Calculation of atom-centered partial charges for heme

Atom-centered partial charges are calculated for the Fe-heme in cytochrome P450cam for use in molecular dynamics simulations of polar substrates bound in the active site of the enzyme. Charges are fit to the electrostatic potential produced by ab initio UHF wavefunctions for an Fe-porphine model. Basis set dependence of these charges is observed using the LANL1DZ, LANL2DZ and augmented 6–31G levels of theory. Upon geometry optimization of the enzyme, these charge sets cause varying degrees of distortion of the porphyrin from its crystallographically observed conformation. Scaling the charges calculated from the augmented 6–31G basis by 75% reduces the heme distortion while preserving reasonable interactions with a polar substrate. A comparison of the calculated charges with other published values is presented.     

A critical role of a facilitatory presynaptic kainate receptor in mossy fiber LTP.

The mechanisms involved in mossy fiber LTP in the hippocampus are not well established. In the present study, we show that the kainate receptor antagonist LY382884 (10 microM) is selective for presynaptic kainate receptors in the CA3 region of the hippocampus. At a concentration at which it blocks mossy fiber LTP, LY382884 selectively blocks the synaptic activation of a presynaptic kainate receptor that facilitates AMPA receptor-mediated synaptic transmission. Following the induction of mossy fiber LTP, there is a complete loss of the presynaptic kainate receptor-mediated facilitation of synaptic transmission. These results identify a central role for the presynaptic kainate receptor in the induction of mossy fiber LTP. In addition, these results suggest that the pathway by which kainate receptors facilitate glutamate release is utilized for the expression of mossy fiber LTP.     

Ethylbenzene hydroxylation by cytochrome P450cam.

The metabolism of ethylbenzene by cytochrome P450cam was analyzed by experimental and theoretical methods. The present experiments indicate that ethylbenzene is hydroxylated almost exclusively at the secondary ethyl carbon with about a 2:1 ratio of R:S product. Several molecular dynamics trajectories were performed with different starting conformations of ethylbenzene in the active site of P450cam. The stereochemistry of hydroxylation predicted from the molecular dynamics simulations was found to be in good agreement with the observed products.     

Analysis of active site motions from a 175 picosecond molecular dynamics simulation of camphor-bound cytochrome P450cam.

The structure and internal motions of the active site residues of camphor-bound cytochrome P450cam have been evaluated on the basis of a 175 psec molecular dynamics simulation. The active site residues generally show very small deviations away from their starting crystal positions. These residues also generally show much smaller fluctuations than for the enzyme as a whole. Phe 87 is dynamically very unusual and is suggested to play a role in substrate movement into and/or out of the active site. The average distance between the heme iron and atoms C5, C6, and C3 of camphor is 5.3, 6.0, and 7.0 A, respectively. This trend is consistent with the experimentally observed stereospecificity of the hydroxylation reaction. On the basis of distance and angle criteria, both 5-exo and 5-endo hydrogen abstraction are predicted to occur during the hydroxylation reaction; although the 5-exo pathway is expected to be 3-fold more likely.     

Energetic and structural aspects of ethidium cation intercalation into DNA minihelices

The enthalpy ΔH for the intercalation of the ethidium cation (EC) into DNA minihelices can be decomposed into (1) an energy of conformational adjustment (i.e., the energy of minihelix extension and unwinding from the B-form to the intercalated form) and (2) EC minihelix intermolecular interactions. In the present study, we have focused our attention mainly on a decomposition of the energetic factors of the EC minihelix intermolecular interactions, while the essential features of the energy of conformational adjustment have been discussed in detail elsewhere by us. The structural features of the various resulting energy-minimized EC-intercalated complexes are compared with each other and the initial x-ray model structure. ΔH is estimated to be in the range of ?12.3 to ?24.0 kcal/mol. This theoretical estimate is qualitatively and quantitatively in agreement with a variety of available experimental data. The energy of conformational adjustment is an energetically unfavorable step, while the energetically favorable contribution of the EC minihelix intermolecular interactions is responsible for the overall favorable nature of the intercalation process involving the EC. On the other hand, the observed preference for intercalation into Pyr(3′–5′)Pur DNA sequences over their isomeric Pur(3′–5′)Pyr sequences is controlled by the energy of conformational adjustment and not by the EC minihelix intermolecular interaction contribution. No base-composition effect is expected at EC concentrations normally found at cellular conditions. Moreover, the structural features of the various EC-intercalated complexes are very similar regardless of minihelix base sequence or composition. These results compare favorably with available evidence. The nature of biologically preferred sites of EC binding with the minihelices is discussed.     

THE INTRARENAL DISTRIBUTION OF TRITIATED PARA-AMINOHIPPURIC ACID DETERMINED BY A MODIFIED TECHNIQUE OF SECTION FREEZE-DRY RADIOAUTOGRAPHY

Section freeze-dry radioautography has been used to examine the intrarenal distribution of a water-soluble organic acid (para-aminohippuric acid (PAH-3H)) under constant-infusion, steady-state conditions in mouse and rat kidney in vivo. The technique described here has the following advantages: (a) Sectioning and freeze-drying are accomplished in a closed cryostat at temperatures below -40°C; (b) Handling of the section is facilitated by mounting of the section-to-be on adhesive-coated Saran Wrap prior to cutting; (c) Unembedded freeze-dried sections are attached to photographic film at ambient temperature in the dark room; (d) Fixation follows completion of radioautographic exposure and precedes photographic development; (e) Permanent close contact is maintained between tissue and film. Morphologic preservation compared favorably with that obtained by optimal fixation techniques, which, however, permit diffusion. Cellular accumulation of PAH-3H during secretion was demonstrated in the proximal tubule under steady-state conditions in vivo. The cellular concentration of PAH-3H was uniform throughout the length of the proximal tubule in mouse and rat kidney.