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Time-Resolved Crystallography with Synchrotron and Free Electron Laser Sources
374a
Tuesday, March 1, 2016
Potassium channels are essential for selective permeation of potassium ions in cell membrane. The selectivity is achieved by the specific part called ‘‘selectivity filter’’, which is composed of several carbonyl groups centripetally toward the center of the pore. X-ray crystallography clearly showed that each potassium ion is favorably coordinated by eight carbonyl oxygens in the selectivity filters of various kinds of potassium channels. Further analysis was performed on a well-known potassium channel, KcsA, where the coordination manners of alkali metal cations in the selectivity filter are somewhat different from each other. In particular, sodium ion is coordinated by four carbonyl groups and the structure of the filter is deformed in the center of the filter. To understand the ion-protein interactions of potassium channels in more detail, the structure around the selectivity filter should be analyzed by other physicochemical methods, such as infrared difference spectroscopy. The infrared difference spectroscopy on KcsA with ion-exchange reaction was firstly reported in 2012 [1]. The significant spectral change was observed in the amide I mode of the peptide carbonyl groups, which were assigned to the selectivity filter and nearby pore helices by mutation [1] and a computational method [2]. The interactions with alkali metal cations were further investigated [3], revealing that the selectivity filter carbonyls coordinating Rbþ or Csþ adopt a conformation similar to those coordinating Kþ, while those coordinating Liþ or Naþ considerably differ from those coordinating Kþ. Similar approach applied on a mammalian potassium channel is now in progress. [1] Y. Furutani et al. J. Phys. Chem. Lett. 3, 3806-3810, 2012 [2] P. Stevenson et al. J. Phys. Chem. B 119, 5824-5831, 2015 [3] Y. Furutani et al. Biophysics and Physicobiology 12, 37-45, 2015 1841-Plat Investigation of M2 Proton Channel in Membrane by Rapid Laser pHJump Technique with TRP Fluorescence as a Probe Ban-Seok Jeong. Chemistry, Emory University, Atlanta, GA, USA. Protonsplay a central role in many important biological processes such as ATP synthesis, viral infection, and cellular transportation. The mechanism and kinetics of these fast proton-related processes could be studied by the rapid laser-induced pH-jump technique, utilizing photo-acids which can release protons in 20 ns. Many researchers have used the laser pH-jump technique to investigate kinetics of protein folding, acid denature, or EGFP chromophore protonation in sub-millisecond time regime, but there has been no laser pH-jump study about membrane proteins to date, although many proton-related processes such as cellular transportation and ATP synthesis are associated with membrane and channel proteins. In this study, therefore, the laser pH-jump experiment on a membrane protein, Influenza A M2 proton channel (M2), was first demonstrated. In the pH-jump experiment with a membrane, it was observed that the local proton concentration on the membrane surface was higher than bulk solution because of the interaction between membrane and photo-acid, resulting in faster protonation kinetics than in bulk solution. Moreover, we first utilized Trp fluorescence as a probe for laser pH-jump, expanding on its capability. By observing the Trp fluorescence change of M2, sub-millisecond time scale kinetics of M2 activation process was measured upon rapid acidification. Trp fluorescence change could be described by a double exponential decay function, indicating that the first exponential is attributed to histidine protonation and the second is associated with subsequent conformational change. The V27A mutant of M2 showed faster overall kinetics than WT due to the lack of valine neck. We believe that our results advance our mechanistic knowledge of the M2 activation and could pave the way for the investigation of dynamics, kinetics, and mechanism of other proton-related processes. 1842-Plat Spectroscopic Studies as a Biophysical Toolbox for Pharmacokinetic Drug Profiling Marlene Lu´cio. Centro de Fı´sica da Universidade do Minho, Braga, Portugal. The rationalization of the drug development process is a requirement both in pharmaceutical industries, and in academic research laboratories. Given the urge to improve the drug discovery process, in vitro screening assays are developed to measure the so called drug property profile which can be useful to medicinal chemists in deciding how to modify structures to improve properties of their drug candidates decreasing development time/cost and delay clinical introduction. Herein it is presented the study of the biophysical interactions of newly synthesized drugs with membrane model systems, using some systematic spectroscopic techniques to establish a pharmacokinetic drug profiling. Liposomes mimicking different body membrane barriers were prepared and further labelled with suitable membrane probes. The interactions of drugs with lipid membranes were used to determine their membrane/water partition coeffi-
cient, which is a predictor of drug affinity to the membranes as well as drug permeability and absorption. Further information about the degree of penetration of the drugs in the lipid membranes was also gained through in depth-dependent fluorescence quenching experiments (steady-state and time-resolved). In addition, it was possible to monitor the fluidity gradient through a bilayer leaflet by measurements of temperature dependent fluorescence anisotropy to evaluate the influence of the drugs changing the cooperativity and the main phase transition temperature which are important to predict toxic effects of drugs at the membrane level. Results obtained provide an insight at the use of spectroscopic techniques as a toolkit for in vitro screening of drug-membrane biophysical interactions to predict with confidence in vivo aspects related with drug absorption and distribution. Acknowledgements M.Lu´cio acknowledges FCT for the exploratory project with the reference IF/ 00498/2012. 1843-Plat Fluorescent Visualization of Cellular Ion Fluxes Lejie Zhang, William Kobertz. Biochemistry and Molecular Pharmacology, Umass Medical School, Worcester, MA, USA. The movement of ions across biological membranes is essential for generating electrical signals, regulating ion homeostasis and cell volume, and maintaining the resting membrane potential. Currently, electrophysiology is the primary tool to detect and measure ion efflux. Although electrical recordings are extremely precise, they do not report on the spatiotemporal flux of a specific ion from the landscape of an entire cell. We have developed an optical approach to detect ion efflux from living cells by covalently attaching small-molecule, fluorescent sensors specifically to the cell’s glycocalyx. Using this fluorescent sensor approach, proton efflux through voltage-gated proton channels (hHv-1) and Cl-/Hþ antiporters (ClC-5) was visualized at the surface of mammalian cells. Furthermore, inward proton fluxes from a Shaker ‘‘omega pore mutant’’ (R362H) were also fluorescently detected, demonstrating that our approach enables the visualization of both ion accumulation and depletion at the extracellular side of the membrane. The nuts and bolts of the labeling procedure and videos of cell surface ion accumulation and depletion will be presented.
Workshop: Time-resolved Crystallography 1844-Wkshp Time-Resolved Crystallography with Synchrotron and Free Electron Laser Sources Keith Moffat. University of Chicago, Chicago, IL, USA. Time-resolved crystallography uses the brief, intense X-ray pulses emitted by synchrotron or hard X-ray free electron laser (FEL) sources to probe the time course of structural changes as they occur in the molecules in a crystal, via pump - probe experiments. Synchrotron sources such as BioCARS sector 14 at the Advanced Photon Source, Argonne National Laboratory, can access the time scale from seconds to 100 picoseconds, where the lower limit is set by the duration of a single X-ray pulse. The new FEL sources such as the Linac Coherent Light Source at Stanford extend this limit to femtoseconds. The characteristics of the X-ray pulses emitted by synchrotron and FEL sources are radically different which necessitates new approaches to the pump - probe experiments and data analysis. For both synchrotron and FEL sources, reaction initiation typically requires light-sensitive systems in which reaction can be initiated by a visible laser pulse - but clearly, not all interesting biological systems are light-sensitive. This raises the question: how can sensitivity to light be conferred on otherwise light-inert systems, by optogenetic approaches? Ultrafast time-resolved crystallography will be illustrated by experiments conducted at synchrotron and FEL sources, that probe the structure of short-lived structural intermediates in the photocycle of the naturally-occurring bacterial blue light photoreceptor known as photoactive yellow protein, PYP. The principles that can be used to confer sensitivity to light on light-inert systems are illustrated by the design and characterization of a blue-light-sensitive histidine kinase. Finally I ask: are these principles equally applicable to experiments in the femtosecond time range at FEL sources. This research has been supported by NIH grants GM111072 and EY024363. 1845-Wkshp Structural Dynamics of Photoactive Yellow Protein Investigated by Time Resolved Serial Femtosecond Crystallography Marius Schmidt. University of Wisconsin at Milwaukee, Milwaukee, WI, USA.
Tuesday, March 1, 2016
Potassium channels are essential for selective permeation of potassium ions in cell membrane. The selectivity is achieved by the specific part called ‘‘selectivity filter’’, which is composed of several carbonyl groups centripetally toward the center of the pore. X-ray crystallography clearly showed that each potassium ion is favorably coordinated by eight carbonyl oxygens in the selectivity filters of various kinds of potassium channels. Further analysis was performed on a well-known potassium channel, KcsA, where the coordination manners of alkali metal cations in the selectivity filter are somewhat different from each other. In particular, sodium ion is coordinated by four carbonyl groups and the structure of the filter is deformed in the center of the filter. To understand the ion-protein interactions of potassium channels in more detail, the structure around the selectivity filter should be analyzed by other physicochemical methods, such as infrared difference spectroscopy. The infrared difference spectroscopy on KcsA with ion-exchange reaction was firstly reported in 2012 [1]. The significant spectral change was observed in the amide I mode of the peptide carbonyl groups, which were assigned to the selectivity filter and nearby pore helices by mutation [1] and a computational method [2]. The interactions with alkali metal cations were further investigated [3], revealing that the selectivity filter carbonyls coordinating Rbþ or Csþ adopt a conformation similar to those coordinating Kþ, while those coordinating Liþ or Naþ considerably differ from those coordinating Kþ. Similar approach applied on a mammalian potassium channel is now in progress. [1] Y. Furutani et al. J. Phys. Chem. Lett. 3, 3806-3810, 2012 [2] P. Stevenson et al. J. Phys. Chem. B 119, 5824-5831, 2015 [3] Y. Furutani et al. Biophysics and Physicobiology 12, 37-45, 2015 1841-Plat Investigation of M2 Proton Channel in Membrane by Rapid Laser pHJump Technique with TRP Fluorescence as a Probe Ban-Seok Jeong. Chemistry, Emory University, Atlanta, GA, USA. Protonsplay a central role in many important biological processes such as ATP synthesis, viral infection, and cellular transportation. The mechanism and kinetics of these fast proton-related processes could be studied by the rapid laser-induced pH-jump technique, utilizing photo-acids which can release protons in 20 ns. Many researchers have used the laser pH-jump technique to investigate kinetics of protein folding, acid denature, or EGFP chromophore protonation in sub-millisecond time regime, but there has been no laser pH-jump study about membrane proteins to date, although many proton-related processes such as cellular transportation and ATP synthesis are associated with membrane and channel proteins. In this study, therefore, the laser pH-jump experiment on a membrane protein, Influenza A M2 proton channel (M2), was first demonstrated. In the pH-jump experiment with a membrane, it was observed that the local proton concentration on the membrane surface was higher than bulk solution because of the interaction between membrane and photo-acid, resulting in faster protonation kinetics than in bulk solution. Moreover, we first utilized Trp fluorescence as a probe for laser pH-jump, expanding on its capability. By observing the Trp fluorescence change of M2, sub-millisecond time scale kinetics of M2 activation process was measured upon rapid acidification. Trp fluorescence change could be described by a double exponential decay function, indicating that the first exponential is attributed to histidine protonation and the second is associated with subsequent conformational change. The V27A mutant of M2 showed faster overall kinetics than WT due to the lack of valine neck. We believe that our results advance our mechanistic knowledge of the M2 activation and could pave the way for the investigation of dynamics, kinetics, and mechanism of other proton-related processes. 1842-Plat Spectroscopic Studies as a Biophysical Toolbox for Pharmacokinetic Drug Profiling Marlene Lu´cio. Centro de Fı´sica da Universidade do Minho, Braga, Portugal. The rationalization of the drug development process is a requirement both in pharmaceutical industries, and in academic research laboratories. Given the urge to improve the drug discovery process, in vitro screening assays are developed to measure the so called drug property profile which can be useful to medicinal chemists in deciding how to modify structures to improve properties of their drug candidates decreasing development time/cost and delay clinical introduction. Herein it is presented the study of the biophysical interactions of newly synthesized drugs with membrane model systems, using some systematic spectroscopic techniques to establish a pharmacokinetic drug profiling. Liposomes mimicking different body membrane barriers were prepared and further labelled with suitable membrane probes. The interactions of drugs with lipid membranes were used to determine their membrane/water partition coeffi-
cient, which is a predictor of drug affinity to the membranes as well as drug permeability and absorption. Further information about the degree of penetration of the drugs in the lipid membranes was also gained through in depth-dependent fluorescence quenching experiments (steady-state and time-resolved). In addition, it was possible to monitor the fluidity gradient through a bilayer leaflet by measurements of temperature dependent fluorescence anisotropy to evaluate the influence of the drugs changing the cooperativity and the main phase transition temperature which are important to predict toxic effects of drugs at the membrane level. Results obtained provide an insight at the use of spectroscopic techniques as a toolkit for in vitro screening of drug-membrane biophysical interactions to predict with confidence in vivo aspects related with drug absorption and distribution. Acknowledgements M.Lu´cio acknowledges FCT for the exploratory project with the reference IF/ 00498/2012. 1843-Plat Fluorescent Visualization of Cellular Ion Fluxes Lejie Zhang, William Kobertz. Biochemistry and Molecular Pharmacology, Umass Medical School, Worcester, MA, USA. The movement of ions across biological membranes is essential for generating electrical signals, regulating ion homeostasis and cell volume, and maintaining the resting membrane potential. Currently, electrophysiology is the primary tool to detect and measure ion efflux. Although electrical recordings are extremely precise, they do not report on the spatiotemporal flux of a specific ion from the landscape of an entire cell. We have developed an optical approach to detect ion efflux from living cells by covalently attaching small-molecule, fluorescent sensors specifically to the cell’s glycocalyx. Using this fluorescent sensor approach, proton efflux through voltage-gated proton channels (hHv-1) and Cl-/Hþ antiporters (ClC-5) was visualized at the surface of mammalian cells. Furthermore, inward proton fluxes from a Shaker ‘‘omega pore mutant’’ (R362H) were also fluorescently detected, demonstrating that our approach enables the visualization of both ion accumulation and depletion at the extracellular side of the membrane. The nuts and bolts of the labeling procedure and videos of cell surface ion accumulation and depletion will be presented.
Workshop: Time-resolved Crystallography 1844-Wkshp Time-Resolved Crystallography with Synchrotron and Free Electron Laser Sources Keith Moffat. University of Chicago, Chicago, IL, USA. Time-resolved crystallography uses the brief, intense X-ray pulses emitted by synchrotron or hard X-ray free electron laser (FEL) sources to probe the time course of structural changes as they occur in the molecules in a crystal, via pump - probe experiments. Synchrotron sources such as BioCARS sector 14 at the Advanced Photon Source, Argonne National Laboratory, can access the time scale from seconds to 100 picoseconds, where the lower limit is set by the duration of a single X-ray pulse. The new FEL sources such as the Linac Coherent Light Source at Stanford extend this limit to femtoseconds. The characteristics of the X-ray pulses emitted by synchrotron and FEL sources are radically different which necessitates new approaches to the pump - probe experiments and data analysis. For both synchrotron and FEL sources, reaction initiation typically requires light-sensitive systems in which reaction can be initiated by a visible laser pulse - but clearly, not all interesting biological systems are light-sensitive. This raises the question: how can sensitivity to light be conferred on otherwise light-inert systems, by optogenetic approaches? Ultrafast time-resolved crystallography will be illustrated by experiments conducted at synchrotron and FEL sources, that probe the structure of short-lived structural intermediates in the photocycle of the naturally-occurring bacterial blue light photoreceptor known as photoactive yellow protein, PYP. The principles that can be used to confer sensitivity to light on light-inert systems are illustrated by the design and characterization of a blue-light-sensitive histidine kinase. Finally I ask: are these principles equally applicable to experiments in the femtosecond time range at FEL sources. This research has been supported by NIH grants GM111072 and EY024363. 1845-Wkshp Structural Dynamics of Photoactive Yellow Protein Investigated by Time Resolved Serial Femtosecond Crystallography Marius Schmidt. University of Wisconsin at Milwaukee, Milwaukee, WI, USA.