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Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Fluorescent Visualization Of Cellular Proton Fluxes, Lejie Zhang Sep 2018

Fluorescent Visualization Of Cellular Proton Fluxes, Lejie Zhang

GSBS Dissertations and Theses

Proton fluxes through plasma membranes are essential for regulating intracellular and extracellular pH and mediating co-transport of metabolites and ions. Although conventional electrical measurements are highly sensitive and precise for proton current detection, they provide limited specificity and spatial information. My thesis focuses on developing optical approaches to visualize proton fluxes from ion channels and transporters.

It has been demonstrated that channel-mediated acid extrusion causes proton depletion at the inner surface of the plasma membrane. Yet, proton dynamics at the extracellular microenvironment are still unclear. In Chapter II, we developed an optical approach to directly measure pH change in this ...


An Examination Of Mhc, Peptide, And Tcr Interactions, Peter Trenh May 2018

An Examination Of Mhc, Peptide, And Tcr Interactions, Peter Trenh

GSBS Dissertations and Theses

T cell receptors (TCR) bind to peptides from various sources on MHC (Major Histocompatibility Complex) molecules. A long-standing goal in the field is to understand the mechanisms of MHC-peptide exchange and MHC-TCR interactions. Here, I present work from three uniquely different systems that address the following: HLA-DR1 conformational stability, self-tolerant mechanisms of TCRs isolated from self-reactive TCR transgenic mice, and TCR cross-reactivity mechanisms between LCMV and VV.

First, I present a crystal structure of HLA-DR1 in complex with A1L9 peptide, a peptide with two amino acid substitutions from the parental peptide. The singly substituted A1 peptide, which has a pocket ...


Three-Dimensional Folding Of Eukaryotic Genomes, Tsung-Han S. Hsieh May 2017

Three-Dimensional Folding Of Eukaryotic Genomes, Tsung-Han S. Hsieh

GSBS Dissertations and Theses

Chromatin packages eukaryotic genomes via a hierarchical series of folding steps, encrypting multiple layers of epigenetic information, which are capable of regulating nuclear transactions in response to complex signals in environment. Besides the 1-dimensinal chromatin landscape such as nucleosome positioning and histone modifications, little is known about the secondary chromatin structures and their functional consequences related to transcriptional regulation and DNA replication. The family of chromosomal conformation capture (3C) assays has revolutionized our understanding of large-scale chromosome folding with the ability to measure relative interaction probability between genomic loci in vivo. However, the suboptimal resolution of the typical 3C techniques ...


Targeted Mutagenesis Of A Therapeutic Human Monoclonal Igg1 Antibody Prevents Gelation At High Concentrations, Paul Casaz, Elisabeth N. Boucher, Rachel Wollacott, Sadettin S. Ozturk, William D. Thomas Jr., Yan Wang May 2014

Targeted Mutagenesis Of A Therapeutic Human Monoclonal Igg1 Antibody Prevents Gelation At High Concentrations, Paul Casaz, Elisabeth N. Boucher, Rachel Wollacott, Sadettin S. Ozturk, William D. Thomas Jr., Yan Wang

UMass Center for Clinical and Translational Science Research Retreat

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or “gelation”. Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >40 mg/mL and the temperature was <6oC. Gel formation was reversible and was affected by salt concentration or pH, suggesting a charge interaction between IgG monomers. However, formulation optimization could not completely prevent gelation at high concentrations so a protein engineering approach was sought to resolve the problem. A comparison of the heavy and light chain amino acid sequences to consensus germline sequences revealed 16 amino acid sequence differences in the framework regions that could be involved with gelation. Restoring the C4 framework sequence to consensus germline residues by targeted mutagenesis resulted in no gel formation at 50 mg/ml at temperatures as low as 0oC. Additional genetic analysis was used to identify the key residue(s) involved in the gelation. A single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine, was found sufficient to prevent gelation, while a double mutation, replacing heavy chain serine 85 and threonine 87 with arginine, increased the temperature at which gel formation initiated. These results indicate that the temperature dependence of gelation may be related to conformational changes near the charged residues or the regions interact with. Our work provided a molecular strategy that can be applied to improve the solubility of other therapeutic antibodies.