- Email: [email protected]
Folding of a Multi-Domain Protein Untangled by Single-Molecule FRET
636a
Wednesday, March 2, 2016
G-quadruplex is a non-canonical DNA structure with a single-stranded DNA composited by tandem repeats of Guanine-rich (G-rich) motifs. This G-rich motif folds into a quadruple-folded structure at the presence of sodium (Naþ), potassium (Kþ) and other cations while it is held together by the Hoogsteen hydrogen bonding network. The stability of G-quadruplex is of great interest due to the stably folded Gquadruplex down regulates the amplification of oncogene promotor, therefore, efforts have been made to stabilize the G-quadruplex structures by binding to antibodies or ligands, which can be potential anti-cancer drugs. G-quadruplex is long believed to be intrinsically stabilized by some monovalent cations, especially the Kþ cation, possibly due to the hydration effect that is sensitive to the ion radius. Physiologically abundant Mg2þ cations are usually considered unimportant or even carrying a negative effect on the stabilization of G-quadruplex based on previous studies carried out in bulk solutions. However, the high DNA concentration required in these measurements may also lead to the interference of the inter- and intra-molecular G-quadruplex, or aggregations. In this work, we utilized single-molecule FRET spectroscopy to study the isolated G-quadruplex molecules and found that the presence of relatively low concentrations of Mg2þ cations alone efficiently promotes the folded structure formation of the human telomere 21 (HT21) and B-cell lymphoma 2 (bcl2) G-quadruplex sequences, compared to the solely presented Kþ cations. In addition, the Mg2þ assisted folding of HT21 shows a preference for the antiparallel configuration. The stabilities of the Mg2þ and Kþ bound G-quadruplex are also addressed by the kinetic analysis of the interconversion between unfolded and various folded conformations. 3137-Pos Board B514 Single-Molecule Studies of Fluorescently-Labelled Polysaccharides Steven D. Quinn1, Charlotte E. Dalton2, Robin A. Jeanneret2, John M. Gardiner2, Steven W. Magennis1. 1 Chemistry, University of Glasgow, Glasgow, United Kingdom, 2Institute of Biotechnology and School of Chemistry, University of Manchester, Manchester, United Kingdom. Together with nucleic acids and proteins, carbohydrates are fundamental building blocks of life that impact molecular and macroscopic functionality. Heparan sulfate (HS) in particular is a linear polysaccharide found on cell surfaces that binds to signalling proteins and growth factors to facilitate, among others, cell proliferation and differentiation. Since HS is known to mediate cell adhesion and control activities of numerous growth and motility factors, it has been implicated in the metastasis of cancer cells, the uptake of viral infections and the early-onset of Alzheimer’s disease. HS oligosaccharides are therefore considered important therapeutic targets for a variety of disease-related pathways. However, the structural requirements for protein binding by HS are largely undefined, in part due to its structural complexity, which includes variable sulfation patterns. In this presentation, we demonstrate the use of an amine tag for the 1:1 fluorescent labelling of structurally-defined chemically synthesized HS oligosaccharides and their detection and applications using advanced single-molecule techniques. This particular approach of labelling and detecting single HS oligosaccharides opens up an exciting platform from which to quantify, in real-time, the dynamic events that exist during interactions with its receptors. 3138-Pos Board B515 Protein Folding Drives Muscle Contraction Jaime A. Rivas Pardo1, Edward C. Eckels1, Ionel Popa1, Pallav Kosuri1, Wolfgang A. Linke2, Julio M. Fernandez1. 1 Department of Biological Sciences, Columbia University, New York, NY, USA, 2Department of Cardiovascular Physiology, Ruhr University Bochum, Bochum, Germany. In the current view of muscle contraction, the power stroke of a myosin motor is the sole source of mechanical energy driving the contraction of muscle. These models exclude titin, the largest protein in the human body, which sets the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin Ig domains occurs in the elastic I band region of intact myofibrils and that the physiological forces on titin fall between 6-8 pN. We utilize magnetic tweezers together with HaloTag anchoring to record the extension of titin Ig domains I10 and I91 for several hours at nanometer resolution in passive force-clamp mode. Titin Ig domains that had been previously unfolded undergo spontaneous stepwise folding at forces below 10 pN. We show that under a pulling force of 8 pN, previously unfolded I10 or I91 domains undergo a stepwise contraction of 13 nm, as
they move from the collapsed to the molten globule state. Thus, folding of a single Ig domain at the physiological force of 8 pN, generates 105 zJ of contractile energy, which is larger than the mechanical energy delivered by the power stroke of a single myosin motor (~40 zJ). From published measurements of passive muscle elasticity, we calculate that both ‘‘soft’’ and ‘‘stiff’’ muscles operate over a range of forces that straddle the folding probability of Ig domains as shown here. We propose that the stretching of an inactivated muscle recruits Ig domains to the unfolded state; activation of the myosin motors reduces the force on titin to harness the energy of Ig domain folding. From this perspective, both the myosin motors and titin operate as an inextricable molecular system for the storage and delivery of mechanical energy. 3139-Pos Board B516 On-Rate Switching under Force Increases the Binding of von Willebrand Factor A1 to GPIba Nathan Hudson1, Jongseong Kim2, Timothy A. Springer1. 1 Program in Cellular and Molecular Medicine, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA, 2Department of Chemistry and the Huck Institute of the Life Sciences, Pennsylvania State University, University Park, PA, USA. Alterations in flow during bleeding trigger the von Willebrand factor (VWF) A1 domain to bind to glycoprotein Iba (GPIba) on platelets, initiating blood clotting. Using a laser tweezers and a Receptor and Ligand in a Single Molecule (ReaLiSM) construct, we find that force can switch A1 and/or GPIba to a different state with a faster on-rate; this provides an additional mechanism for activating VWF binding to platelets. Mutations in VWF induce von Willebrand disease (VWD), a common human bleeding disorder. Our results show that force escalates the effects of VWD mutations with respect to on-and off-rates, explaining pathophysiology. Using the effective concentration of receptor and ligand in ReaLiSM to convert single molecule kon (units of s1) to bulk phase kon (units of s1M1) allows the calculation of an equivalent binding affinity. We find that using extrapolated, zeroforce kon and koff values from the low-force state of the bond to calculate the binding affinity provides remarkably good agreement with bulk phase measurements. 3140-Pos Board B517 Unfolding/Folding of a Multi-Domain Protein Untangled by SingleMolecule FRET Antonie Scho¨ne1, Daryan Kempe2, Michele Cerminara1, Matteo Gabba1,3, Tina Zu¨chner1, Jo¨rg Fitter1,2. 1 Institute of Complex Systems (ICS-5), Forschungszentrum Ju¨lich GmbH, Ju¨lich, Germany, 2I. Physikalisches Institut (IA), AG Biophysik, RWTH Aachen University, Aachen, Germany, 3Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, Netherlands. Deciphering how the amino acid code translates into 3D structures is the key to understand how proteins really work. In this context, the two-domain protein phosphoglycerate kinase (PGK) has proven to be an excellent model for multi-domain proteins. It is known, that both domains of PGK interact during folding. The N-terminal domain only gains its native structure in presence of the C-terminal domain. The C-domain also folds individually but the process is facilitated by the N-domain. In addition, intermediate states are involved. [1,2] A detailed picture of these intermediates and to what extent they are populated is still missing. It is therefore challenging to unravel the mechanisms of tertiary structure formation, especially since subpopulations are hard to identify in ensemble methods. To avoid averaging over all conformations, single-molecule methods are a perfect tool to distinguish and quantify such subpopulations. We established a set of PGK cysteine variants for site-specific labeling with fluorescent dyes for single molecule fluorescence resonance energy transfer (FRET). We verified that secondary and tertiary structures were not affected by cysteine mutations applying circular dichroism (CD) spectroscopy and tryptophan fluorescence. In addition all PGK cysteine mutants were catalytically active. The native states of the double labelled PGK variants were thoroughly characterized by fluorescence correlation spectroscopy (FCS) and single molecule FRET. Our system is designed to follow motions in between and within the individual domains displayed by distance changes of fluorophores during unfolding/folding transitions under denaturing conditions. [1] S. Osva´th, J. J. Sabelko, M. Gruebele, J Mol Biol 2003, 333(1):187. [2] J.-H. Han, S. Batey, A. A. Nickson, S. A. Teichmann, J. Clarke, Nat Rev Mol Cell Bio 2007, 8:319.
Wednesday, March 2, 2016
G-quadruplex is a non-canonical DNA structure with a single-stranded DNA composited by tandem repeats of Guanine-rich (G-rich) motifs. This G-rich motif folds into a quadruple-folded structure at the presence of sodium (Naþ), potassium (Kþ) and other cations while it is held together by the Hoogsteen hydrogen bonding network. The stability of G-quadruplex is of great interest due to the stably folded Gquadruplex down regulates the amplification of oncogene promotor, therefore, efforts have been made to stabilize the G-quadruplex structures by binding to antibodies or ligands, which can be potential anti-cancer drugs. G-quadruplex is long believed to be intrinsically stabilized by some monovalent cations, especially the Kþ cation, possibly due to the hydration effect that is sensitive to the ion radius. Physiologically abundant Mg2þ cations are usually considered unimportant or even carrying a negative effect on the stabilization of G-quadruplex based on previous studies carried out in bulk solutions. However, the high DNA concentration required in these measurements may also lead to the interference of the inter- and intra-molecular G-quadruplex, or aggregations. In this work, we utilized single-molecule FRET spectroscopy to study the isolated G-quadruplex molecules and found that the presence of relatively low concentrations of Mg2þ cations alone efficiently promotes the folded structure formation of the human telomere 21 (HT21) and B-cell lymphoma 2 (bcl2) G-quadruplex sequences, compared to the solely presented Kþ cations. In addition, the Mg2þ assisted folding of HT21 shows a preference for the antiparallel configuration. The stabilities of the Mg2þ and Kþ bound G-quadruplex are also addressed by the kinetic analysis of the interconversion between unfolded and various folded conformations. 3137-Pos Board B514 Single-Molecule Studies of Fluorescently-Labelled Polysaccharides Steven D. Quinn1, Charlotte E. Dalton2, Robin A. Jeanneret2, John M. Gardiner2, Steven W. Magennis1. 1 Chemistry, University of Glasgow, Glasgow, United Kingdom, 2Institute of Biotechnology and School of Chemistry, University of Manchester, Manchester, United Kingdom. Together with nucleic acids and proteins, carbohydrates are fundamental building blocks of life that impact molecular and macroscopic functionality. Heparan sulfate (HS) in particular is a linear polysaccharide found on cell surfaces that binds to signalling proteins and growth factors to facilitate, among others, cell proliferation and differentiation. Since HS is known to mediate cell adhesion and control activities of numerous growth and motility factors, it has been implicated in the metastasis of cancer cells, the uptake of viral infections and the early-onset of Alzheimer’s disease. HS oligosaccharides are therefore considered important therapeutic targets for a variety of disease-related pathways. However, the structural requirements for protein binding by HS are largely undefined, in part due to its structural complexity, which includes variable sulfation patterns. In this presentation, we demonstrate the use of an amine tag for the 1:1 fluorescent labelling of structurally-defined chemically synthesized HS oligosaccharides and their detection and applications using advanced single-molecule techniques. This particular approach of labelling and detecting single HS oligosaccharides opens up an exciting platform from which to quantify, in real-time, the dynamic events that exist during interactions with its receptors. 3138-Pos Board B515 Protein Folding Drives Muscle Contraction Jaime A. Rivas Pardo1, Edward C. Eckels1, Ionel Popa1, Pallav Kosuri1, Wolfgang A. Linke2, Julio M. Fernandez1. 1 Department of Biological Sciences, Columbia University, New York, NY, USA, 2Department of Cardiovascular Physiology, Ruhr University Bochum, Bochum, Germany. In the current view of muscle contraction, the power stroke of a myosin motor is the sole source of mechanical energy driving the contraction of muscle. These models exclude titin, the largest protein in the human body, which sets the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin Ig domains occurs in the elastic I band region of intact myofibrils and that the physiological forces on titin fall between 6-8 pN. We utilize magnetic tweezers together with HaloTag anchoring to record the extension of titin Ig domains I10 and I91 for several hours at nanometer resolution in passive force-clamp mode. Titin Ig domains that had been previously unfolded undergo spontaneous stepwise folding at forces below 10 pN. We show that under a pulling force of 8 pN, previously unfolded I10 or I91 domains undergo a stepwise contraction of 13 nm, as
they move from the collapsed to the molten globule state. Thus, folding of a single Ig domain at the physiological force of 8 pN, generates 105 zJ of contractile energy, which is larger than the mechanical energy delivered by the power stroke of a single myosin motor (~40 zJ). From published measurements of passive muscle elasticity, we calculate that both ‘‘soft’’ and ‘‘stiff’’ muscles operate over a range of forces that straddle the folding probability of Ig domains as shown here. We propose that the stretching of an inactivated muscle recruits Ig domains to the unfolded state; activation of the myosin motors reduces the force on titin to harness the energy of Ig domain folding. From this perspective, both the myosin motors and titin operate as an inextricable molecular system for the storage and delivery of mechanical energy. 3139-Pos Board B516 On-Rate Switching under Force Increases the Binding of von Willebrand Factor A1 to GPIba Nathan Hudson1, Jongseong Kim2, Timothy A. Springer1. 1 Program in Cellular and Molecular Medicine, Boston Children’s Hospital and Harvard Medical School, Boston, MA, USA, 2Department of Chemistry and the Huck Institute of the Life Sciences, Pennsylvania State University, University Park, PA, USA. Alterations in flow during bleeding trigger the von Willebrand factor (VWF) A1 domain to bind to glycoprotein Iba (GPIba) on platelets, initiating blood clotting. Using a laser tweezers and a Receptor and Ligand in a Single Molecule (ReaLiSM) construct, we find that force can switch A1 and/or GPIba to a different state with a faster on-rate; this provides an additional mechanism for activating VWF binding to platelets. Mutations in VWF induce von Willebrand disease (VWD), a common human bleeding disorder. Our results show that force escalates the effects of VWD mutations with respect to on-and off-rates, explaining pathophysiology. Using the effective concentration of receptor and ligand in ReaLiSM to convert single molecule kon (units of s1) to bulk phase kon (units of s1M1) allows the calculation of an equivalent binding affinity. We find that using extrapolated, zeroforce kon and koff values from the low-force state of the bond to calculate the binding affinity provides remarkably good agreement with bulk phase measurements. 3140-Pos Board B517 Unfolding/Folding of a Multi-Domain Protein Untangled by SingleMolecule FRET Antonie Scho¨ne1, Daryan Kempe2, Michele Cerminara1, Matteo Gabba1,3, Tina Zu¨chner1, Jo¨rg Fitter1,2. 1 Institute of Complex Systems (ICS-5), Forschungszentrum Ju¨lich GmbH, Ju¨lich, Germany, 2I. Physikalisches Institut (IA), AG Biophysik, RWTH Aachen University, Aachen, Germany, 3Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Groningen, Netherlands. Deciphering how the amino acid code translates into 3D structures is the key to understand how proteins really work. In this context, the two-domain protein phosphoglycerate kinase (PGK) has proven to be an excellent model for multi-domain proteins. It is known, that both domains of PGK interact during folding. The N-terminal domain only gains its native structure in presence of the C-terminal domain. The C-domain also folds individually but the process is facilitated by the N-domain. In addition, intermediate states are involved. [1,2] A detailed picture of these intermediates and to what extent they are populated is still missing. It is therefore challenging to unravel the mechanisms of tertiary structure formation, especially since subpopulations are hard to identify in ensemble methods. To avoid averaging over all conformations, single-molecule methods are a perfect tool to distinguish and quantify such subpopulations. We established a set of PGK cysteine variants for site-specific labeling with fluorescent dyes for single molecule fluorescence resonance energy transfer (FRET). We verified that secondary and tertiary structures were not affected by cysteine mutations applying circular dichroism (CD) spectroscopy and tryptophan fluorescence. In addition all PGK cysteine mutants were catalytically active. The native states of the double labelled PGK variants were thoroughly characterized by fluorescence correlation spectroscopy (FCS) and single molecule FRET. Our system is designed to follow motions in between and within the individual domains displayed by distance changes of fluorophores during unfolding/folding transitions under denaturing conditions. [1] S. Osva´th, J. J. Sabelko, M. Gruebele, J Mol Biol 2003, 333(1):187. [2] J.-H. Han, S. Batey, A. A. Nickson, S. A. Teichmann, J. Clarke, Nat Rev Mol Cell Bio 2007, 8:319.