Mechanistic Insights into the Folding Mechanism of Region V in Ice-Binding Protein Secreted by Marinomonas primoryensis Revealed by Single-Molecule Force Spectroscopy.

The Gram-negative bacteria Marinomonas primoryensis secrete an ice-binding protein (MpIBP), which is a vital bacterial adhesin facilitating the adaptation and survival of the bacteria in the harsh Antarctic environment. The C-terminal region of MpIBP, known as region V (RV), is the first domain to be exported into the Ca2+-rich extracellular environment and acts as a folding nucleus for the entire adhesin. However, the mechanisms underlying the secretion and folding of RV remain poorly understood. Here, we used optical tweezers (OT) to investigate the secretion and folding mechanisms of RV at the single-molecule level. In the absence of Ca2+, apo-RV remains unstructured, while Ca2+-bound RV folds into a mechanically stable structure. The folding of RV could occur via the formation of an intermediate state. Even though this folding intermediate is "hidden" during the folding process of wild type RV in vitro, it likely forms in vivo and plays an important role in facilitating protein secretion. Additionally, our results revealed that the N-terminal part of the RV can significantly stabilize its C-terminal structure. Our study paves the way for further investigations into the structure and functions of MpIBP that help bacteria survive in challenging environments.

Deep learning for precise axial localization of trapped microspheres in reflective optical systems.

High-precision axial localization measurement is an important part of micro-nanometer optical measurement, but there have been issues such as low calibration efficiency, poor accuracy, and cumbersome measurement, especially in reflected light illumination systems, where the lack of clarity of imaging details leads to the low accuracy of commonly used methods. Herein, we develop a trained residual neural network coupled with a convenient data acquisition strategy to address this challenge. Our method improves the axial localization precision of microspheres in both reflective illumination systems and transmission illumination systems. Using this new localization method, the reference position of the trapped microsphere can be extracted from the identification results, namely the "positioning point" among the experimental groups. This point relies on the unique signal characteristics of each sample measurement, eliminates systematic repeatability errors when performing identification across samples, and improves the localization precision of different samples. This method has been verified on both transmission and reflected illumination optical tweezers platforms. We will bring greater convenience to measurements in solution environments and will provide higher-order guarantees for force spectroscopy measurements in scenarios such as microsphere-based super-resolution microscopy and the surface mechanical properties of adherent flexible materials and cells.