impossible to determine its structure by X-ray diffraction analysis. Up to today, a number of slightly different structures of such complexes have been obtained by experimental and computer methods. Now, artificial intelligence gives us the possibility to predict the structures of intermolecular complexes. In this study, we compare encounter and final complexes obtained by Brownian and molecular dynamics methods, as well as the structures predicted by AlphaFold 3, with NMR and cryo-EM data. Surprisingly, the best match for the plastocyanin electron density obtained by cryo-EM was demonstrated by an AlphaFold 3 structure. The orientation of plastocyanin in this structure almost completely coincides with its orientation obtained by molecular dynamics calculation, and, at the same time, it is different from the orientation of plastocyanin predicted on the basis of NMR data. This is even more unexpected given that only NMR structures for the plastocyanin-cytochrome f complex are available in the PDB database, which was used to train AlphaFold 3.

Imaging Rovibrational Excitation of Scattered YO Molecules in Inelastic Collisions with Kr and Ne.

Energy transfer between atoms and molecules is fundamental to many physical and chemical processes, and understanding the mechanisms and outcomes of energy transfer is crucial for various applications in physics and chemistry. Here, the rovibrational excitation of YO(X 2Σ+) molecules with the collision of Kr and Ne has been studied in the laser-ablation crossed beam and time-sliced ion velocity map imaging setup in combination with the resonance enhanced multiphoton ionization scheme. Significant changes in the angular distribution for different rovibrational excitations of YO molecules are observed with the collision of Kr. The sharp forward distribution for low rovibrational excitation of YO(v' = 0, 1) molecules suggest that the weak attractive potential between Kr and YO is dominant at large impact parameters. Comparatively, the strong sideway distribution for highly rovibrationally excited YO(v' = 1, 2, 3, and 5) is due to rainbow scattering from the stronger attractive potential of Kr···YO at relatively small impact parameters. The more isotropic angular distribution in the highly rovibrationally excited YO(v' = 11) indicates the formation of a short-lived complex. A change in the angular distribution of scattered YO with different rovibrational excitations was also observed in the collisions of Ne. For YO as a heteronuclear diatomic molecule, collisions of the Y- and the O-end of YO with rare gases would affect the contribution of inelastic processes at different impact parameters.

Quantum State-Resolved Nonadiabatic Dynamics of the H+NaF → Na+HF Reaction

The H + NaF reaction is investigated at the quantum state-resolved level using the time-dependent wave-packet method based on a set of accurate diabatic potential energy surfaces. Oscillatory structures in the total reaction probability indicate the presence of the short-lived intermediate complex, attributed to a shallow potential well and exothermicity. Ro-vibrational state-resolved integral cross sections reveal the inverted population distributions of the product. The HF product favors an angular distribution in the forward hemisphere of 30°–60° within the collision energy range from the threshold to 0.50 eV, which is related to the nonlinear approach of the H atom to the NaF molecule. Quantum generalized deflection functions show that the low-J partial waves contribute primarily to the backward scattering, while the high-J partial waves govern the forward scattering. The correlation between the partial wave J and the scattering angle ϑ proves that the reaction follows a predominant direct reaction mechanism.

J Coupling Constants of

Observing pyruvate metabolism in vivo has become a focal point of molecular magnetic resonance imaging. Signal amplification by reversible exchange (SABRE) has recently emerged as a versatile hyperpolarization technique. Tuning of the spin order transfer (SOT) in SABRE is challenging as the small 1H-13C J couplings, in the 13C-pyruvate case, result in SOT being not readily discernible. We demonstrate an experimental method using frequency-selective excitation of parahydrogen-derived polarization SOT sequence (SEPP-SPINEPT); its application led to up to 5700-fold 13C signal gain. In this way, we estimated the lifetime of two Ir-pyruvate SABRE complexes alongside the individual probing of eight small 1H-13C J couplings that connect the hydride protons in these complexes to 1- and 2-13C pyruvate spins, affording values between 0 and 2.69 Hz. Using electronic structure calculations, we define the low-energy structure of the corresponding complexes. Hence, this study demonstrates a novel approach to analyzing the spin topology of short-lived organometallic complexes.

Yeasts associated with mines on tree leaves in the urban areas.

Mines on tree leaves and undamaged leaves were studied to investigate yeast complexes in urban areas (Aesculus hippocastanum, miner - Cameraria ohridella; Betula verrucosa, miner - Caloptilia betulicola; Populus nigra, miner - Lithocolletis populifoliella; Quercus robur, miner - Tischeria companella; Salix caprea, miner - Trachys minuta; Syringa vulgaris, miner - Caloptilia syringella; Tilia cordata, miner - Phyllonorycter issikii; Ulmus laevis, miner - Carpatolechia fugitivella). The abundance and taxonomic structure of yeasts were studied using a surface plating method on solid media (GPY agar). Identification of yeast species was based on the ITS rDNA nucleotide sequence. The average abundance of yeasts during the first stages of mine formation in the internal tissues of leaves was 103cfu/g. After 23-25days, during the last stage of larval metamorphosis before mine destruction, the abundance of yeasts in the mines increased by two orders of magnitude to 105cfu/g. No significant differences were observed in the abundance of yeasts in mines formed by different insects on different trees. A total of twelve yeast species were observed. The fast-growing ascomycetous yeasts Hanseniaspora uvarum and H. occidentalis dominated the mines. On undamaged leaves, the basidiomycetous yeasts Papiliotrema flavescens and Rhodotorula mucilaginosa, typical in the phyllosphere, dominated. The opportunistic yeast Candida parapsilosis was detected in the yeast complexes of all mines examined and was not found on the surface of leaves. Comparison of the relative abundance of yeast species between the studied mines and undamaged leaves using principal component analysis showed that all studied yeast communities in the mines were significantly different from the epiphytic yeast complexes of the undamaged leaves. Thus, miners in urban environments provoke the formation of short-lived endophytic yeast complexes with high abundance of Hanseniaspora. For leaf miners, the yeasts serve primarily as a food source for insect larvae rich in vitamins and amino acids. The adult leaf miners, in turn, contribute to the reproduction of the yeasts and create favorable conditions for their development.

Time Evolution of Ultrasmall Gold Nanoparticle-Protein Interactions.

To date, much effort has been devoted toward the study of protein corona formation onto large gold nanoparticles (GNPs). However, the protein corona concept breaks down for GNPs in the ultrasmall size regime (<3 nm), and, as a result, our understanding of ultrasmall GNP (usGNP)-protein interactions remains incomplete. Herein, we used anionic usGNPs and six different proteins as model systems to systematically investigate usGNP-protein interactions, with particular focus on the time evolution and long-term behavior of complex formation. The different proteins comprised chymotrypsin (Cht), trypsin (Try), thrombin (Thr), serum albumin (HSA), cytochrome c (Cyt c), and factor XII (FXII). We used a range of biochemical and biophysical methods to estimate binding affinities, determine the effects of usGNPs on protein structure and function, assess the reversibility of any protein structural and functional changes, and evaluate usGNP-protein complex stability. Among the main findings, we observed that prolonged (24 h)─but not short-term (10 min)─interactions between proteins and usGNPs permanently altered protein function, including enzyme activities (Try, Thr, and FXIIa), peroxidase-like activity (Cyt c), and ligand-binding properties (HSA). Remarkably, this occurred without any large-scale loss of the native global conformation, implying time-dependent effects of usGNPs on local protein conformation or dynamics. We also found that both short-(10 min) and long-term (24 h) interactions between proteins and usGNPs yielded short-lived complexes, i.e., there was no time-dependent "hardening" of the interactions at the binding interface as usually seen with large GNPs. The present study increases our fundamental understanding of nano-bio interactions in the ultrasmall size regime, which may assist the safe and effective translation of usGNPs into the clinic.

Visualizing the coordination of apurinic/apyrimidinic endonuclease (APE1) and DNA polymerase β during base excision repair

Base Excision Repair (BER) is carried out by a series of proteins that function in a step-by-step process to identify, remove, and replace DNA damage. During BER, the DNA transitions through various intermediate states as it is processed by each DNA repair enzyme. Left unrepaired, these BER intermediates can transition into double-stranded DNA breaks and promote genome instability. Previous studies have proposed a short-lived complex consisting of the BER intermediate, the incoming enzyme, and the outgoing enzyme at each step of the BER pathway to protect the BER intermediate. The transfer of BER intermediates between enzymes, known as BER coordination or substrate channeling, remains poorly understood. Here, we utilize single-molecule total internal reflection fluorescence (TIRF) microscopy to investigate the mechanism of BER coordination between apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase β (Pol β). When preformed complexes of APE1 and the incised abasic site product (APE1 product and Pol β substrate) were subsequently bound by Pol β, the Pol β enzyme dissociated shortly after binding in most of the observations. In the events where Pol β binding was followed by APE1 dissociation during substrate channeling, Pol β remained bound for a longer period of time to allow disassociation of APE1. Our results indicate that transfer of the BER intermediate from APE1 to Pol β during BER is dependent on the dissociation kinetics of APE1 and the duration of the ternary complex on the incised abasic site.

Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.

Imaging the Complex-Forming Reaction Dynamics in Al + CO2 → AlO + CO.

For indirect reactions involving more than one intermediate complex from reactant valley to product valley, the reaction dynamics becomes very complicated for researchers due to competition between pathways. In order to resolve the large discrepancy between theoretical and experimental studies on the linear or bent structures of complexes involved in the title endothermic reaction, we performed a crossed-beam experiment at a large collision energy (Ec) range with mapping of the velocity distributions of Al reactants and AlO products. We found that the reaction proceeds through different complex-forming mechanisms with the increase of Ec: at low Ec near the reaction threshold, only low impact-parameter collisions contribute through a collinear Al-OCO short-lived complex, and at high Ec, the bent-structure complexes, formed by either isomerization or noncollinear collisions, come into play.

Antiviral Activity of CRISPR/Cas9 Ribonucleoprotein Complexes on a Hepatitis B Virus Model In Vivo

Chronic hepatitis B (CHB) is caused by hepatitis B virus (HBV) infection. This disease is a key issue for global health. Modern methods of therapy do not completely eliminate HBV from infected cells and do not cure chronic infection. The CRISPR/Cas9 systems of site-specific nucleases can effectively cleave do not target DNA including viral genomes. The cleavage of the major form of the HBV genome, i.e., covalently closed circular DNA (cccDNA), leads to a robust reduction in viral replication and degradation or mutational inactivation of cccDNA. CRISPR/Cas9-based approaches are one of the most promising ways to achieve a 'sterilizing' cure of CHB, i.e., complete elimination of the virus from the body. Here, the HBV mouse model in vivo has been used to analyze the antiviral activity of the high-specific Cas9 protein and sgRNA targeting HBV genome. We have found that a single injection of short-lived ribonucleoprotein complexes of CRISPR/Cas9 results in a ~10-fold reduction in HBV DNA levels in the serum and liver of mice as early as 48 h after the start of the experiment. The remaining HBV DNAs have been found to harbor rare indel mutations. Developing new antivirals for treating CHB based on CRISPR/Cas9 ribonucleoprotein complexes could substantially reduce the duration of CHB therapy and, potentially, achieve complete elimination of viral infection.

Roaming dynamics of H+C2D2 reaction on fundamental-invariant neural network potential energy surface

We performed extensive quasiclassical trajectory calculations for the H+C2D2→HD+C2D/D2+C2H reaction based on a recently developed, global and accurate potential energy surface by the fundamental-invariant neural network method. The direct abstraction pathway plays a minor role in the overall reactivity, which can be negligible as compared with the roaming pathways. The acetylene-facilitated roaming pathway dominates the reactivity, with very small contributions from the vinylidene-facilitated roaming. Although the roaming pathways proceed via the short-lived or long-lived complex forming process, the computed branching ratio of product HD to D2 is not far away from 2:1, implying roaming dynamics for this reaction is mainly contributed from the long-lived complex-forming process. The resulting angular distributions for the two product channels are also quite different. These computational results give valuable insights into the significance and isotope effects of roaming dynamics in the biomolecular reactions.

The remarkable effects of a ligand nitro substituent in organoplatinum chemistry related to activation of dioxygen or reductive elimination of methane

A comparison is made of the chemistry of platinum with the ligands 2-C5H4NCH2NH-C6H3-2-OH-5-R, when R = H (L) or NO2 (L1) in order to determine the effect of the nitro substituent in L1. With inorganic platinum(II) complexes, L1 acts as a bidentate ligand in [PtCl(SMe2)(κ2-N,N′-L1)]Cl or as a monodentate ligand in trans-[PtI2(SMe2)(κ 1-N-L1)] and, with a methylplatinum unit, it forms the zwitterionic complex [PtMe(SMe2)(κ 2-N,N′-L1-H)], which contains an uncoordinated phenoxide substituent. The reactive dimethylplatinum complex in acetone solution reacts with dioxygen to give the hydroxoplatinum(IV) complex [Pt(OH)Me2(κ 3-N,N′,O-L1-H)], in which the deprotonated L1 acts as a fac-tridentate ligand. The dimethylplatinum complex in CD2Cl2 solution actually exists as a mixture of the three possible fac-hydridodimethylplatinum(IV) isomers [PtHMe2(κ 3-N,N′,O-L1-H)], which slowly undergo reductive elimination of methane or, in the presence of excess D2O, a mixture of isotopomers CHnD4-n. It is proposed that the isomers [PtHMe2(κ 3-N,N′,O-L1-H)] interconvert via shortlived methane complex intermediates [PtMe(CH4)(κ 3-N,N′,O-L1-H)]. No similar reactivity is observed with the ligand L, thus demonstrating the remarkable effect of the nitro substituent.

Imaging Membrane Order and Dynamic Interactions in Living Cells with a DNA Zipper Probe.

The cell membrane is a dynamic and heterogeneous structure composed of distinct sub-compartments. Within these compartments, preferential interactions occur among various lipids and proteins. Currently, it is still challenging to image these short-lived membrane complexes, especially in living cells. In this work, we present a DNA-based probe, termed "DNA Zipper", which allows the membrane order and pattern of transient interactions to be imaged in living cells using standard fluorescence microscopes. By fine-tuning the length and binding affinity of DNA duplex, these probes can precisely extend the duration of membrane lipid interactions via dynamic DNA hybridization. The correlation between membrane order and the activation of T-cell receptor signaling has also been studied. These programmable DNA probes function after a brief cell incubation, which can be easily adapted to study lipid interactions and membrane order during different membrane signaling events.

Diffusive-like redistribution in state-changing collisions between Rydberg atoms and ground state atoms

Exploring the dynamics of inelastic and reactive collisions on the quantum level is a fundamental goal in quantum chemistry. Such collisions are of particular importance in connection with Rydberg atoms in dense environments since they may considerably influence both the lifetime and the quantum state of the scattered Rydberg atoms. Here, we report on the study of state-changing collisions between Rydberg atoms and ground state atoms. We employ high-resolution momentum spectroscopy to identify the final states. In contrast to previous studies, we find that the outcome of such collisions is not limited to a single hydrogenic manifold. We observe a redistribution of population over a wide range of final states. We also find that even the decay to states with the same angular momentum quantum number as the initial state, but different principal quantum number is possible. We model the underlying physical process in the framework of a short-lived Rydberg quasi-molecular complex, where a charge exchange process gives rise to an oscillating electric field that causes transitions within the Rydberg manifold. The distribution of final states shows a diffusive-like behavior.

The disordered DNA-binding domain of p53 is indispensable for forming an encounter complex to and jumping along DNA

The tumor suppressor p53 utilizes a facilitated diffusion mechanism to search for and bind to target DNA sequences. Sub-millisecond single-molecule fluorescence tracking demonstrated that p53 forms a short-lived encounter complex to DNA then converts to the long-lived complex that can move and jump along DNA during the target search. To reveal the role of each DNA-binding domain of p53 in these processes, we investigated two p53 mutants lacking either of two DNA-binding domains; structured core and disordered C-terminal domains, using sub-millisecond single-molecule fluorescence microscopy. We found that the C-terminal domain is required for the encounter complex formation and conversion to the long-lived complex. The long-lived complex is stabilized by the core domain as well as the C-terminal domain. Furthermore, only the C-terminal domain participates in the jump of p53 along DNA at a high salt concentration. We propose that the flexible C-terminal domain of p53 is twined around DNA, which can form the encounter complex, convert to the long-lived complex, and enable p53 to land on DNA after the jump.

Current Methods for Detecting Cell Membrane Transient Interactions.

Short-lived cell membrane complexes play a key role in regulating cell signaling and communication. Many of these complexes are formed based on low-affinity and transient interactions among various lipids and proteins. New techniques have emerged to study these previously overlooked membrane transient interactions. Exciting functions of these transient interactions have been discovered in cellular events such as immune signaling, host–pathogen interactions, and diseases such as cancer. In this review, we have summarized current experimental methods that allow us to detect and analyze short-lived cell membrane protein–protein, lipid–protein, and lipid–lipid interactions. These methods can provide useful information about the strengths, kinetics, and/or spatial patterns of membrane transient interactions. However, each method also has its own limitations. We hope this review can be used as a guideline to help the audience to choose proper approaches for studying membrane transient interactions in different membrane trafficking and cell signaling events.

On Complex Formation between 5-Fluorouracil and β-Cyclodextrin in Solution and in the Solid State: IR Markers and Detection of Short-Lived Complexes by Diffusion NMR.

In this work, the nuclear magnetic resonance (NMR) and IR spectroscopic markers of the complexation between 5-fluorouracil (5-FU) and β-cyclodextrin (β-CD) in solid state and in aqueous solution are investigated. In the attenuated total reflectance(ATR) spectra of 5-FU/β-CD products obtained by physical mixing, kneading and co-precipitation, we have identified the two most promising marker bands that could be used to detect complex formations: the C=O and C-F stretching bands of 5-FU that experience a blue shift by ca. 8 and 2 cm−1 upon complexation. The aqueous solutions were studied by NMR spectroscopy. As routine NMR spectra did not show any signs of complexation, we have analyzed the diffusion attenuation of spin–echo signals and the dependence of the population factor of slowly diffusing components on the diffusion time (diffusion NMR of pulsed-field gradient (PFG) NMR). The analysis has revealed that, at each moment, ~60% of 5-FU molecules form a complex with β-CD and its lifetime is ca. 13.5 ms. It is likely to be an inclusion complex, judging from the independence of the diffusion coefficient of β-CD on complexation. The obtained results could be important for future attempts of finding better methods of targeted anticancer drug delivery.

Reaction between CH&amp;lt;sub&amp;gt;3&amp;lt;/sub&amp;gt;C(O)OOH (peracetic acid) and OH in the gas phase: a combined experimental and theoretical study of the kinetics and mechanism

Abstract. Peracetic acid (CH3C(O)OOH) is one of the most abundant organic peroxides in the atmosphere; yet the kinetics of its reaction with OH, believed to be the major sink, have only been studied once experimentally. In this work we combine a pulsed-laser photolysis kinetic study of the title reaction with theoretical calculations of the rate coefficient and mechanism. We demonstrate that the rate coefficient is orders of magnitude lower than previously determined, with an experimentally derived upper limit of 4×10-14 cm3 molec.−1 s−1. The relatively low rate coefficient is in good agreement with the theoretical result of 3×10-14 cm3 molec.−1 s−1 at 298 K, increasing to ∼6×10-14 cm3 molec.−1 s−1 in the cold upper troposphere but with associated uncertainty of a factor of 2. The reaction proceeds mainly via abstraction of the peroxidic hydrogen via a relatively weakly bonded and short-lived prereaction complex, in which H abstraction occurs only slowly due to a high barrier and low tunnelling probabilities. Our results imply that the lifetime of CH3C(O)OOH with respect to OH-initiated degradation in the atmosphere is of the order of 1 year (not days as previously believed) and that its major sink in the free and upper troposphere is likely to be photolysis, with deposition important in the boundary layer.