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Using Single Molecule Chemo-Mechanical Unfolding to Probe the Effect of Environmental Conditions on Protein Folding
490a
Wednesday, February 15, 2017
X-ray scattering experiments and negative-stain electron microscopy. In contrast to Cd2þ, Pb2þ cannot support the proper fold of C1B but acts successfully as a functional surrogate of Ca2þ in driving protein-membrane association. Our work demonstrates the potential diversity of responses of signaling proteins to toxic metal ions and suggests that molecular mechanisms of Pb2þ and Cd2þ toxicity are distinct. This work was supported by Welch Foundation grant A-1784, NSF CAREER award CHE-1151435, and NIH grant R01 GM108998.
Aggregates, Chaperones, and Mechanical Forces 2409-Pos Board B16 Using Single Molecule Chemo-Mechanical Unfolding to Probe the Effect of Environmental Conditions on Protein Folding Emily J. Guinn, Bharat Jagannathan, Susan Marqusee. California Institute for Quantitative Biosciences, UC-Berkeley, Berkeley, CA, USA. In vivo, proteins function in a complex environment where they are subject to stresses like solutes, temperature and strain which can modulate the protein’s energy landscape. Perturbing these conditions allows one to explore how proteins respond to changes in environment. This also helps to characterize protein energy landscapes because perturbant effects are related to the structure and energetics of the different protein states along the energy landscape. The effect of perturbants on protein stability is related to the structure of the native and denatured state, while the effect of perturbants on protein kinetics is related to the folding pathway. We have developed a technique called chemo-mechanical unfolding where we combine force and chemical denaturant using optical tweezers. We use chemo-mechanical unfolding as well as temperature and point mutations to explore the denatured state and the parallel pathways proteins fold through. We also compare experiments on- and off- the ribosome to determine how the ribosome affects the folding pathway. 2410-Pos Board B17 Protein Aging: Loss of Folding Contraction due to Oxidation of Cryptic Side Chains Jessica Valle Orero1, Jaime Andres Rivas-Pardo1, Rafael Tapia-Rojo1, Ionel Popa2, Daniel J. Echelman1, Julio M. Fernandez1. 1 Biophysical, Columbia University, New York, NY, USA, 2Physics, University of Wisconsin Milwaukee, Milwaukee, WI, USA. Tensegrity is the property of tissues that allows them to regain their shape after a mechanical deformation. Constituent proteins support tensegrity by being able to generate a restoring force at any length. A salient feature of tissue aging is the oxidative modification of its proteins, thus compromising the tensegrity of the system (e.g. sagging skin). Here, we use magnetic tweezers to monitor the folding dynamics of single protein L molecules under force over times scales from hours to days. Mechanically unfolded proteins that are maintained extended for 22 hours entirely lose their ability to fold. This loss of folding is triggered by the exposure of the cryptic side chains to the oxidative environment, as it can be greatly slowed by adding an antioxidant to the solution. This phenomenon compromises the tensegrity of the protein by reducing its extensibility by 40%. We provide an analytical expression that describes the extensibility of a protein under force, combining the entropic elasticity of the polypeptide and the folding collapse. By incorporating an aging factor measured from the loss of protein folding over time, we can predict the loss of tensegrity. Our ability to accurately keep a single protein unfolded for hours to days presents a novel assay for accelerating aging. We anticipate that this will become a useful tool to discern the role of environmental contaminants to understand the loss of tensegrity in exposed tissues. 2411-Pos Board B18 Thermodynamics and Kinetics of Globular Polymers under an Applied Force Samuel Bell, Eugene M. Terentjev. Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom. We examine the problem of a polymer chain, folded into a globule in poor solvent, subjected to a constant tensile force. This system represents a Gibbs thermodynamic ensemble, and is useful for analysing force-clamp AFM measurements - now common in molecular biophysics. Using a basic Flory mean-field theory, we account for surface interactions of monomers with solvent. Under an increasing tensile force a first-order phase transition occurs. The compact globule ruptures and fully extends, in an ‘all-or-nothing’ unfolding event. This contrasts with the regime of imposed extension, first studied by Halperin and Zhulina, where there is a regime of coexistence of a partial globule with an extended chain segment. We relate the transition forces in
this problem to the solvent quality and degree of polymerisation, and find analytical expressions for the energy barriers present in the problem. From these, we analyse the kinetic problem of a force-ramp experiment, showing that the rupture force depends on the rate of loading. We then change the system by inserting a super-hydrophobic ‘core’ at a given point in the polymer chain. This acts as a crude model for a large class of folded biomolecules with hydrophobic or hydrogen-bonded cores. Again, we consider applying a constant tensile force. Introducing a ‘core’ leads to an intrinsic (quenched) stochastic variation in the unfolding rate, even when the positions of the added monomers are fixed along the sequence. This gives rise to non-exponential population dynamics, which is consistent with a variety of experimental data. It does not need any structural disorder of the type thought to be at the origin of non-exponential relaxation laws. 2412-Pos Board B19 Mechano-Induced Unfolding of Von Willebrand Factor: A Clinical Example of Protein Destabilization Camilo A. Aponte Santamaria1, Svenja Lippok2, Judith J. Mittag2, Tobias Obser3, Reinhard Schneppenheim3, Carsten Baldauf4, Frauke Gr€ater1, Ulrich Budde5, Joachim R€adler2. 1 Molecular Biomechanics, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany, 2Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany, 3Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 4Theory, Fritz-Haber-Institut der Max-PlanckGesellschaft, Berlin, Germany, 5MEDILYS Laborgesellschaft mbH, Hemostaseology, Asklepios Klinik Altona, Hamburg, Germany. Protein stability can be quantified, in experiments or simulations, by monitoring changes in free energy induced by single amino-acid mutations relative to changes imposed by the same mutation in a reference unfolded peptide. We here assessed the destabilization by disease mutations of a protein, for which both the folded and unfolded states play key functional roles in hemostasis. Force-induced unfolding of the giant von Willebrand Factor (VWF) multimer-protein exposes a cleavage site for enzymatic proteolysis, a critical down-regulatory mechanism to prevent the formation of large thrombus aggregates. Several naturally occurring mutations modify this process, inducing distinct types of bleeding disorders, by unknown mechanisms. We present the first quantitative description of the dramatic destabilization of VWF caused by a one of such mutations, which strongly accelerates VWF cleavage. Molecular dynamics simulations and free energy calculations revealed this mutation to induce structural, dynamic, and mechanical perturbations in the VWF-A2 domain, thereby destabilizing this domain by ~10 kJ/mol promoting its unfolding. In close agreement, fluorescence correlation spectroscopy (FCS) revealed a 20-fold increase in the cleavage rate for this mutant, compared to the wildtype VWF. Cleavage was found cooperative with a cooperativity coefficient n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites at the same time. Taken together, the enhanced cleavage activity can be readily explained by an increased availability of the cleavage site through A2-domainfold thermodynamic destabilization. Our study therefore puts forward the combination of free energy calculations and FCS, as a powerful way of examining protein stability in a clinically relevant context. Reference: C. Aponte-Santamarı´a, et al. Biophysical Journal. In revision. 2413-Pos Board B20 Mechanical Architecture and Genesis of Bacterial Pilus Domains Revealed by Single-Molecule Force Spectroscopy Alvaro Alonso-Caballero, Raul Perez-Jimenez. CIC nanoGUNE, San Sebastian, Spain. Gram-negative bacteria attach to tissues using long filaments called pili. In uropathogenic Escherichia coli (UPEC), the pilus type-1 is composed by thousands of FimA subunits that form the pilus rod followed by the subunits FimF-FimG-FimH at the tip fibrillum. The mechanical resistance of the pilus is essential for successful attachment to target cells and tissues. With the exception of FimH, the contribution of each subunit to the mechanical architecture of the pilus has not been investigated. Here, we use atomic force spectroscopy to report a complete nanomechanical map of the pilus. We have used protein engineering to redesign the pilus domains in order to replicate the force vector that they sense in vivo. All domains show a very high mechanical stability reaching forces above 400 pN. We have discovered that the domains follow a mechanical hierarchy in which the stability decreases from the pilus rod to the tip. We have also used force-clamp spectroscopy to investigate with single-molecule resolution the mechanism of domain folding from the stretched state to the folded state assisted by periplasmic oxidoreductases and chaperones, DsbA and FimC. We have observed a synergistic effect that allows us to depict a precise kinetic model for the mechanogenesis of the pilus subunits.
Wednesday, February 15, 2017
X-ray scattering experiments and negative-stain electron microscopy. In contrast to Cd2þ, Pb2þ cannot support the proper fold of C1B but acts successfully as a functional surrogate of Ca2þ in driving protein-membrane association. Our work demonstrates the potential diversity of responses of signaling proteins to toxic metal ions and suggests that molecular mechanisms of Pb2þ and Cd2þ toxicity are distinct. This work was supported by Welch Foundation grant A-1784, NSF CAREER award CHE-1151435, and NIH grant R01 GM108998.
Aggregates, Chaperones, and Mechanical Forces 2409-Pos Board B16 Using Single Molecule Chemo-Mechanical Unfolding to Probe the Effect of Environmental Conditions on Protein Folding Emily J. Guinn, Bharat Jagannathan, Susan Marqusee. California Institute for Quantitative Biosciences, UC-Berkeley, Berkeley, CA, USA. In vivo, proteins function in a complex environment where they are subject to stresses like solutes, temperature and strain which can modulate the protein’s energy landscape. Perturbing these conditions allows one to explore how proteins respond to changes in environment. This also helps to characterize protein energy landscapes because perturbant effects are related to the structure and energetics of the different protein states along the energy landscape. The effect of perturbants on protein stability is related to the structure of the native and denatured state, while the effect of perturbants on protein kinetics is related to the folding pathway. We have developed a technique called chemo-mechanical unfolding where we combine force and chemical denaturant using optical tweezers. We use chemo-mechanical unfolding as well as temperature and point mutations to explore the denatured state and the parallel pathways proteins fold through. We also compare experiments on- and off- the ribosome to determine how the ribosome affects the folding pathway. 2410-Pos Board B17 Protein Aging: Loss of Folding Contraction due to Oxidation of Cryptic Side Chains Jessica Valle Orero1, Jaime Andres Rivas-Pardo1, Rafael Tapia-Rojo1, Ionel Popa2, Daniel J. Echelman1, Julio M. Fernandez1. 1 Biophysical, Columbia University, New York, NY, USA, 2Physics, University of Wisconsin Milwaukee, Milwaukee, WI, USA. Tensegrity is the property of tissues that allows them to regain their shape after a mechanical deformation. Constituent proteins support tensegrity by being able to generate a restoring force at any length. A salient feature of tissue aging is the oxidative modification of its proteins, thus compromising the tensegrity of the system (e.g. sagging skin). Here, we use magnetic tweezers to monitor the folding dynamics of single protein L molecules under force over times scales from hours to days. Mechanically unfolded proteins that are maintained extended for 22 hours entirely lose their ability to fold. This loss of folding is triggered by the exposure of the cryptic side chains to the oxidative environment, as it can be greatly slowed by adding an antioxidant to the solution. This phenomenon compromises the tensegrity of the protein by reducing its extensibility by 40%. We provide an analytical expression that describes the extensibility of a protein under force, combining the entropic elasticity of the polypeptide and the folding collapse. By incorporating an aging factor measured from the loss of protein folding over time, we can predict the loss of tensegrity. Our ability to accurately keep a single protein unfolded for hours to days presents a novel assay for accelerating aging. We anticipate that this will become a useful tool to discern the role of environmental contaminants to understand the loss of tensegrity in exposed tissues. 2411-Pos Board B18 Thermodynamics and Kinetics of Globular Polymers under an Applied Force Samuel Bell, Eugene M. Terentjev. Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom. We examine the problem of a polymer chain, folded into a globule in poor solvent, subjected to a constant tensile force. This system represents a Gibbs thermodynamic ensemble, and is useful for analysing force-clamp AFM measurements - now common in molecular biophysics. Using a basic Flory mean-field theory, we account for surface interactions of monomers with solvent. Under an increasing tensile force a first-order phase transition occurs. The compact globule ruptures and fully extends, in an ‘all-or-nothing’ unfolding event. This contrasts with the regime of imposed extension, first studied by Halperin and Zhulina, where there is a regime of coexistence of a partial globule with an extended chain segment. We relate the transition forces in
this problem to the solvent quality and degree of polymerisation, and find analytical expressions for the energy barriers present in the problem. From these, we analyse the kinetic problem of a force-ramp experiment, showing that the rupture force depends on the rate of loading. We then change the system by inserting a super-hydrophobic ‘core’ at a given point in the polymer chain. This acts as a crude model for a large class of folded biomolecules with hydrophobic or hydrogen-bonded cores. Again, we consider applying a constant tensile force. Introducing a ‘core’ leads to an intrinsic (quenched) stochastic variation in the unfolding rate, even when the positions of the added monomers are fixed along the sequence. This gives rise to non-exponential population dynamics, which is consistent with a variety of experimental data. It does not need any structural disorder of the type thought to be at the origin of non-exponential relaxation laws. 2412-Pos Board B19 Mechano-Induced Unfolding of Von Willebrand Factor: A Clinical Example of Protein Destabilization Camilo A. Aponte Santamaria1, Svenja Lippok2, Judith J. Mittag2, Tobias Obser3, Reinhard Schneppenheim3, Carsten Baldauf4, Frauke Gr€ater1, Ulrich Budde5, Joachim R€adler2. 1 Molecular Biomechanics, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany, 2Faculty of Physics and Center for NanoScience, Ludwig Maximilian University, Munich, Germany, 3Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 4Theory, Fritz-Haber-Institut der Max-PlanckGesellschaft, Berlin, Germany, 5MEDILYS Laborgesellschaft mbH, Hemostaseology, Asklepios Klinik Altona, Hamburg, Germany. Protein stability can be quantified, in experiments or simulations, by monitoring changes in free energy induced by single amino-acid mutations relative to changes imposed by the same mutation in a reference unfolded peptide. We here assessed the destabilization by disease mutations of a protein, for which both the folded and unfolded states play key functional roles in hemostasis. Force-induced unfolding of the giant von Willebrand Factor (VWF) multimer-protein exposes a cleavage site for enzymatic proteolysis, a critical down-regulatory mechanism to prevent the formation of large thrombus aggregates. Several naturally occurring mutations modify this process, inducing distinct types of bleeding disorders, by unknown mechanisms. We present the first quantitative description of the dramatic destabilization of VWF caused by a one of such mutations, which strongly accelerates VWF cleavage. Molecular dynamics simulations and free energy calculations revealed this mutation to induce structural, dynamic, and mechanical perturbations in the VWF-A2 domain, thereby destabilizing this domain by ~10 kJ/mol promoting its unfolding. In close agreement, fluorescence correlation spectroscopy (FCS) revealed a 20-fold increase in the cleavage rate for this mutant, compared to the wildtype VWF. Cleavage was found cooperative with a cooperativity coefficient n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites at the same time. Taken together, the enhanced cleavage activity can be readily explained by an increased availability of the cleavage site through A2-domainfold thermodynamic destabilization. Our study therefore puts forward the combination of free energy calculations and FCS, as a powerful way of examining protein stability in a clinically relevant context. Reference: C. Aponte-Santamarı´a, et al. Biophysical Journal. In revision. 2413-Pos Board B20 Mechanical Architecture and Genesis of Bacterial Pilus Domains Revealed by Single-Molecule Force Spectroscopy Alvaro Alonso-Caballero, Raul Perez-Jimenez. CIC nanoGUNE, San Sebastian, Spain. Gram-negative bacteria attach to tissues using long filaments called pili. In uropathogenic Escherichia coli (UPEC), the pilus type-1 is composed by thousands of FimA subunits that form the pilus rod followed by the subunits FimF-FimG-FimH at the tip fibrillum. The mechanical resistance of the pilus is essential for successful attachment to target cells and tissues. With the exception of FimH, the contribution of each subunit to the mechanical architecture of the pilus has not been investigated. Here, we use atomic force spectroscopy to report a complete nanomechanical map of the pilus. We have used protein engineering to redesign the pilus domains in order to replicate the force vector that they sense in vivo. All domains show a very high mechanical stability reaching forces above 400 pN. We have discovered that the domains follow a mechanical hierarchy in which the stability decreases from the pilus rod to the tip. We have also used force-clamp spectroscopy to investigate with single-molecule resolution the mechanism of domain folding from the stretched state to the folded state assisted by periplasmic oxidoreductases and chaperones, DsbA and FimC. We have observed a synergistic effect that allows us to depict a precise kinetic model for the mechanogenesis of the pilus subunits.