Intrinsically Disordered Proteins within the Genome

The hundreds of millions of DNA base pairs within eukaryotic cells are not found free but packed inside the micrometre-sized nuclei through the formation of a macromolecular structure known as chromatin. Chromatin consists of a chain of nucleosomes – nucleoprotein complexes where the DNA makes ∼1.75 turns around a protein octamer core composed of two copies each of H2A, H2B, H3 and H4 histones. A fifth histone H1 binds on the nucleosomal surface close to the entry/exit site of DNA, interacts with linker DNA and aids in chromatin compaction. Enabling the condensation of DNA to fit into the nucleus is however only one-half of chromatin’s role. The three-dimensional spatial organization of chromatin serves a second important role in allowing the capability to exert control over gene expression. The chromatin structure thus serves as an additional layer of complexity above the genome code and permits the transcription of different proteins varying with cell lineages/cycles. The proteins that makeup, modify and read the chromatin structure are particularly enriched in `Intrinsic Disorder’ – a class of proteins lacking a well-defined structure but existing as a dynamic ensemble of rapidly interchanging states. While folded proteins with well-defined structures are amenable to be characterized through standard methods of protein structure determination, the `plasticity’ of the disordered proteins challenges the use of such ensemble averaged techniques. In this thesis, Molecular Dynamics simulations are used to characterize the disordered regions of three proteins that form the core of chromatin structure: histones, linker histones (H1) and heterochromatin protein (HP1). The carboxy-terminal domain of H1 when within the nucleosome, adopts a compact but unstructured conformation that allows its positioning between the two linker DNA strands. In contrast, the amino-terminal domain of H1 undergoes a disorder-to-order transition to an amphiphilic helical conformation. The transition to the amphiphilic helix is however subtype-dependant with the degree of condensation varying with the subtypes' nucleosomal affinity. Finally, the simulations demonstrate that the affinity of HP1 subtypes for the H3 histone is caused by the synergetic effects of both the proteins' unstructured amino-terminal domain and the structured chromodomain

Large-Eddy Simulation of Turbulent Boundary Layers with Spatially Varying Roughness

This dissertation addresses high Reynolds number turbulent boundary layers flows with different inhomogeneous surface roughness distributions using large eddy simulations. The stretched vortex subgrid scale model for the outer flow LES is coupled with a virtual-wall model for the friction velocity with a correction accounting for local roughness effects. A semi-empirical model that describes a fully developed rough-walled turbulent boundary layer with sand-grain roughness length-scale ks = αx that varies linearly with streamwise distance is first developed, with α a dimensionless constant. For large Rex and a free-stream velocity U∞ ~ xm, a simple log-wake model of the local turbulent mean-velocity profile is used that contains a standard mean-velocity correction for the asymptotic, fully rough regime. A two parameter (α; m) family of solutions is obtained for which U∞+ (or equivalently Cf) and boundary-layer measures can be calculated. These correspond to perfectly self-similar boundary-layer growth in the streamwise direction with similarity variable z/ks where z is the wall-normal co-ordinate. Results over a range of α are discussed for cases including the zero-pressure gradient (m = 0) and sink-flow (m = -1) boundary layers. Model trends are supported by high Re wall-modeled LES. Linear streamwise growth of boundary layer measures is confirmed, while for each α, mean-velocity profiles and streamwise turbulent stresses are shown to collapse against z/(αx). Inner scaled velocity defects are shown to collapse against z/Δ, where Δ is the Rotta-Clauser parameter. The present results suggest that these flows may be interpreted as the fully-rough limit for boundary layers in the presence of small-scale, linear roughness. Next, an LES study of a flat-plate turbulent boundary layer at high Re under nonequilibrium flow conditions due to the presence of abrupt changes in surface roughness is presented. Two specific cases, smooth-rough (SR) and rough-smooth (RS) transition are examined in detail. Streamwise developing velocity and turbulent stress profiles are considered and sharp departures from equilibrium flow properties with subsequent relaxation are shown downstream. Relaxation trends are studied using integral parameters and higher-order mean flow statistics with emphasis on Reτ and ks+ dependence. Results are compared with RS experiments at matched Reτ, and show good agreement in terms of recovery rates. Finally, the case of static, impulsive wall-roughness in flows at high Re is addressed using the same LES framework. The initial perturbation from smooth-to-rough appears to dominate the flow behaviour with the length of the impulsive patch showing little effect on recovery rates at matched Reτ and ks+. The resulting trends show good agreement with low Re experiments and support the wall-modeled LES framework as a suitable method for analysing high Re flows in practical applications.</p

Protein disorder-to-order transition enhances the nucleosome-binding affinity of H1.

Intrinsically disordered proteins are crucial elements of chromatin heterogenous organization. While disorder in the histone tails enables a large variation of inter-nucleosome arrangements, disorder within the chromatin-binding proteins facilitates promiscuous binding to a wide range of different molecular targets, consistent with structural heterogeneity. Among the partially disordered chromatin-binding proteins, the H1 linker histone influences a myriad of chromatin characteristics including compaction, nucleosome spacing, transcription regulation, and the recruitment of other chromatin regulating proteins. Although it is now established that the long C-terminal domain (CTD) of H1 remains disordered upon nucleosome binding and that such disorder favours chromatin fluidity, the structural behaviour and thereby the role/function of the N-terminal domain (NTD) within chromatin is yet unresolved. On the basis of microsecond-long parallel-tempering metadynamics and temperature-replica exchange atomistic molecular dynamics simulations of different H1 NTD subtypes, we demonstrate that the NTD is completely unstructured in solution but undergoes an important disorder-to-order transition upon nucleosome binding: it forms a helix that enhances its DNA binding ability. Further, we show that the helical propensity of the H1 NTD is subtype-dependent and correlates with the experimentally observed binding affinity of H1 subtypes, suggesting an important functional implication of this disorder-to-order transition

Waterdock 2.0: Water placement prediction for Holo-structures with a pymol plugin.

Water is often found to mediate interactions between a ligand and a protein. It can play a significant role in orientating the ligand within a binding pocket and contribute to the free energy of binding. It would thus be extremely useful to be able to accurately predict the position and orientation of water molecules within a binding pocket. Recently, we developed the WaterDock protocol that was able to predict 97% of the water molecules in a test set. However, this approach generated false positives at a rate of over 20% in most cases and whilst this might be acceptable for some applications, in high throughput scenarios this is not desirable. Here we tackle this problem via the inclusion of knowledge regarding the solvation structure of ligand functional groups. We call this new protocol WaterDock2 and demonstrate that this protocol maintains a similar true positive rate to the original implementation but is capable of reducing the false-positive rate by over 50%. To improve the usability of the method, we have also developed a plugin for the popular graphics program PyMOL. The plugin also contains an implementation of the original WaterDock.GAR is supported by the Memorial Sloan Kettering Cancer Center, NIH grant P30 CA008748

Waterdock 2.0: Water placement prediction for Holo-structures with a pymol plugin.

Water is often found to mediate interactions between a ligand and a protein. It can play a significant role in orientating the ligand within a binding pocket and contribute to the free energy of binding. It would thus be extremely useful to be able to accurately predict the position and orientation of water molecules within a binding pocket. Recently, we developed the WaterDock protocol that was able to predict 97% of the water molecules in a test set. However, this approach generated false positives at a rate of over 20% in most cases and whilst this might be acceptable for some applications, in high throughput scenarios this is not desirable. Here we tackle this problem via the inclusion of knowledge regarding the solvation structure of ligand functional groups. We call this new protocol WaterDock2 and demonstrate that this protocol maintains a similar true positive rate to the original implementation but is capable of reducing the false-positive rate by over 50%. To improve the usability of the method, we have also developed a plugin for the popular graphics program PyMOL. The plugin also contains an implementation of the original WaterDock.GAR is supported by the Memorial Sloan Kettering Cancer Center, NIH grant P30 CA008748

CD34 and CD117 Stemness of Lineage-Negative Cells Reverses Memory Loss Induced by Amyloid Beta in Mouse Model

A majority of the neurodegenerative disorders including Alzheimer's disease are untreatable and occur primarily due to aging and rapidly changing lifestyles. The rodent Alzheimer's disease models are critical for investigating the underlying disease pathology and screening of novel therapeutic targets in preclinical settings. We aimed to characterize the stemness properties of human umbilical cord blood (hUCB) derived lineage-negative (Lin−) stem cells based on CD34 and CD117 expression as well as surface morphology using flow cytometry and scanning electron microscopy, respectively. The efficacy of the stem cells was tested by its capacity to rescue the injury caused by intrahippocampal delivery of varying doses of amyloid beta. The hUCB Lin− stem cells reversed memory loss due to Aβ42-induced injury more effectively at micromolar concentration, and not picomolar concentration. More studies are required to delineate the underlying molecular events associated with hUCB Lin− stem cells

Deoxyribonucleic Acid Encoded and Size-Defined π-Stacking of Perylene Diimides

Natural photosystems use protein scaffolds to control intermolecular interactions that enable exciton flow, charge generation, and long-range charge separation. In contrast, there is limited structural control in current organic electronic devices such as OLEDs and solar cells. We report here the DNA-encoded assembly of π-conjugated perylene diimides (PDIs) with deterministic control over the number of electronically coupled molecules. The PDIs are integrated within DNA chains using phosphoramidite coupling chemistry, allowing selection of the DNA sequence to either side, and specification of intermolecular DNA hybridization. In this way, we have developed a “toolbox” for construction of any stacking sequence of these semiconducting molecules. We have discovered that we need to use a full hierarchy of interactions: DNA guides the semiconductors into specified close proximity, hydrophobic–hydrophilic differentiation drives aggregation of the semiconductor moieties, and local geometry and electrostatic interactions define intermolecular positioning. As a result, the PDIs pack to give substantial intermolecular π wave function overlap, leading to an evolution of singlet excited states from localized excitons in the PDI monomer to excimers with wave functions delocalized over all five PDIs in the pentamer. This is accompanied by a change in the dominant triplet forming mechanism from localized spin–orbit charge transfer mediated intersystem crossing for the monomer toward a delocalized excimer process for the pentamer. Our modular DNA-based assembly reveals real opportunities for the rapid development of bespoke semiconductor architectures with molecule-by-molecule precision

Photogeneration of Spin Quintet Triplet–Triplet Excitations in DNA-Assembled Pentacene Stacks

Singlet fission (SF), an exciton-doubling process observed in certain molecular semiconductors where two triplet excitons are generated from one singlet exciton, requires correctly tuned intermolecular coupling to allow separation of the two triplets to different molecular units. We explore this using DNA-encoded assembly of SF-capable pentacenes into discrete π-stacked constructs of defined size and geometry. Precise structural control is achieved via a combination of the DNA duplex formation between complementary single-stranded DNA and the local molecular geometry that directs the SF chromophores into a stable and predictable slip-stacked configuration, as confirmed by molecular dynamics (MD) modeling. Transient electron spin resonance spectroscopy revealed that within these DNA-assembled pentacene stacks, SF evolves via a bound triplet pair quintet state, which subsequently converts into free triplets. SF evolution via a long-lived quintet state sets specific requirements on intermolecular coupling, rendering the quintet spectrum and its zero-field-splitting parameters highly sensitive to intermolecular geometry. We have found that the experimental spectra and zero-field-splitting parameters are consistent with a slight systematic strain relative to the MD-optimized geometry. Thus, the transient electron spin resonance analysis is a powerful tool to test and refine the MD-derived structure models. DNA-encoded assembly of coupled semiconductor molecules allows controlled construction of electronically functional structures, but brings with it significant dynamic and polar disorders. Our findings here of efficient SF through quintet states demonstrate that these conditions still allow efficient and controlled semiconductor operation and point toward future opportunities for constructing functional optoelectronic systems