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Title: SIMULATION OF H2A.B CONTAINING HISTONE VARIANT NUCLEOSOME
Creator: Kohestani, Havva
Date: 2019
Description: The H2A.B histone is a highly evolving vertebrate specific variant of the H2A histone family. It has been implicated in increased gene...
Show moreThe H2A.B histone is a highly evolving vertebrate specific variant of the H2A histone family. It has been implicated in increased gene expression, and experiments have shown that incorporation of H2A.B into nucleosomes results in more extended structures with fewer wrapped DNA base pairs. To study the molecular mechanisms of H2A.B, we have performed a series of conventional and enhanced sampling molecular dynamics simulation of H2A.B and canonical H2A containing nucleosomes.Results of adaptively biased molecular simulations show that substitution of canonical H2A with H2A.B results in geometrical changes such as unwrapping of 10 to 15 base pairs of DNA on each side of the nucleosome and an increase in the diameter and radius of gyration, which is in agreement with previous AFM, FRET, and SAXS experiments. DNA unwinding is energetically favorable in H2A.B containing compared to canonical nucleosomes, while in both systems we observe a wide range of sampling over various structures of DNA. H3 histone tails excluded simulations, show the importance and effect of N-terminal residues of H3 histones on attachment of DNA at the entry/exit sites to nucleosome protein core. Clustering and hydrogen bond analysis suggest that introduction of H2A.B to nucleosome systems triggers mechanisms leading to rearrangement of hydrogen bond network which may influence the pattern and intensity of interactions between DNA-protein and protein-protein complexes.
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Title: Detection Of BAXΔ2 Reading Frame Shift Using A Dual Luciferase Reporter System
Creator: Beatty, Evan Alexander
Date: 2019
Description: While initial studies of the pro-apoptotic Bcl-2-associated X protein isoform Δ2 (BAXΔ2) identified the combination of an alternative splicing...
Show moreWhile initial studies of the pro-apoptotic Bcl-2-associated X protein isoform Δ2 (BAXΔ2) identified the combination of an alternative splicing event and a gene-level mutation as the prerequisites for biosynthesis in microsatellite unstable (MSI+) human colon cancer cells, no similar explanation existed to explain the presence of this protein in normal and normal adjacent tissues. To identify an alternative to the gene-level mutation in the absence of an MSI+ phenotype, we utilized a dual luciferase reporter assay designed to observe epigenetic recoding. Plasmid constructs containing the first two exons encoding BAXΔ2 were either transcribed and translated in vitro or transfected into BAX-negative human colon cancer cells. In both cases, assay of the protein products of the reporter genes demonstrate that a low level (2.82% in vitro, 4.43% in vivo) of all translational events which produce the protein product of an upstream reporter gene also produce the protein product of a downstream reporter gene. This occurs despite the two existing in different reading frames as a result of the BAX exons cloned between them. These results confirm that an epigenetic recoding event is able to salvage the BAX reading frame in cases where exon 2 has been excised, and further narrow down the potential mechanism involved to either transcriptional slippage or programmed ribosomal frameshifting.
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Title: ANALYTIC STUDY OF THE CELLULAR FUNCTIONS OF UBL4A
Creator: Zhang, Huaiyuan
Date: 2021
Description: Ubiquitin-like protein 4A (Ubl4A) is a small protein encoded by a “housekeeping” gene that locates on the X chromosome. As a multi-functional...
Show moreUbiquitin-like protein 4A (Ubl4A) is a small protein encoded by a “housekeeping” gene that locates on the X chromosome. As a multi-functional protein, it has roles in a variety of cellular events including anti-tumorigenesis, response to DNA damage, inhibiting the fusion between autophagosome and lysosome, and docking of the tail-anchored proteins to the endoplasmic reticulum. We have previously reported that the newborns from Ubl4A-deficient mice had a high rate of mortality due to defect of AKT-dependent glucose metabolism. At the molecular level, Ubl4A directly binds with the actin related protein (Arp) 2/3 complex to accelerate the building up of the actin branching network, which further promotes the translocation and activation of the Akt, a key kinase for multiple cellular processes, from the cytosol to the plasma membrane.In further exploration of the molecular basis of Ubl4A in cell survival, here, we demonstrated that Ubl4A is critical for mitochondrial fusion and cell survival under nutrient depletion. In WT (wild-type) cells, the association of Ubl4A and the Arp2/3 complex serves as a primed “pool” of the actin branching network near mitochondria and enables mitochondria to fuse quickly for energy conservation upon starvation insult. However, such a “ready-to-go pool” of mitochondria was significantly decreased in the Ubl4A-deficient cells. As the result, the mitochondria became fragmentated, exhibited decreased trans-membrane potential, and accumulated ROS (reactive oxygen species), consequently, initiated mitochondria-mediated apoptosis. In this study, we also observed that Ubl4A-deficient mice displayed type II diabetic phenotype under a high-fat diet feeding. The preliminary results showed that these Ubl4A-deficient mice were more sensitive to glucose intolerance than their WT littermates, most likely owing to a delay in glucose uptake, and/or insulin secretion, both of which require the Arp2/3-actin branching network. We speculated that Ubl4A might be involved in cellular vesicle formation and/or secretion, but further investigation is needed to approve this hypothesis. Taken together, these findings provide a novel function of Ubl4A and further insight into the multi-functional roles of Ubl4A in mammalian cells, as well as the molecular basis for understanding the clinical relevance of Ubl4A in related human diseases.
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Title: Linear Systems Analysis of Molecular Dynamics
Creator: Nicholson, Stanley Anselm
Date: 2023
Description: Most proteins reduce the complexity of atomic motion to stable and coherent structures. Molecular dynamics (MD) has provided swaths of...
Show moreMost proteins reduce the complexity of atomic motion to stable and coherent structures. Molecular dynamics (MD) has provided swaths of trajectory data of proteins. We analyze these trajectories using classical stochastic signal analysis, well established and utilized by engineers. Linear systems analysis operates to uncover linearities given an input and output signal. The coherence function says an input and output are linearly related if and only if the coherence equals one. Analyzing protein motion in the frequency domain allows us to extract a frequency function relating the modes of motion as determined by atomic power spectra. Motivated by biochemistry, we analyze classical interactions like hydrogen bonds and salt bridges and find they act like a linear system, or effective spring. We test our analysis on two protein systems: crambin and the Mu Opioid Receptor (MOR). We extend our results to all pairwise interaction and determine coherent communities of atoms within the MOR. We present various community detection algorithms and demonstrate their validity using common metrics in MD. Identifying rigid and tightly correlated regions of the protein offers great potential in coarse graining protein structure and understanding protein motion.
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$X-Ray Diffraction Studies of Activation and Relaxation In Fast and Slow Rat Skeletal Muscle$

Title: X-Ray Diffraction Studies of Activation and Relaxation In Fast and Slow Rat Skeletal Muscle
Creator: Gong, Henry M.
Date: 2022
Description: The contractile properties of fast-twitch and slow-twitch skeletal muscles are primarily determined by the myosin isoform content and...
Show moreThe contractile properties of fast-twitch and slow-twitch skeletal muscles are primarily determined by the myosin isoform content and modulated by a variety of sarcomere proteins. X-ray diffraction studies of regulatory mechanisms in muscle contraction have focused predominately on fast- or mixed-fiber muscle with slow muscle being much less studied. Here, we used time-resolved x-ray diffraction to investigate the dynamic behavior of the myofilament proteins in relatively pure slow fiber rat soleus (SOL) and pure fast fiber rat extensor digitorum longus (EDL) muscle during twitch and tetanic contractions at optimal lengths (Lo), 95% Lo, and 90% Lo. Before the delivery of stimulation, reduction in muscle length led to decrease in passive tension. The x-ray reflections upon reduction in length showed no transition in the myosin heads from ordered OFF state, where heads are held close to the thick filament backbone, to disordered ON states, where heads are free to bind to thin filament, in both muscles. When stimulation was delivered to both muscles for twitch contractions at Lo, x-ray signatures indicating the transition of myosin heads to ON states were observed in EDL but not in soleus muscle. During tetanic contractions, changes in the disposition of myosin heads as active tension develops is a cooperative process in EDL muscle whereas in soleus muscle this relationship is less cooperative. Moreover, this high cooperativity was maintained in EDL at all lengths tested here, but cooperativity decreased upon reduction in lengths in soleus. The observed reduced extensibility of the thick filaments in soleus muscles as compared to EDL muscles indicate a molecular basis for this behavior. These data indicate that for the EDL thick filament activation is a cooperative strain-induced mechano-sensing mechanism, whereas for the soleus thick filament xiii activation has a more graded response. Lastly, x-ray data collected at different lengths demonstrated that the effect of length on soleus is more pronounce compared to EDL, particularly noticeable in the thick filament during relaxation phase after stimulation ceased. These observations indicate that soleus is more length-dependent than EDL. These different approaches to thick filament regulation in fast- and slow-twitch muscles may be designed to allow for short duration, strong contractions versus sustained finely controlled contractions, respectively.
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Title: STRUCTURE AND DYNAMICS OF MODIFIED NUCLEOSOMES UNDER EPIGENETIC REGULATION
Creator: Kohestani, Havva
Date: 2022
Description: Epigenetic regulations are critical in inducing heritable phenotype changes in biological systems without alternating their core genetic DNA...
Show moreEpigenetic regulations are critical in inducing heritable phenotype changes in biological systems without alternating their core genetic DNA sequences. In vivo, reversible epigenetic mechanisms engage various molecular structures from RNAs to larger proteins. The present thesis investigates the influence of epigenetic regulatory factors such as histone protein variants and small non-coding RNAs on the dynamics and structure of nucleosome core particles. Our results show that a histone substitution is an efficient tool in increasing or decreasing the exposure of DNA to post-translational modification (PTMs) factors or larger molecular assembly elements. Substitution of canonical H2A with H2A.B alters DNA-dimer interface resulting in increased breathing and accessibility of DNA. Replacement of canonical H3 with CENP-A variant impacts the overall core-DNA dynamics with flexibility of DNA entry/ exit sites and more rigid tetramer structure. Histone substitution also affects the micro to macro level molecular communication in the nucleosome system. The long-range correlated motions are weakened in H2A.B compared to canonical NCP. We observed a reduction in effective long-range DNA-DNA and DNA-core allosteric pathways in CENP-A NCP compared to canonical and Widom NCPs. Non-coding RNAs increase the tendency of the H3 tail histones to interact with DNA and induce the structural changes in the initial ideal B-DNA of NCP. Overall, the interaction of epigenetic regulatory factors in the form of protein or nucleic acids shifts the energetic and structural properties of the original nucleosome system. As a result, the chromatin structure is prepared to generate the proper biological response throughout spermatogenesis, chromosome segregation, or PTMs assembly.
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Title: AMPLIFICATION AND PURIFICATION OF RECOMBINANT PRO-DEATH BAXΔ2 PROTEINS FOR STRUCTURE ANALYSIS
Creator: Zhou, Yi
Date: 2020
Description: BaxΔ2 is an isoform of the pro-apoptotic Bax family of proteins, which is an important anti-cancer protein. BaxΔ2 behaves differently from...
Show moreBaxΔ2 is an isoform of the pro-apoptotic Bax family of proteins, which is an important anti-cancer protein. BaxΔ2 behaves differently from Baxα to induce apoptosis. The current computationally predicted model of BaxΔ2 is based on known Baxα structure, which is considered biased. Therefore, the elucidation of the BaxΔ2 crystal structure is critical. The goal of this project was to obtain a sufficient amount of purified recombinant Bax∆2 protein for crystallization. We cloned full-length BaxΔ2 fused with a poly-histidine tag on either N-terminus (His-Bax∆2) or C-terminus (Bax∆2-His) into an inducible bacterial expression vector. We found that His-Bax∆2 proteins were expressed better than Bax∆2-His, which totally inhibit host growth. However, the protein concentration of His-Bax∆2 was still too low to be detected by Coomassie blue staining. To increase His-Bax∆2 expression and avoid cytotoxicity, we further tested different bacterial host cells and applied the chaperone system. However, all attempts could not overcome Bax∆2 cytotoxicity and the protein expression levels were not high enough to be feasible for further large-scale purification. The mechanism underlying how Bax∆2 inhibits bacterial growth is still a mystery because Bax∆2 eukaryotic targets (mitochondria and caspases) do not exist in bacteria. Further experiments are required to explore the mechanism of Bax∆2 cytotoxicity in bacteria, so as to finally optimize and elevate the BaxΔ2 protein yields.
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