Recoil-ion momentum distributions for two-photon double ionization of He and Ne by 44 eV free-electron laser radiation.

Recoil-ion momentum distributions for two-photon double ionization of He and Ne (variant Planck's over omega=44 eV) have been recorded with a reaction microscope at FLASH (the free-electron laser at Hamburg) at an intensity of approximately 1 x 10(14) W/cm2 exploring the dynamics of the two fundamental two-photon-two-electron reaction pathways, namely, sequential and direct (or nonsequential) absorption of the photons. We find strong differences in the recoil-ion momentum patterns for the two mechanisms pointing to the significantly different two-electron emission dynamics and thus provide serious constraints for theoretical models.

Few-photon multiple ionization of ne and ar by strong free-electron-laser pulses.

Few-photon multiple ionization of Ne and Ar atoms by strong vacuum ultraviolet laser pulses from the free-electron laser at Hamburg was investigated differentially with the Heidelberg reaction microscope. The light-intensity dependence of Ne2+ production reveals the dominance of nonsequential two-photon double ionization at intensities of I<6x10(12) W/cm2 and significant contributions of three-photon ionization as I increases. Ne2+ recoil-ion-momentum distributions suggest that two electrons absorbing "instantaneously" two photons are ejected most likely into opposite hemispheres with similar energies.

How do substrates enter and products exit the buried active site of cytochrome P450cam? 1. Random expulsion molecular dynamics investigation of ligand access channels and mechanisms.

Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds and the degradation of xenobiotics. Cytochrome P450cam from Pseudomonas putida has provided a paradigm for the structural understanding of cytochrome P450s. However, the mechanism by which camphor, the natural substrate of cytochrome P450cam, accesses the buried active site is a long-standing puzzle. While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450cam, no such conformational changes have been observed either in different crystal structures or by standard molecular dynamics simulations. Here, a novel simulation method, random expulsion molecular dynamics, is presented, in which substrate-exit channels from the buried active site are found by imposing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field. The random expulsion molecular dynamics method was tested in simulations of the substrate-bound structure of cytochrome P450BM-3, and then applied to complexes of cytochrome P450cam with different substrates and with product. Three pathways were identified, one of which corresponds to a channel proposed earlier on the basis of crystallographic and site-directed mutagenesis data. Exit via the water-filled channel, which was previously suggested to be a product exit channel, was not observed. The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an analysis of crystallographic B-factors. In contrast to large backbone motions (up to 4 A) observed in cytochrome P450BM-3 for the exit of palmitoleic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 A) in conjunction with side-chain rotations. Concomitantly, in almost all the exit trajectories, salt-links that have been proposed to act as ionic tethers between secondary structure elements of the protein, are perturbed.

How do substrates enter and products exit the buried active site of cytochrome P450cam? 2. Steered molecular dynamics and adiabatic mapping of substrate pathways.

Three possible channels by which substrates and products can exit from the buried active site of cytochrome P450cam have been identified by means of random expulsion molecular dynamics simulations. In the investigation described here, we computed estimates of the relative probabilities of ligand passage through the three channels using steered molecular dynamics and adiabatic mapping. For comparison, the same techniques are also applied to investigate substrate egress from cytochrome P450-BM3. The channel in cytochrome P450cam, for which there is the most supporting evidence from experiments (which we name pathway 2a), is computed to be the most probable ligand exit channel. It has the smallest computed unbinding work and force. For this channel, the ligand exits between the F/G loop and the B' helix. Two mechanistically distinct, but energetically similar routes through this channel were observed, showing that multiple pathways along one channel are possible. The probability of ligand exit via the next most probable channel (pathway 3), which is located between the I helix and the F and G helices, is estimated to be less than 1/10 of the probability of exit along pathway 2a. Low-frequency modes of the protein extracted from an essential dynamics analysis of a 1 ns duration molecular dynamics simulation of cytochrome P450cam with camphor bound, support the opening of pathway 2a on a longer timescale. On longer timescales, it is therefore expected that this pathway becomes more dominant than estimated from the present computations.

Secretion and gene expression of metalloproteinases and gene expression of their inhibitors in porcine corpora lutea at different stages of the luteal phase.

We hypothesize that spontaneous regression of corpora lutea (CL) involves short-lasting restructure of luteal tissue with an activation of matrix metalloproteinases (MMPs) and their respective inhibitors (tissue inhibitors of metalloproteinase, TIMPs). This was tested by determining the gene expression of MMP-1, MMP-2, and MMP-9 and respective TIMP-1 and TIMP-2 in luteal tissue from sows at the early, midluteal, and late luteal phase (Days 6-8, Days 9-11, and Days 13-15 of estrous cycle). Gene expression of the three MMPs was low in early, slightly higher in midluteal, and significantly elevated (P < 0.05) in regressing CL. An inverse pattern was found for gene expression of TIMP-1 and TIMP-2. Under culture conditions, the release of MMPs was determined from steroidogenic large luteal cells (LLC). LLC harvested from regressing CL released significantly (P < 0.05) more active MMPs than cells obtained from CL at the early luteal phase. As luteolysis can be induced by prostaglandin F(2alpha) (PGF(2alpha)) and tumor necrosis factor alpha (TNF), we studied their effects on LLC under culture conditions. Treatment of cells with PGF(2alpha) or TNF (10(-7) M or 3 x 10(-9) M, respectively) induced a significantly higher release of MMPs, and gene expression was also significantly stimulated in comparison to that in untreated LLC. The gene expression of TIMPs remained unaffected by either treatment. It is concluded that at the beginning of luteolysis, MMPs are expressed and released in high amounts and that this is essential for the structural regression of the CL.

Towards molecular dynamics simulation of large proteins with a hydration shell at constant pressure.

Molecular dynamics simulation of a large protein in explicit water with periodic boundary conditions is extremely demanding in terms of computation time. Consequently, we have sought approximations of the solvent environment that model its important features. Here, we describe our SAPHYR (Shell Approximation for Protein HYdRation) model in which the protein is surrounded by a shell of water molecules maintained at constant pressure. In addition to the usual pairwise interatomic interactions, these water molecules are subjected to forces approximating van der Waals and dipole-dipole interactions with the implicit surrounding bulk solvent. The SAPHYR model is tested for a system of one argon atom in water and for the protein ubiquitin, and then applied to cytochrome P450cam, a protein with over 400 residues. The results demonstrate that structural and dynamic properties of the simulated systems are improved by use of the SAPHYR model, and that this model provides a significant computational saving over simulations with periodic boundary conditions.

Electrostatic steering and ionic tethering in enzyme-ligand binding: insights from simulations.

To bind at an enzyme's active site, a ligand must diffuse or be transported to the enzyme's surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also influence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-influenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and beta-lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as "ionic tethering." We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme's surroundings even when the substrate is nonpolar.

NMR cross-relaxation investigated by molecular dynamics simulation: a case study of ubiquitin in solution.

A one nanosecond molecular dynamics simulation of ubiquitin in solution has been used for the calculation of the total dipolar, the radial and the reorientational correlation functions of 174 interproton NOEs and the 76 peptide chain NH vectors. The NOEs have been classified according to the structural elements they are associated with. Using multiexponential fits of the raw data spectral densities and cross-relaxation rate constants have been determined. Statistical distributions of correlation function parameters are given. On the basis of these data the assumptions underlying the standard method for distance measurement using NOE enhancements have been scrutinized. The separability of elongation and reorientation is verified for the vast majority of NOEs, but the rigid-body assumption is not supported by the simulation results. Relying on a spectral density expression that neither makes use of the product approximation nor neglects spatially restricted motion, a "bias-free" (with regard to molecular motion) distance measurement method is suggested and compared with the standard method. Errors in distances up to 24% and 50% occur due to the neglect of the dispersion of order parameters and correlation times, respectively. The preconditions for a class-specific calibration method have been investigated. Within the framework of the product approximation a method for decomposing the total cross-relaxation rate constant into contributions from radial and angular motion has been developed and applied. In several cases distance fluctuation contributes significantly to cross-relaxation with both amplitude and time behaviour.

Effects of growth factors and hormones on basal and FSH-stimulated inhibin production by porcine granulosa cells in vitro.

The effect of several growth factors, protein and steroid hormones on follicle stimulating hormone (FSH)-stimulated and basal inhibin secretion by mature porcine granulosa cells (g-cells) in culture was examined in order to elucidate the putative role of growth factors and hormones in the regulation of inhibin secretion by porcine g-cells in vitro. Cells were incubated with the respective hormones over a timespan of 0-144 h and immunoreactive inhibin was measured with a radioimmunoassay against porcine inhibin. Epidermal growth factor (EGF) and human transforming growth factor type beta (TGF-beta) decreased basal and gonadotrophin-stimulated inhibin and progesterone in a dose-dependent manner. In the absence of insulin, insulin-like growth factor type I (IGF-I) caused a 4-fold enhancement of basal inhibin secretion, but inhibin secretion was elevated only to 20% above control in the presence of 500 nM insulin. Porcine platelet-derived growth factor (PDGF) had no significant effect on basal or FSH-induced inhibin secretion by g-cells. In addition, neither gonadotrophin-releasing hormone (GnRH) nor prolactin (PRL), arginine vasopressin (AVP) and oxytocin affected basal or FSH-stimulated inhibin release by porcine g-cells. Oestradiol caused a slight but significant (P less than 0.01) rise of basal inhibin production (158% of control) in the last 2 days of culture (96-144 h) and the effect of androstenedione on basal (158% of control) and FSH-stimulated (140% of control) inhibin release (P less than 0.01) was also only visible on Days 4-6 of culture. In contrast to androstenedione and oestradiol, progesterone did not show any effect during 6 days of culture in a dose range of 10(-5) to 10(-9) M. Like steroids, prostaglandin E2 (PGE2) had a stimulatory effect on basal inhibin production (250% of control) by porcine g-cells, visible on Days 3-6 of culture, but an inhibitory effect on FSH-stimulated release (less than 40% of control). Over all the experiments with different hormones and growth factors, tested in varying doses and over a time span of 0-144 h, there was a strong correlation between progesterone and inhibin secretion by g-cells (0-48 h = 0.78; 48-96 h = 0.92; 96-144 h = 0.92). These results suggest that EGF, TGF-beta, IGF-I, oestradiol and androstendione as well as PGE2 have para- and/or autocrine modulatory effects on basal and FSH-stimulated inhibin secretion by mature porcine g-cells in vitro and further demonstrate that the secretion of the proteohormone inhibin and the steroid progesterone are closely related.