NMR studies of the interaction of tryparedoxin with redox-inactive substrate homologues

Tryparedoxins (TXNs) are trypanothione-dependent peroxiredoxin oxidoreductases involved in hydroperoxide detoxification that have been shown to determine virulence in trypanosomatids. The structure of (15)N,(13)C-doubly-labeled, C-terminally-His-tagged tryparedoxin 1 from Crithidia fasciculata (Cf TXN1) was elucidated by three-dimensional NMR spectroscopy. Global folding was found to be similar to the crystal structure, but regions near the active site, especially the onset of helix alpha1 with the redox-active Cys 43 and helix alpha2 relevant to substrate binding, were less well defined in solution. The redox-inactive inhibitory substrate analogue N(1),N(8)-bis(ophthalmyl)spermidine was used to study the substrate/TXN interaction by two-dimensional (1)H,(15)N NMR spectroscopy. The NMR data complemented by molecular modeling revealed several alternative modes of ligand binding. The results confirm and extend the concept of TXN action and specificity derived from X-ray analysis and site-directed mutagenesis and thus improve the rational basis for inhibitor design.     

Base-pair dynamics in an antiparallel DNA triplex measured by catalyzed imino proton exchange monitored via 1H NMR spectroscopy

Using (1)H NMR spectroscopy, the base-pair opening dynamics of an antiparallel foldback DNA triplex and the corresponding duplex has been characterized via catalyzed imino proton exchange. The triplex system was found to be in an equilibrium between a duplex and a triplex form. The exchange rate between the two forms (i.e., the on/off-rate of the third strand) was measured to be 5 s(-1) at 1 degrees C, and the base-pair dynamics of both forms were investigated separately. Both Watson-Crick and reverse Hoogsteen base pairs were found to have base-pair lifetimes in the order of milliseconds. The stability of the Watson-Crick base pairs was, however, substantially increased in the presence of the third strand. In the DNA triplex, the opening dynamics of the reverse Hoogsteen base pairs was significantly faster than the dynamics of the Watson-Crick pairs. We were able to conclude that, for both Watson-Crick and reverse Hoogsteen base pairs, spontaneous and individual opening from within the closed base triplet is the dominating opening pathway.     

Hydrogen-induced structural changes at the nickel site of the regulatory [NiFe] hydrogenase from Ralstonia eutropha detected by X-ray absorption spectroscopy

For the first time, the nickel site of the hydrogen sensor of Ralstonia eutropha, the regulatory [NiFe] hydrogenase (RH), was investigated by X-ray absorption spectroscopy (XAS) at the nickel K-edge. The oxidation state and the atomic structure of the Ni site were investigated in the RH in the absence (air-oxidized, RH(ox)) and presence of hydrogen (RH(+H2)). Incubation with hydrogen is found to cause remarkable changes in the spectroscopic properties. The Ni-C EPR signal, indicative of Ni(III), is detectable only in the RH(+H2) state. XANES and EXAFS spectra indicate a coordination of the Ni in the RH(ox) and RH(+H2) that pronouncedly differs from the one in standard [NiFe] hydrogenases. Also, the changes induced by exposure to H(2) are unique. A drastic modification in the XANES spectra and an upshift of the K-edge energy from 8339.8 (RH(ox)) to 8341.1 eV (RH(+H2)) is observed. The EXAFS spectra indicate a change in the Ni coordination in the RH upon exposure to H(2). One likely interpretation of the data is the detachment of one sulfur ligand in RH(+H2) and the binding of additional (O,N) or H ligands. The following Ni oxidation states and coordinations are proposed: five-coordinated Ni(II)(O,N)(2)S(3) for RH(ox) and six-coordinated Ni((III))(O,N)(3)X(1)S(2) [X being either an (O,N) or H ligand] for RH(+H2). Implications of the structural features of the Ni site of the RH in relation to its function, hydrogen sensing, are discussed.     

Single fungal kinesin motor molecules move processively along microtubules

Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in allowing some single-headed kinesins to move processively. Neurospora kinesin (NKin) is a phylogenetically distant, dimeric kinesin from Neurospora crassa with high gliding speed and an unusual neck domain. We quantified the processivity of NKin and compared it to human kinesin, HKin, using gliding and fluorescence-based processivity assays. Our data show that NKin is a processive motor. Single NKin molecules translocated microtubules in gliding assays on average 2.14 micro m (N = 46). When we tracked single, fluorescently labeled NKin motors, they moved on average 1.75 micro m (N = 182) before detaching from the microtubule, whereas HKin motors moved shorter distances (0.83 micro m, N = 229) under identical conditions. NKin is therefore at least twice as processive as HKin. These studies, together with biochemical work, provide a basis for experiments to dissect the molecular mechanisms of processive movement.     

Transport of nucleosome core particles in semidilute DNA solutions

We studied the diffusion of native and trypsinized nucleosome core particles (NCPs), in aqueous solution and in concentrated DNA solutions (0.25-100 mg/ml) using fluorescence correlation spectroscopy (FCS). The highest DNA concentrations studied mimic the DNA density inside the cell nucleus. The diffusion coefficient of freely diffusing NCPs depends on the presence or absence of histone tails and is affected by the salt concentration due to the relaxation effect of counterions. NCPs placed in a network of long DNA molecules (30-50 kbp) reveal anomalous diffusion. We demonstrate that NCPs diffusion is in agreement with known particle transport in entangled macromolecular solutions as long as the histone tails are folded onto the particles. In contrast, when these tails are unfolded, the reversible adsorption of NCPs onto the DNA network has to be taken into account. This is confirmed by the fact that removal of the tails leads to reduction of the interaction between NCPs and the DNA network. The findings suggest that histone tail bridging plays an important role in chromatin dynamics.     

Modulation of nuclear pore topology by transport modifiers

The nuclear pore complex (NPC) represents the only pathway for macromolecular communication between the nuclear and cytoplasmic compartments of the cell. Nucleocytoplasmic transport requires the interaction of transport receptors with phenylalanine-glycine (FG)-repeats that line the transport pathway through the NPC. Here we examine the effects of transport receptors and amphipathic alcohols on NPC topology using scanning force microscopy. We show that transport receptors that irreversibly bind FG-repeats increase NPC vertical aspect, whereas transport receptors that weakly interact with FG-repeats increase NPC diameter. Interestingly, small polar alcohols likewise increase NPC diameter. These opposing effects agree with the inhibition or enhancement of nuclear transport, respectively, previously ascribed to these agents.     

Development and validation of a bioreactor for physical stimulation of engineered cartilage

A bioreactor has been developed to apply different regimes of physical stimulation to tissue specimens under highly controlled conditions. The computer-controlled device exposes specimens to compressive deformation at various strains and frequencies, measures the load applied to each sample and allows simultaneous medium stirring at different velocities. Validation tests confirmed the accuracy of the system in (i) its displacement (errors averaged 0.072+/-0.051 microm), and in (ii) setting the contact with the samples utilizing micrometer screws coupled to plungers (errors averaged 1.74+/-0.36% for samples of 1.60-3.18 mm thickness), thus ensuring accurate compressive deformation. The developed bioreactor, which represents an advance in the technology for physical stimulation of tissue specimens, is currently used to apply compressive deformation and hydrodynamic forces to human chondrocytes cultured in biodegradable polymer scaffolds, with the goals of (i) engineering functional grafts for the repair of cartilage defects (ii).     

Biomechanical properties of knee articular cartilage

Structure and properties of knee articular cartilage are adapted to stresses exposed on it during physiological activities. In this study, we describe site- and depth-dependence of the biomechanical properties of bovine knee articular cartilage. We also investigate the effects of tissue structure and composition on the biomechanical parameters as well as characterize experimentally and numerically the compression-tension nonlinearity of the cartilage matrix. In vitro mechano-optical measurements of articular cartilage in unconfined compression geometry are conducted to obtain material parameters, such as thickness, Young's and aggregate modulus or Poisson's ratio of the tissue. The experimental results revealed significant site- and depth-dependent variations in recorded parameters. After enzymatic modification of matrix collagen or proteoglycans our results show that collagen primarily controls the dynamic tissue response while proteoglycans affect more the static properties. Experimental measurements in compression and tension suggest a nonlinear compression-tension behavior of articular cartilage in the direction perpendicular to articular surface. Fibril reinforced poroelastic finite element model was used to capture the experimentally found compression-tension nonlinearity of articular cartilage.     

Structure of a highly phosphorylated lipopolysaccharide core in the Delta algC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3)

Lipopolysaccharides (LPS) were isolated from rough-type mutant strains of Pseudomonas aeruginosa (Delta algC) derived from wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3). Structural studies of the LPS core region with a special focus on the phosphorylation pattern were performed by 2D NMR spectroscopy, including a 1H,(31)P HMQC-TOCSY experiment, MALDI-TOF MS, and Fourier-transform ion cyclotron resonance ESIMS using the capillary skimmer dissociation technique. Both LPS were found to contain two residues each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D-manno-heptose (Hep), one residue of N-(L-alanyl)-D-galactosamine and one O-carbamoyl group (Cm) on the distal Hep residue. The following structures of a tetrasaccharide trisphosphate from strain PAC1R Delta algC and that carrying an additional ethanolamine phosphate group (PEtN) from strain PAO1 Delta algC were elucidated: [carbohydrate structre: see text] where R=P in PAC1R Delta algC and PPEtN in PAO1 Delta algC. To our knowledge, in this work the presence of ethanolamine diphosphate is unambiguously confirmed and its position established for the first time in the LPS core of a rough-type strain of P. aeruginosa. In addition, the structure of the complete LPS core of wild-type strain P. aeruginosa PAO1 was reinvestigated and the position of the phosphorylation sites was revised.