Is there elliptic distortion in the light harvesting complex 2 of purple bacteria?

Single molecule spectroscopy (SMS) revealed an unusually large Gap between two major exciton peaks of the B850 unit of light harvesting complex 2 (LH2), which could be explained assuming elliptic distortion or k = 2 symmetry modulation in the site excitation energy. On the basis of extensive simulation of the SMS data and ensemble line shape, we found that uniform modulation of k = 2 symmetry cannot explain the dependence of intensity ratios on the Gap of the two major peaks, which are available from SMS, nor the ensemble line shape. Alternative models of disorder with k = 1 and k = 2 symmetry correlation are shown to reproduce these data reasonably well and can even explain the Gap distribution when it is assumed that the lower major peak in the SMS line shape is an intensity weighted average of k = 1- and k = 0 states.

Stretched polyethylene films probed by single molecules.

Stretched films of low-density polyethylene (LDPE) doped with 2.3,8.9-dibenzanthanthrene (DBATT) were studied using polarization-selective single-molecule spectroscopy at 1.8 K. By measuring the in-plane component of the electronic transition-dipole moments of individual chromophores, the alignment of dopant molecules is determined without averaging. The distributions of chromophore orientations reveal the presence of two fractions of dopant molecules: those oriented along the stretching direction and randomly oriented molecules. With increasing drawing ratio of the polyethylene films, the ratio of oriented to randomly oriented guest molecules increases, whereas the extent of chromophore orientation, that is, the width of the orientation distribution, remains the same. The results are consistent with the interpretation that oriented chromophores reside on the surfaces of polyethylene crystals, instead of in the amorphous polyethylene regions. Guest molecules in stretched polyethylene are oriented due to the alignment of the crystallites on which they are adsorbed. As such, the shape and width of the distributions of chromophore orientations are determined by the interaction of guest molecules with the crystal surfaces.

Alphaherpesvirus US3-mediated reorganization of the actin cytoskeleton is mediated by group A p21-activated kinases.

The US3 protein is a viral serine/threonine kinase that is conserved among all members of the Alphaherpesvirinae. The US3 protein of different alphaherpesviruses causes dramatic alterations in the actin cytoskeleton, such as the disassembly of actin stress fibers and formation of cell projections, which have been associated with increased intercellular virus spread. Here, we find that inhibiting group A p21-activated kinases (PAKs), which are key regulators in Cdc42/Rac1 Rho GTPase signaling pathways, impairs US3-mediated actin alterations. By using PAK1(-/-) and PAK2(-/-) mouse embryo fibroblasts (MEFs), we show that US3-mediated stress fiber disassembly requires PAK2, whereas US3-mediated cell projection formation mainly is mediated by PAK1, also indicating that PAK1 and PAK2 can have different biological effects on the organization of the actin cytoskeleton. In addition, US3 was found to bind and phosphorylate group A PAKs. Lack of group A PAKs in MEFs was correlated with inefficient virus spread. Thus, US3 induces its effect on the actin cytoskeleton via group A PAKs.

p21-activated kinase regulates mast cell degranulation via effects on calcium mobilization and cytoskeletal dynamics.

Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcepsilonRI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, W(sh)/W(sh) mast cell-deficient mice locally reconstituted with Pak1(-/-) bone marrow-derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1(-/-) BMMCs exhibited a reduction in FcepsilonRI-induced degranulation. Further, Pak1(-/-) BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcepsilonRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases.

Pak1 regulates multiple c-Kit mediated Ras-MAPK gain-in-function phenotypes in Nf1+/- mast cells.

Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by mutations in the NF1 locus, which encodes neurofibromin, a negative regulator of Ras. Patients with NF1 develop numerous neurofibromas, which contain many inflammatory mast cells that contribute to tumor formation. Subsequent to c-Kit stimulation, signaling from Ras to Rac1/2 to the MAPK pathway appears to be responsible for multiple hyperactive mast cell phenotypes; however, the specific effectors that mediate these functions remain uncertain. p21-activated kinase 1 (Pak1) is a downstream mediator of Rac1/2 that has been implicated as a positive regulator of MAPK pathway members and is a modulator of cell growth and cytoskeletal dynamics. Using an intercross of Pak 1(-/-) mice with Nf1(+/-) mice, we determined that Pak1 regulates hyperactive Ras-dependent proliferation via a Pak1/Erk pathway, whereas a Pak1/p38 pathway is required for the increased migration in Nf1(+/-) mast cells. Furthermore, we confirmed that loss of Pak1 corrects the dermal accumulation of Nf1(+/-) mast cells in vivo to levels found in wild-type mice. Thus, Pak1 is a novel mast cell mediator that functions as a key node in the MAPK signaling network and potential therapeutic target in NF1 patients.

Single dibenzoterrylene molecules in an anthracene crystal: main insertion sites.

We present a spectroscopic study of the properties of the two principal insertion sites (at 785.1 and 794.3 nm) of single dibenzoterrylene molecules in anthracene single crystals at cryogenic temperatures. We measured the temperature dependence of the line width, the orientation of the transition dipole moments, and the Stark effect. We performed molecular dynamics simulations, which show that one dibenzoterrylene molecule preferably replaces three anthracene molecules. From simulated annealing, we derive the molecular conformations in the most stable insertion sites and the orientations of the transition dipole moments. The good agreement between the spectroscopic results and the simulations allows us to propose unambiguous structures for the two principal spectroscopic sites.

Single dibenzoterrylene molecules in an anthracene crystal: spectroscopy and photophysics.

We study single dibenzoterrylene molecules in an anthracene single crystal at 1.4 K in two insertion sites at 785.1 and 794.3 nm. The single-molecule zero-phonon lines are narrow (about 30 MHz), intense (the detected fluorescence rates at saturation reach 100,000 counts s(-1)), and very photostable. The intersystem-crossing yield is extremely low (10(-7) or lower). All of these features are hallmarks of an excellent system for high-resolution spectroscopy and nanoscale probing at cryogenic temperatures.

Effect of the venous catheter site on transpulmonary thermodilution measurement variables.

OBJECTIVE: Transpulmonary thermodilution is increasingly used for hemodynamic monitoring of critically ill patients. Injection of a cold saline bolus in the central venous circulation is a prerequisite for transpulmonary thermodilution measurements. Superior vena cava access is typically used for injection. This access, however, is not feasible or available in all intensive care patients (e.g., in burn victims or due to contraindications for Trendelenburg position). The present study investigates whether femoral vein access can be used to obtain clinically acceptable values. DESIGN: Open prospective trial performed between September 2005 and April 2006. SETTINGS: Medical intensive care unit at a university hospital. PATIENTS: Eleven critically ill patients monitored by transpulmonary thermodilution. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: A total of 44 measurements in 11 intensive care patients were performed with the Pulsion PICCO Plus device to compare cardiac output, extravascular lung water index, and global end-diastolic volume index after central venous injection of the cold saline bolus via femoral and jugular venous access. Bland-Altman analysis revealed that catheter insertion site does not relevantly influence cardiac output and extravascular lung water index. The bias between femoral and jugular injection was +0.16 L/min for cardiac output and +0.23 mL/kg for extravascular lung water index. Global end-diastolic volume index values, however, show a constant overestimation of +140.73 mL/m2 after femoral injection, as obtained by Bland-Altman analysis. This overestimation can be explained by a longer mean transit time due to a longer distance of catheter tip and right atrium for a femoral catheter. CONCLUSIONS: Transpulmonary thermodilution measurements with a cold saline bolus via a femoral catheter provide clinically reliable cardiac output and extravascular lung water index values. Concerning global end-diastolic volume index, there is a good correlation as well, but in the interpretation of the results, an overestimation has to be taken into account.

Four-level optical line shape of a single molecule coupled to a single tunneling two-level system.

We propose a density-matrix theory for the four-level system consisting of a single optical two-level system (OTLS) coupled to a single two-level system tunneling along a vibrational coordinate (VTLS). Phonons induce jumping rates of the VTLS, but coherent tunneling has to be considered explicitly as well, because the Born-Oppenheimer potential of the tunnel variable may change upon optical excitation. The OTLS is subject to spontaneous emission and driven by a laser wave with arbitrary strength. Numerical simulations for various coupling cases reproduce limiting behaviors previously discussed separately in the literature, such as motional narrowing, cross transitions, optical saturation and pumping, and nonlinear effects. Our model also perfectly fits recent measurements of the spectra of a single molecule coupled to a single tunneling system in a disordered crystal.

Role of group A p21-activated kinases in activation of extracellular-regulated kinase by growth factors.

The canonical extracellular-regulated kinase (ERK) signaling cascade, consisting of the Ras-Raf-Mek-ERK module, is critically important to many cellular functions. Although the general mechanism of activation of the ERK cascade is well established, additional noncanonical components greatly influence the activity of this pathway. Here, we focus on the group A p21-activated kinases (Paks), which have previously been implicated in regulating both c-Raf and Mek1 activity, by phosphorylating these proteins at Ser(338) and Ser(298), respectively. In NIH-3T3 cells, expression of an inhibitor of all three group A Paks reduced activation of ERK in response to platelet-derived growth factor (PDGF) but not to epidermal growth factor (EGF). Similar results were obtained in HeLa cells using small interference RNA-mediated simultaneous knockdown of both Pak1 and Pak2 to reduce group A Pak function. Inhibition of Pak kinase activity dramatically decreased phosphorylation of Mek1 at Ser(298) in response to either PDGF or EGF, but this inhibition did not prevent Mek1 activation by EGF, suggesting that although Pak can phosphorylate Mek1 at Ser(298), this event is not required for Mek1 activation by growth factors. Inhibition of Pak reduced the Ser(338) phosphorylation of c-Raf in response to both PDGF and EGF; however, in the case of EGF, the reduction in Ser(338) phosphorylation was not accompanied by a significant decrease in c-Raf activity. These findings suggest that Paks are required for the phosphorylation of c-Raf at Ser(338) in response to either growth factor, but that the mechanisms by which EGF and PDGF activate c-Raf are fundamentally different.

Multivariate analysis of single-molecule spectra: surpassing spectral diffusion.

The full exploitation of single-molecule spectroscopy in disordered systems is often hampered by spectral diffusion processes of the optical transitions due to structural fluctuations in the local environment of the probe molecule which leads to temporal averaging of the signal. Multivariate statistical pattern recognition techniques, originally developed for single-molecule cryoelectron microscopy, allow us to retrieve detailed information from optical single-molecule spectra. As an example, we present the phonon side band of the B800 excitations of the light-harvesting 2 (LH2) complex from Rhodospirillum molischianum, revealing the electron-phonon coupling strength for these transitions. The measured Debye-Waller factors, ranging from 0.4 to 0.9, fall in the regime of weak electron-phonon coupling.

The genetics of Pak.

p21-activated kinases (Paks) are a highly conserved family of enzymes that bind to and are activated by small GTPases of the Cdc42 and Rac families. With the notable exception of plants, nearly all eukaryotes encode one or more Pak genes, indicating an ancient origin and important function for this family of enzymes. Genetic approaches in many different experimental systems, ranging from yeast to mice, are beginning to decipher the different functions of Paks. Although some of these functions are unique to a given organism, certain common themes have emerged, such as the activation of mitogen-activated protein kinase (MAPK) cascades and the regulation of cytoskeletal structure through effects on the actin and tubulin cytoskeletons.

Direct observation of tiers in the energy landscape of a chromoprotein: a single-molecule study.

Single-molecule spectroscopic techniques were applied to individual pigments embedded in a chromoprotein. A sensitive tool to monitor structural fluctuations of the protein backbone in the local environment of the chromophore is provided by recording the changes of the spectral positions of the pigment absorptions as a function of time. The data provide information about the organization of the energy landscape of the protein in tiers that can be characterized by an average barrier height. Additionally, a correlation between the average barrier height within a distinct tier and the time scale of the structural fluctuations is observed.

Energy transfer in a single self-aggregated photosynthetic unit.

The primary events of bacterial photosynthesis rely on the interplay of various specialized protein complexes organized in a supramolecular structure commonly termed the photosynthetic unit (PSU), which consists of the photochemical reaction center and of an associated antenna network. Employing single-molecule spectroscopic techniques we have been able to observe the excitation-energy transfer within a single PSU. From these findings we conclude that the building blocks of the PSU spontaneously form stable, functional aggregates in a non-membrane environment.

Single-molecule study of the electronic couplings in a circular array of molecules: light-harvesting-2 complex from Rhodospirillum molischianum.

Applying single-molecule spectroscopic techniques allowed us to determine the mutual angles between the transition-dipole moments associated with optical transitions of the eight bacteriochlorophyll a molecules which form the so-called B800 ring of the light-harvesting-2 complex from Rhodospirillum molischianum. The orientation of the transition-dipole moment is a sensitive probe for the strength of the local intermolecular interactions because of the well-defined arrangement of the individual molecules within the B800 ring. Our data reveal that the strength of the electronic coupling between individual molecules in the ring is subjected to spatial as well as temporal variations.

Spectroscopy on individual light-harvesting 1 complexes of Rhodopseudomonas acidophila.

In this paper the fluorescence-excitation spectra of individual LH1-RC complexes (Rhodopseudomonas acidophila) at 1.2 K are presented. All spectra show a limited number of broad bands with a characteristic polarization behavior, indicating that the excitations are delocalized over a large number of pigments. A significant variation in the number of bands, their bandwidths, and polarization behavior is observed. Only 30% of the spectra carry a clear signature of delocalized excited states of a circular structure of the pigments. The large spectral variety suggests that besides site heterogeneity also structural heterogeneity determines the optical spectrum of the individual LH1-RC complexes. Further research should reveal if such heterogeneity is a native property of the complex or induced during the experimental procedures.