Nanopore Technology for Protein Identification and Functional Analysis

Nanopore technology is emerging as a transformative tool for single-molecule proteomics, both for studying protein function and for identification of proteins. This thesis addresses both of those aspects by applying nanopore sensing to resolve complex biological mechanisms while simultaneously exploring novel methodologies to address limitations in protein identification. Chapter 1 critically reviews the state-of-the-art in nanopore protein analysis. It identifies that nanopore-based fingerprinting strategies are in principle proven, however, open hurdles remain regarding scalability, requirements for conjugation, and the resolution needed for de novo sequencing. Simultaneously, nanopores are already applicable to study protein function within academic research settings, yielding exciting results. This analysis establishes the roadmap for the experimental chapters that follow. Chapter 2 demonstrates the functional analysis of Hsp70 chaperones. Using a nanopore to mimic in vivo protein transport across organelles, we provide the first unambiguous evidence that Hsp70s use the Entropic Pulling mechanism, thereby settling a long-standing debate. Addressing the sample processing bottlenecks in nanopore-based protein fingerprinting (identified in Chapter 1), Chapter 3 introduces a simplified platform for protein identification. We explore the free translocation of full-length proteins through the aerolysin pore, obviating the need for proteolytic digestion or chemical conjugation of the analyte. Finally, Chapter 4 tackles the challenge of de novo sequencing by engineering aerolysin mutants coupled with enzymatic translocation control. While aerolysin has shown promise to reduce the sensing volume in prior static measurement, this dynamic exploration uncovered significant and complex signal convolution in the pore. The investigation suggests that future sequencing efforts should prioritize pores with distinct constriction geometries. Collectively, this work pushes the boundaries of nanopore proteomics. It not only resolves a fundamental biological question about Hsp70s but also rigorously defines the capabilities and limitations of aerolysin-based sensing, paving the way for next-generation protein identification platforms.