Dan Borchardt

Self-Doped Ti3+ Enhanced Photocatalyst for Hydrogen Production under Visible Light

Through a facile one-step combustion method, partially reduced TiO(2) has been synthesized. Electron paramagnetic resonance (EPR) spectra confirm the presence of Ti(3+) in the bulk of an as-prepared sample. The UV-vis spectra show that the Ti(3+) here extends the photoresponse of TiO(2) from the UV to the visible light region, which leads to high visible-light photocatalytic activity for the generation of hydrogen gas from water. It is worth noting that the Ti(3+) sites in the sample are highly stable in air or water under irradiation and the photocatalyst can be repeatedly used without degradation in the activity.

Endosidin2 targets conserved exocyst complex subunit EXO70 to inhibit exocytosis

Significance The exocyst complex is a conserved protein complex that tethers the secretory vesicles to the site of membrane fusion during exocytosis, an essential cellular process that transports molecules, such as protein, to the cell surface or extracellular space. We identified a small molecule that targets the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex to inhibit exocytosis. This compound made it possible to control the dynamics of the exocytosis process in a dosage-dependent manner in different organisms and overcame the mutant lethality and genetic redundancy issues in studying mechanisms of exocyst complex regulation. Further design of molecules with higher affinity and more potent activity may make it possible to use drugs to control human diseases related to exocytosis, such as cancer and diabetes. The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.

Characterization of Chemical Phosphorus Forms in Volcanic Soils Using 31P-NMR Spectroscopy

Abstract Phosphorus (P) is rapidly immobilized in Chilean volcanic soils and its availability to plant is greatly reduced. Chemical forms of P accumulated in soils would eventually determine its availability of plants and its potential to cause pollution in water bodies down stream. To characterize different chemical P species, we carried out chemical fractionation and 31P-Nuclear Magnetic Resonance (NMR) spectroscopy analyses for 10 volcanic ash-derived soils in central southern Chile. Phosphorus in the alkaline extracts of soils at 0–15 cm and 15–30 cm depth was characterized by 31P-NMR. The 31P-NMR results indicate that inorganic-P and monoester-P are the most important P forms in all soils. The diester-P and pyrophosphate were also found in some soils. The patterns of P distribution in the 0–15 and 15–30 cm were essentially the same; the signal intensities were usually stronger in the upper samples. The amounts of organic- and inorganic-P determined by 31P-NMR method correlate with those by the chemical fractionation procedure. Furthermore, the organic P of soils, calculated from NMR signals, correlates with the organic matter content of the soils (r = 0.82, p < 0.01). Those results suggest that the observed 31P-NMR signals represent the P forms in soils.

The N–B Interaction through a Water Bridge: Understanding the Chemoselectivity of a Fluorescent Protein Based Probe for Peroxynitrite

Boronic acid and esters have been extensively utilized for molecular recognition and chemical sensing. We recently reported a genetically encoded peroxynitrite (ONOO(-))-specific fluorescent sensor, pnGFP, based on the incorporation of a boronic acid moiety into a circularly permuted green fluorescent protein (cpGFP) followed by directed protein evolution. Different from typical arylboronic acids and esters, the chromophore of pnGFP is unreactive to millimolar concentrations of hydrogen peroxide (H2O2). The focus of this study is to explore the mechanism for the observed unusual chemoselectivity of pnGFP toward peroxynitrite over hydrogen peroxide by using site-directed mutagenesis, X-ray crystallography, (11)B NMR, and computational analysis. Our data collectively support that a His residue on the protein scaffold polarizes a water molecule to induce the formation of an sp(3)-hybridized boron in the chromophore, thereby tuning the reactivity of pnGFP with various reactive oxygen and nitrogen species (ROS/RNS). Our study demonstrates the first example of tunable boron chemistry in a folded nonnative protein, which offers wide implications in designing selective chemical probes.

DISTRIBUTION OF PHOSPHORUS FORMS IN CHILEAN SOILS AND SEWAGE SLUDGE BY CHEMICAL FRACTIONATION AND 31P-NMR

The disposal of sewage sludge from domestic wastewater treatment plants is a growing problem worldwide. Because of the total P content and other characteristics, it is highly probable that in Chile important amounts of the sewage sludge will be disposed of on agricultural lands. The distribution of P forms in Chilean soils and sewage sludge was studied by chemical fractionation and 31P-NMR spectroscopy. The 0-15 cm depth of soils derived from volcanic materials (one Ultisol and two Andisols), alluvial materials (Mollisol), and sewage sludge from a domestic wastewater treatment plant located in Santiago were considered. The total P concentration is 6 to 18 times higher in the sewage sludge than in soils, but a similar 31P-NMR pattern was found in all samples. The most important signals correspond to inorganic orthophosphate and monoester P, with small signals assigned to pyrophosphate and diester P. The disposal of sewage sludge might not succeed as soil P fertilizer, contributing to an increase in the accumulation of P in volcanic soils, and it might be a potential pollutant in soils and water

EFFECTS OF DIACYLGLYCEROLS ON CONFORMATION OF PHOSPHATIDYLCHOLINE HEADGROUPS IN PHOSPHATIDYLCHOLINE/PHOSPHATIDYLSERINE BILAYERS

The effects of five diacylglycerols (DAGs), diolein, 1-stearoyl,2-arachidonoyl-sn-glycerol, dioctanoylglycerol, 1-oleoyl,2-sn-acetylglycerol, and dipalmitin (DP), on the structure of lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine (4:1 mol/mol) were examined by 2H nuclear magnetic resonance (NMR). Dipalmitoylphosphatidylcholine deuterated at the alpha- and beta-positions of the choline moiety was used to probe the surface region of the membranes. Addition of each DAG except DP caused a continuous decrease in the beta-deuteron quadrupole splittings and a concomitant increase in the alpha-deuteron splittings indicating that DAGs induce a conformational change in the phosphatidylcholine headgroup. Additional evidence of conformational change was found at high DAG concentrations (> or = 20 mol%) where the alpha-deuteron peaks became doublets indicating that the two alpha-deuterons were not equivalent. The changes induced by DP were consistent with the lateral phase separation of the bilayers into gel-like and fluid-like domains with the phosphatidylcholine headgroups in the latter phase being virtually unaffected by DP. The DAG-induced changes in alpha-deuteron splittings were found to correlate with DAG-enhanced protein kinase C (PK-C) activity, suggesting that the DAG-induced conformational changes of the phosphatidylcholine headgroups are either directly or indirectly related to a mechanism of PK-C activation. 2H NMR relaxation measurements showed significant increase of the spin-lattice relaxation times for the region of the phosphatidylcholine headgroups, induced by all DAGs except DP. However, this effect of DAGs did not correlate with the DAG-induced activation of PK-C.

Thermal Isomerization of Cannabinoid Analogues

Thermal isomerization of CBC(an) to THC(an) [nonaromatic analogues of plant cannabinoids cannabichromene (CBC) and Delta(1)-tetrahydrocannabinol (THC), respectively] is predicted in silico and demonstrated experimentally. Density functional theory calculations support a similar isomerization mechanism for the corresponding plant cannabinoids. Docking studies suggest that THC(an), although nonaromatic, has a CB(1) receptor binding affinity similar to that of natural THC.

Comparison of Extraction Procedures Used in Determination of Phosphorus Species by 31P‐NMR in Chilean Volcanic Soils

Abstract Four procedures were employed to extract phosphorus (P) from volcanic soils for 31P‐NMR experiments. The procedures involve 0.5 M NaOH extraction, 0.5 M NaOH and Chelex 100 cation exchange resin extraction, NaOH‐EDTA extraction, and HCl‐NaOH two step sequential extraction with Chelex 100 clean up. Results showed that inorganic‐P, monoester‐P, diester‐P and pyrophosphate were present. Their detection was dependent on the extraction procedure used. The NaOH procedure gives only a broad and vaguely defined signal with poor signal to noise ratio. The incorporation of Chelex 100 in the extraction enhanced the signal to noise ratio and allowed the distinction of inorganic‐P, monoester‐P, diester‐P and pyrophosphate. The two step sequential extraction involving HCl, NaOH, and Chelex 100 significantly improve the signal to noise ratio. The NaOH‐EDTA extraction procedure is efficient only in samples with low OC contents. When soils have low OC content, any of the four extraction procedures can be successfully used. If the OC and the Fe concentration in the extracted solutions are high, the Chelex 100 became essential in clean up the metallic ions. Both the NaOH and Chelex 100 and the HCl‐NaOH‐Chelex produced satisfactory results and the later procedure by far the best resolved spectra.

Spectroscopic evidence for preexisting T- and R-state insulin hexamer conformations.

The insulin hexamer is an allosteric protein exhibiting both positive and negative cooperative homotropic interactions and positive cooperative heterotropic interactions (C. R. Bloom et al., J. Mol. Biol. 245, 324–330, 1995). In this study, detailed spectroscopic analyses of the UV/Vis absorbance spectra of the Co(II)‐substituted human insulin hexamer and the 1H NMR spectra of the Zn(II)‐substituted hexamer have been carried out under a variety of ligation conditions to test the applicability of the sequential (KNF) and the half‐site reactivity (SMB) models for allostery. Through spectral decomposition of the characteristic d → d transitions of the octahedral Co(II)‐T‐state and tetrahedral Co(II)‐R‐state species, and analysis of the 1H NMR spectra of T‐ and R‐state species, these studies establish the presence of preexisting T‐ and R‐state protein conformations in the absence of ligands for the phenolic pockets. The demonstration of preexisting R‐state species with unoccupied sites is incompatible with the principles upon which the KNF model is based. However, the SMB model requires preexisting T‐ and R‐states. This feature, and the symmetry constraints of the SMB model make it appropriate for describing the allosteric properties of the insulin hexamer. Proteins 26:377–390

A NMR METHOD FOR THE ANALYSIS OF MIXTURES OF ALKANES WITH DIFFERENT DEUTERIUM SUBSTITUTIONS

Abstract 13C NMR at 125.76 MHz with 1H and 2H decoupling, 2H NMR at 76.77 MHz with 1H decoupling, and 1H NMR at 500.14 MHz with 2H decoupling were employed as analytical tools to study the complex mixtures of deuterated ethanes resulting from the catalytic H–D exchange of normal ethane with gas-phase deuterium in the presence of a platinum foil. Reference samples consisting of 1:1 binary mixtures of pure normal ethane and ethane-dn (n=1–6) were used to identify the peak positions in the 13C, 2H, and 1H NMR spectra due to each individual isotopomer, and the effect of isotopic substitution on the chemical shifts was determined in each case. While the NMR of all three nuclei worked well for the identification of the individual components of the 1:1 standard mixtures, both 1H and 2H NMR suffered from inadequate resolution when studying complex reaction mixtures because of the broadening of the lines due to 1H–1H (1H NMR) and 2H–2H (2H NMR) couplings. 13C NMR was therefore determined to be the method of choice for the quantitative analysis of the reaction mixtures. Using the 13C NMR results, a correlation that takes into account the primary and secondary isotope substitution effects on chemical shifts was deduced. This equation was used for the identification of the individual components of the mixtures, and integration of the individual observed resonances was then employed for quantification of their composition. This study shows that 13C NMR with 1H and 2H decoupling is a viable procedure for studying mixtures of deuterated ethanes. Furthermore, the additivity of the isotopic effects on chemical shifts and the transferability of the values obtained with ethane to other molecules makes this approach general for the analysis of other isotopomer mixtures.