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3D Motion Maps of TRPV1 Cation Channel Depicted by Diffracted X-ray Tracking Method
Monday, February 13, 2017 the binding patches that are scattered on the protein surface show that their distribution and growth will depend on the protein’s functional group. Finally, in several cases, the binding-site predictions resulting from the cross-docking simulations will lead to the identification of an alternate interface, which corresponds to the interaction with a biomolecular partner that is not included in the original benchmark. Ref: Vamparys et al. (2016), Proteins. doi:10.1002/prot.25086
Protein Dynamics and Allostery II 991-Pos Board B59 3D Motion Maps of TRPV1 Cation Channel Depicted by Diffracted X-ray Tracking Method Kazuhiro Mio1, Keigo Ikezaki2, Hiroshi Sekiguchi3, Muneyo Mio1, Tai Kubo1, Yuji C. Sasaki2. 1 OPERANDO-OIL & molprof, Natl Inst Adv Ind Sci Tech (AIST), Tokyo, Japan, 2Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan, 3Research & Utilization Division, Japan Synchrotron Radiation Research Institute, SPring-8, Sayo-gun, Japan. The TRPV1 is a nonselective cation channel that responds to various signals, including high temperature with threshold at 43 C, acidic conditions, as well as chemical compounds such as capsaicin and allylisothiocyanate. Because TRPV1 is a membrane protein with large molecular mass, crystallography technique has not been succeeded for a long time. However recent progress in the cryoelectron microscopy enabled depiction of the TRPV1 structure in atomic resolution. Cryo-EM and the single particle reconstruction technique showed structures of apo- and ligand binding forms of TRPV1 with various agonists, antagonists and toxins. In spite of accumulation of structural data, gating mechanisms of TRPV1 is not clearly understood yet. Information about stateto-state transition is missing. To understand the dynamics of TRPV1 in channel function, we adopted the Diffracted X-ray Tracking (DXT) technique to this protein. In DXT, individual protein was labeled with gold nanocrystals, and the motion of X-ray diffraction spots from the crystal were investigated as intramolecular movement of TRPV1 in real time. We introduced ‘‘Met tag’’ for labeling nanocrystal and ‘‘His tag’’ for substrate absorption to TRPV1. It was expressed in HEK293 cells, purified, and immobilized on the Ni-NTA coated polyimide substrates. Intramolecular motion was recorded by tracking the movement of diffraction spots from the gold nanocrystals. Molecular dynamics of TRPV1 against capsaicin, pH change, and temperature activation were investigated. 992-Pos Board B60 Balance between Protein Softness and Rigidity Assessed by Inelastic X-ray Scattering Utsab R. Shrestha1, Debsindhu Bhowmik2, Kurt W. Van Delinder1, Eugene Mamontov3, Hugh O’Neill4, Qiu Zhang4, Ahmet Alatas5, Xiang-Qiang Chu1. 1 Department of Physics and Astronomy, Wayne State University, Detroit, MI, USA, 2Computational Science and Engineering Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 3Chemical and Engineering Materials Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 4 Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 5Advanced Photon Source, Argonne National Laboratory, Argonne, IL, USA. Collective excitations due to protein secondary structure have been of considerable interest in the past few years. Such excitations provide the notion of protein softness, and flexibility that are ultimately related to the activity of the protein. However, there has been a lack of evidence for the correlation between the protein structure, collective motions, and the flexibility. Here, we elucidate the protein activity from the perspective of protein softness and flexibility by studying the collective phonon-like excitations in proteins using a state-of-the-art inelastic x-ray scattering (IXS) technique. Both propagating and non-propagating modes of collective phonon-like excitations are observed in proteins on the length scale larger and shorter than the protein secondary structure, respectively. The longitudinal sound velocity of propagating mode of such excitations in protein samples is approximately 2,800 m/s, consistent with the previously reported results. The so-observed non-propagating localized phonon-like excitations give the measure of protein softness and flexibility. Such excitations suggest that protein becomes softer due to breakdown of weak non-covalent bonds (responsible for preserving the native conformation) upon thermal denaturation. In addition, it confirms that protein requires necessary rigidity along with flexibility for the enzyme activity. Furthermore, the drugs like warfarin and ibuprofen that have a strong binding affinity to specific binding sites of the protein, human serum albumin (HSA),
201a
do not affect the protein structure and collective excitations. Such results indicate that the efficiency of HSA upon binding to warfarin and ibuprofen in the plasma remains unaffected in carrying and transporting them to the specific targets. 993-Pos Board B61 Description of Hydration Water in Protein (GFP) Solution Stefania Perticaroli, Georg Ehlers, Christopher Stanley, Eugene Mamontov, Hugh O’Neill, Qui Zhang, Xiaolin Cheng, Dean A.A. Myles, John Katsaras, Jonathan D. Nickels. Oak Ridge National Lab/Univ. of Tennessee, Knoxville, Oak Ridge, TN, USA. The term, hydration water, describes the structurally and dynamically perturbed water surrounding proteins and biomolecules. This population of water has a defining influence on the structure and function of biomolecules, especially proteins; implying a fundamental connection between the dynamical properties of hydration water and many vital biochemical processes including protein folding, protein–ligand recognition, membrane, enzyme function, and DNA stability. This makes the extent of the perturbation (the hydration number, NH) and degree of perturbation (the retardation factor, x) of great practical interest. We present an experimental description of the dynamical perturbation of hydration water around green fluorescent protein in solution using neutron scattering methods. We find that less than two ˚ ) were perturbed, with dynamics a factor of 2–10 times slower shells (~5.5 A than bulk water, depending on their distance from the protein surface and the probe length of the measurement, which neutron scattering allows us to vary. This dependence on probe length demonstrates that hydration water undergoes sub-diffusive motions (for the first hydration shell, for perturbed waters in the second shell), an important difference with neat water which demonstrates diffusive behavior (). 994-Pos Board B62 Second Harmonic Generation as a Method to Identify and Screen for Allosteric Modulators of Protein Targets Joshua Salafsky1, Roman Agafonov2, Elizabeth Donohue Vo3, Katelyn Connell1, Gabriel Mercado1, Tad George1, Frank McCormick3, Dorothee Kern2. 1 Biodesy, Inc., South San Francisco, CA, USA, 2The Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA, 3University of California, San Francisco, San Francisco, CA, USA. Proteins populate a landscape of conformations that changes upon binding native ligands or drugs, altering protein function. Different conformations reveal distinct potential drug target sites, including allosteric sites distal to the active site of the protein. It has been historically difficult to identify allosteric modulators early in the screening process since the structural methods required to reveal binding sites are relatively low throughput. Second Harmonic Generation (SHG), an optical phenomenon exquisitely sensitive to molecular orientation, is used in high throughput, solution-based biophysical assays to measure conformational change. SHG offers a direct and sensitive method to identify and classify compounds, by measuring protein structure in real time and in solution. Moreover, SHG reveals a range of conformations that a protein adopts when bound to different compounds, whether orthosteric or allosteric, which are predicted to produce a specific functional outcome in vivo. Here I show that SHG can be used to target allosteric binding pockets of kRas and Src. The information obtained includes not only potency of ligand interaction, but also distinguishes ligands by the direction and magnitude of conformational change, allowing for classification of analytes by mechanism of binding. SHG offers a unique approach to reveal and define the causal link between target conformation and function. 995-Pos Board B63 Binding Mechanism between CrkII and cAbl Kinase Qingliang Shen, Danyun Zeng, Jae-Hyun Cho. Texas A&M University, College Station, TX, USA. The interaction between non-receptor tyrosine kinase cAbl and signaling adaptor protein CrkII is implicated in diverse cellular processes, such as cell migration, proliferation, and bacterial infection. Because of the modular architecture of their structures, the interaction between cAbl and CrkII can undergo multiple alternative binding modes. This series of interactions starts with the binding of the N-terminal Src homology 3 (nSH3) domain of CrkII to proline-rich motifs (PRMs) in the intrinsically disordered region of cAbl kinase. Despite their importance, the detailed binding mechanism and functional significance of these interactions remains elusive. In this presentation, we report the detailed structure, thermodynamics, and kinetics of the interactions between the nSH3 domain of CrkII and PRMs of cAbl kinase. Our dynamics
Protein Dynamics and Allostery II 991-Pos Board B59 3D Motion Maps of TRPV1 Cation Channel Depicted by Diffracted X-ray Tracking Method Kazuhiro Mio1, Keigo Ikezaki2, Hiroshi Sekiguchi3, Muneyo Mio1, Tai Kubo1, Yuji C. Sasaki2. 1 OPERANDO-OIL & molprof, Natl Inst Adv Ind Sci Tech (AIST), Tokyo, Japan, 2Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan, 3Research & Utilization Division, Japan Synchrotron Radiation Research Institute, SPring-8, Sayo-gun, Japan. The TRPV1 is a nonselective cation channel that responds to various signals, including high temperature with threshold at 43 C, acidic conditions, as well as chemical compounds such as capsaicin and allylisothiocyanate. Because TRPV1 is a membrane protein with large molecular mass, crystallography technique has not been succeeded for a long time. However recent progress in the cryoelectron microscopy enabled depiction of the TRPV1 structure in atomic resolution. Cryo-EM and the single particle reconstruction technique showed structures of apo- and ligand binding forms of TRPV1 with various agonists, antagonists and toxins. In spite of accumulation of structural data, gating mechanisms of TRPV1 is not clearly understood yet. Information about stateto-state transition is missing. To understand the dynamics of TRPV1 in channel function, we adopted the Diffracted X-ray Tracking (DXT) technique to this protein. In DXT, individual protein was labeled with gold nanocrystals, and the motion of X-ray diffraction spots from the crystal were investigated as intramolecular movement of TRPV1 in real time. We introduced ‘‘Met tag’’ for labeling nanocrystal and ‘‘His tag’’ for substrate absorption to TRPV1. It was expressed in HEK293 cells, purified, and immobilized on the Ni-NTA coated polyimide substrates. Intramolecular motion was recorded by tracking the movement of diffraction spots from the gold nanocrystals. Molecular dynamics of TRPV1 against capsaicin, pH change, and temperature activation were investigated. 992-Pos Board B60 Balance between Protein Softness and Rigidity Assessed by Inelastic X-ray Scattering Utsab R. Shrestha1, Debsindhu Bhowmik2, Kurt W. Van Delinder1, Eugene Mamontov3, Hugh O’Neill4, Qiu Zhang4, Ahmet Alatas5, Xiang-Qiang Chu1. 1 Department of Physics and Astronomy, Wayne State University, Detroit, MI, USA, 2Computational Science and Engineering Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 3Chemical and Engineering Materials Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 4 Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 5Advanced Photon Source, Argonne National Laboratory, Argonne, IL, USA. Collective excitations due to protein secondary structure have been of considerable interest in the past few years. Such excitations provide the notion of protein softness, and flexibility that are ultimately related to the activity of the protein. However, there has been a lack of evidence for the correlation between the protein structure, collective motions, and the flexibility. Here, we elucidate the protein activity from the perspective of protein softness and flexibility by studying the collective phonon-like excitations in proteins using a state-of-the-art inelastic x-ray scattering (IXS) technique. Both propagating and non-propagating modes of collective phonon-like excitations are observed in proteins on the length scale larger and shorter than the protein secondary structure, respectively. The longitudinal sound velocity of propagating mode of such excitations in protein samples is approximately 2,800 m/s, consistent with the previously reported results. The so-observed non-propagating localized phonon-like excitations give the measure of protein softness and flexibility. Such excitations suggest that protein becomes softer due to breakdown of weak non-covalent bonds (responsible for preserving the native conformation) upon thermal denaturation. In addition, it confirms that protein requires necessary rigidity along with flexibility for the enzyme activity. Furthermore, the drugs like warfarin and ibuprofen that have a strong binding affinity to specific binding sites of the protein, human serum albumin (HSA),
201a
do not affect the protein structure and collective excitations. Such results indicate that the efficiency of HSA upon binding to warfarin and ibuprofen in the plasma remains unaffected in carrying and transporting them to the specific targets. 993-Pos Board B61 Description of Hydration Water in Protein (GFP) Solution Stefania Perticaroli, Georg Ehlers, Christopher Stanley, Eugene Mamontov, Hugh O’Neill, Qui Zhang, Xiaolin Cheng, Dean A.A. Myles, John Katsaras, Jonathan D. Nickels. Oak Ridge National Lab/Univ. of Tennessee, Knoxville, Oak Ridge, TN, USA. The term, hydration water, describes the structurally and dynamically perturbed water surrounding proteins and biomolecules. This population of water has a defining influence on the structure and function of biomolecules, especially proteins; implying a fundamental connection between the dynamical properties of hydration water and many vital biochemical processes including protein folding, protein–ligand recognition, membrane, enzyme function, and DNA stability. This makes the extent of the perturbation (the hydration number, NH) and degree of perturbation (the retardation factor, x) of great practical interest. We present an experimental description of the dynamical perturbation of hydration water around green fluorescent protein in solution using neutron scattering methods. We find that less than two ˚ ) were perturbed, with dynamics a factor of 2–10 times slower shells (~5.5 A than bulk water, depending on their distance from the protein surface and the probe length of the measurement, which neutron scattering allows us to vary. This dependence on probe length demonstrates that hydration water undergoes sub-diffusive motions (for the first hydration shell, for perturbed waters in the second shell), an important difference with neat water which demonstrates diffusive behavior (). 994-Pos Board B62 Second Harmonic Generation as a Method to Identify and Screen for Allosteric Modulators of Protein Targets Joshua Salafsky1, Roman Agafonov2, Elizabeth Donohue Vo3, Katelyn Connell1, Gabriel Mercado1, Tad George1, Frank McCormick3, Dorothee Kern2. 1 Biodesy, Inc., South San Francisco, CA, USA, 2The Howard Hughes Medical Institute, Brandeis University, Waltham, MA, USA, 3University of California, San Francisco, San Francisco, CA, USA. Proteins populate a landscape of conformations that changes upon binding native ligands or drugs, altering protein function. Different conformations reveal distinct potential drug target sites, including allosteric sites distal to the active site of the protein. It has been historically difficult to identify allosteric modulators early in the screening process since the structural methods required to reveal binding sites are relatively low throughput. Second Harmonic Generation (SHG), an optical phenomenon exquisitely sensitive to molecular orientation, is used in high throughput, solution-based biophysical assays to measure conformational change. SHG offers a direct and sensitive method to identify and classify compounds, by measuring protein structure in real time and in solution. Moreover, SHG reveals a range of conformations that a protein adopts when bound to different compounds, whether orthosteric or allosteric, which are predicted to produce a specific functional outcome in vivo. Here I show that SHG can be used to target allosteric binding pockets of kRas and Src. The information obtained includes not only potency of ligand interaction, but also distinguishes ligands by the direction and magnitude of conformational change, allowing for classification of analytes by mechanism of binding. SHG offers a unique approach to reveal and define the causal link between target conformation and function. 995-Pos Board B63 Binding Mechanism between CrkII and cAbl Kinase Qingliang Shen, Danyun Zeng, Jae-Hyun Cho. Texas A&M University, College Station, TX, USA. The interaction between non-receptor tyrosine kinase cAbl and signaling adaptor protein CrkII is implicated in diverse cellular processes, such as cell migration, proliferation, and bacterial infection. Because of the modular architecture of their structures, the interaction between cAbl and CrkII can undergo multiple alternative binding modes. This series of interactions starts with the binding of the N-terminal Src homology 3 (nSH3) domain of CrkII to proline-rich motifs (PRMs) in the intrinsically disordered region of cAbl kinase. Despite their importance, the detailed binding mechanism and functional significance of these interactions remains elusive. In this presentation, we report the detailed structure, thermodynamics, and kinetics of the interactions between the nSH3 domain of CrkII and PRMs of cAbl kinase. Our dynamics