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Realtime Single Molecular Motion Analysis of Nicotinic Acetylcholine Receptor Alpha 7 by Diffracted X-Ray Tracking Method
222a
Monday, February 29, 2016
1103-Pos Board B80 Revealing the Transport Cycle Dynamics of the Sodium Dependent Sugar Transporter by Double Electron-Electron Resonance and Wide-Angle X-ray Scattering Aviv Paz1, Derek P. Claxton2, Shruti Sharma2, Kelli Kazmier3, Jay Prakash Kumar4, Shannon A. Nolte5, Terrin M. Liwag5, Hassane S. Mchaourab2, Jeff Abramson1,4. 1 Physiology, UCLA, Los Angeles, CA, USA, 2Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA, 3 Chemical and Physical Biology Program, Vanderbilt University Medical Center, Nashville, TN, USA, 4Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, Bangalore, India, 5Molecular Cell and Developmental Biology Program, UCLA, Los Angeles, CA, USA. Dietary sugars have an immense importance in the metabolism of all cells. As such, it is vital to understand their cellular entry and exit pathways through specific transporters. Our research focuses on the sodium dependent sugar transporter from Vibrio parahemolyticus (vSGLT). Currently, this is the best structural model system for the mammalian transporters, which are responsible for sugar absorption in the intestine and reabsorption in the kidney. Recently, this transporter has been the target of various drugs that lower blood-sugar levels by inhibiting sugar reabsorption in the kidney. SGLTs are highly dynamic and switch between a number of conformations to facilitate sugar passage across the cell membrane. There are currently two available x-ray structures for vSGLT in an inward-occluded1 and an inwardopen2 conformation. These two distinct conformations have fundamentally advanced our understanding of sugar transport to an atomic level, but did not encapsulate the dynamics of transport, nor the nature of the outward-open conformation. In order to gain further insight into the ligand dependent dynamics of vSGLT, we have conducted double electron-electron resonance (DEER) and wide-angle x-ray scattering (WAXS) studies under three conditions, each favoring different conformers in the protein: in the absence of sodium and galactose, in the presence of sodium only and in the presence of both sodium and galactose. We will discuss the overall insights gained by these two approaches, the similarities and differences with other transporters that share the Leu-T fold and the structural refinement that included combined energy terms from DEER and WAXS. 1. Faham et al., (2008) Science 321(5890). 2. Watanabe et al., (2010) Nature 468. 1104-Pos Board B81 The Effect of the Protein Dynamical Transition on Intramolecular Vibrations Mengyang Xu1, Katherine A. Niessen1, Yanting Deng1, Nigel S. Michki1, Edward H. Snell2,3, Andrea G. Markelz1. 1 Department of Physics, University at Buffalo, Buffalo, NY, USA, 2 Hauptman-Woodward Medical Research Institute, Buffalo, NY, USA, 3 Department of Structural Biology, University at Buffalo, Buffalo, NY, USA. Protein structural motion is necessary for function. A typical measure of protein flexibility is the average mean squared atomic displacement (MSD). Both X-ray and neutron scattering have found a strong increase in the MSD at 200-220 K for a number of proteins. This increase is often referred to as the dynamical transition (DT) [1]. Function ceases for many proteins below the DT. It was suggested that the DT indicated the temperature at which the motions necessary for function are frozen out. A question has been raised as to how long-range motions are affected by the transition. Specifically, would the long-range intramolecular vibrations associated with conformational changes be present at low frequencies, and then at higher temperatures red shift due to anharmonicity, as is seen in conventional solids? The different DT measurement techniques do not distinguish between local particle excitations and coherent long-range motions. Direct optical measurement of the long-range intramolecular vibrations requires spectroscopy within the energy range of 1-100 cm 1 (n = 0.03-3 THz). Our recently developed technique, CATM (Crystal Anisotropy Terahertz Microscopy) [2], uses polarization dependent absorption on aligned molecules to isolate the long-range intramolecular vibrations from the local excitations. Temperature dependent CATM from 180K to 295K is used to investigate how the DT effects long-range vibrations. We find that a sharp 70 cm-1 band grows in as the temperature increases. We suggest that this new band above DT demonstrates that in fact the surrounding solvent acts as a frozen cage preventing long-range correlated motions, and as the surrounding solvent becomes more mobile, the large-scale motions necessary for function can occur. 1.Doster, W., et al. Physical Review Letters, 2010. 104(9): p. 098101. 2.Acbas, G., et al. Nature Communications, 2014. 5, 3076 http://dx.doi.org/10. 1038/ncomms4076.
1105-Pos Board B82 Realtime Single Molecular Motion Analysis of Nicotinic Acetylcholine Receptor Alpha 7 by Diffracted X-Ray Tracking Method Tai Kubo1, Tomoyuki Baba2, Keigo Ikezaki2, Hiroshi Sekiguchi3, Yuri Nishino4, Atsuo Miyazawa4, Yuji C. Sasaki2,3. 1 Molecular Profiling Research Center for Drug Discovery, 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, 4 Graduate School of Life Sciences, University of Hyogo, Ako-gun, Japan. The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel. Binding of ligands (e.g. acetylcholine, nicotine) to nAChR induces ion flux through the channel formed by pentameric subunits, which leads to the changes of cell excitability, and then triggers the following signaling cascades in the peripheral and the central nervous system. In the previous study we applied Diffracted X-ray Tracking (DXT) method to acetylcholine binding protein (AChBP) and Torpedo nAChR. DXT is a method to track the X-ray diffraction spots from the gold nanocrystal labeled on an individual single protein in real time and real space. We observed molecular fluctuations of the proteins even without ligand, and enhancement of tilting and twisting motions with ligand [Sekiguchi, et al. (2014) Sci. Rep. 4: 6384]. Here, to acquire more precise information on molecular motions of nAChR in the three sub-states (close, open, and desensitization) and their sate-tostate transitions, we applied the DXT for the neuronal nAChR alpha 7. The alpha 7 receptor in the central nervous system plays essential roles in the cholinergic neuro-signal transduction, and it is one of the drug targets for therapeutics of Alzheimer diseases and cognitive impairment associated with schizophrenia (CIAS). Alpha 7 receptor was recombinantly prepared from the cDNA expressed in Xenopus oocytes. To orderly configure the receptors defined tags were introduced at the defined positions; e.g. ‘‘Met tag’’ for labeling with gold nanocrystal in the N-terminal, and ‘‘His tag’’ for absorption on mica substrate in the second cytoplasmic loop of the receptor. Characteristic molecular motion(s) during the three states of the nAChR alpha 7 were revealed by the DXT analysis. Possible transition modeling will be discussed. 1106-Pos Board B83 Structural Dynamics of Hsp90 Resolved by a Novel Multi-Pair FRET Approach Bjoern Hellenkamp1, Philipp Wortmann1, Florian Kandzia2, Martin Zacharias2, Thorsten Hugel1. 1 Institute of Physical Chemistry, University of Freiburg, Freiburg, Germany, 2 Physics department, Technische Universitaet Muenchen, Garching, Germany. Three-dimensional models of proteins at atomic resolution are an essential prerequisite for mechanistic insight into protein function. However, in solution the protein exists as a dynamic equilibrium of global and local conformations. Therefore, a comprehensive understanding of protein function requires knowledge on the structure - dynamics relationship. We developed a novel hybrid approach based on single molecule Fo¨rster Resonance Energy Transfer [1, 2] that integrates hundreds of time resolved intramolecular distances together with x-ray structure information and MD simulations. Simultaneous determination of fluctuations and time resolved anisotropies enables a new level of accuracy and confidence. We demonstrate the power of our method by determining the dynamic structure of the molecular chaperone and heat shock protein Hsp90. Our resulting mean structure for the closed state of Hsp90 resembles ˚ . Beyond that, the corresponding x-ray structure of yeast Hsp90 [3] to 2.5 A we resolved the previously unknown open structure of this multi-domain protein. Surprisingly, the open state shows much larger fluctuations on relevant timescales than the closed state. Finally, we show how this method can be applied to quantify inter-domain dynamics and dynamics of small elements. We anticipate that this dynamic structure determination will solve many long standing questions regarding flexible parts in large proteins and proteinprotein interactions. [1] Muschielok A. et al.; Nature Methods 2008; 5, 965-971. [2] Kalinin S. et al.; Nature Methods 2012; 9(12):1218-1225. [3] Ali M.M. et al.; Nature 2006, 440(7087):1013-1017. 1107-Pos Board B84 Revealing a Heterodimeric Interface between the Members of Two Unrelated Fluorescent Protein Lineages Gary Ch Mo, Jin Zhang. Pharmacology, University of California San Diego, San Diego, CA, USA. Many fluorescent proteins (FPs) were developed from oligomeric predecessors. The use of monomeric FPs prevents oligomerization of the labeled protein of
Monday, February 29, 2016
1103-Pos Board B80 Revealing the Transport Cycle Dynamics of the Sodium Dependent Sugar Transporter by Double Electron-Electron Resonance and Wide-Angle X-ray Scattering Aviv Paz1, Derek P. Claxton2, Shruti Sharma2, Kelli Kazmier3, Jay Prakash Kumar4, Shannon A. Nolte5, Terrin M. Liwag5, Hassane S. Mchaourab2, Jeff Abramson1,4. 1 Physiology, UCLA, Los Angeles, CA, USA, 2Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA, 3 Chemical and Physical Biology Program, Vanderbilt University Medical Center, Nashville, TN, USA, 4Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, Bangalore, India, 5Molecular Cell and Developmental Biology Program, UCLA, Los Angeles, CA, USA. Dietary sugars have an immense importance in the metabolism of all cells. As such, it is vital to understand their cellular entry and exit pathways through specific transporters. Our research focuses on the sodium dependent sugar transporter from Vibrio parahemolyticus (vSGLT). Currently, this is the best structural model system for the mammalian transporters, which are responsible for sugar absorption in the intestine and reabsorption in the kidney. Recently, this transporter has been the target of various drugs that lower blood-sugar levels by inhibiting sugar reabsorption in the kidney. SGLTs are highly dynamic and switch between a number of conformations to facilitate sugar passage across the cell membrane. There are currently two available x-ray structures for vSGLT in an inward-occluded1 and an inwardopen2 conformation. These two distinct conformations have fundamentally advanced our understanding of sugar transport to an atomic level, but did not encapsulate the dynamics of transport, nor the nature of the outward-open conformation. In order to gain further insight into the ligand dependent dynamics of vSGLT, we have conducted double electron-electron resonance (DEER) and wide-angle x-ray scattering (WAXS) studies under three conditions, each favoring different conformers in the protein: in the absence of sodium and galactose, in the presence of sodium only and in the presence of both sodium and galactose. We will discuss the overall insights gained by these two approaches, the similarities and differences with other transporters that share the Leu-T fold and the structural refinement that included combined energy terms from DEER and WAXS. 1. Faham et al., (2008) Science 321(5890). 2. Watanabe et al., (2010) Nature 468. 1104-Pos Board B81 The Effect of the Protein Dynamical Transition on Intramolecular Vibrations Mengyang Xu1, Katherine A. Niessen1, Yanting Deng1, Nigel S. Michki1, Edward H. Snell2,3, Andrea G. Markelz1. 1 Department of Physics, University at Buffalo, Buffalo, NY, USA, 2 Hauptman-Woodward Medical Research Institute, Buffalo, NY, USA, 3 Department of Structural Biology, University at Buffalo, Buffalo, NY, USA. Protein structural motion is necessary for function. A typical measure of protein flexibility is the average mean squared atomic displacement (MSD). Both X-ray and neutron scattering have found a strong increase in the MSD at 200-220 K for a number of proteins. This increase is often referred to as the dynamical transition (DT) [1]. Function ceases for many proteins below the DT. It was suggested that the DT indicated the temperature at which the motions necessary for function are frozen out. A question has been raised as to how long-range motions are affected by the transition. Specifically, would the long-range intramolecular vibrations associated with conformational changes be present at low frequencies, and then at higher temperatures red shift due to anharmonicity, as is seen in conventional solids? The different DT measurement techniques do not distinguish between local particle excitations and coherent long-range motions. Direct optical measurement of the long-range intramolecular vibrations requires spectroscopy within the energy range of 1-100 cm 1 (n = 0.03-3 THz). Our recently developed technique, CATM (Crystal Anisotropy Terahertz Microscopy) [2], uses polarization dependent absorption on aligned molecules to isolate the long-range intramolecular vibrations from the local excitations. Temperature dependent CATM from 180K to 295K is used to investigate how the DT effects long-range vibrations. We find that a sharp 70 cm-1 band grows in as the temperature increases. We suggest that this new band above DT demonstrates that in fact the surrounding solvent acts as a frozen cage preventing long-range correlated motions, and as the surrounding solvent becomes more mobile, the large-scale motions necessary for function can occur. 1.Doster, W., et al. Physical Review Letters, 2010. 104(9): p. 098101. 2.Acbas, G., et al. Nature Communications, 2014. 5, 3076 http://dx.doi.org/10. 1038/ncomms4076.
1105-Pos Board B82 Realtime Single Molecular Motion Analysis of Nicotinic Acetylcholine Receptor Alpha 7 by Diffracted X-Ray Tracking Method Tai Kubo1, Tomoyuki Baba2, Keigo Ikezaki2, Hiroshi Sekiguchi3, Yuri Nishino4, Atsuo Miyazawa4, Yuji C. Sasaki2,3. 1 Molecular Profiling Research Center for Drug Discovery, 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, 4 Graduate School of Life Sciences, University of Hyogo, Ako-gun, Japan. The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel. Binding of ligands (e.g. acetylcholine, nicotine) to nAChR induces ion flux through the channel formed by pentameric subunits, which leads to the changes of cell excitability, and then triggers the following signaling cascades in the peripheral and the central nervous system. In the previous study we applied Diffracted X-ray Tracking (DXT) method to acetylcholine binding protein (AChBP) and Torpedo nAChR. DXT is a method to track the X-ray diffraction spots from the gold nanocrystal labeled on an individual single protein in real time and real space. We observed molecular fluctuations of the proteins even without ligand, and enhancement of tilting and twisting motions with ligand [Sekiguchi, et al. (2014) Sci. Rep. 4: 6384]. Here, to acquire more precise information on molecular motions of nAChR in the three sub-states (close, open, and desensitization) and their sate-tostate transitions, we applied the DXT for the neuronal nAChR alpha 7. The alpha 7 receptor in the central nervous system plays essential roles in the cholinergic neuro-signal transduction, and it is one of the drug targets for therapeutics of Alzheimer diseases and cognitive impairment associated with schizophrenia (CIAS). Alpha 7 receptor was recombinantly prepared from the cDNA expressed in Xenopus oocytes. To orderly configure the receptors defined tags were introduced at the defined positions; e.g. ‘‘Met tag’’ for labeling with gold nanocrystal in the N-terminal, and ‘‘His tag’’ for absorption on mica substrate in the second cytoplasmic loop of the receptor. Characteristic molecular motion(s) during the three states of the nAChR alpha 7 were revealed by the DXT analysis. Possible transition modeling will be discussed. 1106-Pos Board B83 Structural Dynamics of Hsp90 Resolved by a Novel Multi-Pair FRET Approach Bjoern Hellenkamp1, Philipp Wortmann1, Florian Kandzia2, Martin Zacharias2, Thorsten Hugel1. 1 Institute of Physical Chemistry, University of Freiburg, Freiburg, Germany, 2 Physics department, Technische Universitaet Muenchen, Garching, Germany. Three-dimensional models of proteins at atomic resolution are an essential prerequisite for mechanistic insight into protein function. However, in solution the protein exists as a dynamic equilibrium of global and local conformations. Therefore, a comprehensive understanding of protein function requires knowledge on the structure - dynamics relationship. We developed a novel hybrid approach based on single molecule Fo¨rster Resonance Energy Transfer [1, 2] that integrates hundreds of time resolved intramolecular distances together with x-ray structure information and MD simulations. Simultaneous determination of fluctuations and time resolved anisotropies enables a new level of accuracy and confidence. We demonstrate the power of our method by determining the dynamic structure of the molecular chaperone and heat shock protein Hsp90. Our resulting mean structure for the closed state of Hsp90 resembles ˚ . Beyond that, the corresponding x-ray structure of yeast Hsp90 [3] to 2.5 A we resolved the previously unknown open structure of this multi-domain protein. Surprisingly, the open state shows much larger fluctuations on relevant timescales than the closed state. Finally, we show how this method can be applied to quantify inter-domain dynamics and dynamics of small elements. We anticipate that this dynamic structure determination will solve many long standing questions regarding flexible parts in large proteins and proteinprotein interactions. [1] Muschielok A. et al.; Nature Methods 2008; 5, 965-971. [2] Kalinin S. et al.; Nature Methods 2012; 9(12):1218-1225. [3] Ali M.M. et al.; Nature 2006, 440(7087):1013-1017. 1107-Pos Board B84 Revealing a Heterodimeric Interface between the Members of Two Unrelated Fluorescent Protein Lineages Gary Ch Mo, Jin Zhang. Pharmacology, University of California San Diego, San Diego, CA, USA. Many fluorescent proteins (FPs) were developed from oligomeric predecessors. The use of monomeric FPs prevents oligomerization of the labeled protein of