Dielectric and Ultrasonic Studies of Macromolecular Growth During Polymerization

Measurements by dielectric spectroscopy, ultrasonics and calorimetry ofseveral low viscosity monomeric liquids undergoing spontaneous chemical reaction, to form three new, linear chain polymers under isothermal conditions, have been used to determine how the number ofcovalent bonds formed during the growth of a linear chain affects the dielectric and ultrasonic properties, their respective relaxation times, and their spectral shape. The dielectric properties changed in the following manner. During this reaction, the static permittivity decreased and the relaxation time increased towards limiting values. As the number of covalent bonds increased towards the Avogadro number, the change in the complex permittivity as measured for a fixed frequency was phenomenologically similar to that observed on varying the frequency, although the exact formalisms in both cases differed. In both cases the relaxation function could be well described by a stretched exponential or sum ofexponentials, characterized by a temperature and system dependent exponent that decreased as the state of the system changed from a monomeric liquid to a fully reacted polymer. At later stages of chemical reaction a second relaxation process at higher frequencies is revealed. The dielectric manifestation of the irreversible process of covalent bond formation is remarkably similar to that observed on supercooling a molecular or polymeric liquid. Longitudinal velocity and attenuation of ultrasonic waves travelling through the three molecular liquids at different temperatures have been measured as its molecules combine irreversibly to form large entities and thereby decrease the diffusivity and increase the configurational restrictions to their dynamics. From these data, the longitudinal modulus and compliance are calculated, and the molecular relaxation time and related properties are deduced and interpreted in terms ofthe number of covalent bonds formed, by a formalism that connects the size ofthe molecules in the liquid with its elastic behaviour. This relaxation time increases monotonically with increase in the molecule's size, tending to infinity as the number ofcovalent bonds formed approaches Avogadro's number. The complex plane plots ofthe modulus and compliance have a shape which is described by a skewed arc function, with a temperature dependent exponent ϒ, that ranges in values from 0.33- 0.31 for modulus and 0.39-0.45 for compliance. Departure from this shape is shown to be due to contributions from non-zero shear viscosity for relatively small size of molecules, and contributions from a faster, or sub Tg-relaxation process when the molecular size is large, which is similar to the behaviour for the dielectric properties. Simulation of the data suggests that this sub Tg-relaxation process, which is progressively more separated from the main relaxation process as the molecular size increases, contributes significantly to the high frequency elastic properties. The measured longitudinal modulus has been deconvoluted to show that the increase in the bulk modulus, and not the shear modulus, dominates the elastic properties when the molecular size increases. Comparison ofthe calculated relaxation times for the longitudinal modulus and compliance with the dielectric relaxation time show that the compliance and dielectric data change in a remarkably similar manner with increasing time of chemical reaction, which is unexpected owing to their different mechanisms. In the last part of this work, the dipolar diffusion in the glassy and supercooled liquid states of 9 additional molecular liquids and oftheir linear chain or network polymerized states formed by condensation-polymerization at different temperatures and times have been studied by measuring the dielectric properties for a fixed ac frequency of 1 kHz. The study showed that as the extent of polymerization increased with increasing isothermal temperature of polymerization, the sub-Tg relaxation peak due to localized molecular motions in the molecular state became gradually extinct, and a corresponding peak at a higher temperature evolved and reached its maximum height. The temperature of the sub-Tg relaxation peak in the polymerized state differed from that of the α-relaxation peak of the supercooled molecular liquid by as much as 70K, but, in several cases, the two temperatures were similar. Reasons for the latter occurrence are given in phenomenogical terms. It is concluded that the localized relaxation modes of the polar segments of the macromolecule are not related to the modes of molecular diffusion in the monomeric liquid state above its Tg. The localized relaxation characteristic of the glassy molecular state persists in the incompletely polymerized state, where it is seen as a ϒ-relaxation.Doctor of Philosophy (PhD

Characterization of yeast histone H3-specific type B histone acetyltransferases identifies an ADA2-independent Gcn5p activity

BACKGROUND: The acetylation of the core histone NH(2)-terminal tails is catalyzed by histone acetyltransferases. Histone acetyltransferases can be classified into two distinct groups (type A and B) on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4. RESULTS: To better understand the role of histone acetylation in the assembly of chromatin structure, we have identified additional type B histone acetyltransferase activities specific for histone H3. One such activity, termed HatB3.1, acetylated histone H3 with a strong preference for free histones relative to chromatin substrates. Deletion of the GCN5 and ADA3 genes resulted in the loss of HatB3.1 activity while deletion of ADA2 had no effect. In addition, Gcn5p and Ada3p co-fractionated with partially purified HatB3.1 activity while Ada2p did not. CONCLUSIONS: Yeast extracts contain several histone acetyltransferase activities that show a strong preference for free histone H3. One such activity, termed HatB3.1, appears to be a novel Gcn5p-containing complex which does not depend on the presence of Ada2p

Pompe Disease

Course Code: Biochemistry 5614BiochemistryData AnalyticsMolecular Genetic

An overview of chromatin modifications

The last 15 years have witnessed tremendous progress in elucidating the roles of chromatin modifications in transcription regulation, DNA repair, replication, recombination, and other genomic processes. In this issue of Biopolymers, a series of reviews will summarize recent advances in our understanding of chromatin modifying enzymes and explore unresolved questions with respect to their regulation and functions in gene expression and other nuclear processes. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 95–97, 2013.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94507/1/22158_ftp.pd

Linker Histone H1 and H3K56 Acetylation are Antagonistic Regulators of Nucleosome Dynamics

H1 linker histones are highly abundant proteins that compact nucleosomes and chromatin to regulate DNA accessibility and transcription. However, the mechanisms that target H1 regulation to specific regions of eukaryotic genomes are unknown. Here we report fluorescence measurements of human H1 regulation of nucleosome dynamics and transcription factor (TF) binding within nucleosomes. H1 does not block TF binding, instead it suppresses nucleosome unwrapping to reduce DNA accessibility within H1-bound nucleosomes. We then investigated H1 regulation by H3K56 and H3K122 acetylation, two transcriptional activating histone post translational modifications (PTMs). Only H3K56 acetylation, which increases nucleosome unwrapping, abolishes H1.0 reduction of TF binding. These findings show that nucleosomes remain dynamic, while H1 is bound and H1 dissociation is not required for TF binding within the nucleosome. Furthermore, our H3K56 acetylation measurements suggest that a single-histone PTM can define regions of the genome that are not regulated by H1

Expanded binding specificity of the human histone chaperone NASP

NASP (nuclear autoantigenic sperm protein) has been reported to be an H1-specific histone chaperone. However, NASP shares a high degree of sequence similarity with the N1/N2 family of proteins, whose members are H3/H4-specific histone chaperones. To resolve this paradox, we have performed a detailed and quantitative analysis of the binding specificity of human NASP. Our results confirm that NASP can interact with histone H1 and that this interaction occurs with high affinity. In addition, multiple in vitro and in vivo experiments, including native gel electrophoresis, traditional and affinity chromatography assays and surface plasmon resonance, all indicate that NASP also forms distinct, high specificity complexes with histones H3 and H4. The interaction between NASP and histones H3 and H4 is functional as NASP is active in in vitro chromatin assembly assays using histone substrates depleted of H1

The human histone chaperone sNASP interacts with linker and core histones through distinct mechanisms

Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones