Folding and Mechanical Response of Single Proteins Probed at High Spatio-Temporal Resolution

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Proteins are essential to life. For humans to walk, hear, or touch, proteins must generate and respond to forces after folding into their correct three-dimensional structure. Yet, traditional biochemical techniques do not measure a protein's response to force, hindering progress in the emerging field of mechanobiology. Inside a cell, force can induce subtle change in a protein's structure, fully unfold a protein, or disrupt the interaction between two proteins. These effects all have biological consequence. To exert a force on individual proteins and measure these tiny signals, this project will use novel atomic force microscope (AFM) cantilevers, which are micron-scale, diving-board like force sensors. This project will help elucidate the response of proteins to force, a critical but understudied signaling mechanism in biology. More generally, studying the process of protein folding with these novel cantilevers will provide insight into how proteins fold, an ongoing challenge despite five decades of effort. By using the tools of physics and nanoscience to solve exciting problems in biology, this project will provide excellent interdisciplinary training for high school and colleges students just starting their research career. As statistics are vital to interpreting experiments but are poorly understood by many young researchers, this project will also generate novel training tools focused on statistics and data analysis using biological examples. These interactive education simulations will be developed in conjunction with the CU-Boulder PhET program and thereby leverage their expertise in such simulations and world-wide distribution.The twin scientific goals are (i) to characterize structural transitions in two widely studied mechano-sensitive proteins, titin's I27 domain and the focal adhesion kinase, since previous studies have either yielded conflicting results or failed to resolve a predicted mechanically induced transition; and (ii) measure a model two-state protein's folding transition time with 1-microsecond resolution. The key technical hurdle is that traditional AFM studies lack the spatial precision to resolve subtle changes in protein structure and lack the temporal precision to characterize the conformational dynamics of a traditional globular protein during the brief time the protein moves along its transition path. This project will overcome these obstacles by applying a state-of-the-art combination of force precision, stability, and time resolution enabled by focused-ion beam modification of AFM cantilevers. This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences and the Physics of Living Systems Program in the Division of Physics.