Wolfram Saenger - Academia.edu (original) (raw)

Papers by Wolfram Saenger

Journal of Biomolecular Structure & Dynamics, 1989

Journal of Computational Chemistry, 1987

Page 1. Cooperative Effects in Extended Hydrogen Bonded Systems Involving 0-H Groups. Ab Initio S... more Page 1. Cooperative Effects in Extended Hydrogen Bonded Systems Involving 0-H Groups. Ab Initio Studies of the Cyclic S4 Water Tetramer J. E. H. Koehler and W. Saenger Znstitut fur Kristallographie, Freie Uniuersitat Berlin, Takustr. ...

Biochemistry, 2005

The oxygen-evolving photosystem II core complexes (PSIIcc) from the thermophilic cyanobacterium T... more The oxygen-evolving photosystem II core complexes (PSIIcc) from the thermophilic cyanobacterium Thermosynechococcus elongatus (PSIIccTe) and the higher plant Spinacia oleracea (PSIIccSo) have been isolated from the thylakoid membrane by solubilization with n-dodecyl--D-maltoside, purified and characterized by gel permeation chromatography (GPC), dynamic light scattering (DLS), and analytical ultracentrifugation (AUC). DLS suggests that PSIIcc from both organisms exists as a monomer in dilute solution and aggregates with increasing protein concentration. In contrast to DLS, GPC and AUC showed that PSIIcc of both organisms occur as monomers and dimers, and it became clear from our studies that calibration of GPC columns with soluble proteins leads to wrong estimates of the molecular masses of membrane proteins. At a PSIIcc protein concentration of 0.2 mg/mL, molar masses, M, of 756 ( 18 kDa and 710 ( 15 kDa for dimeric PSIIccTe and PSIIccSo, respectively, were determined by analytical ultracentrifugation. At very low protein concentrations, at or below 0.05 mg/ mL, the dimeric form of PSIIccTe partially dissociates (20-30%) to form monomers. On the basis of these studies 3-dimensional crystals of PSIIccTe were obtained that contain dimers in the asymmetric unit [Zouni, A. et al. (2001) Nature 409, 739-743]. Using synchrotron radiation the crystals diffract to a resolution of 3.8 Å, which has been improved recently to 3.

Biochemistry, 2001

Fluorescence correlation spectroscopy (FCS) was used to characterize the interaction of fluoresce... more Fluorescence correlation spectroscopy (FCS) was used to characterize the interaction of fluorescence labeled single-stranded DNA (ssDNA) with hexameric RepA DNA-helicase (hRepA) encoded by plasmid RSF1010. The apparent dissociation constants, Kd(app), for the equilibrium binding of 12mer, 30mer, and 45mer ssDNA 5'-labeled with BFL to hRepA dimer in the presence of 0.5 mM ATPgammaS at pH 5.8 and 25 degrees C were determined to be 0.58 +/- 0.12, 0.52 +/- 0.07, and 1.66 +/- 0.32 microM, respectively. Binding curves are compatible with one binding site for ssDNA present on hRepA dimer, with no indication of cooperativity. At pH 7.6 in the presence of ATPgammaS and at pH 5.8 in the absence of ATPgammaS, complex formation between ssDNA and hRepA was too weak for measuring complete binding curves by FCS. Under these conditions, the dissociation constant, Kd(app), is in the range between 10 and 250 microM. The kinetics of complex formation at pH 5.8 are faster than the time resolution (approximately 10-20 s) of FCS experiments under pseudo-first-order conditions, with respect to BFL-ssDNA. Photon correlation spectroscopy (PCS) experiments yielded, within the experimental error range, the same values for the apparent hydrodynamic radii, R(h), of hRepA dimer and its complex with ssDNA as determined by FCS (R(h) = 6.6 +/- 1 nm). hRepA starts to aggregate under acidic conditions (<pH 6.0) which are optimal for ssDNA binding. CD spectra taken at pH 5.8 in the presence of ATPgammaS showed a structural change induced by ssDNA binding to hRepA which is not visible at pH 7.6 and with ADP as nucleotide cofactor.

European journal of biochemistry / FEBS, 1988

The crystal and molecular structure of proteinase K was determined by X-ray diffraction data to 0... more The crystal and molecular structure of proteinase K was determined by X-ray diffraction data to 0.15-nm resolution. The enzyme belongs to the subtilisin family with an active-site catalytic triad Asp39--His69--Ser224 but is a representative of a subgroup with a free Cys73 close to and 'below' the active His69. Besides this Cys72, proteinase K has two disulfide bonds, Cys34--Cys123 and Cys178--Cys249, which contribute to the stability of the tertiary structure consisting of an extended central parallel beta-sheet decorated by six alpha-helices, three short antiparallel beta-sheets, 18 beta-turns and involving several internal, structurally important water molecules. Proteinase K exhibits two Ca2+-binding sites, one very strong and the other weak, which were the sites of the heavy atoms (Pb2+, Sm3+) used to solve the crystal structure. The weak binding site is liganded to the N and C termini, Thr16 and Asp260, and is only incompletely coordinated by oxygen ligands. The strong ...

Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2007

PLoS ONE, 2009

Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase pl... more Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD) of H. pylori DnaB (HpDnaB) helicase at 2.2 Å resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.

Journal of Inclusion Phenomena and Macrocyclic Chemistry - J INCL PHENOM MACROCYCL CHEM, 1983

a-Cyclodextrin, a torus shaped molecule with a 5 Å wide central cavity, forms a number of deep gr... more a-Cyclodextrin, a torus shaped molecule with a 5 Å wide central cavity, forms a number of deep green, blue and black crystals when complexed with iodine/metal iodide. In contrast, ß-cyclodextrin, having a 6 Å cavity produces only one type of reddish-brown crystal, no matter what metal iodide is used. The complex (ß-cyclodextrin)2 ·KI7·9H2O displays space groupP21 (pseudo-C2) with cell constantsa=19.609(5),b=24.513(7),c=15.795(6)Å, ß=109.50(2)°,Z=4. The crystal structure was solved inC2 on the basis of 3022 absorption corrected CuKa (Ni-filter) X-ray intensities and refined by full matrix least squares toR=17%. This relatively highR-factor is due to many weak reflections (pseudo-C2) and considerable disorder exhibited by water and iodine. In the complex, ß-cyclodextrin adopts a ‘round’ shape with O(2)...O(3) interglucose hydrogen bonds formed and all O(6) hydroxyls pointing away from the cavity. Two molecules are arranged head-to-head to produce a dimer, and dimers are stacked such t...

European Journal of Biochemistry, 2001

In a series of four racemic phenoxyalkyl-alkyl carbinols, 1-phenoxy-2-hydroxybutane (1) is enanti... more In a series of four racemic phenoxyalkyl-alkyl carbinols, 1-phenoxy-2-hydroxybutane (1) is enantioselectively acetylated by Burkholderia cepacia (formerly Pseudomonas cepacia) lipase with an E value $ 200, whereas for the other three racemates E was found to be # 4. To explain the high preference of B. cepacia lipase for (R)-(1)-1, a precursor of its transition state analogue with a tetrahedral P-atom, (R P ,S P )-O-(2R)-(1-phenoxybut-2-yl)-methylphos-phonic acid chloride was prepared and crystallized in complex with B. cepacia lipase. The X-ray structure of the complex was determined, allowing to compare the conformation of the inhibitor with results of molecular modelling.

Science, 1994

The crystal structure of beta-D-cellotetraose shows the same molecule packing as cellulose II, wi... more The crystal structure of beta-D-cellotetraose shows the same molecule packing as cellulose II, with two antiparallel molecules in the unit cell: For cellulose II, the orientation of the C6-O6 bonds has been described as gauche-trans and trans-gauche, respectively, for the two antiparallel molecules, which otherwise have identical conformations. In contrast, in beta-D-cellotetraose all C6-O6 bonds are gauche-trans, but the conformations of the two antiparallel molecules are different. Energy minimization and molecular dynamics studies suggest that the structure of cellulose II should be reinvestigated in light of these findings.

Science, 2006

Oxidation of water to dioxygen is catalyzed within photosystem II (PSII) by a Mn4Ca cluster, the ... more Oxidation of water to dioxygen is catalyzed within photosystem II (PSII) by a Mn4Ca cluster, the structure of which remains elusive. Polarized extended X-ray absorption fine structure (EXAFS) measurements on PSII single crystals constrain the Mn4Ca cluster geometry to a set of three similar high-resolution structures. Combining polarized EXAFS and X-ray diffraction data, the cluster was placed within PSII taking into account the overall trend of the electron density of the metal site and the putative ligands. The structure of the cluster from the present study is unlike either the 3.0 or 3.5 Angstrom resolution X-ray structures, and other previously proposed models.

Proteins: Structure, Function, and Genetics, 1988

Proteinase K, the extracellular serine endopeptidase (E.C.3.4.21.14) from the fungus Tritirachium... more Proteinase K, the extracellular serine endopeptidase (E.C.3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl ketone to the active site of proteinase K was first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 A and 5.0 A resolution. The protein inhibitor complex was refined by restrained least-squares minimization with the data between 10.0 and 1.8 A. The final R factor was 19.1%, and the model contained 2,018 protein atoms, 28 inhibitor atoms, 125 water molecules, and two Ca2+ ions. The peptide portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.

Proteins: Structure, Function, and Bioinformatics, 2004

Protein Science, 2008

Although there is X-ray crystallographic evidence that the interaction between major histocompati... more Although there is X-ray crystallographic evidence that the interaction between major histocompatibility complex (MHC, in humans HLA) class I molecules and T cell receptors (TCR) or killer cell Ig-like receptors (KIR) may be accompanied by considerable changes in the conformation of selected residues or even entire loops within TCR or KIR, conformational changes between receptor-bound and -unbound MHC class I molecules of comparable magnitude have not been observed so far. We have previously determined the structure of the MHC class I molecule HLA-A1 bound to a melanoma antigen-encoding gene (MAGE)-A1-derived peptide in complex with a recombinant antibody fragment with TCR-like specificity, Fab-Hyb3. Here, we compare the X-ray structure of HLA-A1:MAGE-A1 with that complexed with Fab-Hyb3 to gain insight into structural changes of the MHC molecule that might be induced by the interaction with the antibody fragment. Apart from the expulsion of several water molecules from the interface, Fab-Hyb3 binding results in major rearrangements (up to 5.5 Å ) of heavy chain residues Arg65, Gln72, Arg145, and Lys146. Residue 65 is frequently and residues 72 and 146 are occasionally involved in TCR binding-induced conformational changes, as revealed by a comparison with MHC class I structures in TCR-liganded and -unliganded forms. On the other hand, residue 145 is subject to a reorientation following engagement of HLA-Cw4 and KIR2DL1. Therefore, conformational changes within the HLA-A1:MAGE-A1:Fab-Hyb3 complex include MHC residues that are also involved in reorientations in complexes with natural ligands, pointing to their central importance for the peptide-dependent recognition of MHC molecules.

Proceedings of the National Academy of Sciences, 1999

Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent ant... more Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a ␤-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Gln-Pro-Ser-Asn, with the second, third, and sixth position present in the D-configuration. The gene cluster from B. subtilis ATCC6633 specifying the biosynthesis of mycosubtilin was identified. The putative operon spans 38 kb and consists of four ORFs, designated fenF, mycA, mycB, and mycC, with strong homologies to the family of peptide synthetases. Biochemical characterization showed that MycB specifically adenylates tyrosine, as expected for mycosubtilin synthetase, and insertional mutagenesis of the operon resulted in a mycosubtilin-negative phenotype. The mycosubtilin synthetase reveals features unique for peptide synthetases as well as for fatty acid synthases: (i) The mycosubtilin synthase subunit A (MycA) combines functional domains derived from peptide synthetases, amino transferases, and fatty acid synthases. MycA represents the first example of a natural hybrid between these enzyme families. (ii) The organization of the synthetase subunits deviates from that commonly found in peptide synthetases. On the basis of the described characteristics of the mycosubtilin synthetase, we present a model for the biosynthesis of iturin lipopeptide antibiotics. Comparison of the sequences flanking the mycosubtilin operon of B. subtilis ATCC6633, with the complete genome sequence of B. subtilis strain 168 indicates that the fengycin and mycosubtilin lipopeptide synthetase operons are exchanged between the two B. subtilis strains.

Philosophical Transactions of the Royal Society B: Biological Sciences, 2002

The structure of photosystem I at 3.8 A c resolution illustrated the main structural elements of ... more The structure of photosystem I at 3.8 A c resolution illustrated the main structural elements of the wateroxidizing photosystem II complex, including the constituents of the electron transport chain. The location of the Mn cluster within the complex has been identi ed for the rst time to our knowledge. At this resolution, no individual atoms are visible, however, the electron density of the Mn cluster can be used to discuss both the present models of the Mn cluster as revealed from various spectroscopic methods and the implications for the mechanisms of water oxidation. Twenty-six chlorophylls from the antenna system of photosystem II have been identi ed. They are arranged in two layers, one close to the stromal side and one close to the lumenal side. Comparing the structure of the antenna system of photosystem II with the chlorophyll arrangement in photosystem I, which was recently determined at 2.5 A c resolution shows that photosystem II lacks the central domain of the photosystem I antenna, which is discussed in respect of the repair cycle of photosystem II due to photoinhibition.

Nature, 2005

Oxygenic photosynthesis in plants, algae and cyanobacteria is initiated at photosystem II, a homo... more Oxygenic photosynthesis in plants, algae and cyanobacteria is initiated at photosystem II, a homodimeric multisubunit proteincofactor complex embedded in the thylakoid membrane 1 . Photosystem II captures sunlight and powers the unique photo-induced oxidation of water to atmospheric oxygen 1,2 . Crystallographic investigations of cyanobacterial photosystem II have provided several medium-resolution structures (3.8 to 3.2 Å ) 3-6 that explain the general arrangement of the protein matrix and cofactors, but do not give a full picture of the complex. Here we describe the most complete cyanobacterial photosystem II structure obtained so far, showing locations of and interactions between 20 protein subunits and 77 cofactors per monomer. Assignment of 11 b-carotenes yields insights into electron and energy transfer and photoprotection mechanisms in the reaction centre and antenna subunits. The high number of 14 integrally bound lipids reflects the structural and functional importance of these molecules for flexibility within and assembly of photosystem II. A lipophilic pathway is proposed for the diffusion of secondary plastoquinone that transfers redox equivalents from photosystem II to the photosynthetic chain. The structure provides information about the Mn 4 Ca cluster, where oxidation of water takes place. Our study uncovers near-atomic details necessary to understand the processes that convert light to chemical energy.

Febs Letters - FEBS LETT, 1984

A proteolytic core of the Escherichia coli single-stranded DNA-binding protein (SSB) has been cry... more A proteolytic core of the Escherichia coli single-stranded DNA-binding protein (SSB) has been crystallized from phosphate buffer. Crystals suitable for X-ray data collection display monoclinic space group C2 with a = 106.8, b = 62.3, c = 100.2 A, p = 112" and contain one tetramer of proteolysis product SSB*-A per asymmetric unit. Two other crystal forms have been obtained in the presence of the inhibitor diisopropylfluorophosphate.

$Research paper thumbnail of beta.-D-Cellotetraose Hemihydrate as a Structural Model for Cellulose II. An X-ray Diffraction Study$

Journal of the American Chemical Society, 1995