| 1. |
- Andersson, Magnus, et al.
(author)
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A proposed time-resolved X-ray scattering approach to track local and global conformational changes in membrane transport proteins
- 2008
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In: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 16:1, s. 21-28
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Journal article (peer-reviewed)abstract
- Time-resolved X-ray scattering has emerged as a powerful technique for studying the rapid structural dynamics of small molecules in solution. Membrane-protein-catalyzed transport processes frequently couple large-scale conformational changes of the transporter with local structural changes perturbing the uptake and release of the transported substrate. Using light-driven halide ion transport catalyzed by halorhodopsin as a model system, we combine molecular dynamics simulations with X-ray scattering calculations to demonstrate how small-molecule time-resolved X-ray scattering can be extended to the study of membrane transport processes. In particular, by introducing strongly scattering atoms to label specific positions within the protein and substrate, the technique of time-resolved wide-angle X-ray scattering can reveal both local and global conformational changes. This approach simultaneously enables the direct visualization of global rearrangements and substrate movement, crucial concepts that underpin the alternating access paradigm for membrane transport proteins.
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| 2. |
- Andersson, Magnus, et al.
(author)
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Structural Dynamics of Light-Driven Proton Pumps
- 2009
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In: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 17:9, s. 1265-1275
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Journal article (peer-reviewed)abstract
- Bacteriorhodopsin and proteorhodopsin are simple heptahelical proton pumps containing a retinal chromophore covalently bound to helix G via a protonated Schiff base. Following the absorption of a photon, all-trans retinal is isomerized to a 13-cis conformation, initiating a sequence of conformational changes driving vectorial proton transport. In this study we apply time-resolved wide-angle X-ray scattering to visualize in real time the helical motions associated with proton pumping by bacteriorhodopsin and proteorhodopsin. Our results establish that three conformational states are required to describe their photocycles. Significant motions of the cytoplasmic half of helix F and the extracellular half of helix C are observed prior to the primary proton transfer event, which increase in amplitude following proton transfer. These results both simplify the structural description to emerge from intermediate trapping studies of bacteriorhodopsin and reveal shared dynamical principles for proton pumping.
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| 3. |
- Backmark, Anna, 1979, et al.
(author)
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Affinity tags can reduce merohedral twinning of membrane protein crystals
- 2008
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In: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; D64, s. 1183-1186
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Journal article (peer-reviewed)abstract
- This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
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| 4. |
- Davidsson, Jan, et al.
(author)
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Structural determination of a transient isomer of CH2I2 by picosecond x-ray diffraction
- 2005
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In: Physical Review Letters. - 0031-9007 .- 1079-7114. ; 94:24
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Journal article (peer-reviewed)abstract
- Ultrafast time-resolved spectroscopic studies of complex chemical reactions in solution are frequently hindered by difficulties in recovering accurate structural models for transient photochemical species. Time-resolved x-ray and electron diffraction have recently emerged as techniques for probing the structural dynamics of short lived photointermediates. Here we determine the structure of a transient isomer of photoexcited CH2I2 in solution and observe the downstream reactions of the initial photoproducts. Our results illustrate how geminate recombination proceeds via the formation of a transient covalent bond onto the iodine atom remaining with the parent molecule. Further intramolecular rearrangements are thus required for the CH2I-I isomer to return to CH2I2. The generation of I-3(-) from those iodine radicals escaping the solvent cage is also followed with time.
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| 5. |
- Fischer, Gerhard, 1978, et al.
(author)
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Crystal structure of a yeast aquaporin at 1.15 angstrom reveals a novel gating mechanism.
- 2009
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In: PLoS biology. - : Public Library of Science (PLoS). - 1545-7885 .- 1544-9173. ; 7:6
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Journal article (peer-reviewed)abstract
- Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 A resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.
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| 6. |
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| 7. |
- Georgiou, P., et al.
(author)
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Picosecond calorimetry: Time-resolved x-ray diffraction studies of liquid CH2Cl2
- 2006
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In: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 124:23, s. 234507-
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Journal article (peer-reviewed)abstract
- Liquid phase time-resolved x-ray diffraction with 100 ps resolution has recently emerged as a powerful technique for probing the structural dynamics of transient photochemical species in solution. It is intrinsic to the method, however, that a structural signal is observed not only from the photochemical of interest but also from the embedding solvent matrix. To experimentally characterize the x-ray diffraction signal deriving from the solvent alone we performed time-resolved diffraction studies of a pure liquid sample over a time domain from -250 ps to 2.5 mu s. Multiphoton excitation was used to rapidly heat liquid CH2Cl2 using UV pulses of 100 fs duration. A significant x-ray diffraction signal is visible prior to the onset of thermal expansion, which characterizes a highly compressed superheated liquid. Liquid CH2Cl2 then expands as a shock wave propagates through the sample and the temporal dependence of this phenomenon is in good agreement with theory. An unexpectedly slow initial release of energy into the liquid as heat is observed from multiphoton excited CH2Cl2, revealing the presence of a metastable state of multiphoton excited CH2Cl2.
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| 8. |
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| 9. |
- Gordon, Euan, 1971, et al.
(author)
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Effective high-throughput overproduction of membrane proteins in Escherichia coli
- 2008
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In: Protein Expression and Purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 62:1, s. 1-8
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Journal article (peer-reviewed)abstract
- Structural biology is increasingly reliant on elevated throughput methods for protein production. In particular, development of efficient methods of heterologous production of membrane proteins is essential. Here, we describe the heterologous overproduction of 24 membrane proteins from the human pathogen Legionella pneumophila in Escherichia coli. Protein production was performed in 0.5 ml cultures in standard 24-well plates, allowing increased throughput with minimal effort. The effect of the location of a histidine purification tag was analyzed, and the effect of decreasing the length of the N- and C-terminal extensions introduced by the Gateway cloning strategy is presented. We observed that the location and length of the purification tag significantly affected protein production levels. In addition, an auto-induction protocol for membrane protein expression was designed to enhance the overproduction efficiency such that, regardless of the construct used, much higher expression was achieved when compared with standard induction approaches such as isopropyl-β-d-thiogalactopyranoside (IPTG). All 24 targets were produced at levels exceeding 2 mg/l, with 18 targets producing at levels of 5 mg/l or higher. In summary, we have designed a fast and efficient process for the production of medically relevant membrane proteins with a minimum number of screening parameters.
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| 10. |
- Gourdon, Pontus Emanuel, 1978, et al.
(author)
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Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump
- 2008
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In: Protein Expression and Purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 58:1, s. 103-113
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Journal article (peer-reviewed)abstract
- Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0 mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5 mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.
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