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Gramicidin A forms a variety of structures dependent upon its environment. In organic solvents, it forms either unstructured monomers or a series of highly structured dimers (species 1-4). Two of these dimers have been isolated and their structures determined by 2D NMR. An HPLC technique was developed to isolate species 4 in dioxane. Distance geometry was used to generate backbone and sidechain structures from 218 experimental constraints. Species 4 in dioxane is a right-handed parallel $\beta$ double helix with approximately 5.7 residues per turn, a length of 27 A, and a pore width between 2 and 3 A. In ethanol, all four dimeric species exist and interconvert. Addition of benzene to ethanol solution was found to shift the conformational equilibrium to greatly favor species 3. This isolated species 3 conformation was determined to high resolution. A total of 702 experimental constraints were generated. Backbone and sidechain structures were generated via a unique distance geometry/simulated annealing routine, wherein the backbone structure was determined first, and then held rigid while the sidechain structures were determined. Species 3 in benzene/ethanol is a left-handed antiparallel $\beta$ double helix with approximately 5.7 residues per turn, a length of 36-37 A, and a pore width between 2 and 3 A. A qualitative estimate of sidechain dynamics was obtained through analysis of NOE violations and coupling constant data. This conformation can be inserted into a lipid bilayer where it undergoes a transformation to a right-handed single-stranded head-to-head $\beta$-helical dimer which acts as a monovalent cation channel. The transformation pathway is discussed. The species 3 conformation is one of the highest resolution NMR structures of a peptide or protein to date, and is compared with one of the most highly refined crystal structures, that of gramicidin / species 3 out of benzene/ethanol. A surprising degree of agreement is found, particularly in the backbone, but also in the sidechains. Dynamics in solution is correlated with disorder in the crystal for several sites. Similarities and differences are interpreted in light of differences in the environment and/or the experimental and computational methods. / Source: Dissertation Abstracts International, Volume: 54-03, Section: B, page: 1280. / Major Professor: Timothy A. Cross. / Thesis (Ph.D.)--The Florida State University, 1993.

The backbone motions of the lipid membrane spanning polypeptide gramicidin A in its channel conformation have been examined by solid state nuclear magnetic resonance spin-lattice relaxation experiments. The spin relaxation parameters are interpreted in light of a detailed model of local motions. It is found that localized motions of the peptide backbone linkage planes occur with significant amplitudes on the nanosecond timescale. Peptide plane librations on this timescale are two orders of magnitude slower than has been predicted by molecular dynamics simulations and have not been considered explicitly in computational studies of the channel. This timescale is on the same order as rates of ion translocation through the channel lumen and raises the interesting possibility of a direct correlation between dynamics and function. / Source: Dissertation Abstracts International, Volume: 54-08, Section: B, page: 4035. / Major Professor: T. A. Cross. / Thesis (Ph.D.)--The Florida State University, 1993.

The determination of protein structures is central to the field of structural biology. Although techniques exist and are well developed for the determination of proteins in solution and proteins that are readily crystallized, techniques for the determination of protein structures in the solid state are just now being developed. Described here is such a technique. The structure of the membrane spanning, monovalent cation channel gramicidin A is solved through the use of experimentally determined Solid State Nuclear Magnetic Resonance orientational constraints. The structure is solved for the peptide in its native conformation in a fully hydrated lipid bilayer by taking advantage of the wealth of experimental observations. The resulting structure is computationally refined by a method described here using a simulated annealing protocol which defines a penalty function based on the experimental observations and the CHARMM energy. This refinement strategy produces a structure that meets the experimental data and has a reasonable global energy. / Source: Dissertation Abstracts International, Volume: 57-01, Section: B, page: 0181. / Major Professor: Timothy A. Cross. / Thesis (Ph.D.)--The Florida State University, 1995.

A characterization of nicotinic acetycholine-activated ion channels in insects was carried out in dissociated neurons in primary culture from the central nervous systems of the larval fruit fly, Drosophila melanogaster, larval and embryonic house fly, Musca domestica, and the nymphal locust, Schistocerca gregaria using whole-cell and cell-attached patch-clamp techniques. The single ion channel currents observed in cell-attached patches were very similar between the four insect preparations. These channels exhibited characteristics similar to channels described in both vertebrate neurons and muscle, as well as other invertebrate preparations. / There were two major classes with average single channel conductances of 32 pS and 57 pS. The 32pS insect channel had longer open times than the 57 pS channel. The channel activity occurred in bursts (openings separated by brief closures) lasting from 1 to 5 ms. The 57 pS channel openings also occurred in bursts, though they were more frequently isolated fast openings of less than 1 ms duration. / Activation of fly 32 pS acetylcholine channels was examined more closely by looking at their concentration- and voltage-dependence. The open and closed interval durations of this channel were independent of voltage. Two separate types of dose-response experiments were performed. Both sets of curves indicated multiple ligand binding steps for the activation of insect acetylcholine channels. Analysis of closed intervals and clusters (groups of bursts separated by long closures) across a concentration series revealed an unusual inactive state and heterogeneous kinetic behavior within the 32 pS channel class. Tentative estimates of rates for the channel conformational changes were made. / Insect neuronal acetylcholine channels were affected by several established nicotinic pharmaceutical agents. However, the pharmacological profile of these channels showed some unique features. They were resistant to activation by the nicotinic agonist suberyldicholine, though both carbachol and nicotine were capable of opening these channels. Hexamethonium and decamethonium were approximately equi-effective. The insect channels were also blocked by both $\alpha$- and $\kappa$-bungarotoxin. / Source: Dissertation Abstracts International, Volume: 52-03, Section: B, page: 1259. / Major Professor: Christopher J. Lingle. / Thesis (Ph.D.)--The Florida State University, 1991.

Patulin is an antibiotic and mycotoxin synthesized from acetyl- and malonyl-CoA during the stationary growth phase of a number of fungi including Penicillium patulum. The investigation of the metabolic regulation of patulin production and of the patulin biosynthetic scheme has been hindered by the lack of a rapid and sensitive technique for the analysis of culture medium. A technique utilizing reversed-phase HPLC and methanol gradient elution in the presence of the ion-pair reagent, tetrabutylammonium phosphate, was developed to allow the quantitation of medium metabolites during the course of a culture. This technique was determined to be at least 100-fold more sensitive than an earlier GC-based technique. With slight modifications, the HPLC technique was adapted for use in the detection and quantitation of a number of pathway enzymes in vitro. The utility of the HPLC enzyme assay technique was demonstrated during the development of a protocol for the purification of one of these enzymes, m-hydroxybenzyl alcohol dehydrogenase. Hydrophobic interaction, anion exchange, and size exclusion FPLC steps were included in this protocol. Pure dehydrogenase enzyme had a native molecular weight of 105-120 kDa and a subunit weight of 32 kDa. No cooperativity was detected during a kinetic analysis with m-hydroxybenzaldehyde substrate. The dehydrogenase obeyed classical Michaelis-Menten kinetics except at high aldehyde substrate concentrations where substrate inhibition was observed. Using the HPLC assay technique, we detected a cell-free microsomal activity capable of converting gentisyl alcohol (K$\sb{\rm m}\sp\prime$ = 170 $\mu$M), but not gentisaldehyde, to phyllostine and neopatulin in the absence of added cofactor. This activity converted toluquinol (K$\sb{\rm m}\sp\prime$ = 240 $\mu$M) to a new metabolite identified as ($-$)desoxyphyllostine. The substrates for / this microsomal activity were determined to be the hydroquinones and O$\sb2$ rather the quinones and H$\sb2$O$\sb2$. Attempts at purification of this enzyme failed, but a characterization of partially purified enzyme detected inhibition by 1,10-phenanthroline, EDTA, and PCMBS, but not by CO or KCN. / Source: Dissertation Abstracts International, Volume: 49-12, Section: B, page: 5115. / Major Professor: Robley J. Light. / Thesis (Ph.D.)--The Florida State University, 1988.

An analytical approach for the determination of chemical shift tensor orientations with respect to molecular frame is described. Advantage is taken of the known orientation of the unique axis of the heteronuclear dipolar interaction between $\sp{15}$N-$\sp{13}$C in doubly labeled samples through observations of the dipolar-coupled $\sp{15}$N and $\sp{13}$C chemical shift powder patterns. Certain singularities in the powder pattern spectrum have a unique dependence on a single rotational variable and hence an analytical solution can be found from the frequency differences of the singularities. Using this approach the orientations of the amide $\sp{15}$N and carbonyl $\sp{13}$C chemical shift tensors with respect to the molecular frame have been determined by solid state NMR for sites in the gramicidin A transmembrane channel. The results show that the magnitudes of the $\sp{15}$N chemical shift tensor are different for various sites in gramicidin A and the orientations of this tensor are similar for most sites. Both magnitudes and orientations of the $\sp{13}$C chemical shift tensor are similar for a variety of sites with the exception of the $\sigma\sb{22}$ element of Gly$\sb2$. The orientation of the $\sp{14}$N electric field gradient tensor with respect to the molecular frame has been also studied based on the orientation of the $\sp{13}$C chemical shift tensor which has been determined for the site of interest. Armed with the $\sp{15}$N-$\sp{13}$C$\sb1$ and $\sp{15}$N-$\sp1$H nuclear dipolar interactions as well as the chemical shift interactions for two specific sites in an oriented preparation of gramicidin A, it is shown here that the torsion angles in the polypeptide backbone of the gramicidin A transmembrane channel in a lipid environment can be determined analytically. For the first time an analytical solution for polypeptide backbone torsion / angles have been achieved from solid state NMR spectroscopic data. With the use of the three nuclear spin interactions and the consideraction of bond angle constraints, the torsion angle solution set can be reduced from 1024 possibilities to just two pairs of torsion angles for the Ala$\sb3$ site. Both of the possible structural solutions for the relative orientation of the peptide planes on either side of Ala$\sb3$ demonstrated a right-handed $\beta$-type helix. / Source: Dissertation Abstracts International, Volume: 51-07, Section: B, page: 3285. / Major Profesor: Timothy A. Cross. / Thesis (Ph.D.)--The Florida State University, 1990.

The non-random covalent binding to DNA by the intercalators 8-azido-ethidium, 7-azido-actinomycin D, anti-(+)-benzo (a) pyrene 7,8-diol 9,10-epoxide, and anti-($-$)-benzo (a) pyrene 7,8-diol 9,10-epoxide was investigated using techniques analogous to DNA sequencing. A computer-assisted methodology was subsequently developed that allowed the characterization of all sequence preferences of covalent binding up to the quartet level. All intercalators exhibited some preferences in binding. 7-azido-actinomycin D was most sequence selective and 8-azido-ethidium was least sequence selective. Next-nearest neighboring bases exert a major influence on the reactivities of the intercalators. The magnitude of the next-nearest neighbor influence is almost as great as the nearest-neighbor influence. There were certain sequence preferences of covalent binding shared by all intercalators. The sequence preference analysis indicated that 7-azido-actinomycin D and 8-azido-ethidium were excellent probes for evaluating the reversible binding of the parent actinomycin D and ethidium molecules. There were some differences in the sequence preferences of covalent binding by the two benzo (a) pyrene diol epoxide enantiomers. The potent carcinogenicity of the anti-(+)-isomer may be caused by the adduct at the exocyclic amine of guanine bases, since this isomer forms more of these adducts than the anti-($-$)-isomer. / Source: Dissertation Abstracts International, Volume: 51-07, Section: B, page: 3284. / Major Professor: Randolph L. Rill. / Thesis (Ph.D.)--The Florida State University, 1990.

A variety of methods have been used to explore the suitability of Ac-MP-8 as a water soluble, non-aggregating ferric heme model for the peroxidases. In particular, resonance Raman spectroscopy has been used to investigate the structure of resting Ac-MP-8 over the entire pH 2-12 range as well as that of the high valent intermediates formed on its interaction with hydroperoxides. Both B- and Q-band excitation have been employed to observe the skeletal stretching frequencies of the porphyrin ring. The neutral form of resting Ac-MP-8 is best described as a thermal mixture of six coordinate high-spin and intermediate-spin species since two sets of porphyrin skeletal stretching frequencies are observed in the 1300-1700 cm$\sp{-1}$ region at 296$\sp\circ$K. The axial ligand are His and water. The acid form of Ac-MP-8 exists in a high-spin five- and six-coordinate equilibrium with water or buffer as axial ligands. The alkaline form of Ac-MP-8, however, is predominantly a five-coordinate high-spin species with only the imidazolate form of His as an axial ligand. / The addition of two equivalents of hydroperoxide to Ac-MP-8 at pH 7.0 gives a green intermediate whose optical spectrum resembles that of HRP compound I and other $\rm\sp2A\sb{2u}$ porphyrin $\pi$-cation radicals. The resonance Raman spectrum of this species exhibits bands at 1640, 1575, 1491, 1372 cm$\sp{-1}$ that are assignable to the porphyrin skeletal modes $\nu\sb{10}$, $\nu\sb2$, $\nu\sb3$, and $\nu\sb4$, respectively. The shift in vibrational frequencies on formation of Ac-MP-8 compound I match the trends predicted for an $\sp2$A$\sb{\rm 2u}$ $\pi$-cation radical. When the same reaction is carried out above pH 10.5, a red species is formed. Its optical spectrum strongly resembles HRP compound II with a Soret band at 407 nm and a well-formed doublet at 520 and 548 nm. The resonance Raman spectrum of this red species is a typical of a normal six-coordinate, low-spin porphyrin with $\nu\sb{10}$, $\nu\sb2$, $\nu\sb3$, $\nu\sb4$ observed at 1641, 1591, 1509, 1377 cm$\sp{-1}$, respectively. A $\nu$(Fe(IV)=O) band is observed at 783 cm$\sp{-1}$ that is confirmed by its shift to 743 cm$\sp{-1}$ when $\rm H\sb2\sp{18}O\sb2$ is used in the reaction. Thus, Ac-MP-8 and its high valent intermediates are good models for the resting form of peroxidases and their catalytic intermediates. / Source: Dissertation Abstracts International, Volume: 53-03, Section: B, page: 1350. / Major Professor: Harold E. Van Wart. / Thesis (Ph.D.)--The Florida State University, 1992.

A detailed experimental description of molecular dynamics requires an accurate description of the global correlation time. For the gramicidin cation-selective channel the local dynamics of the polypeptide backbone are thought to play a very significant role in the functional process of this channel which occurs on the 10-100 nsec time scale. Here, the global correlation time is found from fully deuterated Ala$\sb3$ and Ala$\sb5$ $\sp2$H NMR powder pattern spectra to be 0.92 msec at 309 K. This surprisingly long correlation time may reflect the high viscosity of the peptide environment or possibly the higher molecular weight of a peptide-lipid or peptide-peptide aggregate. / High resolution dynamic and structural characterizations have been achieved for each of the alanine, valine, and leucine sidechains of the gramicidin channel while solubilized in hydrated lipid bilayers. The characterizations have been achieved by $\sp2$H solid state NMR. Spectra of unoriented hydrated samples recorded at 278 K provide the sidechain dynamic information since the global motion is eliminated at this temperature. The measured quadrupole splittings from the aligned samples provide orientational constraints for determining the sidechain structures. Librational amplitudes for each site throughout the sidechain have also been characterized. Val$\sb6$ and Val$\sb8$ are shown to be fixed in rotameric states. However, Val$\sb1$ and Val$\sb7$ undergo three-state jump motions in which the populations among the three $\chi\sb1$ rotameric states are unequal. For the temperature range between 278 and 309 K there is no evidence for changes in the conformational state populations. Evidence shows that slight changes of the backbone structure occur when the temperature falls below the liquid-crystalline to gel phase. / The overall gramicidin structure is a triangular shape when viewed from either end. The role of Val$\sb6$ and Leu$\sb{12}$, and Val$\sb8$ and Leu$\sb{14}$ sidechains is to stabilize the Trp$\sb{13}$ and Trp$\sb{15}$ indole ring conformation, respectively. The Leu$\sb4$ sidechain stabilizes the Trp$\sb{11}$ indole ring. The Leu$\sb{10}$ sidechain possibly interacts with the ethanolamine group. This gramicidin structure shows a significantly different shape from the gramicidin structure determined by solution NMR in SDS micelles. / Source: Dissertation Abstracts International, Volume: 55-11, Section: B, page: 4751. / Major Professor: Timothy A. Cross. / Thesis (Ph.D.)--The Florida State University, 1994.