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Title: Kinetic and Structural Characterization of the Vibrio cholerae Flavin Transferase ApbE
Creator: Fang, Xuan
Date: 2019
Description: Cholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the...
Show moreCholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the antibiotic-resistance of its causative agent, V. cholerae. The resistance is mainly supported by a transmembrane electrochemical gradient of Na+ produced by the respiratory complex Na+-NQR coupled with an internal electron transfer pathway. The assembly and function of Na+-NQR is fulfilled by ApbE, the only known flavin transferase which covalently attaches two FMN molecules to the complex as part of its electron transport chain. Hence, ApbE is closely associated with the cause of antibiotic resistance. Because it does not have any human homologues, ApbE becomes an excellent drug target. In this work, we have investigated the physical properties of the enzyme and clarified its substrate specificity and pH dependence. For instance, our experiments indicate that divalent cations are essential for ApbE function, and that the selectivity depends largely on the size and the coordination sphere of the cation. Our data also show that ApbE regulation by pH, ADP and potassium is an important mechanism that enhances the adaptation, survival and colonization of V. cholerae in the small intestine. Moreover, pH dependence, mutagenesis, and steady-state kinetic studies have led us to identify the conserved His257 as a residue with dual roles: substrate binding and catalysis. Furthermore, bi-substrate kinetic studies have also revealed that ApbE follows a random Bi Bi mechanism. Together with structural studies, we propose a reaction mechanism where His257 functions as a base, shedding light into the understanding of the ApbE family.
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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: Biophysical and Computational Characterization of CinDel Edits of Dystrophin
Creator: Stojkovic, Vladimir
Date: 2022
Description: Duchenne muscular dystrophy (DMD) is a degenerative genetic disease caused by a genetic defect that results in the absence of dystrophin, a...
Show moreDuchenne muscular dystrophy (DMD) is a degenerative genetic disease caused by a genetic defect that results in the absence of dystrophin, a protein with an important stabilizing role in muscle cells. DMD causes progressive muscle degeneration leading to the loss of ambulation, and typically results in death before the third decade of life. Treatments for DMD aim to restore dystrophin expression and typically do so by producing edited or modified dystrophins. The only FDA approved therapy, exon skipping, produces dystrophin edits at exon boundaries but emerging therapeutic approaches like gene replacement therapy and CRISPR-Cas9-based gene editing techniques like CinDel allow for greater flexibility and are not constrained to exon boundary edits. However, understanding of what makes a “good”, functional edit is limited so it is not clear how to make use of this increased flexibility to produce optimal edits which are believed to be necessary for robust treatment. In an effort to improve understanding of the biophysics of these non-exon edits, we have embarked on a mixed experimental and computational study of a set of CinDel edits in the D19-D21 region of the dystrophin central rod domain. First, we have conducted an Alphafold structure prediction-based screen of a subset of possible edits in this region and selected one edit for follow-up characterization. We then compared this computationally-selected edit to three other heuristically designed edits experimentally and computationally by molecular dynamics simulations. We found that the computationally selected edit is significantly more thermodynamically stable than the other edits in the cohort. This edit also generally exhibited more favorable properties in MD simulations across multiple measures such as helicity, STR-junction unwinding and conformational variability.
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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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