Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm−1 and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s−1 under zero force up to 15 s−1 under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.

Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by 13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680.+ primary donor chlorophyll

We report 13C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo- CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance 13C provide information on the electronic structure of the primary electron donor P680 (chlorophyll a molecules absorbing around 680 nm) and on the pz spin density pattern in its oxidized form, P680⨥. Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P680⨥. PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P680 by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.

Information about movement direction obtained from synchronous activity of motor cortical neurons

Although neuronal synchronization has been shown to exist in primary motor cortex (MI), very little is known about its possible contribution to coding of movement. By using cross-correlation techniques from multi-neuron recordings in MI, we observed that activity of neurons commonly synchronized around the time of movement initiation. For some cell pairs, synchrony varied with direction in a manner not readily predicted by the firing of either neuron. Information theoretic analysis demonstrated quantitatively that synchrony provides information about movement direction beyond that expected by simple rate changes. Thus, MI neurons are not simply independent encoders of movement parameters but rather engage in mutual interactions that could potentially provide an additional coding dimension in cortex.

Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods

A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships.

Contribution of Malic Enzyme, Pyruvate Kinase, Phosphoenolpyruvate Carboxylase, and the Krebs Cycle to Respiration and Biosynthesis and to Intracellular pH Regulation during Hypoxia in Maize Root Tips Observed by Nuclear Magnetic Resonance Imaging and Gas Chromatography-Mass Spectrometry1

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by 13C incorporation from [1-13C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. 13C-Nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.