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Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy Investigations of the Structure and Orientation of Membrane Proteins in Oriented Lipid Bilayers
Monday, February 29, 2016
Platform: Membrane Protein Structure and Folding II 951-Plat Measuring Reversible CLC-ec1 Dimerization In Membranes by Single Molecule Photobleaching Rahul Chadda1, Larry Friedman2, Mike Rigney2, Luci-Kolmakova Partensky2, Jeff Gelles2, Janice L. Robertson1. 1 University of Iowa, Iowa City, IA, USA, 2Brandeis Univeristy, Waltham, MA, USA. ClC-ec1 is a bacterial Cl-/Hþ antiporter that exists as a homodimer with each subunit containing a structurally independent pathway for ion transport. Mutations at the dimerization interface I201W/I422W (WW) destabilize the dimer in detergent while preserving function. In this study, we have applied single fluorescent molecule imaging and photobleaching to study the dimerization of an intermediately stable mutant; I422W (IW) in phospholipid membranes. WT CLC-ec1 and its mutants were purified from E. coli into detergent and labelled with Cy5-maleimide at a unique surface exposed cysteine residue. The dye/protein ratio for all samples were ~0.66. Cy5 labelled proteins were reconstituted in Alexa Fluor 488 SDP-ester labelled 2:1 POPE/POPG liposomes at 0.03 % fluorophore/lipid. Dialyzed liposomes were then freeze-thawed 7 times to form large multilamellar membranes, and then incubated at 25 C to facilitate protein mixing. Prior to imaging on a multi-color TIR-FM these membranes were extruded through a 400 nm filter. Protein reconstitution in liposomes follows a Poisson distribution resulting in liposomes containing either 0, 1, 2 or more protein molecules. We counted the overall occupancy of liposomes using two-color colocalization and counting the number of labelled protein particles in each liposome using step-wise photobleaching of Cy5. We determined the photo-bleaching distribution for dimeric WT, monomeric WW and intermediately stable IW. Upon increasing protein/lipid mole fraction from 1.5 x 10-9 to 7.5 x 10-4, we find that IW evolves from a monomer to dimer distribution. By carrying out a least-squares fit to the monomer and expected dimer distribution, we calculate Fdimer, I422W(X) that shows a density dependent increase that fits to a dimerization isotherm with KD=2x10-6 protein/lipid. The dimers dissociate into monomers upon diluting proteoliposomes with empty membranes suggesting that we are observing the reversible dimerization reaction in membranes. 952-Plat Mapping the Energy Landscape for Second Stage Folding of a Single Membrane Protein Duyoung Min1,2, Robert E. Jefferson1, James U. Bowie1, Tae-Young Yoon2. 1 Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, USA, 2Department of Physics, KAIST, Daejeon, Korea, Republic of. Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, E. coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (delta G = 6.5 kT) but a large unfolding barrier (21.3 kT) that can maintain the protein in a folded state for long periods of time (t1/2 ~ 3.5 hrs). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure. 953-Plat Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy Investigations of the Structure and Orientation of Membrane Proteins in Oriented Lipid Bilayers Luke S. Evans1, Rohanah Hussain2, Giuliano Siligardi2, Philip T.F. Williamson1. 1 Biological Sciences, University of Southampton, Southampton, United Kingdom, 2B23, Diamond Light Source, Oxfordshire, United Kingdom. Oriented SRCD spectroscopy is a challenging but revealing tool for investigating the structure of membrane proteins in lipid bilayers. Using the B23 beamtime at Diamond Light Source, Oxfordshire, UK, we have been developing methods for the preparation and measurement of oriented SRCD samples to characterise the structure of integral membrane proteins and their orientation with respect to the lipid bilayer. In the work presented here we outline two
191a
methods for the preparation of oriented membrane proteins. The first relies on mechanical orientation obtained by macroscopically aligning bilayers on a Suprasil quartz support. The second exploits magnetic fields to align bicelle membrane mimetics with the membrane director either parallel or perpendicular to the direction of propagation of the light beam. The merits and drawbacks of these two methods for the determination of membrane protein orientation will be discussed. As a model membrane protein for these measurements we are using the transmembrane domain (TMD) of a putative glycosyltransferase Fukutin-I (FkI), whose mislocalization has been linked to the onset of Fukuyama Muscular Dytrophy. The localisation of Fk-I in the late ER/Golgi is thought to be mediated through changes in oligomeric state and structure induced by the surrounding lipids. Using oriented samples for both SRCD and solid-state NMR we are beginning to understand how the distinct lipid composition within the late ER/Golgi may govern the retention of this protein in these compartments. 954-Plat Mechanisms of Assembly and Covalent Flavinylation in Complex II Chrystal Starbird1, Elena Maklashina2, Sany Rajagukguk2, Gary Cecchini2, Tina Iverson1. 1 Vanderbilt University, Nashville, TN, USA, 2University of California, San Francisco, California, USA, San Francisco, CA, USA. Complex II is an essential metabolic enzyme that functions in both the Krebs cycle and the electron transport chain in mitochondria, coupling succinate oxidation to fumarate with ubiquinone reduction to ubiquinol. Covalent FAD attachment to the flavoprotein subunit of this heterotetrameric complex is essential for succinate oxidation. Mutations that abrogate this covalent linkage result in pheochromocytoma-paraganglioma syndrome in humans although the underlying biochemistry is currently undefined. Several recently identified assembly factors are required for covalent flavin attachment, and mutations in the assembly factors can recapitulate clinical symptoms associated with Complex II deficiency. Understanding the mechanism of covalent flavin attachment is vital to understanding the function of Complex II and how this unique enzyme has adapted utilization of an FAD cofactor to couple two essential respiratory processes. Our work uses a combination of structural, biochemical and biophysical methods to investigate covalent flavinylation of Escherichia coli Complex II homologs, a process which may be mechanistically conserved from bacteria to mammals. Initial structural characterization of Escherichia coli Complex II homologs bearing mutations that prevent covalent linkage has provided insight into the potential role of inter-domain stability in the mechanism of covalent flavinylation. Additionally, site-specific photoaffinity crosslinking identified the binding location of the SdhE assembly factor on the E. coli Complex II flavoprotein, which suggests that this evolutionarily conserved assembly factor acts as a chaperone during covalent flavinylation. 955-Plat Probing the Structure and Binding of KCNE1 to the Voltage-Gated Potassium Channel KCNQ1 using Pulsed EPR Spectroscopy Gary A. Lorigan, Indra D. Sahu, Andrew Craig, Rongfu Zhang, Robert M. McCarrick. Chemistry and Biochemistry, Miami University, Oxford, OH, USA. Pulsed Electron Paramagnetic Resonance (EPR) spectroscopic techniques coupled with site-directed spin-labeling (SDSL) can provide important structural information on complicated biological systems such as membrane proteins. We are developing EPR techniques such as Double Electron-Electron Resonance (DEER) and Electron Spin Echo Envelope Modulation (ESEEM) for studying membrane proteins. Voltage-gated ion channels are essential for the electrical excitability of neurons, muscles and other excitable cells. KCNE1 is a single transmembrane protein that modulates the activity of voltage gated potassium ion channels (Kv). In the human heart, KCNE1 (E1) interacts with KCNQ1 (Q1) and decreases the rate of channel activation, increases conductance, and generates a slowly activating Kþ current critical for cardiac repolarization. Mutations on either KCNE1 or KCNQ1 genes in E1/Q1 complex can lead to long QT syndrome. Despite the biological significance of the Q1/E1 interaction, its exact nature is not fully understood. CWEPR, ESEEM and DEER measurements will be used to probe the structure and binding of KCNQ1 with KCNE1. ESEEM spectroscopy was used to probe the secondary structure of different regions of 2H labeled E1. DEER was also used to measure distances between the spin labels attached on E1 and Q1 separately in the Q1/E1 complex to determine the interacting sites in detail. These results provide direct evidence of binding of Q1 with E1 and will be very useful for determining the structural model of the Q1/E1 complex.
Platform: Membrane Protein Structure and Folding II 951-Plat Measuring Reversible CLC-ec1 Dimerization In Membranes by Single Molecule Photobleaching Rahul Chadda1, Larry Friedman2, Mike Rigney2, Luci-Kolmakova Partensky2, Jeff Gelles2, Janice L. Robertson1. 1 University of Iowa, Iowa City, IA, USA, 2Brandeis Univeristy, Waltham, MA, USA. ClC-ec1 is a bacterial Cl-/Hþ antiporter that exists as a homodimer with each subunit containing a structurally independent pathway for ion transport. Mutations at the dimerization interface I201W/I422W (WW) destabilize the dimer in detergent while preserving function. In this study, we have applied single fluorescent molecule imaging and photobleaching to study the dimerization of an intermediately stable mutant; I422W (IW) in phospholipid membranes. WT CLC-ec1 and its mutants were purified from E. coli into detergent and labelled with Cy5-maleimide at a unique surface exposed cysteine residue. The dye/protein ratio for all samples were ~0.66. Cy5 labelled proteins were reconstituted in Alexa Fluor 488 SDP-ester labelled 2:1 POPE/POPG liposomes at 0.03 % fluorophore/lipid. Dialyzed liposomes were then freeze-thawed 7 times to form large multilamellar membranes, and then incubated at 25 C to facilitate protein mixing. Prior to imaging on a multi-color TIR-FM these membranes were extruded through a 400 nm filter. Protein reconstitution in liposomes follows a Poisson distribution resulting in liposomes containing either 0, 1, 2 or more protein molecules. We counted the overall occupancy of liposomes using two-color colocalization and counting the number of labelled protein particles in each liposome using step-wise photobleaching of Cy5. We determined the photo-bleaching distribution for dimeric WT, monomeric WW and intermediately stable IW. Upon increasing protein/lipid mole fraction from 1.5 x 10-9 to 7.5 x 10-4, we find that IW evolves from a monomer to dimer distribution. By carrying out a least-squares fit to the monomer and expected dimer distribution, we calculate Fdimer, I422W(X) that shows a density dependent increase that fits to a dimerization isotherm with KD=2x10-6 protein/lipid. The dimers dissociate into monomers upon diluting proteoliposomes with empty membranes suggesting that we are observing the reversible dimerization reaction in membranes. 952-Plat Mapping the Energy Landscape for Second Stage Folding of a Single Membrane Protein Duyoung Min1,2, Robert E. Jefferson1, James U. Bowie1, Tae-Young Yoon2. 1 Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, USA, 2Department of Physics, KAIST, Daejeon, Korea, Republic of. Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, E. coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (delta G = 6.5 kT) but a large unfolding barrier (21.3 kT) that can maintain the protein in a folded state for long periods of time (t1/2 ~ 3.5 hrs). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure. 953-Plat Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy Investigations of the Structure and Orientation of Membrane Proteins in Oriented Lipid Bilayers Luke S. Evans1, Rohanah Hussain2, Giuliano Siligardi2, Philip T.F. Williamson1. 1 Biological Sciences, University of Southampton, Southampton, United Kingdom, 2B23, Diamond Light Source, Oxfordshire, United Kingdom. Oriented SRCD spectroscopy is a challenging but revealing tool for investigating the structure of membrane proteins in lipid bilayers. Using the B23 beamtime at Diamond Light Source, Oxfordshire, UK, we have been developing methods for the preparation and measurement of oriented SRCD samples to characterise the structure of integral membrane proteins and their orientation with respect to the lipid bilayer. In the work presented here we outline two
191a
methods for the preparation of oriented membrane proteins. The first relies on mechanical orientation obtained by macroscopically aligning bilayers on a Suprasil quartz support. The second exploits magnetic fields to align bicelle membrane mimetics with the membrane director either parallel or perpendicular to the direction of propagation of the light beam. The merits and drawbacks of these two methods for the determination of membrane protein orientation will be discussed. As a model membrane protein for these measurements we are using the transmembrane domain (TMD) of a putative glycosyltransferase Fukutin-I (FkI), whose mislocalization has been linked to the onset of Fukuyama Muscular Dytrophy. The localisation of Fk-I in the late ER/Golgi is thought to be mediated through changes in oligomeric state and structure induced by the surrounding lipids. Using oriented samples for both SRCD and solid-state NMR we are beginning to understand how the distinct lipid composition within the late ER/Golgi may govern the retention of this protein in these compartments. 954-Plat Mechanisms of Assembly and Covalent Flavinylation in Complex II Chrystal Starbird1, Elena Maklashina2, Sany Rajagukguk2, Gary Cecchini2, Tina Iverson1. 1 Vanderbilt University, Nashville, TN, USA, 2University of California, San Francisco, California, USA, San Francisco, CA, USA. Complex II is an essential metabolic enzyme that functions in both the Krebs cycle and the electron transport chain in mitochondria, coupling succinate oxidation to fumarate with ubiquinone reduction to ubiquinol. Covalent FAD attachment to the flavoprotein subunit of this heterotetrameric complex is essential for succinate oxidation. Mutations that abrogate this covalent linkage result in pheochromocytoma-paraganglioma syndrome in humans although the underlying biochemistry is currently undefined. Several recently identified assembly factors are required for covalent flavin attachment, and mutations in the assembly factors can recapitulate clinical symptoms associated with Complex II deficiency. Understanding the mechanism of covalent flavin attachment is vital to understanding the function of Complex II and how this unique enzyme has adapted utilization of an FAD cofactor to couple two essential respiratory processes. Our work uses a combination of structural, biochemical and biophysical methods to investigate covalent flavinylation of Escherichia coli Complex II homologs, a process which may be mechanistically conserved from bacteria to mammals. Initial structural characterization of Escherichia coli Complex II homologs bearing mutations that prevent covalent linkage has provided insight into the potential role of inter-domain stability in the mechanism of covalent flavinylation. Additionally, site-specific photoaffinity crosslinking identified the binding location of the SdhE assembly factor on the E. coli Complex II flavoprotein, which suggests that this evolutionarily conserved assembly factor acts as a chaperone during covalent flavinylation. 955-Plat Probing the Structure and Binding of KCNE1 to the Voltage-Gated Potassium Channel KCNQ1 using Pulsed EPR Spectroscopy Gary A. Lorigan, Indra D. Sahu, Andrew Craig, Rongfu Zhang, Robert M. McCarrick. Chemistry and Biochemistry, Miami University, Oxford, OH, USA. Pulsed Electron Paramagnetic Resonance (EPR) spectroscopic techniques coupled with site-directed spin-labeling (SDSL) can provide important structural information on complicated biological systems such as membrane proteins. We are developing EPR techniques such as Double Electron-Electron Resonance (DEER) and Electron Spin Echo Envelope Modulation (ESEEM) for studying membrane proteins. Voltage-gated ion channels are essential for the electrical excitability of neurons, muscles and other excitable cells. KCNE1 is a single transmembrane protein that modulates the activity of voltage gated potassium ion channels (Kv). In the human heart, KCNE1 (E1) interacts with KCNQ1 (Q1) and decreases the rate of channel activation, increases conductance, and generates a slowly activating Kþ current critical for cardiac repolarization. Mutations on either KCNE1 or KCNQ1 genes in E1/Q1 complex can lead to long QT syndrome. Despite the biological significance of the Q1/E1 interaction, its exact nature is not fully understood. CWEPR, ESEEM and DEER measurements will be used to probe the structure and binding of KCNQ1 with KCNE1. ESEEM spectroscopy was used to probe the secondary structure of different regions of 2H labeled E1. DEER was also used to measure distances between the spin labels attached on E1 and Q1 separately in the Q1/E1 complex to determine the interacting sites in detail. These results provide direct evidence of binding of Q1 with E1 and will be very useful for determining the structural model of the Q1/E1 complex.