Lasers, Balls and Nanomotors

Abstract

FtsH is a universally conserved, membrane-bound AAA+ protease critical for the quality control of membrane proteins. While the nanomechanics of homologous proteases have been widely studied, the nanomechanical profile of FtsH, remains largely unexplored. This thesis investigates the hyperthermophilic FtsH variant from Aquifex aeolicus using single trap optical tweezers.
First, we validated the functional integrity of detergent-solubilized AaFtsH through bulk biochemical assays. Wedemonstrated robust proteolytic activity against both disordered (β-casein) and structured (titin-I27 variants) at the experimental optical trapping temperature of 50°C.
To distinguish sub-nanometer motor steps from the high thermal fluctuations and instrumental drift inherent to high-temperature experiments, we conducted a rigorous system noise analysis and validation. We demonstrated that standard step-finding algorithms can be optimized through a novel Allan Variance-based Kalafut-Visscher step-fitting (AVKV) method and released it as an open-source Python toolkit. This workflow objectively determines optimal resampling bandwidth, ensuring that step detection is driven by statistical rigor.
Using this validated AVKV framework, we resolved distinct translocation events by FtsH character ized by a mean step size of 1.15±0.64 nm and a step dwell time of 0.29±0.28 s. Notably, repeated unfolding patterns were not observed for the titin-V13P substrate, likely due to a combination of mutation and elevated temperature rendering the substrate unstable.
Collectively, this work provides the first single-molecule observation of processive stepping by AaFtsH and establishes a standardized, reproducible computational framework for analyzing optical tweezers data in high-noise environments.