Understanding the local conformations of DNA at the level of individual nucleic acid bases is important for the study of the mechanism of DNA sequence-dependent behavior. Here we apply linear absorption, circular dichroism (CD), and fluorescence spectroscopy to study the DNA local base conformation using 6-methyl Isoxanthopterin (6-MI) labeled DNA. We interpret excitation–emission peak shift (EES) measurements of the 6-MI, both as a ribonucleotide monophosphate in solution and as a site-specific substituent for guanine in various DNA constructs, by implementing a simple two-state model. We show that the spectroscopic properties of the 6-MI probe in DNA can be used to obtain detailed information about local base conformations and conformational heterogeneity and fluctuations. Based on these findings, we apply a simple theoretical model to calculate CD of 6-MI substituted DNA constructs. We find that our model can be used to extract basesequence- dependent information about the local conformation of the 6-MI probe as modulated by the local base or base-pair environment. We next apply 6-MI to probe the ligand insertion of small molecules to duplex DNA, further extending the potential of 6- MI as a useful reporter of local nucleic acid base conformation. These studies served to establish a new level of sophistication in qualitatively analyzing 6-MI structural behavior in terms of local base stacking and unstacking conformations. v This dissertation contains previously published and unpublished co-authored material.

Au Yuen-yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 140-152). / Abstracts in English and Chinese. / Abstract --- p.iii / Acknowledgements --- p.v / Chapter Chapter One: --- General Background and Introduction --- p.1 / Chapter 1 -1 --- Introduction --- p.1 / Chapter 1-2 --- Three-Bond Coupling Constants (3J) --- p.1 / Chapter 1-2-1 --- Development of the Karplus Equation --- p.1 / Chapter 1-2-2 --- Application of3J in the Conformational Analysis of Nucleic Acid --- p.4 / Chapter 1-2-3 --- Problem of Accuracy for3 J Measurement --- p.7 / Chapter 1-3 --- Two-Bond Coupling Constants (2J) --- p.7 / Chapter 1-3-1 --- The Use of the Projection Method --- p.7 / Chapter 1-3-2 --- 2J Coupling Constant Involving Hydrogen Bonds --- p.8 / Chapter 1-4 --- One-Bond Coupling Constants (1J) --- p.10 / Chapter 1-5 --- Conclusion --- p.11 / Chapter Chapter Two: --- Experimental Section / Chapter 2-1 --- Introduction --- p.12 / Chapter 2-2 --- Heteronuclear Multiple-Quantum Coherence (HMQC) Experiment --- p.12 / Chapter 2-3 --- Experimental Section --- p.15 / Chapter 2-3-1 --- Sample Preparation --- p.15 / Chapter 2-3-2 --- NMR Spectroscopy --- p.16 / Chapter Chapter Three: --- Theory of Nuclear Spin-Spin Coupling Constants --- p.18 / Chapter 3-1 --- Introduction --- p.18 / Chapter 3-2 --- Application of Finite Perturbation Theory on Nuclear Spin-Spin Coupling --- p.18 / Chapter 3-3 --- Methodology --- p.22 / Chapter Chapter Four: --- DFT and NMR Study of1JCH Coupling Constants --- p.28 / Chapter 4-1 --- Introduction --- p.28 / Chapter 4-2 --- Nomenclature and Definition of Structural Parametersin DNA and RNA --- p.28 / Chapter 4-2-1 --- "Nomenclature, Symbols and Atomic Numbering Schemes" --- p.28 / Chapter 4-2-2 --- Definition of Torsion Angles and their Rangesin Nucleotides --- p.31 / Chapter 4-2-3 --- Description of the Furanose Ring --- p.31 / Chapter 4-3 --- Results and Discussion --- p.37 / Chapter 4-3-1 --- Basis Set Effect --- p.37 / Chapter 4-3-2 --- Relative Conformational Energy Profiles --- p.37 / Chapter 4-3-3 --- Comparison of the Dependence of 1JCH Coupling Constants on Conformational Changes With and Without the DNA Backbone --- p.40 / Chapter 4-3-4 --- Effect of Backbone 3'- and 5'-Phosphate --- p.42 / Chapter 4-3-5 --- Effect of Glycosidic Torsion Anglex --- p.49 / Chapter 4-3-6 --- Effect of Ring Conformation with Fixed Glycosidic Torsion Anglex --- p.52 / Chapter 4-3-7 --- Effect of Torsion Angle α --- p.52 / Chapter 4-3-8 --- Effect of Torsion Angle β --- p.53 / Chapter 4-3-9 --- Effect of Torsion Angle γ --- p.56 / Chapter 4-3-10 --- Effect of Torsion Angle ε --- p.59 / Chapter 4-3-11 --- Effect of Torsion Angle ζ --- p.61 / Chapter 4-3-12 --- Effect of Base Pairing --- p.65 / Chapter 4-3-13 --- Effect of Base Stacking from the (n-1) and (n+1) Base --- p.65 / Chapter 4-3-14 --- Comparison of Experimental and Theoretical Data --- p.68 / Chapter 4-4 --- Conclusion --- p.74 / Chapter Chapter Five: --- DFT Study of 2JCH and 3JCH Coupling Constants --- p.79 / Chapter 5-1 --- Introduction --- p.79 / Chapter 5-2 --- Results and Discussion on 2JCH Coupling Constants --- p.79 / Chapter 5-2-1 --- Effect of Backbone 3'- and 5'-Phosphate --- p.79 / Chapter 5-2-2 --- Effect of Ring Conformation with Fixed Glycosidic Torsion Anglex --- p.82 / Chapter 5-2-3 --- Effect of Glycosidic Torsion Anglex --- p.87 / Chapter 5-2-4 --- Effect of Torsion Angleγ --- p.87 / Chapter 5-2-5 --- Effect of Torsion Angle ε --- p.90 / Chapter 5-2-6 --- Effect of Base Pairing --- p.90 / Chapter 5-2-7 --- Effect of Base Stacking from the (n-1) and (n+1) Base --- p.90 / Chapter 5-3 --- Results and Discussion on 3JCH Coupling Constants --- p.95 / Chapter 5-3-1 --- Effect of Backbone 3'- and 5'-Phosphate --- p.95 / Chapter 5-3-2 --- Effect of Ring Conformation with Fixed Glycosidic Torsion Anglex --- p.95 / Chapter 5-3-3 --- "Effect of Different Torsion Angles (X,α,β,γ,ε,and ζ)" --- p.100 / Chapter 5-3-4 --- Effect of Base Pairing --- p.100 / Chapter 5-3-5 --- Effect of Base Stacking from the (n-1) and (n+1) Base --- p.105 / Chapter 5-4 --- Conclusion --- p.105 / Chapter Chapter Six: --- Conclusion --- p.111 / Appendix A Product Operator Formalism on HMQC Pulse Scheme --- p.113 / Appendix B Finite Perturbation Theory --- p.115 / Appendix C Supplementary Figures of Chapter Four --- p.118 / Appendix D Some of the NMR Spectra --- p.134 / References --- p.140

Nous avons déterminé, par diffusion de neutrons aux petits angles, la conformation et la dynamique de polymères cristaux liquides dans la phase nématique ou isotrope. En phase isotrope les polymères, linéaires ou en peigne adoptent une conformation gaussienne. En phase nématique les polymères linéaires ou greffés en haltère sont confinés dans de très longs cylindres. Pour les linéaires cela implique les repliements en épingle à cheveux prévus par P.G. de Gennes. La conformation des polymères en peigne est faiblement anisotrope. L'étude de la dynamique à l'état fondu de ces polymères non enchevêtrés a révélé l'existence d'amas dus à des interactions temporaires entre les mésogènes. La taille de ces amas est supérieure aux dimensions d'une chaîne mais inférieure à celle de l'échantillon. La relaxation de la conformation est décrite en détail.

Alzheimer’s disease (AD) is a neurodegenerative disorder that is one of such diseases associated with protein aggregation. Aβ is the main protein component of senile plaques in AD, and is neurotoxic when aggregated. In particular, soluble oligomeric forms of Aβ are closely related to neurotoxicity. In this dissertation, we examine the differences in Aβ aggregation intermediates, and final structures formed when only a simple modification in Aβ aggregation conditions is made, the presence or absence of mixing during aggregation. We show that intermediates in the aggregation pathway show significantly different structural rearrangements. The protein stabilities of Αβ species show that spherical aggregates corresponding to the most toxic Αβ species change their structure the most rapidly in denaturant, and that in general, increased toxicity correlated with decreased aggregate stability. In Alzheimer’s disease, even delaying Aβ aggregation onset or slowing its progression might be therapeutically useful, as disease onset is late in life. Small heat shock proteins (sHsps) may be useful for prevention of Αβ aggregation, since sHsps can interact with partly folded intermediate states of proteins to prevent incorrect folding and aggregation. In this research, several small heat shock proteins (sHsps) are tested to prevent Aβ aggregation and toxicity. sHsps used in this research are Hsp17.7, Hsp27, and Hsp20. All types of Hsp20, Hsp20-MBP, His-Hsp20 and His-Hsp20 without 11 residues in C-terminus, can prevent Aβ1-40 aggregation. Hsp20 also prevents Aβ toxicity in the same concentration ranges of it aggregation prevention activity. Hsp17.7 and Hsp27, however, can inhibit Αβ1-40 aggregation but not toxicity. A number of experiments to examine the mechanism of Hsp20 suggest that multivalent binding of sHsp to Aβ is necessary for the toxicity prevention activity. Conclusively, different Aβ incubation conditions in vitro can affect the rate of Aβ fibril formation, the morphology, the toxicity and the conformation of intermediates in the aggregation pathway. Hsp20 rather than other sHsps may be a useful molecular model for the drug design of the next generation of Aβ aggregation inhibitors to be used in the treatment of AD.

The Cre sequence (ACGT) is a site responsible for the binding of specific transcription factors that determine the activation of genes. Due to its major role in gene transcription, it has become a subject of immense research. The binding of transcription factors to the Cre binding site has been determined to be dependent on DNA conformation. In this study, the effects of flanking sequence around the Cre binding site on the conformation and the dynamics of DNA were investigated. The Cre binding site was studied in its native form with differing flanking sequences to determine the BI/BII profile (conformation) and the magnitude of the energy transition barrier (dynamics) between the BI and BII conformations of each phosphate step of the following three dodecamer sequences: CreACAG, CreGGAG, and CreTATA. In order to obtain the BI/BII profile of each phosphate step, 2D 31P-NMR NOESY and HSQC experiments at various temperatures were utilized. Based of the basic principles of kinetics, the lower the energy barrier between the two conformations, the easier the transition between the BI and BII conformation. Therefore, it was hypothesized that low and high %BII character lead to a large energy barrier (high ∆G‡ values), whereas average %BII character leads to a small energy barrier (low ∆G‡ values). The results of the 2D 31P-NMR experiments of the three dodecamer sequences confirmed this relationship between the %BII character and the magnitude of the energy barrier (∆G‡). However, further conformation and dynamics studies must be conducted to further understand the correlation.

Data were analyzed on 2 to 6 yr old cows to evaluate sire and family effects. Cows were produced in the McGregor Genomics Project from 13 embryo transfer (ET) full sib families (n = 188, F2 Nellore-Angus (NA)) and 4 half sib natural service (NS) families (n = 114, out of 1/2 Brahman 1/2 British dams) from the same 4 F1 NA sires. The ET and NS cows were analyzed separately and together as paternal half sibs (PHS). Daughters of bull 437J had the highest calving rate and weaning rate; daughters of 551G were the lowest in ET, and daughters of 297J were the lowest in NS. Calves out of daughters of 551G were the heaviest at birth; those from of daughters of 437J were the lightest in NS and PHS. Calves out of daughters of 297J were the heaviest at weaning in ET and PHS, and those from daughters of 432H were the lightest. Calves from daughters of 297J and 437J gained the most weight and those from daughters of 432H gained the least. Daughters of 297J and 551G had longer and larger diameter teats and lower udder support scores (more pendulous) than daughters of 432H and 437J. Daughters of 437J had the highest body condition score (BCS); daughters of 551G were the lowest in ET and NS. Calves from daughters of 297J had the highest BCS at weaning. Those out of daughters of 551G had the lightest WWT, those out of daughters of 437J were the heaviest in NS and PHS, and those out of daughters of 432H were the heaviest in ET. Daughters of 437J and 551G scored the highest for disposition (least docile) in ET and PHS, and daughters of 432H were lowest. The regression of WWT on weaning age was 0.82 plus/minus 0.07 in ET, 0.71 plus/minus 0.08 in NS, and 0.78 plus/minus 0.05 kg/d in PHS. There appears to be sufficient variation within and between these full sib and half sib families for use in QTL analysis for major genes affecting cow productivity in NA crosses.

Collagen is an important structural protein in living organisms and plays an indispensable role in connecting cells and tissues, such as in musculature, bone, and ligament. The stability and conformation of collagen are, however, strongly influenced by ambient temperature and constitutes an interesting subject of study. Thermally induced conformation change of collagen has been investigated by techniques such as differential scanning calorimetry (DSC) and second harmonic generation. DSC is a powerful method in uncovered important thermal dynamics properties including phase change, enthalpy, and thermal stability of the collagen. However, due to its collective nature, no localized information can be found. For comparison, second harmonic generation, which reflects structural symmetry, can be combined with laser scanning microscopy to investigate localized variation. It has been shown in previous studies that the thermal stability of collagen is strongly influenced by the water content within collagen. For comparison, we are investigating the conformational change of collagen under a vacuum stat with second harmonic microscopy so as to isolate environmental effects, particularly those from water and oxygen. In this way, we have found the conformational change of collagen takes place at a much higher temperature and activation energy. Additionally, the high spatial resolution achieved also allows many further possibilities.

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No description available.

Alzheimer’s disease (AD) is a neurodegenerative disorder that is one of such diseases associated with protein aggregation. Aβ is the main protein component of senile plaques in AD, and is neurotoxic when aggregated. In particular, soluble oligomeric forms of Aβ are closely related to neurotoxicity. In this dissertation, we examine the differences in Aβ aggregation intermediates, and final structures formed when only a simple modification in Aβ aggregation conditions is made, the presence or absence of mixing during aggregation. We show that intermediates in the aggregation pathway show significantly different structural rearrangements. The protein stabilities of Αβ species show that spherical aggregates corresponding to the most toxic Αβ species change their structure the most rapidly in denaturant, and that in general, increased toxicity correlated with decreased aggregate stability. In Alzheimer’s disease, even delaying Aβ aggregation onset or slowing its progression might be therapeutically useful, as disease onset is late in life. Small heat shock proteins (sHsps) may be useful for prevention of Αβ aggregation, since sHsps can interact with partly folded intermediate states of proteins to prevent incorrect folding and aggregation. In this research, several small heat shock proteins (sHsps) are tested to prevent Aβ aggregation and toxicity. sHsps used in this research are Hsp17.7, Hsp27, and Hsp20. All types of Hsp20, Hsp20-MBP, His-Hsp20 and His-Hsp20 without 11 residues in C-terminus, can prevent Aβ1-40 aggregation. Hsp20 also prevents Aβ toxicity in the same concentration ranges of it aggregation prevention activity. Hsp17.7 and Hsp27, however, can inhibit Αβ1-40 aggregation but not toxicity. A number of experiments to examine the mechanism of Hsp20 suggest that multivalent binding of sHsp to Aβ is necessary for the toxicity prevention activity. Conclusively, different Aβ incubation conditions in vitro can affect the rate of Aβ fibril formation, the morphology, the toxicity and the conformation of intermediates in the aggregation pathway. Hsp20 rather than other sHsps may be a useful molecular model for the drug design of the next generation of Aβ aggregation inhibitors to be used in the treatment of AD.