Structural Biology Commons^™

D101n: A Unique And Ill-Understood Familial Als-Related Sod1 Mutant, Analisa Lott

Honors Program Theses and Research Projects

Background: Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease, with 90% of documented cases considered sporadic and about 10% showing a hereditary link; such cases are deemed as familial ALS (fALS). Superoxide dismutase (SOD), a copper-zinc binding enzyme that protects cells from damaging free radicals, has been linked to fALS with certain toxic gain of function mutations, such as the D101N mutant. The human SOD1 D101N mutant has been shown to be associated with rapidly progressing neurodegeneration yet is less prone to aggregation than similar mutants, but its deviation from wild-type (WT) SOD1 remains misunderstood.

Objective: Although the D101N …

Structure-Based Targeting Of The Nemo:Ikk Interaction For Canonical Nf-Κb Inhibition, Amy Kennedy

Dartmouth College Ph.D Dissertations

The NF-κB pathway is important for cell survival and proliferation, inflammation, and innate immunity, and its dysregulation is a common theme in many cancers, autoimmune disorders, and other disease states. The protein-protein interaction between the scaffolding protein NEMO and the kinase IKK in the NF-κB pathway represents a compelling target for selective NF-κB inhibition because it occurs only in the canonical branch of the NF-κB pathway. Disruption of the NEMO:IKK interaction has been established for decades in the literature as a safe and effective way to selectively inhibit overactivation of the canonical branch of the NF-κB pathway. The benchmark in …

Structural Basis For Reversible Glucan Phosphorylation By Glucan Dikinases And Glucan Phosphatases, Tiantian Chen

Theses and Dissertations--Molecular and Cellular Biochemistry

Plants store glucose in the form of starch, an insoluble macromolecule that is synthesized and degraded diurnally. Glucan phosphatases play a critical role in starch breakdown, working with glucan dikinases in a cyclic phosphorylation and dephosphorylation. Herein, we present a novel glucan phosphatase Like SEX Four3 (LSF3) from Physcomitrella patens. Structural and enzymatic studies demonstrate that LSF3 is a dimeric glucan phosphatase that displays robust specific glucan phosphatase activity. Dimerization is shown to be essential for LSF3 stability and function. Structure-guided bioinformatic analysis shows that LSF3 is found in early land plants and green algae.

Building upon our understanding of …

Structure Of Clostridium Perfringens Type Iv Pili, Alexander R. Meyer

Honors Program: Senior Projects (Public)

Type IV pili (T4P) are thin, hair-like bacterial appendages composed of protein subunits polymerized into a helical fiber. T4P perform diverse functions such as host cell adhesion, biofilm formation, natural competence, and twitching motility. While T4P are well characterized in Gram-negative bacteria, they have more recently been found in Gram-positive bacteria as well. In this work we aimed to solve the crystal structure of the type IV major pilin protein PilA2 from Clostridium perfringens, the predominant pilus subunit which makes up about 99% of the pilus fiber. We report expression, purification, and crystallization conditions which are sufficient for X-ray …

Characterization Of Human Pyrroline-5-Carboxylate Reductase Enzymes Responsible For L-Proline Biosynthesis, Sagar Patel

Department of Biochemistry: Dissertations, Theses, and Student Research

Pyrroline-5-carboxylate reductases (EC 1.5.1.2) are important housekeeping enzymes of L-proline biosynthesis, which generate L-proline and influence redox cycling of NAD(P)H/NAD(P)⁺ to support cellular growth in all domains of life. Structural evidence from X-ray crystal structures of HsPYCR1 (PDB codes 5UAT, 5UAU, and 5UAV) shows both NADPH bound in the N-terminal Rao-Rossmann fold motif and an important hydrogen bond or proton donor role for Thr238 with L-P5C. The Thr238Ala mutation results in 10-fold loss in catalytic efficiency with varied L-P5C relative to the wild-type enzyme, thus indicating Thr238’s potential hydrogen bond and proton donation to L-P5C is critical for catalysis. …

Method Development For Structural Assessment Of Nanolipoprotein Particles With And Without Cross-Linked Lipids, Emma J. Mullen, Wei He, Sean Gilmore, Matthias Frank, Matthew Coleman, Megan Shelby

STAR Program Research Presentations

Membrane proteins make up approximately 30% of the cellular proteome and account for over 60% of pharmaceutical targets.1 Determining the structures of this class of proteins is critical to our understanding of disease states and will advance rational drug design. But membrane proteins have limited solubility, rarely form large crystals that diffract well, and often misfold outside of a bilayer, hindering crystallographic studies.1 Nanolipoprotein particles (NLPs) have arisen as a platform to readily solubilize membrane proteins while mimicking a native lipid environment. NLPs consist of a discoidal phospholipid bilayer encircled by an apolipoprotein belt. In an effort to optimize and …

Crystal Structure Of Yersinia Pestis Virulence Factor Yfea Reveals Two Polyspecific Metal-Binding Sites, Christopher D. Radka, Lawrence J. Delucas, Landon S. Wilson, Matthew B. Lawrenz, Robert D. Perry, Stephen G. Aller

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Gram-negative bacteria use siderophores, outer membrane receptors, inner membrane transporters and substrate-binding proteins (SBPs) to transport transition metals through the periplasm. The SBPs share a similar protein fold that has undergone significant structural evolution to communicate with a variety of differentially regulated transporters in the cell. In Yersinia pestis, the causative agent of plague, YfeA (YPO2439, y1897), an SBP, is important for full virulence during mammalian infection. To better understand the role of YfeA in infection, crystal structures were determined under several environmental conditions with respect to transition-metal levels. Energy-dispersive X-ray spectroscopy and anomalous X-ray scattering data show that …

Purification, Optimization, And Growth Of New Delhi Metallo-Β-Lactamase-1 Protein Crystals Mixed With Nz218 Inhibitor, Brandon M. Wills

Celebration of Learning

New Delhi metallo-β-lactamase-1 is a problematic gene found in certain strains of bacteria that cause them to become antibiotic resistant to nearly all known antibiotics. While some antibiotics are available to treat patients with a bacterial infection, most are toxic or do not have 100% success rates. With that being said, it is imperative that we search for a molecule that is successfully able to inhibit the effects of this gene every time. Such a discovery would help tremendously with new antibiotic drug development and also prevent further damage by these dangerous bacteria. In this presentation, I will describe the …

Neuropilin In The Vascular System: Mechanistic Basis Of Angiogenesis, Hou-Fu Guo

Theses and Dissertations--Molecular and Cellular Biochemistry

The vascular system is critical for maintaining homeostasis in all vertebrates. Structural studies of Neuropilin (Nrp), an essential angiogenic receptor, have defined its role in regulating angiogenesis, the formation of new vessels from the existing vasculature. Utilizing biochemical and biophysical tools we describe the ability of Nrp to function as a co-receptor for the VEGFR receptor tyrosine kinase. Two families of Nrp-1 ligands, Vascular Endothelial Growth Factor A (VEGF-A) and Semaphorin3F (Sema3F), physically compete for binding to the Nrp-1 b1 domain, and have opposite roles. VEGF-A is a potent pro-angiogenic cytokine while Sema3F is an angiogenesis inhibitor. Using coupled structural …

Structural Basis Of Substrate Recognition In Thimet Oligopeptidase And Development Of Nanoparticles For Therapeutic Enzyme Delivery, Jonathan Mark Wagner

Theses and Dissertations--Molecular and Cellular Biochemistry

Neuropeptidases are responsible for degradation of signaling peptides in the central nervous system and periphery. Some neuropeptidases have also been shown to play a role as part of the cell’s hydrolytic machinery responsible for breaking down proteins and peptides into amino acids, and these enzymes therefore influence small peptide availability for antigen presentation. A better understanding of how neuropeptidases recognize their substrates could lead to therapeutics that modulate the activity of these important enzymes. Alternatively, re-engineering these enzymes to selectively hydrolyze undesirable peptides could make them attractive as therapeutics themselves. A key question in understanding the activity of these enzymes …