Single-molecule force spectroscopies, including AFM and magnetic tweezers, are powerful methods for examining the folding process of proteins and other biomolecules as they adopt their functional shapes. With single-molecule force spectroscopies, sparsely populated intermediates and the transitions between them can be studied in detail. Molecular dynamics (MD) simulations provide a complementary method which assists in the interpretation of experimental data. Using Upside, our new ultra-fast MD approach for modeling protein folding and dynamics, we studied the force-induced unfolding of two membrane proteins, bacteriorhodopsin and GlpG, detailing how the different applications of force spectroscopies investigate different regions of the energy surface.
The cover image of the October 15 issue of Biophysical Journal features a depiction of the membrane protein GlpG embedded in a bicelle (right) adjacent to an energy landscape, surrounded by renderings of partially unfolded conformations. The landscape, generated using principle component analysis, depicts the various unfolding pathways and numerous intermediate states observed in our simulations. The three major pathways (red lines) differ by whether unfolding begins from the middle or the amino- or carboxy-terminus of the protein. This emphasizes the key finding in our study: GlpG unfolds against force along different routes with multiple steps, rather than in a cooperative manner along a single pathway.
A major contribution of our study is the introduction of a fast computational tool to simulate a variety of forced-induced unfolding measurements of membrane and soluble proteins with a resolution comparable to the highest resolution measurements. Our tool bridges the huge gap of pulling velocity between experiment and standard all-atom MD simulations (~100 nm/s versus ~1 m/s). We find that the mode of applying force can greatly alter the perception of the energy surface, an important concept that should be considered when designing experiments and interpreting unfolding data. The cover image highlights the multiplicity of the unfolding pathways of GlpG under force, furthering our understanding of how membrane proteins and other biomolecules fold. Further information on our research on protein folding and dynamics can be found at http://sosnick.uchicago.edu/index.html
- Zongan Wang, John M Jumper, Karl F Freed, Tobin R Sosnick