Abstract:Antibodies are key molecules for the immune system of vertebrates. The Y-shaped IgGs exhibit C2-symmetry; their Fc stem is connected to two identical Fab arms binding antigens. The Fc part is recognized by the complement
“…Note that forces become negative values when a certain terminal COM moves more than a target position (defined by the dummy point) due to the thermal fluctuation. This could be considered to be evidence that the pulling speed in this work is low enough to probe a realistic response (although a pulling speed of 500 mm/s in this study is still much higher than those used in experiments whose usual highest pulling speed reaches 0.01 mm/s in atomic force microscopy 36 , 37 , or at most 0.1 mm/s 38 ). Figure 5 Force profiles as a function of D EE for different systems, ( A ) (red) and (blue) and ( B ) (green) and (black).…”
von Willebrand Factor (vWF) is a large multimeric protein that binds to platelets and collagen in blood clotting. vWF A2 domain hosts a proteolytic site for ADAMTS13 (A Disintegrin and Metalloprotease with a ThromboSpondin type 1 motif, member 13) to regulate the size of vWF multimers. This regulation process is highly sensitive to force conditions and protein-glycan interactions as the process occurs in flowing blood. There are two sites on A2 domain (N1515 and N1574) bearing various N-linked glycan structures. In this study, we used molecular dynamics (MD) simulation to study the force-induced unfolding of A2 domain with and without a single N-linked glycan type on each site. The sequential pullout of β-strands was used to represent a characteristic unfolding sequence of A2. This unfolding sequence varied due to protein-glycan interactions. The force-extension and total energy-extension profiles also show differences in magnitude but similar characteristic shapes between the systems with and without glycans. Systems with N-linked glycans encountered higher energy barriers for full unfolding and even for unfolding up to the point of ADAMTS13 cleavage site exposure. Interestingly, there is not much difference observed for A2 domain structure itself with and without glycans from standard MD simulations, suggesting roles of N-glycans in A2 unfolding through long-ranged protein-glycan interactions.
“…Note that forces become negative values when a certain terminal COM moves more than a target position (defined by the dummy point) due to the thermal fluctuation. This could be considered to be evidence that the pulling speed in this work is low enough to probe a realistic response (although a pulling speed of 500 mm/s in this study is still much higher than those used in experiments whose usual highest pulling speed reaches 0.01 mm/s in atomic force microscopy 36 , 37 , or at most 0.1 mm/s 38 ). Figure 5 Force profiles as a function of D EE for different systems, ( A ) (red) and (blue) and ( B ) (green) and (black).…”
von Willebrand Factor (vWF) is a large multimeric protein that binds to platelets and collagen in blood clotting. vWF A2 domain hosts a proteolytic site for ADAMTS13 (A Disintegrin and Metalloprotease with a ThromboSpondin type 1 motif, member 13) to regulate the size of vWF multimers. This regulation process is highly sensitive to force conditions and protein-glycan interactions as the process occurs in flowing blood. There are two sites on A2 domain (N1515 and N1574) bearing various N-linked glycan structures. In this study, we used molecular dynamics (MD) simulation to study the force-induced unfolding of A2 domain with and without a single N-linked glycan type on each site. The sequential pullout of β-strands was used to represent a characteristic unfolding sequence of A2. This unfolding sequence varied due to protein-glycan interactions. The force-extension and total energy-extension profiles also show differences in magnitude but similar characteristic shapes between the systems with and without glycans. Systems with N-linked glycans encountered higher energy barriers for full unfolding and even for unfolding up to the point of ADAMTS13 cleavage site exposure. Interestingly, there is not much difference observed for A2 domain structure itself with and without glycans from standard MD simulations, suggesting roles of N-glycans in A2 unfolding through long-ranged protein-glycan interactions.
“…Single-molecule experiments have allowed biophysicists to quantitatively describe forces in molecular processes ( 7 , 20 ). Typically, AFM ( 6 , 7 , 21 ) or optical tweezers ( 6 , 7 , 22 ) were used to manipulate biomolecules. Stretching polymers such as polyethylene glycol, DNA, RNA, and polypeptide chains (in protein unfolding experiments) using AFM and optical tweezers provided insights into polymer physics experimentally and were combined with and/or stimulated theory ( 14 , 23 ) providing access to equilibrium energetics through the Jarzynski theorem ( 24 ).…”
Section: Resultsmentioning
confidence: 99%
“…A dream experiment for single-molecule biophysicists would be to pull an ion channel open—using an AFM (atomic force microscopy) tip ( 6 ) or an optical tweezer ( 7 ) — and directly measure the forces and energies involved in the process. This has, to the best of my knowledge, remained impossible, and such experiments would not only be extremely difficult but might be flawed by imprecisions in the calibration of the system, in vectorial pulling, and difficulties in measuring low piconewton (pN) forces.…”
The canonical gating mechanism of tetrameric cation channels involves the spreading of the pore-lining helices at the so-called bundle-crossing gate. Despite a wealth of structural information, we lack a physical description of the gating process. Here, I took advantage of an entropic polymer stretching physical model and MthK structures to derive the forces and energies involved in pore-domain gating. In MthK, the Ca
2+
-induced conformational change in the RCK domain alone opens the bundle-crossing gate through pulling via unfolded linkers. In the open conformation, the linkers serve as entropic springs between the RCK domain and bundle-crossing gate that store an elastic potential energy of 3.6
k
B
T and exert 9.8 pN (piconewton) radial pulling force to keep the gate open. I further derive that the work to load the linkers to prime the channel for opening is up to 3.8
k
B
T, exerting up to 15.5 pN to pull the bundle-crossing open. Opening of the bundle-crossing leads to a release of 3.3
k
B
T spring potential energy. Thus, the closed/RCK-apo and the open/RCK-Ca
2+
conformations are separated by a barrier of several
k
B
T. I discuss how these findings relate to the functional properties of MthK and suggest that given the architectural conservation of the helix–pore-loop–helix pore-domain among all tetrameric cation channels, these physical parameters might be quite general.
“…However, this does not mean that SMD simulations provide incorrect values [67,100]. With the development of high-speed force spectroscopy (HS-FS), the gap between experiments and simulations tends to vanish and a good agreement between simulations and experiments has been observed for protein unfolding [12]. From the computational point of view, using coarse grained (CG) models may provide simulations at experimental speeds with reasonable computational time and resources.…”
“…Importantly, AFM works under liquid conditions allowing characterization of biological samples. The flexible cantilever system is sensitive to picometer deflection changes and has associated a spring constant that can be as low as ~6 pN/nm and, using micrometer size high-speed AFM cantilevers, the reachable time resolution is <1 microsecond [12,13]. Thus, it is an excellent force sensor that allows manipulation of the sample and force application.…”
The mechanical properties of cells and of subcellular components are important to obtain a mechanistic molecular understanding of biological processes. The quantification of mechanical resistance of cells and biomolecules using biophysical methods matured thanks to the development of nanotechnologies such as optical and magnetic tweezers, the biomembrane force probe and atomic force microscopy (AFM). The quantitative nature of force spectroscopy measurements has converted AFM into a valuable tool in biophysics. Force spectroscopy allows the determination of the forces required to unfold protein domains and to disrupt individual receptor/ligand bonds. Molecular simulation as a computational microscope allows investigation of similar biological processes with an atomistic detail. In this chapter, we first provide a step-by-step protocol of force spectroscopy including sample preparation, measurement and analysis of force spectroscopy using AFM and its interpretation in terms of available theories. Next, we present the background for molecular dynamics (MD) simulations focusing on steered molecular dynamics (SMD) and the importance of bridging of computational tools with experimental techniques
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