by Chi Ching Mak. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 122-130). / Contents --- p.i / Acknowledgments --- p.iii / Abstract --- p.iv / Abbreviations --- p.v / List of publications originated from this thesis --- p.vi / Chapter I --- Introduction to Dendrimer Chemistry / Chapter I-1 --- Background --- p.1 / Chapter I-2 --- Comparison of Dendrimer and Linear Polymer --- p.6 / Chapter I-3 --- Synthesis --- p.8 / Chapter I-3-1 --- Divergent-growth Approach --- p.8 / Chapter I-3-2 --- Convergent-growth Approach --- p.10 / Chapter I-3-3 --- Accelerated Approach --- p.11 / Chapter II --- Functional Dendrimers --- p.14 / Chapter III --- Optically Active Dendrimers / Chapter III-1 --- Background --- p.18 / Chapter III-2 --- Architecture of Tartaric Acid-based Optically Active Dendrimers --- p.23 / Chapter III-3 --- Results and Discussion --- p.24 / Chapter III-3-1 --- Syntheses and Structure Elucidations of all (L)-Tartaric Acid-based Dendrimers 41 and 42 --- p.24 / Chapter III-3-2 --- Syntheses and Structure Elucidations of Optically Active Layer-block Dendrimers 43 and 44 --- p.29 / Chapter III-3-3 --- Structure-Optical Rotation Relationships --- p.33 / Chapter III-3-4 --- "Syntheses and Characterization of Higher Generation, Optically Active Dendritic Fragments" --- p.37 / Chapter III-3-5 --- Conclusions --- p.43 / Chapter IV --- Catalytic Dendrimers / Chapter IV-1 --- Background --- p.44 / Chapter IV-2 --- Syntheses and Characterization of Dendritic Bis(oxazoline) Copper(II) Catalyst --- p.47 / Chapter IV-2-1 --- Architecture --- p.47 / Chapter IV-2-2 --- Syntheses of the Bis(oxazoline) Core Precursors --- p.49 / Chapter IV-2-3 --- Syntheses of the Achiral Dendritic Sectors --- p.51 / Chapter IV-2-4 --- Syntheses of Dendritic Ligands --- p.52 / Chapter IV-3 --- Kinetics and Selectivity of Dendritic Bis(oxazoline)-Copper(II) Complex Catalyzed Diels-Alder Reaction --- p.55 / Chapter IV-3-1 --- Introduction --- p.55 / Chapter IV-3-2 --- Reaction Mechanism and Reactivity Kinetics --- p.56 / Chapter IV-3-3 --- Substrate Selectivity --- p.63 / Chapter IV-4 --- Syntheses and Properties of Optically Active Dendritic Bis(oxazoline) Ligands and Their Copper(II) Complexes Catalysts --- p.65 / Chapter IV-4-1 --- Background --- p.65 / Chapter IV-4-2 --- Syntheses and Characterization of Chiral Dendritic Bis(oxazoline) Ligands 109-112 --- p.67 / Chapter IV-4-3 --- Chiroptical Properties --- p.72 / Chapter IV-4-4 --- Enantioselectivity of the Metal-catalyzed Diels-Alder Reaction --- p.73 / Chapter IV-4-5 --- Conclusions --- p.74 / Chapter V --- Summary --- p.75 / Chapter VI --- Experimental --- p.77 / References --- p.122 / Spectra --- p.131

by Feng Xue. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 76-84). / ACKNOWLEDGMENTS --- p.I / ABSTRACT --- p.II / TABLE OF CONTENTS --- p.III / LIST OF TABLES --- p.V / LIST OF FIGURES --- p.VI / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter 1. --- supramolecular chemistry --- p.1 / Chapter 2. --- Infinite multi-dimensional architecture --- p.10 / Chapter 3. --- Chemistry of pseudohalides --- p.15 / Chapter 4. --- Chemistry of hexamethylenetetramine --- p.20 / Chapter 5. --- Objectives of the present research --- p.24 / Chapter CHAPTER 2: --- EXPERIMENTAL --- p.25 / Chapter 1. --- Preparation --- p.25 / Chapter 1.1 --- Materials --- p.25 / Chapter 1.2 --- Preparation of the compounds --- p.25 / Chapter 2. --- X-ray crystallography --- p.26 / Chapter CHAPTER 3: --- DESCRIPTION OF CRYSTAL STRUCTURES --- p.28 / Chapter 1. --- isostructural compounds K2[FEII(CN)6] ´Ø 2[(CH2)6n4H] ´Ø 4H2 O(1) and K2[FEIII(CN)6] ´Ø [(CH2)6N4H] ´Ø [(CH2)6N4] ´Ø 4H2O (2) --- p.28 / Chapter 2. --- Crystal structure of NA2[FEIII(CN)6] ´Ø [(CH2)6N4H] ´Ø [(CH2)6N4] ´Ø 5H2O (3) --- p.33 / Chapter 3. --- Crystal structure of K3[FEIII(CN)6] ´Ø 2[(CH2)6N4] ´Ø 4H2O (4) --- p.37 / Chapter 4. --- Crystal Structure of ferricyanic acid-HMT adduct H3[FEIII(CN)6] ´Ø 2[(CH2)6N4] ´Ø 2H2O (5) --- p.43 / Chapter 5. --- "Crystal structure of cadmium chloride-HMT adduct, Cd2[C6H12N4H2]Cl6´Ø 3H2O (6)" --- p.48 / Chapter 6. --- "Crystal structure of cadmium bromide-HMT adduct, CdBr2 ´Ø 2[(CH2)6N4] ´Ø 2H2O (7)" --- p.53 / Chapter 7. --- "Crystal structure of cadmium iodide-HMT adduct, 3CdI2 ´Ø 2[(CH2)6N4] ´Ø 4H2O (8)" --- p.57 / Chapter 8. --- Crystal structure of cadmium thiocyanate-HMT adduct Cd(SCN)2 ´Ø 1(CH2)6N4] ´Ø MeOH (9) --- p.61 / Chapter 9. --- Crystal structure of cobalt cyanate-HMT adduct Co(NCO)2 ´Ø [(CH2)6N4] ´Ø 2H2O (10) --- p.67 / Chapter CHAPTER 4: --- SUMMARY AND DISCUSSIONS --- p.72 / REFERENCES --- p.76 / TABLE A1 Crystallographic data for compounds 1 ~5 --- p.85 / TABLE A2 Crystallographic data for compounds 6~10 --- p.86 / APPENDIX 1 Atomic Coordinates (Ax 104) and Equivalent Isotropic Thermal Parameters (A 2x103) --- p.87 / APPENDIX 2 Anisotropic thermal parameters (A2x 103) --- p.92 / APPENDIX 3 H-atom coordinates (x 104) and isotropic thermal parameters (A2x 103) --- p.96

No description available.

The use of polymer brushes (long polymer chains anchored at their end to a surface or an interface) as a robust approach to control surface properties has generated significant interest in recent years. The stretched conformation of polymer brushes results in unique aggregation, phase, and dynamic behaviors, therefore, they have been used to stabilize colloidal particles and applied in numerous innovative biomedical applications: targeted magnetic hyperthermia, targeted drug delivery, and genotyping. The main goal of this thesis is to shed light on the key factors that affect the formation of these brushes in solution on solid surfaces. In Chapter 3, attenuated total reflectance infrared spectroscopy (ATR-IR) is used to directly measure the rates of the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions between alkyne-terminated polystyrene and poly(n-butyl acrylate) and azide-functional substrates in the good solvent DMF. Four regimes of behavior are observed: initially, the reaction rate is diffusion-controlled scaling with t^1/2; in the crossover regime at the onset of chain overlap, the rate scales with ln(t); the rate then accelerates briefly; and finally, in the terminal or penetration-limited regime, the logarithm of areal density scales linearly with time. Kinetic behavior in the diffusion-limited, crossover, and penetration-limited regimes corresponds well to the predictions of Ligoure and Leibler. The blob model suggests that the acceleration in rate is due to lateral chain contraction during the mushroom to brush transition. A theory is presented which predicts that the areal density at saturation should scale as Σsaturation ∼ MW^1.2 for good solvents, and experimentally we find MW^(−0.93±0.04) scaling. In Chapter 4, the effect of symmetry of the CuAAC reaction is investigated for the reaction of end-functional polystyrene and solid surfaces modified with self-assembled monolayers (SAMs). The polymer grafting density on azide-functional substrates is about two times higher than the polymer density on alkyne-functional surfaces. This asymmetry in the reaction density is caused by the difference in the mobility of the alkyne groups between the two systems. While the reaction stoichiometry requires one alkyne and one azide, the reaction mechanism involves two alkyne groups and one azide group in the formation of a stable triazole ring. When the alkyne groups are on the surfaces, their mobility is significantly reduced, preventing the formation of the triazole rings and consequently decreasing the amount of polymer grafted. Increasing the alkynes’ mobility by either extending the thickness of the alkyne monolayer or adding free 1-pentyne improves the polymer density on alkyne-functional silica substrates. The presence of free 1-pentyne also increases the polymer density on alkyne-functional wafers containing a preexisting polymer brush. This study shows that the placement of each functional group in the CuAAC reaction is important in surface modification applications. In Chapter 5, a universal model to quantify the amount of tails vs. loops during brush formation of telechelic polymers is proposed. This model involves the synthesis of telechelic polymers bearing a degradable unit in the middle of each chain via ATRP. Several reaction schemes are suggested for the synthesis of the required bi-functional ATRP initiators with degradable units. The amount of singly (tails) vs. doubly (loops) bound chains is quantified by comparing the brush heights, measured by ellipsometry, before and after degradation.

Single-particle cryogenic electron microscopy (cryo-EM) has become a powerful mainstay tool in high resolution structural biology thanks to advances in hardware, software and sample preparation technology. In my thesis, I utilized this technique to unravel the function of various challenging biological macromolecules. My first focus was bacterial ribosomal biogenesis: understanding how bacteria assemble their ribosomes. Ribosomes are the factories of the cell, responsible for manufacturing all proteins. Ribosomes themselves are huge, with the bacterial version made of 52 proteins and 4566 RNA nucleotides. How these components assemble has long been a mystery. Early groundbreaking work sketched out a biogenesis pathway using purified components in vitro – but under non-physiological conditions. We sought to understand how the bacterial ribosome – specifically the large subunit 50S – is built inside the cell. To achieve this, we engineered a conditional knock-out bacterial strain that lacked one specific ribosomal protein (L17). This caused the cells to accumulate incomplete intermediates along the 50S biogenesis pathway. These intermediates were purified and examined with mass spectrometry and single-particle cryo-EM. Two major hurdles arose in this project: firstly, the biogenesis intermediates exhibited a preferred orientation when vitrified for cryo-EM analysis. This means that instead of showing many different views required for reconstruction of the 3D structure, the intermediates only adopted one view on the cryo-EM grid. To overcome this problem, we engineered a method to induce additional views on the microscope by tilting the stage. Using another test protein that also exhibited preferred orientation (hemagglutinin), we optimized and characterized this new tilt methodology and showed it was generally applicable to overcoming preferred orientation, regardless of type of specimen. We also created a software tool, called 3DFSC (3dfsc.salk.edu), for other microscopists to calculate the degree of directional anisotropy in their structures due to preferred orientation. Using this tilt strategy finally enabled the structural elucidation of our 50S intermediates. The second challenge in the project was the large amount of heterogeneity present in the sample. Through hierarchical 3D classification schemes using the latest software tools, we obtained 14 different 50S intermediate structures, all from imaging a single cryo-EM grid. By analyzing the missing components of each intermediate, and corroborating these observations with mass spectrometry data, we outlined the first in vivo 50S assembly pathway, and showed that ribosome assembly occurs step-wise and in parallel pathways. My second focus was on pushing the resolution limits of single-particle cryo-EM using adeno-associated virus (AAV) serotype 2 homogeneous virus-like particles (VLPs) that lack DNA. Exploiting several technical advances to improve resolution, including use of gold grids, per-particle CTF refinement, and correction for Ewald sphere curvature, we managed to obtain a 1.86 Å resolution reconstruction of the AAV2L336C variant VLP, the highest resolution icosahedral virus reconstruction solved by single-particle cryo-EM to date. Using our structure, we were able to show improvements using Ewald sphere curvature correction and shed light on the mechanistic basis as to why the L336C mutation resulted in defects in genome packaging and infectivity compared to the WT viral particles. My third focus was the understanding of small membrane proteins involved in infectious diseases. Membrane proteins are a challenge to work with due to the need for them to be extracted from the lipid bilayer for studies as compared to soluble proteins. Infectious diseases have a huge burden on society, with the top three infectious agents accounting for 2.7 million deaths in 2016. The third most deadly infectious disease is malaria, a mosquito-borne parasite which kills 450,000 people annually. One drug used early on for treating malaria was chloroquine but its usefulness waned due to development of resistance. Chloroquine resistance is mediated by the chloroquine resistance transporter (PfCRT). Although small (49 kDa) for single-particle cryo-EM, we solved its structure by using fragment antibody technology to add mass and help with image alignment and 3D reconstruction. The 3.2 Å structure resembles other drug metabolite transporters, and the chloroquine resistance mutations map to a ring around the central cavity, suggesting this central pore as the drug binding site. Tuberculosis (TB) is the top killer, above malaria and HIV/AIDS, being responsible for 1.3 million deaths. In TB, a common antibiotic target is the bacterium’s cell wall synthesis machinery. One family of such enzymes is the arabinosyltransferases, which synthesize the critical arabinose sugars. Using single-particle cryo-EM, we solved two high resolution structures of one such essential enzyme, AftD. Due to the low yield of the protein, a picoliter automated sample dispensing robot was crucial to allow for initial cryo-EM analysis. We then performed mutagenesis studies in M. smegmatis, a TB model organism, which uncovered the critical amino acid residues in the active site and determined that a bound acyl-carrier-protein was likely involved in allosteric inhibition of AftD’s active site. Another member of the family, EmbB, is the target of a widely used frontline TB drug called ethambutol. We have solved the high resolution structures of the apo and putative drug-bound states of EmbB, allowing us to map out, for the first time, both the active site and drug-resistance mutations of this crucial enzyme. The atomic structures of the functional pockets of Mycobacterial AftD and malarial PfCRT will hopefully enable structure-based drug design to improve existing drugs or potentially even develop new treatments against these infectious maladies. In conclusion, the continual and breathtaking improvements in single-particle cryo-EM methodology has been instrumental in allowing the elucidation of the aforementioned biological macromolecules from ribosome biogenesis intermediates, to AAV2 vehicle, Plasmodium drug resistance transporter to mycobacterial glycosyltransferases – structures of which help explain biological function.

Methods are presented to acquire data from analytical ultracentrifugation experiments by computer using the absorption optical scanning system of the Beckman Model-E ultracentrifuge. A computer program was written which analyzes sedimentation velocity experiments by the van Holde - Weischet method and by the second moment method. The van Holde - Weischet method allows a high resolution analysis of sedimentation velocity data by eliminating the effects of diffusion on the shape of the moving boundary to provide sedimentation coefficients for a heterogeneous composition of a sample. The second moment method obtains the sedimentation coefficient by calculating the second moment point, by which the sedimentation coefficient is defined. Since it is impractical to manually analyze sedimentation velocity data by this method, these computer programs make an important analysis method available to the researcher. Using this computer program, it is now possible to analyze data to a higher resolution and accuracy than manual analysis of stripchart recordings would permit. Moreover, the time required for the analysis is greatly reduced. Data from sedimentation equilibrium experiments are analyzed by x² minimization. Further, a program was written for the acquisition of data to measure diffusion coefficients from quasi elastic light scattering experiments with a Langley Ford correlator. The analysis of autocorrelation spectra from light scattering experiments is performed by the Levenberg - Marquardt method, which allows fitting of data to nonlinear models. The model used allows the analysis of multicomponent systems by fitting to a sum of exponentials and a baseline. Traditional analysis of autocorrelation data by hand was limited to least squares fitting of the data to a linear model of one component without an optimized baseline, often an unrealistic approximation of the system. Analysis of autocorrelation data by nonlinear curve fitting increases both the accuracy and amount of data that can be analyzed. The development of the PPOL-1 208-n series of plasmids and of the miniplasmid pMX is described. These plasmids were designed to allow studies of in vitro transcription and chromatin structure after reconstitution with histones. The plasmids themselves were analyzed by sedimentation and diffusion studies using the computer programs. Sedimentation data is presented which suggests a new method for rapid estimation of S₀ (the sedimentation coefficient at zero concentration) for molecules which show a concentration dependency of the sedimentation coefficient. This is accomplished by linearly extrapolating van Holde Weischet distributions to zero concentration. Manual analysis of sedimentation velocity experiments to determine nonideality contributions required several experiments, computer analysis can provide this information in a single experiment due to the increased resolution of the method. Diffusion data for this plasmid DNA is used to demonstrate the feasibility of the multicomponent analysis presented here. Also, sedimentation measurements were carried out on reconstituted chromatin and on the effects of ethidium bromide on reconstituted chromatin. The programs were used to demonstrate significant changes in chromatin structure upon ethidium bromide binding. These changes involved the reduction of S of reconstituted plasmids upon addition of ethidium bromide as well as a reduction of heterogeneity of the sample. The data indicates the possibility of a forced exchange of nucleosomes between plasmids, as well as conformational changes in the chromatin structure. / Graduation date: 1993

Proteins and DNA are essential to life as we know it and understanding their function is understanding their structure and dynamics. The importance of the latter is being appreciated more in recent years and has led to the development of novel interdisciplinary techniques and approaches to studying protein function. Three techniques to study protein structure and dynamics have been used and combined in different ways in the context of this thesis and have led to a better understanding of the three systems described herein. X-ray crystallography is the oldest and still arguably most popular technique to study macromolecular structures. Nuclear magnetic resonance (NMR) spectroscopy is a not much younger technique that is a powerful tool not only to probe molecular structure but also dynamics. The last technique described herein are molecular dynamics (MD) simulations, which are only just growing out of their infancy. MD simulations are computer simulations of macromolecules based on structures solved by X-ray crystallography or NMR spectroscopy, that can give mechanistic insight into dynamic processes of macromolecules whose amplitudes can be estimated by the former two techniques. MD simulations of the model protein GB3 (B3 immunoglobulin-binding domain of streptococcal protein G) were conducted to identify origins of discrepancies between order parameters derived from different sets of MD simulations and NMR relaxation experiments.The results highlight the importance of time scales as well as sampling when comparing MD simulations to NMR experiments. Discrepancies are seen for unstructured regions like loops and termini and often correspond to nanosecond time scale transitions between conformational substates that are either over- or undersampled in simulation. Sampling biases can be somewhat remedied by running longer (microsecond time scale) simulations. However, some discrepancies persist over even very long trajectories. We show that these discrepancies can be due to the choice of the starting structure and more specifically even differences in protonation procedures. A test for convergence on the nanosecond time scale is shown to be able to correct for many of the observed discrepancies. Next, MD simulations were used to predict in vitro thermostability of members of the bacterial Ribonuclease HI (RNase H) family of endonucleases. Thermodynamic stability is a central requirement for protein function and a goal of protein engineering is improvement of stability, particularly for applications in biotechnology. The temperature dependence of the generalized order parameter, S, for four RNase H homologs, from psychrotrophic, mesophilic and thermophilic organisms, is highly correlated with experimentally determined melting temperatures and with calculated free energies of folding at the midpoint temperature of the simulations. This study provides an approach for in silico mutational screens to improve thermostability of biologically and industrially relevant enzymes. Lastly, we used a combination of X-ray crystallography, NMR spectroscopy and MD simulations to study specificity of the interaction between Drosophila Hox proteins and their DNA target sites. Hox proteins are transcription factors specifying segment identity during embryogenesis of bilaterian animals. The DNA binding homeodomains have been shown to confer specificity to the different Hox paralogs, while being very similar in sequence and structure. Our results underline earlier findings about the importance of the N-terminal arm and linker region of Hox homeodomains, the cofactor Exd, as well as DNA shape, for specificity. A comparison of predicted DNA shapes based on sequence alone with the shapes observed for different DNA target sequences in four crystal structures when in complex with the Drosophila Hox protein AbdB and the cofactor Exd, shows that a combined ”induced fit”/”conformational selection” mechanism is the most likely mechanism by which Hox homeodomains recognize DNA shape and achieve specificity. The minor groove widths for all sequences is close to identical for all ternary complexes found in the different crystal structures, whereas predicted shapes vary between the different DNA sequences. The sequences that have shown higher affinity to AbdB in vitro have a predicted DNA shape that matches the observed DNA shape in the ternary complexes more closely than the sequences that show low in vitro affinity to AbdB. This strongly suggests that the AbdB-Exd complex selects DNA sequences with a higher propensity to adopt the final shape in their unbound form, leading to higher affinity. An additional AbdB monomer binding site with a strongly preformed binding competent shape is observed for one of the oligomers in the reverse complement strand of one of the canonical (weak) Hox-Exd complex binding site. The shape preference seems strong enough for AbdB monomer binding to compete with AbdB-Exd dimer binding to that same oligomer, suggested by the presence of both binding modes in the same crystal. The monomer binding site is essentially able to compete with the dimer binding site, even though binding with the cofactor is not possible, because its shape is very close to the ideal shape. A comparison of different crystal structures solved herein and in the literature as well as a set of molecular dynamics simulations was performed and led to insights about the importance of residues in the Hox N-terminal arm for the preference of certain Hox paralogs to certain DNA shapes. Taken together all these insights contribute to our understanding of Hox specificity in particular as well as protein-DNA interactions in general.