Genetic code reprogramming enables us to assign nonproteinogenic amino acids to codons in place of proteinogenic amino acids according to the genetic code, which allows us to express desired nonstandard peptides using an in vitro translation system. Here, we discuss FIT (Flexible In vitro Translation) system, which is a method that facilitates genetic code reprogramming by employing flexizymes. Furthermore, we also report a method called RaPID (Random non-standard Peptide Integrated Discovery) system, which is an integration of FIT system with an in-vitro mRNA display. RaPID system allows us to select peptides with high binding affinities from highly diverse nonstandard peptide libraries and is quickly becoming a valuable new tool for drug discovery.
To understand the mechanism underlying the one-dimensional (1D) Brownian motion of proteins, we analyzed the behaviors of counterions along microtubules using the electroorientation method and compared these with the 1D Brownian motion of charged nanoparticles. Both results demonstrated that during 1D Brownian motion, each positively charged nanoparticle behaves as a polycation constrained within the electrostatic field around the microtubules. Owing to the polyelectrolytic nature of microtubules, nanoparticles can move along microtubules with a diffusion constant independent of the charge density of the microtubules. The study highlights the possibility that 1D Brownian motion of proteins is based on a similar, nonspecific charge-dependent mechanism.
F1-ATPase (F1) is a rotary motor protein which couples ATP hydrolysis/synthesis to rotary motion in a reversible fashion. Compared to other motor proteins, F1 is unique for its high efficiency and reversibility in converting chemical energy into mechanical work. To elucidate its marvelous mechanism, we developed the novel single-molecule manipulation technique with magnetic tweezers and determined the timing of Pi release, which was the last unknown piece of the puzzle of chemomechanical coupling scheme of F1. The established fundamental chemomechanical coupling scheme gave us a clue for high reversibility between catalysis and mechanical work.
Bacterial flagellar motor is an ion-driven supramolecular nanomachine embedded in the cell envelope. Rotor-stator interaction that couples to the specific ion translocation through the stator channel is the nature of torque generation. To produce fully functional motor, multiple stator complexes must be incorporated around the rotor at appropriate places. However, such stator assembly mechanism has not been investigated by the structural point of view. Here we describe stator assembly and activation mechanism revealed from the crystal structure of a motor component located in the periplasmic space, suggesting the dynamic conformational changes in the stator during its assembly-coupled activation.