| Proteins are very important class of biological macromolecules and play important roles in metabolism, cell communication, individual growth, development and etc. Single molecule force spectroscopy is widely used in characterization of conformational change of protein in physiological environment. On one hand, in biological processes, proteins bind with different binding partners, such as metal ions, DNA, small organic molecules and other proteins, and after binding with ligands, proteins undergo conformational changes. The conformational changes due to the binding with ligands are very important for protein function and signal transduction. Because proteins make slightly change of the shape of its binding surface for adaption to bind with its ligand, the flexibility is the key factor for the ability of interaction with different binding partners. Our study of a PAS domain (human ARNT PAS-B domain) which can bind with different ligands reveals its high flexibility. This allows the PAS-B domain to extend by up to 75% of its resting end-to-end distance without unfolding. We propose that the structural flexibility of PAS-B domains may provide a unique mechanism for them to recruit diverse binding partners. Moreover, we found that the ARNT PAS-B domain unfolds in two distinct pathways via a kinetic partitioning mechanism. One the other hand, many biomacromolecules regulate its structure and biological functions via mechanical force. They generate, sense, transduce and response to mechanical signals. Besides the tensile stress between different proteins within a complex, the proteins which have multi domains also have inner stress. It is still not known if the inner stress influences the function of the function. Our single molecule AFM study on gelsolin demonstrates that the calcium-binding affinity of the sixth domain of gelsolin increases significantly by tensile force. A stretching force of 10 pN increases the calcium-binding affinity of G6 by a factor of ～6. In the deactivation state of gelsolin, occupation of any of binding sites by a calcium ion will begin the activation process that drives conformational reorganization and concomitantly creates inter-domain strain, leading to the C-terminal tail under tension. Consequently, gelsolin displays cooperativity in binding calcium ions that is mediated through changes in structure and, we propose, resultant changes in strain.The main contents of this thesis are arranged as follows:In chapter 1, I introduced the development of single molecule force spectroscopy and the principle of single molecule AFM technique. Then, I introduced the theory of the single molecule biophysics. I made a brief review of the mechanical unfolding of protein and the interaction of protein-ligand and protein-protein, and illustrate the mechanism of force signal transduction, the force-induced allosteric, and the importance of the force in biological processes. Then I listed several excellent works done by single molecule force spectroscopy to make a brief review of the development of the single molecule AFM.In chapter 2, we used smAFM experiment to study a PAS domain. Per-ARNT-Sim (PAS) domains serve as versatile binding motifs in many signal-transduction proteins and are able to respond to a wide spectrum of chemical or physical signals. Despite their diverse functions, PAS domains share a conserved structure. It has been suggested that the structure of PAS domains is flexible and thus adaptable to many binding partners. However, direct measurement of the flexibility of PAS domains has not yet been provided. Here, we quantitatively measure the mechanical unfolding of a PAS domain, ARNT PAS-B, using single-molecule atomic force microscopy. Our force spectroscopy results indicate that the structure of ARNT PAS-B can be unraveled under mechanical forces as low as～30 pN due to its broad potential well for the mechanical unfolding transition of ～2 nm. This allows the PAS-B domain to extend by up to 75%of its resting end-to-end distance without unfolding. Moreover, we found that the ARNT PAS-B domain unfolds in two distinct pathways via a kinetic partitioning mechanism. Sixty-seven percent of ARNT PAS-B unfolds through a simple two-state pathway, whereas the other 33% unfolds with a well-defined intermediate state in which the C-terminal β-hairpin is detached. We propose that the structural flexibility and force-induced partial unfolding of PAS-B domains may provide a unique mechanism for them to recruit diverse binding partners and lower the free-energy barrier for the formation of the binding interface.In chapter 3, we used smAFM experiment to reveal the force-enhanced binding of calcium ions by gelsolin. Force is increasingly recognized as an important element in controlling biological processes. Forces can deform native protein conformations leading to protein-specific effects. Protein-protein binding affinities may be decreased, or novel protein-protein interaction sites may be revealed, on mechanically stressing one or more components. Here we demonstrate that the calcium-binding affinity of the sixth domain of the actin-binding protein gelsolin (G6) can be enhanced by mechanical force. Our kinetic model suggests that the calcium-binding affinity of G6 increases exponentially with force, up to the point of G6 unfolding. This implies that gelsolin may be activated at lower calcium ion levels when subjected to tensile forces. The demonstration that cation-protein binding affinities can be force-dependent provides a new understanding of the complex behaviour of cation-regulated proteins in stressful cellular environments, such as those found in the cytoskeleton-rich leading edge and at cell adhesions.In chapter 4, we used smAFM experiment to study the relationship between the ligand binding and anisotropic effects. Force has been increasingly recognized as an important element for the control of a large variety of biological processes. Force can change the conformation of a protein or even trigger the unfolding, leading to many force specific effects. With the development of single molecule force spectroscopy techniques, the mechanical properties of single protein molecules have been studied in great details. Especially, with the development of double-cysteine crosslinking method, force can be applied to a protein from different orientations. It was revealed that the mechanical stability of proteins is anisotropic and depends on the direction of applied forces. In parallel, it also has been revealed that ligand binding can efficiently modulate the mechanical stability of proteins. However, it is still unknown how the mechanical stability of a protein at different pulling directions is regulated by ligand binding. Here, we studied the anisotropic response of SH3 protein in three different directions with and without its ligand using atomic force microscopy (AFM) based single molecule force spectroscopy. We found that the change of the mechanical stability at three different pulling directions was distinct, and correlated with the original mechanical stability. The unfolding force increased ranging from 5% to 80% at a pulling speed of 400 nm/s. Detailed kinetic analysis revealed three different types of effects caused by ligand binding. First, in 2 of the mutants, the cysteine mutations affect the binding of the ligand with SH3, thus, the unfolding force do not change. Second, the stability of the proteins will increase after the proteins bind with RLP2. However, the increment of stability has no relationship with the original stability. Third, the unfolding distance of the protein does not change with the binding of ligand, which means that the unfolding pathways of these proteins keep unchanged. This finding serves as the basis for the understanding of many force-regulated biological processes and could inspire the engineering of complex force-responsive materials for biological applications.In chapter 5,1 make a conclusion of my Ph.D. work, and propose a future plan for my single molecule study. |
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