Relating protein structure and mechanics in cells using coherent Raman imaging

• Applicant: Professor Dr. Sapun Parekh
• Subject Area: Biophysics
• Term: from 2014 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 255099641

Protein structure and function ultimately regulate all cellular function. It is clear that point mutations or biochemical environmental factors are sufficient to disrupt protein structure and cause dysfunctional effects directly related to pathogenesis. Over the last decade, mechanical forces have also been shown to impact protein confirmation. The ability to exert pico-Newton mechanical forces to single molecules in vitro has verified and inspired new theories regarding conformational stability and kinetics of unfolding both at rest and under tensile force for mechanosensitive proteins. While in vitro mechanobiology and structural biology of proteins has matured quite fast, very little is known about structural biology of mechanosensitive proteins in vivo. Unfortunately, single molecule techniques have limited application in the intracellular environment, and it is unclear if mechanical force has the same effect on protein stability, and more subtle protein structure, in vivo as that seen in vitro. Quantifying the changes in protein structure within cells, secondary, tertiary, and quaternary would provide critical information about biophysical connection between mechanical loads, protein structure/function, and cell function. For example, measurements of intracellular protein secondary structure under load would allow tracking of structural transitions (i.e. alpha-helical to beta-sheet), which are not accessible from single molecule experiments and may be pertinent in vivo. This project details a plan to measure spatially-resolved protein secondary structure of mechanosensitive intermediate filament (IF) proteins under tensile loads using a nonlinear spectroscopic imaging technique. IFs are part of the load-bearing cytoskeleton and have a defined secondary structure that is predicted to change under loads. However, this has never been observed in vivo or in vitro. The information gleaned here will provide a unique view of the impact that forces play on protein structure in vivo.

• DFG Programme: Research Grants