Grant: Discovery Grants Program - Individual

Proteins possess nanomechanical properties that are uniquely suitable for their biological functions across different length scales, from single molecules to organisms. In living cells, proteins serve as basic working elements to sense, generate and bear mechanical forces, which are recognized as important physical cues in regulating cellular behaviors. Understanding how such mechano-proteins are designed to perform their mechanical functions not only important in elucidating fundamental biophysical principles underlying various biological processes, but may also pave ways to exploiting proteins as building blocks for the bottom-up construction of functional biomaterials with tailored mechanical properties for applications in material sciences and biomedical engineering. Our research will focus on the study of protein mechanics and engineering at the single molecule level. The long term goals of our research program are: 1) to develop force spectroscopy-based enabling technologies to address challenges in life sciences and material sciences; and 2) to rationally tailor mechanical properties of protein-based biomaterials by programming the molecular sequence, and thus nanomechanical properties, of elastomeric proteins at the single-molecule level. Here we propose to combine single molecule atomic force microscopy (AFM), optical tweezers with protein engineering, and traditional biophysical techniques to elucidate the complete folding mechanism of metalloproteins at single molecule level, develop new generation of protein-based force sensors that are fully calibrated and report the force experienced by individual proteins during mechanobiological processes, and to use protein folding as a driving force to engineer protein biomaterials that can generate mechanical work. Building upon the recent technical advance we achieved in AFM to monitor protein folding-unfolding near equilibrium, we will use single molecule AFM to probe the folding mechanisms of two small metalloproteins rubredoxin and ferredoxin, which are challenging to study using traditional methods. These studies will allow us to directly monitor the folding of these metalloproteins in real time, and critically examine the role played by metal in the folding of metalloproteins. These studies will help elucidate the detailed folding mechanisms of both metalloproteins. To meet the demands of mechanobiology and materials sciences, we will develop the new generation of protein force sensors based on protein unfolding. We will engineer a series of force sensor proteins with different and well-defined unfolding forces and use them to precisely quantify the force experienced by individual proteins during mechanobiological processes. Moreover, we will use calcium-triggered protein folding as a novel mechanism to generate mechanical work and actuation at both single molecule and macroscopic levels, and engineer protein-based actuators for applications in mechanobiology and material sciences.