Regulation of Chorismate Mutase, an Enzyme from Aromatic Amino Acid Synthesis, in Early Plants

From the Washington University Undergraduate Research Digest: WUURD, Volume 11, 2015-2016. Published by the Office of Undergraduate Research, Joy Zalis Kiefer Director of Undergraduate Research and Assistant Dean in the College of Arts & Sciences; Lindsey Paunovich, Editor; Kristin Sobotka, Editor; Jennifer Kohl. Mentor: Joe Je

LILAC: Designing and Implementing a Light-Sensitive Actin-Binding Peptide

Cytoskeletal structure and dynamics are key for cells to function. Not only does the cytoskeleton give static support to the cell, its self-organization and restructuring are critical for cytokinesis, muscle movement, lamellipodia driven cell movement and more. Visualization of the actin cytoskeleton is necessary for various fields of biological research, and there are several actin labels available. For live-cell imaging, the most popular label is Lifeact, a 17 amino-acid peptide derived from actin binding protein (ABP) 140 in yeast. Despite its use in over 7,000 studies, Lifeact has several known concentration-dependent side effects, likely due to its competitive binding with important ABPs, cofilin and myosin. I addressed this problem by creating a light-sensitive version of Lifeact that has low affinity in the dark, but high affinity for actin when excited by blue light. Using previous designs of optogenetic tools as a guide, I designed LILAC (Light-Induced Label for ACtin) by strategically integrating Lifeact into the reversibly unfolded J$\alpha$ helix of AsLOV2, a photosenstive protein from oat. Reversible, light-induced recruitment of LILAC to filamentous actin was validated both in live cells and with purified proteins. Pre-illumination subtraction and correlative imaging allow for enhanced visualization of actin, and the recovery time constant can be modulated with buffer conditions. I also utilized LILAC to pattern actin filaments \textit{in vitro}, a tool that could be applied to elucidate how components of the actin cytoskeleton are integrated spatiotemporally to form complex structures. In my thesis, I describe the development and application of LILAC as an imaging and patterning tool, though a wide array of other applications remain

Structure-informed microbial population genetics elucidate selective pressures that shape protein evolution

Comprehensive sampling of natural genetic diversity with metagenomics enables highly resolved insights into the interplay between ecology and evolution. However, resolving adaptive, neutral, or purifying processes of evolution from intrapopulation genomic variation remains a challenge, partly due to the sole reliance on gene sequences to interpret variants. Here, we describe an approach to analyze genetic variation in the context of predicted protein structures and apply it to a marine microbial population within the SAR11 subclade 1a.3.V, which dominates low-latitude surface oceans. Our analyses reveal a tight association between genetic variation and protein structure. In a central gene in nitrogen metabolism, we observe decreased occurrence of nonsynonymous variants from ligand-binding sites as a function of nitrate concentrations, revealing genetic targets of distinct evolutionary pressures maintained by nutrient availability. Our work yields insights into the governing principles of evolution and enables structure-aware investigations of microbial population genetics