The protease (alpha)-chymotrypsin, specific for uncharged hydrophobic residues, was used to probe the fine structural topography of the nucleosome core. The consequences of proteolytic cleavages at specific histone sites were assessed in terms of effects upon the overall nucleohistone structural integrity. / Electrophoresis of histones from partially proteolyzed cores on 18% polyacrylamide slab gels containing SDS showed that H3 was rapidly digested by chymotrypsin, yielding a single product (CP1) approximately 112 residues long. Extensive digestion yielded a stable limit pattern of four major histone fragments ranging in size from 102-80 residues long. Comparative electrophoresis performed on gels containing 8 M urea, 5% acetic acid, and 6 mM triton X-100 established that other core histones were essentially resistant to digestion until the majority of H3 was degraded. Second dimension electrophoresis showed that CP1 migrated near H3 in the trition-acid-urea system. Also resolved by second dimension electrophoresis were two additional products that migrated near H4 and CP1 (CP2' and CP3'), respectively, in the SDS-dimension. / High pressure liquid chromatography peptide analyses confirmed that the major chymotryptic product, CP1, was derived from H3 and had an intact carboxy terminus. HPLC analyses of dansyl amino acids derived from CP1 made it possible to assign leucine 20 as the preferential chymotryptic cleavage site in H3. This is the only one of 72 possible chymotryptic sites in core histones that clearly is topographically accessible in native cores. Few and perhaps no sites are accessible in the other core histones. / Cleavage at leucine 20 in H3 promoted structural unfolding and induced changes in the core DNA winding angle. Circular dichroism and Raman spectroscopy, thermal denaturation, DNAse I digestion, and nucleo-histone electrophoresis studies were used to characterize these changes and indicated that non-basic histone-DNA interactions were essential for the maintenance of core structure and stability. / Source: Dissertation Abstracts International, Volume: 43-07, Section: B, page: 2091. / Thesis (Ph.D.)--The Florida State University, 1982.

Lumichrome (7,8-dimethylalloxazine) undergoes excited state double proton transfer in two solvents: acetic acid and pyridine. This research extends the mechanistic understanding of this process. Evidence is presented that points to two different mechanisms in the two solvent systems. In acetic acid, the one-step, concerted double proton transfer mechanism is supported. In pyridine, a two-step mechanisms is proposed. In the first step, dissociation occurs in the ground state, with the formation of the lumichrome anion and the pyridinium cation. In the second step, excited state single proton transfer takes place, from the pyridinium cation to the lumichrome anion, forming the tautomer species. / In other solvents, e.g., alcohols, ethers, excited state proton transfer is not observed. In order to explain this it is proposed that the first ground state pK(,a) (defined as the dissociation constant in aprotic solvents) of lumichrome is highly dependent on the hydrogen-bond strength of the solvent, becoming more acidic in weakly hydrogen bonding solvents. If this is the case, then lumichrome exists in neutral and anionic forms in equilibirum in these solvents, and the cyclic hydrogen bonding required for proton transfer cannot occur. / 7-azaindole has long been known to undergo excited state proton transfer in hydrocarbon and alcohol solutions. We have demonstrated the occurrence of an analogous phototautomerization in the purine bases adenine and guanine, which are structurally similar to 7-azaindole. However, this proton transfer does not occur in hydrocarbon and alcohol solvents. Rather, as in the case of lumichrome, acetic acid and pyridine catalyze tautomerization in the purine bases. From these results we have drawn certain interesting conclusions as to the role of the solvent in excited state proton transfer. In hydroxyl-type solvents, such as alcohols and acetic acid, the pK(,a) of the solvent must match as closely as possible the pK(,a) of the molecule in question in order for proton transfer to occur. In pyridine, the more acidic the molecule, the more efficient is phototautomerization. / Source: Dissertation Abstracts International, Volume: 45-09, Section: B, page: 2784. / Thesis (Ph.D.)--The Florida State University, 1984.

Results of the characterization of a soluble chromatin fraction that is released by brief digestion with micrococcal nuclease are described. Both transcriptionally active sequences and newly replicated sequences are highly enriched in this rapidly released, soluble (RRS) chromatin. RRS fractionation permits 7 to 20 fold enrichments of transscriptionally active sequences and 15 fold enrichments of newly replicated DNA (i.e. compared to the insoluble or P fraction). This functionally important chromatin is more accessible or sensitive to nuclease than the bulk of the chromatin. Greater accessibility is probably a natural consequence of a necessity to interact with regulatory molecules, polymerases, or packaging proteins. RRS chromatin is enriched in HMG 1, 2, 14 and 17 and many other non-histone chromosomal proteins (NHCP). RRS chromatin contains a generally basic subset of NHCP, and RRS chromatin mononucleosomes contain a very basic subset of these. RRS chromatin nucleosomes are highly enriched in a protein we have termed HL4. HL4 was isolated; its amino acid composition indicates it is not a breakdown product of histones or common NHCP. HL4 appears to replace H4 in the nucleosome core. A protein with mobility similar to HL4 also appears in chicken erythrocyte (CE) RRS chromatin. RRS chromatin from CE is very similar to mouse myeloma (MM) RRS chromatin in its protein component and enrichment in transcriptionally active sequences, but differs from the MM system in its low yields and faster sedimenting fractions. Histones 3 and 4 of soluble fraction are less acetylated than in bulk chromatin, but the NHCP of the soluble fraction are highly acetylated. RRS contains many unusual nucleosomes which can be separated into distinct classes. The ability to separate these unusual nucleosomes permits a wide range of future experiments. RRS chromatin provides new insights into functionally important NHCP and the fine structure of active genes. / Source: Dissertation Abstracts International, Volume: 42-01, Section: B, page: 0042. / Thesis (Ph.D.)--The Florida State University, 1981.

Natural abundance carbon-13 nmr studies of myosin in solution, its proteolytic fragments S1 (globular head) and rod (alpha-helical tail), of its filaments and of LiBr-denatured myosin are reported. Solution spectra, T(,1)'s and NOE's were measured at 37.7 MHz, 20(DEGREES)C with scalar decoupling ((gamma)(,H)B(,2)/2(pi) = 2 kHz). If native proteins were rigid only S1 would have observable intensity. In fact 28% of myosin and rod, 56% of S1 and 54% of denatured myosin carbons are measurable. The rest are broadened beyond detection (> 2 kHz) by ('13)C-('1)H dipolar coupling (aliphatic and non-protonated) and by anisotropic chemical shift (CSA) interactions (non-protonated carbons). Observable carbons possess at least one component of motion faster than 10('-7)s which effectively averages the couplings. 10% of myosin, 9% of rod and 56% of S1 alpha (backbone) carbons; 20, 13, and 56% (resp.) of aliphatic sidechain carbons; 24, 25 and 56% of carbonyl (backbone + sidechain) carbons have this motion. Alpha region T(,1)'s (0.1 to 0.3 s) and nonminimal NOE's (1.4 to 2.3) place fast motion in the range 10('-9) to 10('-7)s. Analysis of alpha relaxation using models for diffusive motion indicates that a wide distribution of correlation times is required to fit most alpha data. Thus the fast motion of backbone carbons is anisotropic. Intensities and relaxation of carbonyls compared to aliphatics are consistent with substantial contribution due to dipolar interactions in carbonyls. / High power double resonance experiments at 15.1 MHz, 5(DEGREES) were done with Dr. D. A. Torchia, to investigate motion in myosin filaments. Dramatic increase in intensity with dipolar decoupling ((gamma)(,H)B(,2)/2(pi) = 55 kHz) using a 2s delay time indicates that most (80%) aliphatics are strongly coupled due to rigidity but still have short T(,1)'s, more characteristic of amorphous than crystalline regions. Large NOE's strongly indicate motion components near 10('-8)s. Thus strongly coupled carbons have highly anisotropic motion. Cross polarization (CP) spectra show that most alphas and sidechain aliphatics are much more rigid than the remainder of aliphatics. CP linewidths indicate that aliphatic motion ( 10('-4)s) does not. / Source: Dissertation Abstracts International, Volume: 42-11, Section: B, page: 4285. / Thesis (Ph.D.)--The Florida State University, 1982.

A detailed spectroscopic study was carried out on 3-hydroxyflavone in order to elucidate the mechanism which is responsible for the anomalous fluorescence of flavonols in the green-yellow region. A combination of absorption and luminescence measurements indicated that the green emission in 3-hydroxyflavone is due to excited state tautomerization of the molecule by an intramolecular proton transfer mechanism. Excitation spectra, deuteration studies, and comparison with the luminescence behavior of the corresponding methylated analogue confirmed the proton transfer mechanism. In a rigid hydrocarbon or ether glass at 77 K, the green emission is replaced by a predominant UV-violet fluorescence of the normal molecule, in the usual vibrational envelope inversion relationship with the absorption spectrum. The extraordinary viscosity dependence of the tautomerization process has been explained in terms of the mechanical viscous-flow-barrier model of Dellinger and Kasha. Luminescence measurements were performed at various temperatures between 25 K and ambient temperature. The gradual changes in the ratio of normal vs. tautomer emission appeared to follow the expectations according to the above model. / The studies on 3-hydroxyflavone were extended to other flavonols, having additional hydroxy substituents. The general findings were similar to 3-hydroxyflavone, with characteristic minor differences. The molecules 5-hydroxyflavone and 7-hydroxyflavone were also investigated, in order to compare their luminescence behavior with that of 3-hydroxyflavone. The three molecules were found to exhibit very distinct and contrasting luminescence properties. Both ground and excited state ionization were indicated in 7-hydroxyflavone, while in 5-hydroxyflavone a non-adiabatic proton tranfer mechanism, leading to rapid radiationless deactivation of the excited state, seems very probable. / A study was undertaken on the pigment contents of near-white cultivars of Hemerocallis in order to provide guidelines on hybridization programs directed toward production of blue cultivars. The cultivars were divided into four classes, chiefly on the basis of absorption profiles of their methanolic extracts. Flavonols were found in one class only, while carotenoids and hydroxycinnamic acids were present in all classes. / Finally, an attempt was made to characterize synthetic anthocyanin- copigment complexes on the basis of absorption and luminescence measurements. / Source: Dissertation Abstracts International, Volume: 41-12, Section: B, page: 4363. / Thesis (Ph.D.)--The Florida State University, 1980.

This series of experiments systematically evaluates the effect of phospholipid headgroup structures on the interaction between rhodopsin and phospholipids. Three types of experiments are reported. / First, the effect of rhodopsin incorporation on the DMPC and DMPS gel-to-liquid-crystalline phase transition is analyzed with ESR techniques. Partial, binary phase diagrams of the DMPS- and DMPC-rhodopsin systems at pH = 7.0 are constructed by studying the partitioning of Tempo between polar and hydrophobic domains as a function of temperature and system composition. A main result of this analysis is that rhodopsin broadens and reduces the amplitude of the DMPS phase transition to a much smaller extent than the DMPC phase transition. When interpreted in terms of theoretical treatments of integral protein-lipid interactions, this indicates that rhodopsin has a lower affinity for DMPS than DMPC. / Second, ESR experiments involving nitroxide-labeled phosphatidylcholine, phosphatidylserine and phosphatidic acid are reported. / Third, the effect of the major headgroup classes of phospholipids on the conformational stability of rhodopsin is investigated. / This series of experiments demonstrate that the structure of the fatty acid chains is as important as the headgroup structure in determining the stabilization ability of a phospholipid. / Source: Dissertation Abstracts International, Volume: 42-09, Section: B, page: 3550. / Thesis (Ph.D.)--The Florida State University, 1981.

The structural motif and backbone dynamics of the gramicidin-A transmembrane channel in a membrane environment have been investigated using solid-state $\sp{15}$N NMR. Recent determinations of the $\sp{15}$N chemical shift anisotropy tensor with respect to the molecular frame enable the quantitative evaluation of the $\sp{15}$N chemical shift resonances obtained from oriented dimyristoylphosphatidylcholine (DMPC) bilayer samples containing specific-site $\sp{15}$N-labeled gramicidin. / Spectra obtained from oriented samples in the liquid-crystalline phase have been used to verify the $\beta$-type hydrogen-bonding pattern of the helical backbone, and to determine that in these DMPC bilayer preparations the gramicidin channel is right-handed. In addition, these data place constraints on the C$\sb\alpha$-C$\sb\alpha$ axis orientation of individual peptide planes relative to the helix axis. / Spectra obtained from oriented samples in the gel phase have been analyzed to yield a spatial model for local motion. This model includes the axis of motion, the mean orientation, and the maximum amplitude of displacement for individual peptide planes. Specific sites in the first turn of the amino terminus were investigated, with emphasis on the Ala$\sb3$ and Leu$\sb4$ linkages for which the orientation of the $\sp{15}$N tensor with respect to the molecular frame has been determined. The effects of two well defined smectic layer defect structures, parabolic focal conics (PFC's) and oily streaks, are included in the spectral simulations. It is concluded that in the absence of ions, large amplitude motions are not present in the peptide planes of the first turn of the helix. A detailed characterization of bilayer surface geometry is presently the limiting factor in the use of this technique for probing the spatial extent of local motions in integral membrane proteins. / Source: Dissertation Abstracts International, Volume: 51-04, Section: B, page: 1604. / Major Professor: Timothy Albert Cross. / Thesis (Ph.D.)--The Florida State University, 1990.

Histone genes differ from other eucaryotic genes in that they lack introns and are not polyadenylated. The levels of histone mRNAs are coordinately regulated with DNA synthesis. mRNA stability is a major determinant of histone mRNA levels and histone mRNAs are rapidly degraded when DNA synthesis is inhibited. This thesis examines sequence requirements for both processing and degradation of histone mRNA. The highly conserved terminal stem-loop of the histone mRNA is necessary and sufficient to mediate regulated degradation and is also necessary for processing the 3$\sp\prime$ end of the histone mRNA. Histone mRNAs are degraded during translation and the stem-loop must be less than 300 nucleotides downstream of the termination codon for proper regulation of degradation. No particular sequence in the 4-base loop of the stem-loop is necessary for proper degradation since RNAs with changes in the sequence of these bases are regulated normally. A disruption of the base pair at the top of the stem however abolishes regulation of degradation and the mRNA is stable when DNA synthesis is inhibited. Changes in the sequence of the bases of the loop also caused the mRNA to be processed inefficiently. Taken together, these data suggest that degradation occurs on the ribosome and that the stem-loop structure is specifically recognized. A wild type loop sequence is not sufficient for efficient processing since a gene with the terminal stem-loop at the end of histone mRNA has multiple functions in histone mRNA metabolism. A link between the lack of introns in histone genes and lack of polyadenylation of histone mRNAs has been demonstrated. Thus introduction of intron(s) into the histone H2a gene interferes with 3$\sp\prime$ end formation resulting in substantial reduction in the amount of histone mRNA with a terminal stem-loop and a parallel increase in the amount of / polyadenylated histone mRNA. It is proposed that this occurs because a nascent spliceosome directs 3$\sp\prime$ end formation. / Source: Dissertation Abstracts International, Volume: 51-04, Section: B, page: 1605. / Major Professor: William F. Marzluff. / Thesis (Ph.D.)--The Florida State University, 1990.

Liquid-crystalline phase formation is concentrated DNA solutions with the ionic strengths of 0.01, 0.3 and 1 M Na$\sp{+}$ was investigated using phosphorus-31 NMR spectroscopy and optical microscopy. The phase diagrams for isotropic to liquid-crystalline transitions were determined for all three ionic strengths from $\sp{31}$P NMR data and were found to be in good agreement with theoretical predictions of P. J. Flory (Proc. Roy. Soc. A 234, 73; 1956) and Stroobants et al. (Macromolecules 19, 2232; 1986). The critical DNA concentration required for the anisotropic phase formation was found to be weakly dependent on ionic strength, indicating that the effective DNA radius is not strongly dependent on ionic strength. / Two types of mesophases were formed in solutions of all ionic strengths investigated: a weakly birefringent, precholesteric phase and a cholesteric phase. In addition, concentrated solutions with DNA concentrations exceeding 250 mg/mL in 0.01 M and 0.3 M Na$\sp{+}$ buffer exhibited microscopic textures similar to the textures observed in smectic phases formed in solutions of small molecules, indicating a possible two-dimensional ordering of DNA helices. / The sodium-DNA interactions in solutions with DNA concentrations in the range of 10-300 mg/mL and ionic strengths of 0.01 and 1 M Na$\sp{+}$ were investigated using sodium-23 NMR. The longitudinal relaxation rate of bound ions in the range of DNA concentrations of 10-200 mg/mL was found to be $\sim$200 Hz in 0.01 M Na$\sp{+}$ buffer and $\sim$380 Hz in 1 M Na$\sp{+}$ buffer. The relaxation rate was larger in samples which exhibited cholesteric ordering of DNA molecules. / Quadrupole splitting was observed in samples in which the cholesteric phase first appeared: at 190 mg/mL in 0.01 M Na$\sp{+}$ and 250 mg/mL in 1 M Na$\sp{+}$ buffer. The magnitude of quadrupole splitting decreased with increasing DNA concentration in 0.01 M Na$\sp{+}$ buffer and remained relatively constant in 1 M Na$\sp{+}$ buffer. In addition, the quadrupole splitting changed sign when the temperature was increased from 20 to 60 $\sp\circ$C. / Source: Dissertation Abstracts International, Volume: 49-08, Section: B, page: 2979. / Major Professor: Randolph Lynn Rill. / Thesis (Ph.D.)--The Florida State University, 1988.

The purpose of this investigation was to determine if there can be transport of vibrational energy along alpha helices by means of a soliton mechanism. This has been proposed by A. S. Davydov, and, if proven correct, would be highly significant for the interpretation of structure and function in biological macromolecules. / The approach necessary to generate dynamical solutions with soliton character for the helix, is one of forming a reduced Hamiltonian for the amide I vibrational modes, i.e. replacing 'background' modes, to which they are coupled, with quantum mechanical averages. As a result the total wavefunction has a semiclassical form, i.e. a product of background and 'primary' (amide I) wavefunctions. / Since such product forms for the wavefunctions of interacting systems are not generally valid we sought a way to describe the composite system exactly. This we achieved by means of two simple transformations of the total Hamiltonian, which allowed the extraction of dynamically significant terms. The simulations were performed on a Cyber 205, by means of a highly vectorized algorithm for the transformed Hamiltonian. / The results of these studies were that no soliton-like behavior was observed. Energy, initially localized, spreads down the helix more slowly, but the width of the excitation packet is wider than if there was no background coupling. / Source: Dissertation Abstracts International, Volume: 49-08, Section: B, page: 2979. / Major Professor: William C. Rhodes. / Thesis (Ph.D.)--The Florida State University, 1988.