Structural mechanism of the dynein power stroke.

Dyneins are large microtubule motor proteins required for mitosis, intracellular transport and ciliary and flagellar motility. They generate force through a power-stroke mechanism, which is an ATP-consuming cycle of pre- and post-power-stroke conformational changes that cause relative motion between different dynein domains. However, key structural details of dynein's force generation remain elusive. Here, using cryo-electron tomography of intact, active (that is, beating), rapidly frozen sea urchin sperm flagella, we determined the in situ three-dimensional structures of all domains of both pre- and post-power-stroke dynein, including the previously unresolved linker and stalk of pre-power-stroke dynein. Our results reveal that the rotation of the head relative to the linker is the key action in dynein movement, and that there are at least two distinct pre-power-stroke conformations: pre-I (microtubule-detached) and pre-II (microtubule-bound). We provide three-dimensional reconstructions of native dyneins in three conformational states, in situ, allowing us to propose a molecular model of the structural cycle underlying dynein movement.

Proton radiography in three dimensions: a proof of principle of a new technique.

PURPOSE: Monte Carlo simulations were used to investigate a range of phantom configurations to establish enabling three-dimensional proton radiographic techniques. METHODS: A large parameter space of stacked phantom geometries composed of tissue inhomogeneity materials such as lung, bone, and cartilage inserted within water background were simulated using a purposefully modified version of TOPAS, an application running on top of the GEANT4 Monte Carlo code. The phantoms were grouped in two classes, one with the inhomogeneity inserted only half-way in the lateral direction and another with complete inhomogeneity insertion. The former class was used to calculate the track count and the energy fluence of the protons as they exit the phantoms either having traversed the inhomogeneity or not. The latter class was used to calculate one yield value accounting for loss of protons due to physical processes only and another yield value accounting for deliberately discarded protons due to large scattering angles. A graphical fingerprinting method was developed to determine the inhomogeneity thickness and location within the phantom based on track count and energy fluence information. Two additional yield values extended this method to the general case which also determines the inhomogeneity material and the phantom thickness. RESULTS: The graphical fingerprinting method was manually validated for two, and automatically tested for all, tissue materials using an exhaustive set of inhomogeneity geometries for 16 cm thick phantoms. Unique recognition of test phantom configurations was achieved in the large majority of cases. The method in the general case was further tested using an exhaustive set of inhomogeneity and phantom tissues and geometries where the phantom thicknesses ranged between 8 and 24 cm. Unique recognition of the test phantom configurations was achieved only for part of the phantom parameter space. The correlations between the remaining false positive recognitions were analyzed. CONCLUSIONS: The concept of 3D proton radiography for tissue inhomogeneities of simple geometries was established with the current work. In contrast to conventional 2D proton radiography, the main objective of the demonstrated 3D technique is not proton range. Rather, it is to measure the depth and thickness of an inhomogeneity located in an imaged geometry. Further work is needed to extend and apply the method to more complex geometries.

The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella.

Cilia and flagella are highly conserved microtubule (MT)-based organelles with motile and sensory functions, and ciliary defects have been linked to several human diseases. The 9 + 2 structure of motile axonemes contains nine MT doublets interconnected by nexin links, which surround a central pair of singlet MTs. Motility is generated by the orchestrated activity of thousands of dynein motors, which drive interdoublet sliding. A key regulator of motor activity is the dynein regulatory complex (DRC), but detailed structural information is lacking. Using cryoelectron tomography of wild-type and mutant axonemes from Chlamydomonas reinhardtii, we visualized the DRC in situ at molecular resolution. We present the three-dimensional structure of the DRC, including a model for its subunit organization and intermolecular connections that establish the DRC as a major regulatory node. We further demonstrate that the DRC is the nexin link, which is thought to be critical for the generation of axonemal bending.

Photoinduced charge separation in pyrenedicarboxamide-linked DNA hairpins.

The synthesis and photophysical properties of the dihydroxypropylamide derivative of pyrene-1,6-dicaboxamide, its aniline dyad, and DNA conjugates are reported. The dicarboxamide serves as a hairpin linker for bis(oligonucleotide) conjugates having short base pair stems. The dihydroxypropyl derivative has a large fluorescence quantum yield and long singlet decay time, as determined by fluorescence and time-resolved broad band pump-probe spectroscopy. The aniline dyad undergoes exergonic charge separation with formation of a radical ion pair which decays via charge recombination. The highly characteristic transient absorption spectrum of the pyrene anion radical is used to monitor the dynamics of its formation and decay. The dicarboxamide-linked hairpin conjugates undergo charge separation with adjacent guanine and adenine bases. Charge separation with guanine is accompanied by efficient pyrene fluorescence quenching. In contrast, reversible charge separation with adenine results in multiple exponential fluorescence decay. The energetics and dynamics of charge separation are compared with those of other arenedicarboxamide DNA hairpin linkers.

Getting to guanine: mechanism and dynamics of charge separation and charge recombination in DNA revisited.

The mechanism and dynamics of charge separation and charge recombination in synthetic DNA hairpins possessing a stilbenedicarboxamide linker and a single guanine-cytosine base pair have been reinvestigated. The combination of femtosecond broad-band pump probe spectroscopy, nanosecond transient absorption experiments, and picosecond fluorescence decay measurements permits analysis of the formation and decay of the stilbene anion radical. Reversible hole injection resulting in the formation of the stilbene-adenine contact radical ion pair is found to occur on the picosecond time scale. The mechanism for charge separation across two or more base pairs is revised from single step superexchange to a multi-step process: hole injection followed by hole transport and hole trapping. The mechanism of charge recombination remains assigned to a superexchange process.

Reversible bridge-mediated excited-state symmetry breaking in stilbene-linked DNA dumbbells.

The excited-state behavior of synthetic DNA dumbbells possessing stilbenedicarboxamide (Sa) linkers separated by short A-tracts or alternating A-T base-pair sequences has been investigated by means of fluorescence and transient absorption spectroscopy. Electronic excitation of the Sa chromophores results in conversion of a locally excited state to a charge-separated state in which one Sa is reduced and the other is oxidized. This symmetry-breaking process occurs exclusively via a multistep mechanism-hole injection followed by hole transport and hole trapping-even at short distances. Rate constants for charge separation are strongly distance-dependent at short distances but become less so at longer distances. Disruption of the A-tract by inversion of a single A-T base pair results in a pronounced decrease in both the rate constant and efficiency of charge separation. Hole trapping by Sa is highly reversible, resulting in rapid charge recombination that occurs via the reverse of the charge separation process: hole detrapping, hole transport, and charge return to regenerate the locally excited Sa singlet state. These results differ in several significant respects from those previously reported for guanine or stilbenediether as hole traps. Neither charge separation nor charge recombination occur via a single-step superexchange mechanism, and hole trapping is slower and detrapping faster when Sa serves as the electron donor. Both the occurrence of symmetry breaking and reversible hole trapping by a shallow trap in a DNA-based system are without precedent.

Ultrafast energy delocalization and electron transfer dynamics in 2-aminopurine-containing trinucleotides.

The fate of electronically excited states in DNA base stacks is of tremendous importance for subsequent photochemical damage reactions in the genome. In this study we present a femtosecond broadband pump-probe study on the adenine isomer 2-aminopurine (Ap) incorporated into trinucleotides. After selective excitation of Ap we can monitor energy delocalization between neighboring Ap moieties as well as excited state electron transfer, depending on the sequence of the trinucleotide. Our results establish the time scale for intrastand excimer formation and reveal the lifetime of the excimer state.

Electronic energy delocalization and dissipation in single- and double-stranded DNA.

The mechanism that nature applies to dissipate excess energy from solar UV light absorption in DNA is fundamental, because its efficiency determines the vulnerability of all genetic material to photodamage and subsequent mutations. Using femtosecond time-resolved broadband spectroscopy, we have traced the electronic excitation in both time and space along the base stack in a series of single-stranded and double-stranded DNA oligonucleotides. The obtained results demonstrate not only the presence of delocalized electronic domains (excitons) as a result of UV light absorption, but also reveal the spatial extent of the excitons.

Base pair motions control the rates and distance dependencies of reductive and oxidative DNA charge transfer.

In 1999, Wan et al. [Proc. Natl. Acad. Sci. USA 96, 6014-6019] published a pioneering paper that established the entanglement between DNA base pair motions and the transfer time of the charge carrier. The DNA assemblies contained an ethidium covalently bound via a flexible alkyl chain to the 5' hydroxyl group of the DNA backbone. Although covalently attached, the loose way in which the ethidium was linked to DNA allowed for large degrees of conformational freedom and thus raised some concern with respect to conformational inhomogeneity. In this letter, we report studies on a different set of ethidium DNA conjugates. In contrast to the "Caltech systems," these conjugates contain ethidium tightly incorporated (as a base pair surrogate) into the DNA base stack, opposite to an abasic site analog. Despite the tight binding, we found that charge transfer from the photoexcited ethidium base pair surrogate across two or more base pairs is several orders of magnitude slower than in case of the DNA systems bearing the tethered ethidium. To further broaden the scope of this account, we compared (oxidative) electron hole transfer and (reductive) electron transfer using the same ethidium chromophore as a charge donor in combination with two different charge acceptors. We found that both electron and hole transfer are characterized by similar rates and distance dependencies. The results demonstrate the importance of nuclear motions and conformational flexibility and underline the presence of a base gating mechanism, which appears to be generic to electronic transfer processes through pi-stacked nucleic acids.

Dynamics and mechanism of bridge-dependent charge separation in pyrenylurea-nitrobenzene pi-stacked protophanes.

Herein are reported the synthesis, structure, and electronic properties of a series of tertiary di- and polyarylureas possessing pyrene and nitrobenzene end groups separated by a variable number of internal phenylenediamine bridging groups. These molecules adopt folded "protophane" structures in which the adjacent arenes are loosely pi-stacked. The behavior of both the pyrene and nitrobenzene singlet states has been investigated by means of femtosecond broadband pump-probe spectroscopy, and the transients have been assigned on the basis of comparison to reference molecules. Femtosecond time resolution permits direct observation of the fast internal conversion process for both the pyrene and nitrobenzene upper singlet states, as well as the intersystem crossing of nitrobenzene. The ultrafast (ca. 100 fs) charge separation of the donor-acceptor urea having no bridging group is attributed to an internal conversion process. The slower charge separation and charge recombination of the donor-acceptor urea having a single bridging group occur via a bridge-mediated superexchange process. Addition of a second bridging unit results in a role reversal for the pyrene singlet state, which now serves as an excited-state acceptor with the bridging units serving as the electron donors. The change in the directionality of electron transfer upon addition of a second bridging phenylenediamine is a consequence of a decrease in the bridge oxidation potential as well as a decrease in the rate constant for single-step superexchange electron transfer.

Crossover from superexchange to hopping as the mechanism for photoinduced charge transfer in DNA hairpin conjugates.

The mechanism and dynamics of photoinduced charge separation and charge recombination have been investigated in synthetic DNA hairpins possessing donor and acceptor stilbenes separated by one to seven A:T base pairs. The application of femtosecond broadband pump-probe spectroscopy, nanosecond transient absorption spectroscopy, and picosecond fluorescence decay measurements permits detailed analysis of the formation and decay of the stilbene acceptor singlet state and of the charge-separated intermediates. When the donor and acceptor are separated by a single A:T base pair, charge separation occurs via a single-step superexchange mechanism. However, when the donor and acceptor are separated by two or more A:T base pairs, charge separation occurs via a multistep process consisting of hole injection, hole transport, and hole trapping. In such cases, hole arrival at the electron donor is slower than hole injection into the bridging A-tract. Rate constants for charge separation (hole arrival) and charge recombination are dependent upon the donor-acceptor distance; however, the rate constant for hole injection is independent of the donor-acceptor distance. The observation of crossover from a superexchange to a hopping mechanism provides a "missing link" in the analysis of DNA electron transfer and requires reevaluation of the existing literature for photoinduced electron transfer in DNA.

Ultrafast proton-coupled electron-transfer dynamics in pyrene-modified pyrimidine nucleosides: model studies towards an understanding of reductive electron transport in DNA.

5-(Pyren-1-yl)-2'-deoxyuridine (PydU) and 5-(Pyren-1-yl)-2'-deoxycytidine (PydC) were used as model nucleosides for DNA-mediated reductive electron transport (ET) in steady-state fluorescence and femtosecond time-resolved transient absorption spectroscopy studies. Excitation of the pyrene moiety in PydU and PydC leads to an intramolecular electron transfer that yields the pyrenyl radical cation and the corresponding pyrimidine radical anion (dU.- and dC.-. By comparing the excited state dynamics of PydC and PydU, we derived information about the energy difference between the two pyrimidine radical anion states. To determine the influence of protonation on the rates of photoinduced intramolecular ET, the spectroscopic investigations were performed in acetonitrile, MeCN, and in water at different pH values. The results show a significant difference in the basicity of the generated pyrimidine radical anions and imply an involvement of proton transfer during electron hopping in DNA. Our studies revealed that the radical anion dC.- is being protonated even in basic aqueous solution on a picosecond time scale (or faster). These results suggest that protonation of dC.- may also occur in DNA. In contrast, efficient ET in PydU could only be observed at low pH values (< 5). In conclusion, we propose--based on the free energy differences and the different basicities--that only dT.- but not dC.- can participate as an intermediate charge carrier for excess electron migration in DNA.