The Mechanical Properties Of Gelsolin Revealed By Single Molecular Force Spectroscopy

Force is very important in life science, many biological processes require the positive or negative interaction forces between the biological elements, such as muscle contraction, membrane fusion, cell division, cell crawling, cell adhesion, organelle transport, protein translocation, protein degradation, protein folding and unfolding. However, the forces generated in the intra-or inter molecular are so low that we have to measure and control them using sensitive instruments. Using the atomic-force-microscope based single molecule force spectroscopy technique, more and more biological molecules are explored by scientists at the single molecule level, following by more and more surprises and challenges.Cytoskeleton plays a very important role in cell’s life, which can crutch the membrane. And the translation of cytoplasm from the gel state to the sol state should owe to the cytoskeleton. In this process, the important part of the cytoskeleton, F-actin, is modulated by the gelsolin protein family by binding, severing and capping actin filament or nucleating the G-actin. Under the regulation of gelsolin protein, the F-actin is in a continuous state of assembly and disassembly to control the cell motility, and force participates in all these processes. Nevertheless, a common trend is emerging that several proteins that regulate the force-generating actin polymerization machine are themselves subjected to force regulation. So we studied the mechanical properties of gelsolin family proteins using atomic force microscope based single molecule force spectroscopy technique.In Chapter I, we introduced the techniques that are used to study the mechanical properties of gelsolin protein and the structure and function of gelsolin protein.In Chapter Ⅱ, we demonstrated that the calcium-binding affinity of the actin-binding protein gelsolin domain G6is enhanced by mechanical force. Using a recently developed single molecule-binding assay based on atomic force microscopy, we established that the calcium-binding affinity of G6increases exponentially with the applied force, up to the point of G6unfolding. This implies that gelsolin will be activated at lower calcium ion levels when subjected to tensile forces and suggests a basis for enhanced cooperativity during multi-cation induced activation. The demonstration that cation-protein binding affinities can be force-dependent provides a new paradigm in understanding the complex interactions of cation-regulated proteins in stressful cellular environments, such as those found in the cytoskeleton-rich leading edge and at cell adhesions.In Chapter Ⅲ, we then studied the headpiece domain (Hp35) of villin, another example that belongs to gelsolin family. Hp35folds at sub-microsecond time scale with low folding cooperativity. Although it has been extensively investigated, still relatively little is known about its folding mechanism. Here using single molecule force spectroscopy and steered molecule dynamics simulation, we studied the unfolding of Hp35under external force. Our results showed that Hp35unfolds at extremely low force without well-defined unfolding transition state. Subsequently, we probed the structure of unfolded Hp35using the persistence length obtained in the force spectroscopy. We found that the persistence length of unfolded Hp35is around0.72nm, more than40%longer than typical unstructured proteins, suggesting that there are significant amount of residual secondary structures in the unfolded Hp35. Molecular dynamics simulation further confirmed this finding and revealed that many native contacts are maintained in Hp35even its two ends have been extended up to8nm. Our results therefore suggested that retaining significant amount of secondary structure in the unfolded state of Hp35may be an efficient way to reduce the entropic cost for the formation of tertiary structure and increase the folding speed, despite that the folding cooperativity is compromised.In order to describe the dynamic information of proteins, we need to pull the protein molecule with different loading rates. In Chapter IV, in the single molecule force spectroscopy experiments we found that if the pulling speed was high enough, the movement of the cantilever can be affected by the hydrodynamic force besides the force exerted by the tethered protein. On the other hand, hydrodynamic force is ubiquitous in biology. Many cellular processes, such as diffusion of biomacromolecules, movement of molecular motors and conformational dynamics of proteins, are subjected to hydrodynamic forces because of the high viscosities of cellular environments. However, it is still unknown how hydrodynamic forces are related to the physical properties of different viscogens. Here, using the atomic-force-microscope based force spectroscopy technique, we directly measured the hydrodynamic forces acting on a moving cantilever in various viscogen solutions. We found that the hydrodynamic force is not only dependent on the viscosity, but also related to the molecular weight of viscogens. Contour-intuitively, at the same macroscopic viscosity, the hydrodynamic force rises with the increase of molecular weight of viscogens, although the local microscopic viscosity of the solution decreases. This finding provides insights into the origin of hydrodynamic forces in biomolecule solutions and could inspire many force-spectroscopy based techniques to directly measure the molecular weight and conformational changes of biomacromolecules in biological settings.In summary, in this thesis we studied the mechanical properties of gelsolin and vilin using single molecule atomic force microscopy, from which many new insights on their folding and cation binding were revealed. We found that force can enhance the binding of metal ions to protein for the first time. We showed that some small proteins could fold through on-route intermediate states to speed up the entire folding process. Using a moving cantilever tip as an example, we showed that hydrodynamic forces acting on a moving object is related to both the viscosity of the solution and the molecular weight of the viscogen. Together, these findings will contribute to the general understandings on how forces regulate biological processes in vivo.