Reversible Unfolding and Folding of the Metalloprotein Ferredoxin Revealed by Single-Molecule Atomic Force Microscopy

Plant type [2Fe-2S] ferredoxins function primarily as electron transfer proteins in photosynthesis. Studying the unfolding–folding of ferredoxins in vitro is challenging, because the unfolding of ferredoxin is often irreversible due to the loss or disintegration of the iron–sulfur cluster. Additionally, the in vivo folding of holo-ferredoxin requires ferredoxin biogenesis proteins. Here, we employed atomic force microscopy-based single-molecule force microscopy and protein engineering techniques to directly study the mechanical unfolding and refolding of a plant type [2Fe-2S] ferredoxin from cyanobacteria Anabaena. Our results indicate that upon stretching, ferredoxin unfolds in a three-state mechanism. The first step is the unfolding of the protein sequence that is outside and not sequestered by the [2Fe-2S] center, and the second one relates to the force-induced rupture of the [2Fe-2S] metal center and subsequent unraveling of the protein structure shielded by the [2Fe-2S] center. During repeated stretching and relaxation of a single polyprotein, we observed that the completely unfolded ferredoxin can refold to its native holo-form with a fully reconstituted [2Fe-2S] center. These results demonstrate that the unfolding–refolding of individual ferredoxin is reversible at the single-molecule level, enabling new avenues of studying both folding–unfolding mechanisms, as well as the reactivity of the metal center of metalloproteins in vitro

Angular Trapping of Spherical Janus Particles

Developing angular trapping methods, which will enable optical tweezers to rotate a micronized bead, is of great importance for the studies of biomacromolecules during a wide range of torque-generation processes. Here we report a novel controlled angular trapping method based on composite Janus particles. We used a chemically synthesized Janus particle, which consists of two hemispheres made of polystyrene (PS) and poly(methyl methacrylate) (PMMA) respectively, as a model system to demonstrate this method. Through computational and experimental studies, we demonstrated the feasibility to control the rotation of a Janus particle in a linearly polarized laser trap. Our results showed that the Janus particle aligned its two hemisphere's interface parallel to the laser propagation direction as well as the laser polarization direction. In our experiments, the rotational state of the particle can be easily and directly visualized by using a CMOS camera, and does not require complex optical detection system. The rotation of the Janus particle in the laser trap can be fully controlled in real time by controlling the laser polarization direction. Our newly developed angular trapping technique has the great advantage of easy implementation and real time controllability. Considering the easy chemical synthesis of Janus particles and implementation of the angular trapping, this novel method has the potential of becoming a general angular trapping method. We anticipate that this new method will significantly broaden the availability of angular trapping in the biophysics community, and expand the scope of the research that can be enabled by the angular trapping approach