High-Pressure Small-Angle X-ray Scattering: Recent discoveries have illustrated a significant portion of earth’s biosphere, potentially as large as 80%, exists under conditions of extreme pressure (>10 MPa or 100 atm) yet the biological mechanisms of adaptation to life under such conditions is poorly understood. Moreover, pressure is a unique thermodynamic variable and therefore provides a powerful biophysical lens into protein structure, stability, ligand binding events, and other biophysical processes. This thesis presents important advancements in high-pressure small-angle X-ray scattering (HP-SAXS), focusing on the development of novel instrumentation and methodologies to probe biomolecular structures under extreme pressure conditions. Two experimental configurations will be discussed: (1) a so-called static HP-cell capable of data acquisition at pressures up to 400 MPa with high reproducibility and minimal background interference and (2) a chromatography-coupled SAXS system capable of reaching pressures of 100 MPa over a pressure range of approximately 4-ß55 °C. These configurations utilize modern material design, such as diamond windows and corrosion-resistant alloys, optimizing reproducible structural measurements of biological materials particularly for non-expert users. This work significantly expands the capacity of HP-SAXS to investigate pressure-dependent structural changes, contributing to the understanding of molecular adaptation in extreme environments and enhancing biophysical methodologies for broader applications in structural biology. Aspartate transcarbamoylase: For nearly seven decades, aspartate transcarbamoylase (ATCase) from Escherichia coli, a heterododecamer comprising two catalytic trimers and three regulatory dimers, has been a classical model for enzyme allostery. Despite extensive research, the precise molecular mechanism of allosteric regulation by nucleotides has remained elusive. In this work, we provide a comprehensive biochemical and structural characterization of E. coli ATCase, revealing novel insights into its allosteric regulation. Using activity assays, SAXS, cryo-EM, and crystallography, we demonstrate that ATCase generally follows a two-state model in response to substrate binding, from a T (inactive) to R-state (active), but exhibits distinct R-state ensembles depending on the identity of the bound nucleotides. Remarkably, ATP and GTP induce a novel conformational intermediate that expands the enzyme in the absence of both substrates, bypassing the energy-intensive T-to-R transition and decoupling the catalytic trimers. This discovery challenges the classical Monod-Wyman-Changeux (MWC) model and uncovers a sophisticated regulatory mechanism ensuring balanced nucleotide synthesis. Our findings not only resolve longstanding questions but also provide novel insight into the molecular mechanism of allosteric regulation in E. coli ATCase.