Human immunodeficiency virus type 1 (HIV-1), the pathogen responsible for the onset of acquired immuno-deficiency syndrome (AIDS) in humans has reached pandemic proportions. To this day, no cure is available for infection with this virus and the only treatment option for this chronic infection is the life-long adherence to anti-retroviral therapy. Efforts in the quest to control the worldwide AIDS pandemic include the search for an effective anti-HIV-1 vaccine. Providing hope in this endeavor are a few monoclonal antibodies possessing broad neutralizing characteristics (bnmAbs) that have been isolated from the sera of rare patients that have a delayed progression to AIDS. In this thesis, one of these bnmAbs, 2F5 is extensively characterized at the atomic level to better understand its binding and neutralization mechanism. In total, 27 crystal structures of the 2F5 Fab’ in complex with various peptides representing its linear gp41 membrane proximal external region (MPER) epitope are presented. Furthermore, expression of the 2F5 Fab in a bacterial system allowed to design mutants of the 2F5 Fab and therefore investigate the implication of specific domains of 2F5 in mediating binding and neutralization. Atomic level characterization of this immune complex revealed a somewhat promiscuous recognition of 2F5 for its 664DKW666 epitope as long as the following characteristics were conserved: the aspartate’s negative charge, the hydrophobic alkyl-pi stacking arrangement between the beta-turn lysine and tryptophan, and the positive charge of the former. Moreover, it was demonstrated that 2F5 has an elongated and flexible complementary determining region 3 loop of the heavy chain (CDR H3), which is required for neutralization and is involved in secondary binding interactions other than to its core linear epitope. These contributions will significantly help in guiding the structure-based design of an HIV-1 vaccine looking to elicit 2F5-like antibody responses.

The thesis presented here describes the steps that were taken in an attempt to solve the protein structure of MenD via molecular replacement and multiple wavelength anomalous dispersion. The introduction provides background on menaquinone biosynthesis and the role of MenD in this metabolic pathway. Also, a detailed discussion of the DC Family of enzymes, a subgroup of ThDP dependent enzymes, which MenD is a part of, is included.<p> Utilizing various software packages a 1.9 Å data set was processed and analyzed in an attempt to provide a molecular replacement result. When molecular replacement was deemed incapable of solving the phase problem of the data set, the production of SeMet protein was attempted to allow for MAD phasing.<p> A homology model of MenD was produced using the program Modeller with benzaldehyde lyase as a template. A structure based sequence alignment was done with all DC Family enzymes with structures published. Then a second structure based sequence alignment was done to compare the same set to the Modeller model. This was done to gain a deeper understanding of MenD and how it interacts with its cofactors ThDP and Mg2+. Furthermore, these results were used to implicate potential active site residues.

Human immunodeficiency virus type 1 (HIV-1), the pathogen responsible for the onset of acquired immuno-deficiency syndrome (AIDS) in humans has reached pandemic proportions. To this day, no cure is available for infection with this virus and the only treatment option for this chronic infection is the life-long adherence to anti-retroviral therapy. Efforts in the quest to control the worldwide AIDS pandemic include the search for an effective anti-HIV-1 vaccine. Providing hope in this endeavor are a few monoclonal antibodies possessing broad neutralizing characteristics (bnmAbs) that have been isolated from the sera of rare patients that have a delayed progression to AIDS. In this thesis, one of these bnmAbs, 2F5 is extensively characterized at the atomic level to better understand its binding and neutralization mechanism. In total, 27 crystal structures of the 2F5 Fab’ in complex with various peptides representing its linear gp41 membrane proximal external region (MPER) epitope are presented. Furthermore, expression of the 2F5 Fab in a bacterial system allowed to design mutants of the 2F5 Fab and therefore investigate the implication of specific domains of 2F5 in mediating binding and neutralization. Atomic level characterization of this immune complex revealed a somewhat promiscuous recognition of 2F5 for its 664DKW666 epitope as long as the following characteristics were conserved: the aspartate’s negative charge, the hydrophobic alkyl-pi stacking arrangement between the beta-turn lysine and tryptophan, and the positive charge of the former. Moreover, it was demonstrated that 2F5 has an elongated and flexible complementary determining region 3 loop of the heavy chain (CDR H3), which is required for neutralization and is involved in secondary binding interactions other than to its core linear epitope. These contributions will significantly help in guiding the structure-based design of an HIV-1 vaccine looking to elicit 2F5-like antibody responses.

The thesis presented here describes the steps that were taken in an attempt to solve the protein structure of MenD via molecular replacement and multiple wavelength anomalous dispersion. The introduction provides background on menaquinone biosynthesis and the role of MenD in this metabolic pathway. Also, a detailed discussion of the DC Family of enzymes, a subgroup of ThDP dependent enzymes, which MenD is a part of, is included.<p> Utilizing various software packages a 1.9 Å data set was processed and analyzed in an attempt to provide a molecular replacement result. When molecular replacement was deemed incapable of solving the phase problem of the data set, the production of SeMet protein was attempted to allow for MAD phasing.<p> A homology model of MenD was produced using the program Modeller with benzaldehyde lyase as a template. A structure based sequence alignment was done with all DC Family enzymes with structures published. Then a second structure based sequence alignment was done to compare the same set to the Modeller model. This was done to gain a deeper understanding of MenD and how it interacts with its cofactors ThDP and Mg2+. Furthermore, these results were used to implicate potential active site residues.

No description available.

Thesis (M.S.)--University of Florida, 2003. / Title from title page of source document. Includes vita. Includes bibliographical references.

Malaria is a global health concern accounting for approximately 219 million cases and an estimated 660 000 deaths in 2010. The most fatal strain of malarial parasite, Plasmodium falciparum is found to contain 3 Adenylate Kinases (PfAK1, PfAK2 and PfGAK). Adenylate Kinases are important enzymes that essentially catalyze and regulate energy metabolism processes. PfAK1 and PfAK2 catalyze the reversible MG2+ reaction ATP + AMP ←→ 2ADP whereas, the PfGAK catalyzes the Mg2+ dependent reaction GTP+AMP ←→ ADP+GDP. Of all malarial strains, only the Plasmodium falciparum Adenylate Kinase 2 (PfAK2) was found to contain a N-myristoylation sequence and subsequently formed a stable heterodimer with Plasmodium falciparum N-myristoyl transferase (PfNMT). The myristoylation of PfAK2 by PfNMT is believed to help transport PfAK2 to the parasitophorous vacuole membrane (PVM) so that the enzyme can perform its essential functions. With these enzymes being key components in the parasite’s survival, the structural study of these enzymes would provide a lot of insight into targeting these proteins for drug design that would effectively kill the parasite without affecting the human host. In this study, PfAK1 was able to be expressed, purified and crystallized with a dataset collected at 4.3Å. PfGAK was expressed and purified. A GTP analogue called GP5A was used to soak the purified PfGAKand the PfGAK bound to GP5A was crystallized and diffracted. Moreover, PfAK2 and PfNMT was successfully expressed and co-purified. The purified PfAK2-PfNMT heterodimer are undergoing crystal screening for possible crystallization conditions. / published_or_final_version / Physiology / Master / Master of Philosophy

No description available.

published_or_final_version / Chemistry / Master / Master of Philosophy

No description available.