Quasi-harmonic method for studying very low frequency modes in proteins

A quasi-harmonic approximation is described for studying very low frequency vibrations and flexible paths in proteins. The force constants of the empirical potential function are quadratic approximations to the potentials of mean force; they are evaluated from a molecular dynamics simulation of a protein based on a detailed anharmonic potential. The method is used to identify very low frequency (～1 cm^?1) normal modes for the protein pancreatic trypsin inhibitor. A simplified model for the protein is used, for which each residue is represented by a single interaction center. The quasi-harmonic force constants of the virtual internal coordinates are evaluated and the normal-mode frequencies and eigenvectors are obtained. Conformations corresponding to distortions along selected low-frequency modes are analyzed.     

Photokilling of bacteria by the natural dye curcumin

Curcumin is a yellow-orange compound derived from the root of Curcuma longa (Zingiberaceae family), that has been used as a medicine, spice and coloring agent. Curcumin has proved nontoxic in a number of cell culture and whole animal studies. Curcumin has, however, been reported to have bactericidal effects at very high concentrations. When illuminated, curcumin exerted potent phototoxic effects in micromolar amounts. Gram-negative bacteria displayed greater resistance to curcumin phototoxicity relative to Gram-positive bacteria. Oxygen was required for curcumin phototoxicity. Curcumin binding to cells was not required for photokilling; the reactive intermediate therefore must be relatively long-lived. The mechanism(s) of curcumin phototoxicity may involve hydrogen peroxide production. Singlet excited oxygen was not detected.Publication no 35 of the Center for Photochemical Sciences     

Mechanism of formation of bound alpha-iminoglutarate from alpha-ketoglutarate in the glutamate dehydrogenase reaction. A chemical basis for ammonia recognition

Two mechanisms have been postulated for the formation of bound alpha-iminoglutarate intermediate during the glutamate dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate; one involves the nucleophilic attack of ammonia on a covalently bound Schiff base in the enzyme-NADPH-alpha-ketoglutarate complex, and the other involves the reaction of ammonia with the carbonyl group of alpha-ketoglutarate in the ternary complex. We have measured the rates of carbonyl oxygen exchange in the complex to unambiguously distinguish between these two mechanisms. We find that the loss of label in the carbonyl oxygen-labeled ternary complex is at least 10(5) times slower than the rate of the reductive amination reaction. Therefore, the former mechanism cannot be operative. We also find that (i) the carbonyl oxygen exchange in free alpha-ketoglutarate proceeds without any significant catalysis by its gamma-carboxylate group; (ii) this exchange reaction has energy parameters which are comparable to those observed for the hydration of simple aliphatic ketones; and (iii) the carbonyl oxygen exchange in bound alpha-ketoglutarate is slower than that in the free keto acid over a wide pH range. We conclude that the oxygen exchange in the free and bound alpha-ketoglutarate must occur via a gem-diol intermediate. The observation that the enzyme inhibits the reaction of water with alpha-ketoglutarate while it catalyzes the reaction of ammonia with the same keto acid points to an extraordinary recognition of ammonia by the enzyme. We interpret this observation by assuming that the enzyme-NADPH-alpha-ketoglutarate complex exists in two forms, a predominant form which is produced rapidly upon mixing the components together and an unstable form which is produced in trace amounts from the predominant form via a gem-diol intermediate. These two forms are presumed to differ in the spatial relationship of the carbonyl group to the enzyme functional groups. The carbonyl group in the unstable form is assumed to be surrounded by the same enzyme groups as the iminium ion is in the bound iminoglutarate complex. We ascribe the remarkable catalysis of the ammonia reaction and the inhibition of the water reaction by the enzyme to the opposing interactions of the iminium and carbonyl groups with these surrounding enzyme groups.     

Thermodynamic parameters for the glutamate dehydrogenase catalyzed alpha-imino acid-alpha-amino acid interconversion

Delta 1-Piperidine 2-carboxylic acid, an alpha-imino acid, is reduced by 1,4-dihydropyridines to pipecolic acid, an alpha-amino acid, and the corresponding pyridinium ions. This nonenzymatic reaction occurs only in the direction of pipecolic acid production. Glutamate dehydrogenase catalyzes this reaction when the reductant is NADPH and gives as products L-pipecolic acid and NADP+. The reaction velocity for the enzyme-catalyzed reaction is measurable in either direction. The pH-independent equilibrium constant, Keq, for the reduction of the imino acid by NADPH to give pipecolic acid anion and NADP+ was determined from the equilibrium conditions and the pKa values of pipecolic acid (10.72) and of the cyclic imino acid (8.10). The value of Keq was found to be 175 +/- 30; the values of delta G0, delta H0 and delta S0 are -3.1 +/- 0.1 kcal/mol, 5 +/- 1 kcal/mol and 27 +/- 4 e.u., respectively. The data indicate that the reactants are far more solvated than the products and that there must be a large degree of solvent reorganization during the course of the reaction. If these thermodynamic parameters apply to the redox step of the enzyme-catalyzed glutamate reaction, then the burst phase which results upon mixing the enzyme, L-glutamate and NADP+ in stoichiometric amounts must contain a hidden nonredox step of large delta H0 value to account for the curved Arrhenius plot observed for this phase (A.H. Colen, R.T. Medary and H.F. Fisher, Biopolymers 20 (1981) 879).     

Alport Syndrome mutations in type IV tropocollagen alter molecular structure and nanomechanical properties

Alport Syndrome is a genetic disease characterized by breakdown of the glomerular basement membrane (GBM) around blood vessels in the kidney, leading to kidney failure in most patients. It is the second most inherited kidney disease in the US, and many other symptoms are associated with the disease, including hearing loss and ocular lesions. Here we probe the molecular level structure–property relationships of this disease using a bottom-up computational materiomics approach implemented through large-scale molecular dynamics simulation. Since the GBM is under constant mechanical loading due to blood flow, changes in mechanical properties due to amino acid mutations may be critical in the symptomatic GBM breakdown seen in Alport Syndrome patients. Through full-atomistic simulations in explicit solvent, the effects of single-residue glycine substitution mutations of varying clinical severity are studied in short segments of type IV tropocollagen molecules. The segments with physiological amino acid sequences are equilibrated and then subjected to tensile loading. Major changes are observed at the single molecule level of the mutated sequence, including a bent shape of the structures after equilibration (with the kink located at the mutation site) and a significant alteration of the molecules’ stress–strain responses and stiffnesses. These results suggest that localized structural changes at amino acid level induce severe alterations of the molecular properties. Our study opens a new approach in pursuing a bottom-up multi-scale analysis of this disease.     

Atypical cytologic features of medullary carcinoma of the thyroid. A review of 12 cases.

A total of 12 cases of medullary carcinoma of the thyroid were reviewed with regard to atypical cytologic features. They included clusters of cells with a follicular arrangement, papillary fronds, spindle-shaped cells, pleomorphic nuclei, nucleoli and intranuclear inclusions. Congo red staining on selected destained smears was positive for amyloid in three cases. Histology was also available in 12 cases. Two cases were incorrectly diagnosed as papillary carcinoma and one as follicular carcinoma on cytology. There were no false-positive diagnoses.     

Maleated thermoplastic starch by reactive extrusion

Novel maleated thermoplastic starch (MTPS) with both improved processing and reactivity useful in the melt-blending with biodegradable polyester was prepared through in situ reactive modification of thermoplastic starch (TPS) with maleic anhydride (MA) as esterification agent. Glycerol was used as plasticizer. Physico-chemical parameters of MTPS were determined at different MA contents, while keeping both the content in glycerol (20 wt% by starch), and the processing temperature constant (150 °C). Soxhlet extraction attested for the complete incorporation of glycerol into the starch backbone during the maleation process at low content in MA. In addition, two-dimensional liquid-phase NMR measurements attested for the preferential esterification of starch backbone at C6, together with the occurrence of some hydrolysis and glucosidation reactions. Such reactions promoted by MA moieties reduced the intrinsic viscosity of the MTPS, expecting an improvement in its processability. WAXS diffraction analyses confirmed the complete disruption of the granular structure of native starch in MTPS during the reactive extrusion processing.