Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Cell Biology
First Advisor
Gupta, Kallol
Abstract
Membrane proteins (MPs) stand as pivotal regulators of cellular functions, orchestrating intricate processes vital for organismal homeostasis. However, traditional methodologies employed to study MPs, such as detergent extraction, often fail to accurately preserve the native membrane environment. This deficiency results in the loss of critical biological information, hindering our understanding of MP structure, function, and interactions. To address these limitations, I employ innovative strategies centered around the utilization of membrane active polymers (MAPs). MAPs offer a promising alternative to conventional detergent-based extraction methods by enabling the preservation of the native bilayer environment during MP extraction. Through their unique chemistry, MAPs partition the membrane into native nanodiscs, effectively encapsulating MPs within a structure that mimics their natural surroundings. This preservation of the spatial arrangement of MPs and their surrounding molecular environment is essential for maintaining the biological relevance of extracted proteins. However, the translation of MAPs from synthesis to practical application in biological systems presents challenges. The effective utilization of MAPs in protein extraction requires extensive optimization efforts for effective use in protein extraction. Addressing these challenges necessitates a comprehensive understanding of MAP properties and their interactions with membranes and proteins. This thesis seeks to bridge the gap between synthesis and application of MAPs by introducing innovative strategies to enhance solubilization efficiency and establish standardized testing protocols for newly developed polymers. By streamlining benchmarking processes, effective MAPs are identified, laying the groundwork for widespread adoption. Additionally, a protein-centric approach to solubilization is outlined, utilizing a high-throughput proteomics pipeline to systematically evaluate extraction efficiency across diverse MAPs in a protein-specific manner. This pipeline offers insights into polymer-protein dynamics, enhancing understanding of MP solubilization mechanisms. Additionally, I apply my robust solubilization platforms exploring several applications database-guided solubilization including near-endogenous protein extraction and multi-MP co-complex purification. Leveraging techniques like total internal reflection fluorescence (TIRF) microscopy, I developed a platform that allows for detailed investigations into MP spatial distribution, hierarchical organization, and dynamics in their native environment. By demonstrating MAP versatility and utility interfaced with different analytical techniques, these applications underscore the transformative potential of this approach to studying MPs. Within the context of the platforms presented in this thesis, the potential of MAPs to revolutionize MP studies by preserving the native environment during extraction holds significant promise. By bridging the gap between synthetic chemistry and biological application, MAPs offer a pathway towards more accurate and insightful investigations into the roles of MPs in cellular function and disease. Through continued research and optimization, MAP-based methodologies have the potential to deepen investigations into MP organization, interactions, and dynamics, thereby advancing our understanding of membrane biology.
Recommended Citation
Brown, Caroline, "Capturing Membrane Snapshots: A Quantitative Guide for Spatially-Resolved Extraction of Membrane Proteins on Native Membranes" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1306.
https://elischolar.library.yale.edu/gsas_dissertations/1306
