Search results
(1 - 4 of 4)
- 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.
Show less
- 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.
Show less
- 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.
Show less
- 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.
Show less