Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item FORCE RESPONSE OF CELL- ADHESION COMPLEXES AND HELICASES(2014) Chakrabarti, Shaon; Thirumalai, Devarajan; Jarzynski, Christopher; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Rapidly increasing technological prowess has led to the development of increasingly precise experiments to track biological systems at the single molecule level. The ability to apply measured amounts of external forces in such experiments, has added an extra probe to a scientist's arsenal of tools, allowing detailed investigations into the response of molecules that were not possible even a few years ago. However, the emerging raw single-molecule data tends to be of limited use in the absence of careful theories that can analyze and make sense of such data. This thesis focuses on understanding single-molecule force spectroscopy data on two important biological systems--cell adhesion complexes called selectins and integrins, and nucleic-acid unwinding motors known as helicases. Selectins and integrins are receptors expressed in blood vessels, that bind to specific ligands on leukocytes, initiating a process of absorption of leukocytes from the blood flow. The microscopic details of the selectin-ligand interactions that allow this process to occur, is hotly debated and a topic of intense current research. Over the last few years, it has been established that certain selectin-ligand lifetimes show a surprising `catch-bond' behavior, where the lifetime under force first increases before decreasing as expected. In this thesis, we build a structural model to explain this phenomenon and quantitatively explain a number of experimental results. Our work suggests that a loop region on the selectin receptor domain undergoes an allosteric conformational change, allowing the receptor to bind more tightly to the ligand. Force enhances this allosteric conformational change, thus resulting in an initial increase in lifetime of the complex. We provide quantitative support for this model, and also precise predictions of the outcomes of multiple mutation experiments. Helicases are molecular motors that hydrolyze nucleoside triphosphate (NTP) to carry out various kinds of cellular activities related to nucleic-acid metabolism. The particular aspect of certain helicases that we focus on in this thesis, is the NTP driven unwinding of double strand nucleic acids. Based on whether or not the helicase destabilizes the duplex base pairs while unwinding, helicases are classified as `active' or `passive', with different physical properties associated with each type. We develop a mathematical technique to analyze the velocities and processivities of such helicases, and predict a surprising universal behavior of the processivity under external forces. Our analysis suggests that partner proteins (invariably required for efficient unwinding of nucleic acids in vivo) have coevolved with helicases to increase the processivity, as opposed to the velocity, of all types of helicases. Finally, we establish the unwinding mechanism of the T-7 helicase, thereby providing insight into the unwinding mechanisms of a whole family (SF-4) of helicases.Item PURIFICATION AND CHARACTERIZATION OF THE RECD PROTEIN-HOMOLOGUE FROM DEINOCOCCUS RADIODURANS(2004-12-06) Wang, Jianlei; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In many gram-negative bacteria, RecBCD enzyme is found to be responsible for double strand DNA break repair through homologous recombination. The AddAB enzyme, a RecBCD analog, is found in some gram-positive bacteria and functions in a similar way as RecBCD. A few bacteria appear to lack both RecBCD and AddAB enzymes entirely. One such organism is the bacterium Deinococcus radiodurans. This remarkable organism is able to survive in the presence of very high levels of radiation or DNA-damaging chemicals, levels that would overwhelm the DNA repair capacity of most other organisms. Interestingly, the D. radiodurans genome does have an open reading frame that would encode a protein that is homologous to the E. coli RecD protein. The amino acid sequence of this D. radiodurans RecD-like protein suggests that it is a helicase and therefore could function in some aspect of DNA repair, as does its E. coli homologue. However, the RecD protein of D. radiodurans must serve a different and novel function compared to the E. coli RecD protein. The D. radiodurans RecD protein can be expressed at high levels in E. coli and is readily purified by chromatography on a nickel column followed by single-stranded DNA-cellulose. The purified protein exhibits DNA-dependent ATPase and DNA helicase activities. The helicase activity requires at least a 10 nucleotide single strand overhang at the 5'-end of the double strand DNA substrate to start unwinding. The helicase assay shows that D. radiodurans RecD-like protein unwinds dsDNA substrates catalytically, but with low processivity, even with the help of single strand binding proteins (SSB) from either E. coli or D. radiodurans. These results show that D. radiodurans RecD-like protein is a DNA helicase that moves with 5'-3' polarity on single-stranded DNA. The E. coli RecD protein was shown recently to unwind dsDNA with the same 5'-3' polarity. The low processivity of the D. radiodurans RecD-like protein suggests that it may function in a complex with other proteins. The identity of these proteins is not known. We have also generated insertion mutations that are likely to disrupt all of the recD gene copies in the D. radiodurans genome after multiple generations growing in media with antibiotics. The in vivo effects of the insertion mutation, such as the growth curve and the sensitivity to UV radiation and DNA damaging chemicals, were studied.