Ligand trajectories trapped within a docking site or within an internal cavity near the active site of proteins are important issues toward the elucidation of the mechanism of reaction of such ...complex systems, in which activity requires the shuttling of oriented ligands to and from their active site. The ligand motion within ba3-cytochrome c oxidase from Thermus thermophilus has been investigated by measuring time-resolved step-scan Fourier transform infrared difference spectra of photodissociated CO from heme a3 at ambient temperature. Upon photodissociation, 15-20% of the CO is not covalently attached to CuB but is trapped within a docking site near the ring A of heme a3 propionate. Two trajectories of CO that are distinguished spectroscopically and kinetically (vCO = 2131 cm-1, td = 10-35 μs and vCO = 2146 cm-1, td = 85 μs) are observed. At later times (td = 110 μs) the docking site reorganizes about the CO and quickly establishes an energetic barrier that facilitates equilibration of the ligand with the protein solvent. The time-dependent shift of the CO trajectories we observe is attributed to a conformational motion of the docking site surrounding the ligand. The implications of these results with respect to the ability of the docking site to constrain ligand orientation and the reaction dynamics of the docking site are discussed herein.
A reversible temperature-dependent high-spin to low-spin transition with
= -60 °C has been observed in the resonance Raman spectra of the equilibrium reduced and photoreduced heme
of the thermophilic
...heme-copper oxidoreductase. The transition is based on the frequency shifts of the spin-state marker bands
(C
C
) and
(C
C
) and is attributed to the displacement of the heme iron along the heme normal as a consequence of the Fe-Np repulsion at temperature below -40 °C which will increase the ligand field strength forcing the pairing of d electrons into the lower energy orbitals.
Stent-induced vascular injury is manifested by removal of the endothelium and phenotypic changes in the underlying medial smooth muscle cells layer. This results in pathological vascular remodelling ...primarily contributed to smooth muscle cell proliferation and leads to vessel re-narrowing; neointimal hyperplasia. Current drug-eluting stents release non-selective anti-proliferative drugs such as paclitaxel from the stent surface that not only inhibit growth of smooth muscle cells but also delay endothelial healing, potentially leading to stent thrombosis. This highlights the need for novel bioactive stent coating candidates with the ability to target key events in the pathogenesis of in-stent restenosis. Citric acid, a molecule with anti-coagulant properties, was investigated against L-ascorbic acid, an antioxidant molecule reported to preferentially promote endothelial growth, and paclitaxel, a typically used anti-proliferative stent coating. Citric acid was found to exhibit growth supporting properties on endothelial cells across a range of concentrations that were significantly better than the model stent coating drug paclitaxel and better than the ascorbic acid which inhibited endothelial proliferation at concentrations ≥100 μg/ml. It was demonstrated that a citric acid-paclitaxel combination treatment significantly improves cell viability in comparison to paclitaxel only treated cells, with endothelial cells exhibiting greater cell recovery over smooth muscle cells. Furthermore, cell treatment with citric acid was found to reduce inflammation in a lipopolysaccharide (LPS)-induced in vitro inflammation model by significantly reducing interleukin 6 expression. Thus, this study demonstrates that citric acid is a promising candidate for use as a coating in stents and other endovascular devices.
Display omitted
•Novel stent platforms to selectively promote EC proliferation are needed.•Citric acid is a potential anti-inflammatory stent coating candidate.•Citric acid may promote endothelialization relevant to stent post-implantation.•Citric acid and paclitaxel combinations are potential therapeutic stent coatings.
Nanosecond time-resolved step-scan FTIR spectroscopy (nTRS 2 -FTIR) has been applied to literally probe the active site of the carbon monoxide (CO)-bound thermophilic ba3 heme-copper oxidoreductase ...as it executes its function. The nTRS 2 - snapshots of the photolysed heme a3 Fe-CO/CuB species captured a “transition state” whose side chains prevent the photolysed CO to enter the docking cavity. There are three sets of ba3 photoproduct bands of docked CO with different orientation exhibiting different kinetics. The trajectories of the “docked” CO at 2122, 2129 and 2137cm−1 is referred to in the literature as B2, B1 and B0 intermediate states, respectively. The present data provided direct evidence for the role of water in controlling ligand orientation in an intracavity protein environment.
•Detection of the B0, B1 and B2 intermediate states in ba3 oxidoreductase.•Nanosecond structural changes in the heme a3 rings D– and –A propionates.•H2O/D2O controls the orientation of “docked’ carbon monoxide.
A novel device for capillary‐free mounting of protein crystals is described. A controlled stream of air allows an accurate adjustment of the humidity at the crystal. The crystal is held on the tip of ...a micropipette. With a video system (CCD camera), the two‐dimensional shadow projections of crystals can be recorded for optical analysis. Instead of the micropipette, a standard loop can also be used. Experiments and results for different crystal systems demonstrate the use of this method, also in combination with shock‐freezing, to improve crystal order. Working with oxygen‐free gases offers the possibility of crystal measurements under anaerobic conditions. Furthermore, the controlled application of arbitrary volatile substances with the gas stream is practicable.
Following hints from X-ray data (Ostermeier C et
al., 1997, Proc Natl Acad Sci USA 94:10547–10553;
Yoshikawa S et al., 1998, Science 280:1723–1729),
chemical evidence is presented from four distantly ...related
cytochrome-c oxidases for the existence of a
copperB-coordinated His240–Tyr244)
cross-link at the O2-activating Heme
Fea3–CuB center in the
catalytic subunit I of the enzyme. The early evolutionary
invention of this unusual structure may have prevented
demaging bull OH-radical release at e−-transfer
to dioxygen and thus have enabled O2 respiration.
Antihistamines are capable of blocking mediator responses in allergic reactions including allergic rhinitis and dermatological reactions. By incorporating various H1 receptor antagonists into a lipid ...cubic phase network, these active ingredients can be delivered locally over an extended period of time owing to the mucoadhesive nature of the system. Local delivery can avoid inducing unwanted side effects, often observed after systematic delivery. Lipid-based antihistamine delivery systems are shown here to exhibit prolonged release capabilities. In vitro drug dissolution studies investigated the extent and release rate of two model first-generation and two model second-generation H1 antagonist antihistamine drugs from two monoacyglycerol-derived lipid models. To optimize the formulation approach, the systems were characterized macroscopically and microscopically by small-angle X-ray scattering and polarized light to ascertain the mesophase accessed upon an incorporation of antihistamines of varying solubilities and size. The impact of encapsulating the antihistamine molecules on the degree of mucoadhesivity of the lipid cubic systems was investigated using multiparametric surface plasmon resonance. With the ultimate goal of developing therapies for the treatment of allergic reactions, the ability of the formulations to inhibit mediator release utilizing RBL-2H3 mast cells with the propensity to release histamine upon induction was explored, demonstrating no interference from the lipid excipient on the effectiveness of the antihistamine molecules.
Display omitted
Lipid cubic phase (LCP) formulations enhance the intestinal solubility and bioavailability of hydrophobic drugs by reducing precipitation and facilitating their mass transport to the ...intestinal surface for absorption. LCPs with an ester linkage connecting the acyl chain to the glycerol backbone (monoacylglycerols), are susceptible to chemical digestion by several lipolytic enzymes including lipases, accelerating the release of hydrophobic agents from the lipid bilayers of the matrix. Unlike regular enzymes that transform soluble substrates, lipolytic enzymes act at the interface of water and insoluble lipid. Therefore, compounds that bind to this interface can enhance or inhibit the activity of enzymes to varying extent. Here, we explore how the lipolysis rate can be tuned by the interfacial interaction of porcine pancreatic lipase with monoolein LCPs containing a known lipase inhibitor, tetrahydrolipstatin. Release of the Biopharmaceutical Classification System (BCS) class IV drug, paclitaxel, from the inhibitor-modified LCP was examined in the presence of lipase and its effectors colipase and calcium. By combining experimental dynamic digestion studies, thermodynamic measurements and molecular dynamics simulations of the competitive inhibition of lipase by tetrahydrolipstatin, we reveal the role and mode of action of lipase effectors in creating a precisely-balanced degradation-controlled LCP release system for the poorly soluble paclitaxel drug.
PDF
