Georg Büldt

Moscow Institute of Physics and Technology, Moskva, Moscow, Russia

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Publications (98)585.3 Total impact

  • Crystal Growth & Design 11/2015; DOI:10.1021/acs.cgd.5b01061 · 4.89 Impact Factor
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    ABSTRACT: Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.
    Nature Structural & Molecular Biology 04/2015; 22(5). DOI:10.1038/nsmb.3002 · 13.31 Impact Factor
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    ABSTRACT: Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein's fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies.
    PLoS ONE 12/2014; 9(12):e112873. DOI:10.1371/journal.pone.0112873 · 3.23 Impact Factor
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    ABSTRACT: X-ray-radiation-induced alterations to protein structures are still a severe problem in macromolecular crystallography. One way to avoid the influence of radiation damage is to reduce the X-ray dose absorbed by the crystal during data collection. However, here it is demonstrated using the example of the membrane protein bacteriorhodopsin (bR) that even a low dose of less than 0.06 MGy may induce structural alterations in proteins. This dose is about 500 times smaller than the experimental dose limit which should ideally not be exceeded per data set ( i.e. 30 MGy) and 20 times smaller than previously detected specific radiation damage at the bR active site. To date, it is the lowest dose at which radiation modification of a protein structure has been described. Complementary use was made of high-resolution X-ray crystallography and online microspectrophotometry to quantitatively study low-dose X-ray-induced changes. It is shown that structural changes of the protein correlate with the spectroscopically observed formation of the so-called bR orange species. Evidence is provided for structural modifications taking place at the protein active site that should be taken into account in crystallographic studies which aim to elucidate the molecular mechanisms of bR function.
    Acta Crystallographica Section D Biological Crystallography 10/2014; 70(10). DOI:10.1107/S1399004714017295 · 2.67 Impact Factor
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    ABSTRACT: We report a time-resolved fluorescence anisotropy study of ribosome-bound nascent chains (RNCs) of calmodulin (CaM), a prototypical member of the EF-hand family of calcium-sensing proteins. As shown in numerous studies, in vitro protein refolding can differ substantially from biosynthetic protein folding, which takes place cotranslationally and depends on the rate of polypeptide chain elongation. A challenge in this respect is to characterize the adopted conformations of nascent chains before their release from the ribosome. CaM RNCs (full-length, half-length, and first EF-hand only) were synthesized in vitro. All constructs contained a tetracysteine motif site-specifically incorporated in the first N-terminal helix; this motif is known to react with FlAsH, a biarsenic fluorescein derivative. As the dye is rotationally locked to this helix, we characterized the structural properties and folding states of polypeptide chains tethered to ribosomes and compared these with released chains. Importantly, we observed decelerated tumbling motions of ribosome-tethered and partially folded nascent chains, compared to released chains. This indicates a pronounced interaction between nascent chains and the ribosome surface, and might reflect chaperone activity of the ribosome.
    ChemBioChem 05/2014; 15(7). DOI:10.1002/cbic.201400014 · 3.09 Impact Factor
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    Biophysical Journal 01/2014; 106(2):669a. DOI:10.1016/j.bpj.2013.11.3708 · 3.97 Impact Factor
  • Valentin Borshchevskiy · Georg Büldt ·
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    ABSTRACT: Arrestin proteins regulate cellular signalling cascades initiated by ubiquitous G-protein-coupled receptors. Crystal structures reveal that two arrestins undergo similar structural changes on activation. See Letters p.137 & p.142
    Nature 04/2013; 497(7447). DOI:10.1038/nature12096 · 41.46 Impact Factor
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    ABSTRACT: The complex of sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII) mediates negative phototaxis in halobacteria Natronomonas pharaonis. Upon light activation NpSRII triggers, by means of NpHtrII, a signal transduction chain homologous to the two component system in eubacterial chemotaxis. Here we report on the crystal structure of the ground state of the mutant NpSRII-D75N/NpHtrII complex in the space group I212121. Mutations of this aspartic acid in light-driven proton pumps dramatically modify or/and inhibit protein functions. However, in vivo studies show that the similar D75N mutation retains functionality of the NpSRII/NpHtrII complex. The structure provides the molecular basis for the explanation of the unexpected observation that the wild and the mutant complexes display identical physiological response on light excitation.
    Journal of photochemistry and photobiology. B, Biology 03/2013; 123C:55-58. DOI:10.1016/j.jphotobiol.2013.03.008 · 2.96 Impact Factor
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    ABSTRACT: SynonymsPhoborhodopsinDefinitionSensory rhodopsin II belongs to the microbial rhodopsins, which constitute a family of seven-helix membrane proteins with the chromophore retinal. Members of this family are distributed throughout the Bacteria, Archaea, and Eukaryota. These photoactive proteins use a common structural design for two distinct functions: light-driven ion transport and phototaxis. The sensors start a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis. The connecting membrane protein between the photoreceptor and the following cytoplasmic signal cascade is formed by a transducer molecule that binds tightly and specifically to its cognate receptor by means of two transmembrane helices (TM1 and TM2) (Gordeliy et al. 2002).Basic CharacteristicsThe discovery of purple membrane from Halobacterium salinarum and its constituent bacteriorhodopsin (bR) more than 40 years ago (Oesterhel and Stoeckenius ...
    Encyclopedia of Biophysics, 01/2013: pages 2312-2315; , ISBN: 978-3-642-16711-9
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    ABSTRACT: We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i) the stabilization of membrane proteins in the meso phase, (ii) the control of hydration level and additive concentration by vapor diffusion. The new technology (iii) significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv) direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR) crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII) from Halobacterium salinarum for the first time.
    PLoS ONE 04/2012; 7(4):e35458. DOI:10.1371/journal.pone.0035458 · 3.23 Impact Factor
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    ABSTRACT: Visual arrestin specifically binds to photoactivated and phosphorylated rhodopsin and inactivates phototransduction. In contrast, the p44 splice variant can terminate phototransduction by binding to nonphosphorylated light-activated rhodopsin. Here we report the crystal structure of bovine p44 at a resolution of 1.85 Å. Compared to native arrestin, the p44 structure reveals significant differences in regions crucial for receptor binding, namely flexible loop V-VI and polar core regions. Additionally, electrostatic potential is remarkably positive on the N-domain and the C-domain. The p44 structure represents an active conformation that serves as a model to explain the 'constitutive activity' found in arrestin variants.
    Journal of Molecular Biology 03/2012; 416(5):611-8. DOI:10.1016/j.jmb.2012.01.028 · 4.33 Impact Factor
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    Biophysical Journal 01/2012; 102(3):268a. DOI:10.1016/j.bpj.2011.11.1473 · 3.97 Impact Factor
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    ABSTRACT: Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the "phosphate-sensor" of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins.
    Proceedings of the National Academy of Sciences 11/2011; 108(46):18690-5. DOI:10.1073/pnas.1015461108 · 9.67 Impact Factor
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    ABSTRACT: The molecular mechanism of transmembrane signal transduction is still a pertinent question in cellular biology. Generally, a receptor can transfer an external signal via its cytoplasmic surface, as found for G-protein-coupled receptors such as rhodopsin, or via the membrane domain, such as that in sensory rhodopsin II (SRII) in complex with its transducer, HtrII. In the absence of HtrII, SRII functions as a proton pump. Here, we report on the crystal structure of the active state of uncomplexed SRII from Natronomonas pharaonis, NpSRII. The problem with a dramatic loss of diffraction quality upon loading of the active state was overcome by growing better crystals and by reducing the occupancy of the state. The conformational changes in the region comprising helices F and G are similar to those observed for the NpSRII-transducer complex but are much more pronounced. The meaning of these differences for the understanding of proton pumping and signal transduction by NpSRII is discussed.
    Journal of Molecular Biology 08/2011; 412(4):591-600. DOI:10.1016/j.jmb.2011.07.022 · 4.33 Impact Factor
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    ABSTRACT: The structure-function relationship of the light-driven proton pump bacteriorhodopsin in the purple membrane was investigated by neutron diffraction, X-ray synchrotron radiation, and optical spectroscopy with pH-indicators. Based on experimental data and molecular modelling, a molecular mechanism explaining proton pumping is discussed.
    Membrane Proteins: Structures, Interactions and Models, 07/2011: pages 69-84;
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    ABSTRACT: Bacteriorhodopsin (bR) provides light-driven vectorial proton transport across a cell membrane. Creation of electrochemical potential at the membrane is a universal step in energy transformation in a cell. Published atomic crystallographic models of early intermediate states of bR show a significant difference between them, and conclusions about pumping mechanisms have been contradictory. Here, we present a quantitative high-resolution crystallographic study of conformational changes in bR induced by X-ray absorption. It is shown that X-ray doses that are usually accumulated during data collection for intermediate-state studies are sufficient to significantly alter the structure of the protein. X-ray-induced changes occur primarily in the active site of bR. Structural modeling showed that X-ray absorption triggers retinal isomerization accompanied by the disappearance of electron densities corresponding to the water molecule W402 bound to the Schiff base. It is demonstrated that these and other X-ray-induced changes may mimic functional conformational changes of bR leading to misinterpretation of the earlier obtained X-ray crystallographic structures of photointermediates.
    Journal of Molecular Biology 06/2011; 409(5):813-25. DOI:10.1016/j.jmb.2011.04.038 · 4.33 Impact Factor
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    ABSTRACT: We show that optical tweezers are a valuable tool to study the co-translational folding of a nascent polypeptide chain at the ribosome in real-time. The aim of this study was to demonstrate that a stable and intact population of ribosomes can be tethered to polystyrene beads and that specific hook-ups to the nascent polypeptide chain by dsDNA handles, immobilized on a second bead, can be detected. A rupture force of the nascent chain in the range of 10-50 pN was measured, which demonstrates that the system is anchored to the surface in a stable and specific way. This will allow in numerous future applications to follow protein folding using much lower forces.
    FEBS letters 06/2011; 585(12):1859-63. DOI:10.1016/j.febslet.2011.04.045 · 3.17 Impact Factor
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    ABSTRACT: Here we report a successful use of a recently developed isoprenoid-chained lipid family for in meso crystallization of membrane proteins. The isoprenoid-chained lipid 1-O-(3,7,11,15-tetramethylhexadecyl)-β–d-xyloside (β-XylOC16+4) used as a host lipid for in meso crystallization provided high quality bacteriorhodopsin (bR) crystals (P63 space group) diffracting to high resolution and characterized by low twinning ratio. β-XylOC16+4 has an isoprenoid chain with methyl branches at each 4th position and a xylose group in the water-soluble part. These peculiarities make the lipid clearly distinguishable in the bR crystalline lattice and provides a unique opportunity to study the role of the host lipid in the in meso crystallization. We conclude that β-XylOC16+4 may have a general application for in meso crystallization for a wide range of membrane proteins. The cubic phase of β-XylOC16+4 is present over a wide range of temperatures and is stable at low temperature (down to about 8 °C). This opens up the possibility of using temperature as a tool for the optimization of in meso crystallization with additional advantages for the crystallization of membrane proteins at lower temperatures where the proteins of interest may be more stable.
    Journal of Crystal Growth 11/2010; 312(22-312):3326-3330. DOI:10.1016/j.jcrysgro.2010.08.018 · 1.70 Impact Factor
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    ABSTRACT: Sensory rhodopsin I (SRI) from Halobacterium salinarum mediates both positive and negative phototaxis in a light-dependent manner. SRI photoactivation elicits extensive structural changes which are transmitted to the cognate transducer (HtrI). The atomic structure of the SRI-HtrI complex has not been solved yet and, therefore, details on the interaction which define the binding site between receptor and transducer are missing. The related complex SRII-HtrII from Natronobacterium pharaonis exhibits a hydrogen bond between the receptor Y199 and transducer N54. This bond has been suggested to mediate signal relay in the SRII-HtrII system. Our previous results on the SRI-HtrI complex indicated that HtrI N53 forms a hydrogen bond at the cytoplasm-proximity of the membrane. Here, based on kinetic and spectroscopic data, we demonstrate that Y210 of SRI is functionally significant for the signal relay in the SRI-HtrI complex. Each of the tyrosine residues Y197, Y208, Y210 and Y213 were conservatively exchanged for phenylalanine but only the Y210F mutation led to the disappearance of the infrared band of the terminal amide C=O of N53. From this FT-IR spectroscopic result, we conclude that Y210 of SRI and N53 of HtrI interact via a hydrogen bond which is crucial for the signal transfer from the light receptor to the transducer.
    Biophysical chemistry 03/2010; 150(1-3):23-8. DOI:10.1016/j.bpc.2010.02.017 · 1.99 Impact Factor
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    ABSTRACT: It's not easy being green: Real-time visualization of labeled ribosomes and de novo synthesized green fluorescent protein molecules using single-molecule-sensitive fluorescence microscopy demonstrates that the mutant GFPem is produced with a characteristic time of five minutes. Fluorescence of the fastest GFP molecules appears within one minute (see picture).
    Angewandte Chemie International Edition 01/2010; 48(10):1758-61. DOI:10.1002/anie.200806070 · 11.26 Impact Factor

Publication Stats

4k Citations
585.30 Total Impact Points


  • 2011-2014
    • Moscow Institute of Physics and Technology
      Moskva, Moscow, Russia
  • 1996-2012
    • Forschungszentrum Jülich
      • • Institute of Complex Systems (ICS)
      • • Molecular Biophysics (ICS-5)
      Düren, North Rhine-Westphalia, Germany
  • 2003
    • Chonbuk National University
      • Department of Chemistry
      Seoul, Seoul, South Korea
  • 2002
    • Max Planck Institute of Molecular Physiology
      Dortmund, North Rhine-Westphalia, Germany
  • 1986-1995
    • Freie Universität Berlin
      • • Department of Physics
      • • Division of Geophysics
      Berlín, Berlin, Germany
  • 1976-1995
    • Universität Basel
      • Department of Biophysical Chemistry
      Bâle, Basel-City, Switzerland