Lifetime Of Intermediate Research Articles

Phototriggered Release of a Leaving Group in Ketoprofen Derivatives via a Benzylic Carbanion Pathway, But not via a Biradical Pathway

2-Acetoxymethyl-2-(3-benzoylphenyl)propionic acid (KP-OAc) was used as a model to elucidate the solvent-mediated photochemistry mechanism of Ketoprofen (KP). In solutions with a low concentration of water, KP-OAc exhibits a benzophenone-like photochemistry, reacting with water molecules through some reaction to form a ketyl radical intermediate. In neutral solutions with a high concentration of water or acidic solutions, KP-OAc undergoes a photodecarboxylation reaction with the assistance of water molecules or with the catalysis of perchloric acid to directly generate a biradical intermediate that cannot induce the phototrigger reaction to release the AcO(-) group. Therefore, the lifetime of the biradical intermediate of KP-OAc is almost same as that of the biradical intermediate formed from KP in the same kinds of solutions. However, the photodecarboxylation of KP-OAc in phosphate buffer solution directly produces the benzylic carbanion intermediate, which can induce the phototrigger reaction to release the AcO(-) group. Therefore, the lifetime of the biradical intermediate of KP-OAc is significantly shorter than the lifetime of the biradical intermediate of KP in phosphate buffer solution. Interestingly, the investigation of the photochemistry of KP-OAc not only verifies the solvent-mediated photochemistry mechanism of KP but also provides some new insight into the potential of using this kind of platform for phototrigger applications. The biradical intermediate is not the key species leading to the phototrigger reaction but the benzylic carbanion species is the key reactive intermediate that can mediate the phototrigger reaction of KP-OAc. Therefore, a change in the pH of the solutions can be utilized to switch on and switch off the photorelease reactions of KP derivative phototrigger compounds.

The EF Loop in Green Proteorhodopsin Affects Conformation and Photocycle dynamics

The proteorhodopsin family consists of retinal proteins of marine bacterial origin with optical properties adjusted to their local environments. For green proteorhodopsin, a highly specific mutation in the EF loop, A178R, has been found to cause a surprisingly large redshift of 20 nm despite its distance from the chromophore. Here, we analyze structural and functional consequences of this EF loop mutation by time-resolved optical spectroscopy and solid-state NMR. We found that the primary photoreaction and the formation of the K-like photo intermediate is almost pH-independent and slower compared to the wild-type, whereas the decay of the K-intermediate is accelerated, suggesting structural changes within the counterion complex upon mutation. The photocycle is significantly elongated mainly due to an enlarged lifetime of late photo intermediates. Multidimensional MAS-NMR reveals mutation-induced chemical shift changes propagating from the EF loop to the chromophore binding pocket, whereas dynamic nuclear polarization-enhanced 13C-double quantum MAS-NMR has been used to probe directly the retinylidene conformation. Our data show a modified interaction network between chromophore, Schiff base, and counterion complex explaining the altered optical and kinetic properties. In particular, the mutation-induced distorted structure in the EF loop weakens interactions, which help reorienting helix F during the reprotonation step explaining the slower photocycle. These data lead to the conclusion that the EF loop plays an important role in proton uptake from the cytoplasm but our data also reveal a clear interaction pathway between the EF loop and retinal binding pocket, which might be an evolutionary conserved communication pathway in retinal proteins.

Sub-Inhibitory Concentrations of Human α-defensin Potentiate Neutralizing Antibodies against HIV-1 gp41 Pre-Hairpin Intermediates in the Presence of Serum

Human defensins are at the forefront of the host responses to HIV and other pathogens in mucosal tissues. However, their ability to inactivate HIV in the bloodstream has been questioned due to the antagonistic effect of serum. In this study, we have examined the effect of sub-inhibitory concentrations of human α-defensin HNP-1 on the kinetics of early steps of fusion between HIV-1 and target cells in the presence of serum. Direct measurements of HIV-cell fusion using an enzymatic assay revealed that, in spite of the modest effect on the extent of fusion, HNP-1 prolonged the exposure of functionally important transitional epitopes of HIV-1 gp41 on the cell surface. The increased lifetime of gp41 intermediates in the presence of defensin was caused by a delay in the post-coreceptor binding steps of HIV-1 entry that correlated with the marked enhancement of the virus' sensitivity to neutralizing anti-gp41 antibodies. By contrast, the activity of antibodies to gp120 was not affected. HNP-1 appeared to specifically potentiate antibodies and peptides targeting the first heptad repeat domain of gp41, while its effect on inhibitors and antibodies to other gp41 domains was less prominent. Sub-inhibitory concentrations of HNP-1 also promoted inhibition of HIV-1 entry into peripheral blood mononuclear cells by antibodies and, more importantly, by HIV-1 immune serum. Our findings demonstrate that: (i) sub-inhibitory doses of HNP-1 potently enhance the activity of a number of anti-gp41 antibodies and peptide inhibitors, apparently by prolonging the lifetime of gp41 intermediates; and (ii) the efficiency of HIV-1 fusion inhibitors and neutralizing antibodies is kinetically restricted. This study thus reveals an important role of α-defensin in enhancing adaptive immune responses to HIV-1 infection and suggests future strategies to augment these responses.

Anionic Lipids Are Required for Vesicular Stomatitis Virus G Protein-mediated Single Particle Fusion with Supported Lipid Bilayers

Viral glycoproteins mediate fusion between viral and cellular membranes upon binding to cognate receptors and/or experiencing low pH. Although activation of viral glycoproteins is thought to be necessary and sufficient for fusion, accumulating evidence suggests that additional cellular factors, including lipids, can modulate the fusion process. Understanding the role of lipids in virus entry via endocytosis is impeded by poor accessibility and the highly diverse nature of endosomes. Here we imaged fusion of single retroviral particles pseudotyped with the vesicular stomatitis virus (VSV) G protein with dextran-supported lipid bilayers. Incorporation of diffusible fluorescent labels into the viral membrane and the viral interior enabled detection of the lipid mixing (hemifusion) and content transfer (full fusion) steps of VSV G-mediated fusion at low pH. Although single virus fusion with supported bilayers made of zwitterionic lipids could not be detected, inclusion of anionic lipids, phosphatidylserine, and bis(monoacylglycero)phosphate (BMP), greatly enhanced the efficiency of hemifusion and permitted full fusion. Importantly, lipid mixing always preceded the opening of a fusion pore, demonstrating that VSV G-mediated fusion proceeds through a long-lived hemifusion intermediate. Kinetic analysis of lipid and content transfer showed that the lags between lipid and content mixing defining the lifetime of a hemifusion intermediate were significantly shorter for BMP-containing compared with PS-containing bilayers. The strong fusion-enhancing effect of BMP, a late endosome-resident lipid, is consistent with the model that VSV initiates fusion in early endosomes but releases its core into the cytosol after reaching late endosomal compartments.

Single-molecule DNA repair in live bacteria

Cellular DNA damage is reversed by balanced repair pathways that avoid accumulation of toxic intermediates. Despite their importance, the organization of DNA repair pathways and the function of repair enzymes in vivo have remained unclear because of the inability to directly observe individual reactions in living cells. Here, we used photoactivation, localization, and tracking in live Escherichia coli to directly visualize single fluorescent labeled DNA polymerase I (Pol) and ligase (Lig) molecules searching for DNA gaps and nicks, performing transient reactions, and releasing their products. Our general approach provides enzymatic rates and copy numbers, substrate-search times, diffusion characteristics, and the spatial distribution of reaction sites, at the single-cell level, all in one measurement. Single repair events last 2.1 s (Pol) and 2.5 s (Lig), respectively. Pol and Lig activities increased fivefold over the basal level within minutes of DNA methylation damage; their rates were limited by upstream base excision repair pathway steps. Pol and Lig spent >80% of their time searching for free substrates, thereby minimizing both the number and lifetime of toxic repair intermediates. We integrated these single-molecule observations to generate a quantitative, systems-level description of a model repair pathway in vivo.

Effect of coordination ability of axial ligands on the stability and catalytic activity of chloro(tetramesitylporphyrinato)manganese(III) and lifetime of Mn-oxo intermediate

Some nitrogen donors, acetate and bromide ions were used as co-catalyst in the epoxidation reaction of cis-stilbene by meta-chloroperbenzoic acid catalyzed by chloro(tetramesitylporphyrinato)manganese(III). Cyclic voltammetry was used for comparison of coordination ability of these axial ligands. Stability of manganese porphyrin complex and lifetime of Mn-oxo intermediate species were compared using UV–vis spectroscopy in the presence of these axial ligands. It was shown that increasing coordination ability of these axial ligands has positive effects on the stability of the catalyst and also on the stability of Mn-oxo intermediate species but has a negative effect on the catalytic activity of the manganese porphyrin complex.

Deuterium isotope effects in the polyatomic reaction of O(1D2) + CH4 → OH + CH3

The scattering distributions of state-selected CH3 products are measured for the O((1)D2) reaction with CH4 using a crossed molecular beam ion imaging method at collision energies of 0.9-6.8 kcal mol(-1). The results are compared with the reaction with CD4 to examine the isotope effects. The scattering distributions exhibit contributions from both the insertion and abstraction pathways, respectively, on the ground- and excited-state potential energy surfaces. Insertion is the main pathway, and it provides a strongly forward-enhanced angular distribution of methyl radicals. Abstraction is a minor pathway, causing backward scattering of methyl radicals with a discrete speed distribution. From the collision energy dependence of the abstraction/insertion ratio, the barrier height for the abstraction pathway is estimated to be 0.7 ± 0.3 and 0.8 ± 0.1 kcal mol(-1) for O((1)D2) with CH4 and CD4, respectively. The insertion pathway of the O((1)D2) reaction with CH4 has a narrower angular width in the forward scattering and a larger insertion/abstraction ratio than the reaction with CD4, which indicates that the insertion reaction with CH4 has a larger cross section and a shorter reaction time than the reaction with CD4. Additionally, while the insertion reaction with CD4 exhibits strong angular dependence of the CD3 speed distribution, CH3 exhibits considerably smaller dependence. The result suggests that, although intramolecular vibrational redistribution (IVR) within the lifetime of the methanol intermediate is restrictive in both isotopomers, relatively more extensive IVR occurs in CD3OD than CH3OH, presumably due to the higher vibrational state density.

Controlled tin catalyzed hydrolysis of 3-acryloxypropyltrimethoxysilane with mono- and multi-functional mercaptans

Mechanistic investigations were initiated to further our understanding of the reaction pathways for hydrolysis and condensation of organofunctional alkoxysilanes in organic media. Previous work has shown that addition of mercaptans to solutions that contain dibutyltin dilaurate (DBTDL), which functions as a catalyst, and 3-acryloxypropyltrimethoxysilane can greatly reduce the rate of silane hydrolysis and condensation. Gas chromatography/mass spectrometry (GC/MS) hydrolysis studies were carried out to understand how mercaptan structure and concentration affected the relative hydrolysis rate of the acrylate silane in solution. Detailed nuclear magnetic resonance spectroscopy (NMR) and electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry (MS) were carried out on simple mixtures of DBTDL and select mercaptans, and with varying mercaptan concentrations, to elucidate which significant tin complexes can form. These studies showed that mercaptans readily undergo ligand exchange reactions with the labile carboxylate groups of DBTDL and that an excess of mercaptans reduced the amount of proposed active catalyst species. It was found that an excess of mercaptan functional group was necessary to prevent significant and rapid hydrolysis of the organofunctional silane in organic media. A minimum of 1:4 DBTDL carboxylate group:mercaptan functional group was required for most mercaptans examined. A tetramercaptan, pentaerythritol tetrakis(3-mercaptopropionate), was shown to be effective in deactivating the DBTDL catalyst, even at relatively low concentrations of tetramercaptan. The findings in this work provide further support for the proposed mechanism for catalyzed hydrolysis and condensation. The tin metal was determined to be predominately 4-fold coordinated in the various tin mercaptide compounds studied. Evidence for transient tin hydroxide reactive intermediates was detected using mass spectrometry, as upon the addition of water, oligomeric condensation products were observed. However, the lifetime of these reactive intermediates precluded their direct observation by either mass spectrometry or NMR. Oligomeric and cyclic tin mercaptide species were shown to form when multifunctional mercaptans were combined with DBTDL in solution.

Effects of the frontier orbitals on the electrochemical and electrochemiluminescent properties of the bis-cyclometalated iridium(III) complexes with different ligands

The electrochemical and electrochemiluminescent (ECL) properties of six bis-cyclometalated iridium(III) complexes (ppy)2Ir(N-phenylacetamide) (1), (ppy)2Ir(N-phenylbenzamide) (2), (ppy)2Ir(N-naphthylbenzamide) (3), (ppy)2Ir(N-phenylmethacrylamide) (4), (pq)2Ir(N-phenylmethacrylamide) (5) and (pq)2Ir(acac) (6) were studied in acetonitrile, where ppy is phenylpyridine anion, pq is phenylquinoline anion and acac is acetylacetone anion. These complexes exhibited quasi-reversible one-electron oxidation waves with the oxidation potentials varied from 0.95 to 1.51V (vs. SCE) and the first irreversible reduction peaks fell into the range of −1.58 to −1.86V. The effects of the different ligands on the redox potentials and electrochemical reversibility were elucidated by the energies and compositions of the frontier orbitals. The relative ECL efficiencies of the complexes 1–6 were 0.032, 0.020, 0.014, 0.041, 0.11 and 26.0 respectively when referenced to Ru(bpy)32+ and the differences in ECL efficiency were rationalized by the density functional theory (DFT) calculations. The energy gap between the lowest unoccupied molecular orbital (LUMO) of anodic product (M+) and the deprotonated product (TPA) determined the electron transfer efficiency to generate the excited states. Other factors such as the lifetime of the radical intermediates, the oxidation potential gaps between the M and TPA, the amount of M+ generation and the luminescence efficiency (Φem) were also considered in comparison of different ECL systems.

A crossed beam study of 18O(3P)+NO2 and 18O(1D)+NO2: Isotope exchange and O2+NO formation channels

The products and dynamics of the reactions (18)O((3)P)+NO(2) and (18)O((1)D)+NO(2) have been investigated using crossed beams and provide new constraints on the structures and lifetimes of the reactive nitrogen trioxide intermediates formed in collisions of O((3)P) and O((1)D) with NO(2). For each reaction, two product channels are observed - isotope exchange and O(2)+NO formation. From the measured product signal intensities at collision energies of ∼6 to 9.5 kcal/mol, the branching ratio for O(2)+NO formation vs. isotope exchange for the O((3)P)+NO(2) reaction is 52(+6/-2)% to 48(+2/-6)%, while that for O((1)D)+NO(2) is 97(+2/-12)% to 3(+12/-2)%. The branching ratio for the O((3)P)+NO(2) reaction derived here is similar to the ratio measured in previous kinetics studies, while this is the first study in which the products of the O((1)D)+NO(2) reaction have been determined experimentally. Product energy and angular distributions are derived for the O((3)P)+NO(2) isotope exchange and the O((1)D)+NO(2)→O(2)+NO reactions. The results demonstrate that the O((3)P)+NO(2) isotope exchange reaction proceeds by an NO(3)∗ complex that is long-lived with respect to its rotational period and suggest that statistical incorporation of the reactant (18)O into the product NO(2) (apart from zero point energy isotope effects) likely occurs. In contrast, the (18)O((1)D)+NO(2)→O(2)+NO reaction proceeds by a direct "stripping" mechanism via a short-lived (18)O-O-NO∗ complex that results in the occurrence of (18)O in the product O(2) but not in the product NO. Similarly, (18)O is detected in O(2) but not NO for the O((3)P)+NO(2)→O(2)+NO reaction. Thus, even though the product energy and angular distributions for O((3)P)+NO(2)→O(2)+NO derived from the experimental data are uncertain, these results for isotope labeling under single collision conditions support previous kinetics studies that concluded that this reaction proceeds by an asymmetric (18)O-O-NO∗ intermediate and not by a long-lived symmetric NO(3)∗ complex, as earlier bulk isotope labeling experiments had concluded. Applicability of these results to atmospheric chemistry is also discussed.

Quasi-classical trajectory study of the H + CO2 → HO + CO reaction on a new ab initio based potential energy surface

A detailed quasi-classical trajectory study of the H + CO(2) → HO + CO reaction is reported on an accurate potential energy surface based on ab initio data. The influence of the vibrational and rotational excitations of CO(2) was investigated up to the collision energy of 2.35 eV. It was found that the total reaction integral cross section increases monotonically with the collision energy, consistent with experimental results. The excitation of the CO(2) bending vibration enhances the reaction, while the excitation in its asymmetric stretching vibration inhibits the reaction. The calculated thermal rate constants are in excellent agreement with experiment. At the state-to-state level, the rotational state distributions of the HO product are in good agreement with experimental results, while those for the CO product are much hotter than measurements. The calculated differential cross sections are dominated by forward scattering, suggesting that the lifetime of the HOCO intermediate may not be sufficiently long to render the reaction completely statistical.

'Self-healing' dyes: intramolecular stabilization of organic fluorophores

Response to NMETH-{type:entrez-nucleotide,attrs:{text:C14399,term_id:1569106,term_text:C14399}}C14399: A battery of new mechanistic investigations will be required to provide a full explanation of the observation that individual, small-molecule compounds, referred to here as 'protective agents', have the capacity to promote dramatic enhancements to overall fluorophore performance1. As Tinnefeld and Cordes suggest, cycles of reduction (red) and oxidation (ox) - and/or the reverse -may confer a 'self-healing' property to the fluorogenic center. This could indeed be achieved by a single neighboring molecule, like Trolox, facilitating the fluorophore's rapid return to the ground state from relatively long-lived, non-fluorescent, radical intermediates through 'ping-pong' red-ox chemistry. However, this model requires that the rates of red-ox cycling be properly matched for benefits to be achieved. Further insights into the prevalence and lifetimes of charged intermediates for each molecule will need to be quantified to validate this model. Alternative mechanisms for photostabilization, which may play a significant or even dominant role, will also need to be carefully considered and quantified. Previous investigations, largely motivated by the once vigorous dye-laser industry, suggest that meaningful contributions to fluorophore performance may arise from changes in the rates of internal conversion and/or the introduction of mechanisms for triplet-triplet energy transfer and exciplex-type relaxation pathways2. Triplet-triplet energy transfer, a mechanism that would also fall into the 'self-healing' category, could provide substantial benefits to fluorophore performance by reducing the lifetime of non-fluorescent triplet states from which photobleaching and/or radical states can occur. Given our estimates of the triplet energy and red-ox potential of cyclooctatetraene (unpublished data), this mechanism may potentially be the dominant pathway of cyanine fluorophore photostabilization. Triplet-triplet energy transfer can be differentiated from red-ox-type chemistries by quantifying changes in triplet state lifetimes and charged, intermediate species. Other pathways may also exist that are distinct from the 'self-healing' variety. For example, a 'self-protecting' mechanism that reduces the probability of transitions into damaged states in the first place would also give rise to enhancements in fluorophore performance. Single-molecule imaging efforts can play an important role in the characterization of fluorophore performance by giving direct access to the frequency and duration of fluorescent and non-fluorescent states as well as a fluorophore’s total photon output prior to blinking or photobleaching. However, a quantitative understanding of the distinct mechanisms for enhancing fluorophore performance can only be delineated with confidence when married with bulk electrochemical and spectroscopic investigations. These combined approaches, while requiring large quantities of material, could provide grounded insights into the impact and relative weighting of 'self-healing' and 'self-protecting' mechanisms for distinct fluorophore types. Progress on this front will enable both further improvements in the performances of known protective agents and the search for new compounds with similar or improved properties. Developments of this kind may ultimately facilitate the design and synthesis of new classes of next-generation fluorophores spanning the visible spectrum that exhibit enhancements in performance even greater than those observed for the cyanine class (see Correspondence by Altman et al) and tailored properties for distinct experimental demands.

Study of the antioxidant mechanisms of Trolox and eugenol with 2,2′-azobis(2-amidinepropane)dihydrochloride using ultra-high performance liquid chromatography coupled with tandem mass spectrometry

The study of antioxidant mechanisms is a difficult task that involves the monitoring and identification of unknown intermediate and final products. Most of the time, the lifetime of intermediates is too short to allow their isolation and subsequent identification by nuclear magnetic resonance (NMR). The developments of ultra-high performance liquid chromatography (UHPLC) coupled with the advances in the acquisition rates of mass spectrometry could facilitate the research on antioxidant mechanisms. This work is based on the reaction involved in the Oxygen Radical Antioxidant Capacity (ORAC) and Total Radical trapping Antioxidant Parameter (TRAP) assays. Hence, the reaction between 2,2'-azobis(2-amidinepropane)dihydrochloride (AAPH) radicals and an antioxidant was carried out in the thermostatized autosampler of a chromatographic device. Then, the reaction media were injected every six minutes, and the compounds were separated by UHPLC and detected by mass spectrometry in scan mode. Nine consecutive injections were registered in a unique file, then the evolution of the reaction for one hour in a single run was monitored. In this way, the reaction mechanisms of Trolox and eugenol with AAPH were studied, leading to the detection of nine and thirteen different compounds, respectively. An exhaustive analysis of the spectra obtained in product ion scan mode led to the identification of the compounds. Most of them were species previously found in the literature, but others have never been reported, so tentative structures were suggested. All this allowed the proposal of several steps within the antioxidant mechanisms of Trolox and eugenol, showing the great performance of UHPLC-MS/MS to complement the use of NMR in antioxidant mechanistic studies.

RNA molecules with conserved catalytic cores but variable peripheries fold along unique energetically optimized pathways

Functional and kinetic constraints must be efficiently balanced during the folding process of all biopolymers. To understand how homologous RNA molecules with different global architectures fold into a common core structure we determined, under identical conditions, the folding mechanisms of three phylogenetically divergent group I intron ribozymes. These ribozymes share a conserved functional core defined by topologically equivalent tertiary motifs but differ in their primary sequence, size, and structural complexity. Time-resolved hydroxyl radical probing of the backbone solvent accessible surface and catalytic activity measurements integrated with structural-kinetic modeling reveal that each ribozyme adopts a unique strategy to attain the conserved functional fold. The folding rates are not dictated by the size or the overall structural complexity, but rather by the strength of the constituent tertiary motifs which, in turn, govern the structure, stability, and lifetime of the folding intermediates. A fundamental general principle of RNA folding emerges from this study: The dominant folding flux always proceeds through an optimally structured kinetic intermediate that has sufficient stability to act as a nucleating scaffold while retaining enough conformational freedom to avoid kinetic trapping. Our results also suggest a potential role of naturally selected peripheral A-minor interactions in balancing RNA structural stability with folding efficiency.

Functional characterization of the human myosin-7a motor domain

Myosin-7a participates in auditory and visual processes. Defects in MYO7A, the gene encoding the myosin-7a heavy chain, are causative for Usher syndrome 1B, the most frequent cause of deaf-blindness in humans. In the present study, we performed a detailed kinetic and functional characterization of the isolated human myosin-7a motor domain to elucidate the details of chemomechanical coupling and the regulation of motor function. A rate-limiting, slow ADP release step causes long lifetimes of strong actin-binding intermediates and results in a high duty ratio. Moreover, our results reveal a Mg2+-sensitive regulatory mechanism tuning the kinetic and mechanical properties of the myosin-7a motor domain. We obtained direct evidence that changes in the concentration of free Mg2+ ions affect the motor properties of human myosin-7a using an in vitro motility assay system. Our results suggest that in a cellular environment, compartment-specific fluctuations in free Mg2+ ions can mediate the conditional switching of myosin-7a between cargo moving and tension bearing modes.

Photolysis dynamics of diastereoselectivity in the photocyclization of o-ethoxybenzophenone and o-2,2,2-trifluoroethoxybenzophenone

The photochemical reactions of o-ethoxybenzophenone (R-1) and o-2,2,2-trifluoroethoxybenzophenone (R-2) in cyclohexane solutions of 0.7M pyridine have been investigated using time-resolved laser flash photolysis to understand diastereoselectivity in the photocyclization of the two compounds. Whereas the formation times of biradical intermediates (7ns) are the same for the two compounds, the lifetime of the biradical intermediate of R-2 (200ns) is eight times larger than that of R-1 (25ns), explaining why diastereoselectivity is much lower in the photocyclization of R-2.

Observation of a chemically labile, noncovalent enzyme intermediate in the reaction of metal‐dependent Aquifex pyrophilus KDO8PS by time‐resolved mass spectrometry

The direct detection of intermediates in enzymatic reactions can yield important mechanistic insights but may be difficult due to short intermediate lifetimes and chemical instability. Using a rapid-mixing device coupled with electrospray ionization time-of-flight mass spectrometry, the noncovalent hemiketal intermediate in the reaction of metal-dependent 3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P) synthase from Aquifex pyrophilus was observed in the millisecond time range. Using single turnover conditions, the noncovalent complexes of enzyme with Cd(2+):phosphoenolpyruvate, Cd(2+):phosphate, Cd(2+):KDO8P, and Cd(2+):intermediate complexes were resolved. The intermediate complex is present during times ranging from 50-630 ms, indicating that the intermediate builds up at the ambient temperatures of the experiment. This represents the first direct detection of the intermediate with a native metal-dependent KDO8PS, and further demonstrates that time-resolved mass spectrometry is a useful tool in mechanistic studies of enzymatic reactions.

Transient tether between the SRP RNA and SRP receptor ensures efficient cargo delivery during cotranslational protein targeting

Kinetic control of macromolecular interactions plays key roles in biological regulation. An example of such control occurs in cotranslational protein targeting by the signal recognition particle (SRP), during which the SRP RNA and the cargo both accelerate complex assembly between the SRP and SRP receptor FtsY 10(2)-fold. The molecular mechanism underlying these rate accelerations was unclear. Here we show that a highly conserved basic residue, Lys399, on the lateral surface of FtsY provides a novel RNA tetraloop receptor to mediate the SRP RNA- and cargo-induced acceleration of SRP-FtsY complex assembly. We propose that the SRP RNA, by using its tetraloop to interact with FtsY-Lys399, provides a transient tether to stabilize the early stage and transition state of complex formation; this accelerates the assembly of a stable SRP-FtsY complex and allows the loading of cargo to be efficiently coupled to its membrane delivery. The use of a transient tether to increase the lifetime of collisional intermediates and reduce the dimension of diffusional search represents a novel and effective mechanism to accelerate macromolecular interactions.

Myosin complexed with ADP and blebbistatin reversibly adopts a conformation resembling the start point of the working stroke

The powerstroke of the myosin motor is the basis of cell division and bodily movement, but has eluded empirical description due to the short lifetime and low abundance of intermediates during force generation. To gain insight into this process, we used well-established single-tryptophan and pyrene fluorescent sensors and electron microscopy to characterize the structural and kinetic properties of myosin complexed with ADP and blebbistatin, a widely used inhibitor. We found that blebbistatin does not weaken the tight actin binding of myosin.ADP, but unexpectedly it induces lever priming, a process for which the gamma-phosphate of ATP (or its analog) had been thought necessary. The results indicate that a significant fraction of the myosin.ADP.blebbistatin complex populates a previously inaccessible conformation of myosin resembling the start of the powerstroke.

Caught in the act: the lifetime of synaptic intermediates during the search for homology on DNA

Homologous recombination plays pivotal roles in DNA repair and in the generation of genetic diversity. To locate homologous target sequences at which strand exchange can occur within a timescale that a cell’s biology demands, a single-stranded DNA-recombinase complex must search among a large number of sequences on a genome by forming synapses with chromosomal segments of DNA. A key element in the search is the time it takes for the two sequences of DNA to be compared, i.e. the synapse lifetime. Here, we visualize for the first time fluorescently tagged individual synapses formed by RecA, a prokaryotic recombinase, and measure their lifetime as a function of synapse length and differences in sequence between the participating DNAs. Surprisingly, lifetimes can be ∼10 s long when the DNAs are fully heterologous, and much longer for partial homology, consistently with ensemble FRET measurements. Synapse lifetime increases rapidly as the length of a region of full homology at either the 3′- or 5′-ends of the invading single-stranded DNA increases above 30 bases. A few mismatches can reduce dramatically the lifetime of synapses formed with nearly homologous DNAs. These results suggest the need for facilitated homology search mechanisms to locate homology successfully within the timescales observed in vivo.