TY  - JOUR
A1  - Lipman, Everett A.
A1  - Schuler, Benjamin
A1  - Bakajin, Olgica
A1  - Eaton, William A.
T1  - Single-molecule measurement of protein folding kinetics
N2  - In order to investigate the behavior of single molecules under conditions far from equilibrium, we have coupled a microfabricated laminar-flow mixer to a confocal optical system. This combination enables time-resolved measurement of Foerster resonance energy transfer after an abrupt change in solution conditions. Observations of a small protein show the evolution of the intramolecular distance distribution as folding progresses. This technique can expose subpopulations, such as unfolded protein under conditions favoring the native structure, that would be obscured in equilibrium experiments.
Y1  - 2003
UR  - http://www.sciencemag.org/content/301/5637/1233
SN  - 0036-8075
ER  - 
TY  - JOUR
A1  - Tolan, Dean R.
A1  - Schuler, Benjamin
A1  - Beernink, Peter T.
A1  - Jaenicke, Rainer
T1  - Thermodynamic analysis of the dissociation of the aldolase tetramer substituted at one or both of the subunit interfaces
N2  - The fructose-1,6-bis(phosphate) aldolase isologous tetramer tightly associates through two different subunit interfaces defined by its 222 symmetry. Both single- and double-interfacial mutant aldolases have a destabilized quaternary structure, but there is little effect on the catalytic activity. These enzymes are however thermolabile. This study demonstrates the temperature-dependent dissociation of the mutant enzymes and determines the dissociation free energies of both mutant and native aldolase. Subunit dissociation is measured by sedimentation equilibrium in the analytical ultracentrifuge. At 25C the tetramerdimer dissociation constants for each single-mutant enzyme are similar, about 10 -6 M. For the double-mutant enzyme, sedimentation velocity experiments on sucrose density gradients support a tetramermonomer equilibrium. Furthermore, sedimentation equilibrium experiments determined a dissociation constant of 10- 15 M3 for the double-mutant enzyme. By the same methods the upper limit for the dissociation constant of wild-type aldolase A is approximately 10-28 M3, which indicates an extremely stable tetramer. The thermodynamic values describing monomer-tetramer and dimer-tetramer equilibria are analyzed with regard to possible cooperative interaction between the two subunit interfaces.
Y1  - 2003
UR  - http://www.reference-global.com/doi/abs/10.1515/BC.2003.162
ER  - 
TY  - JOUR
A1  - Biswas, Shyamasri
A1  - Kayastha, Arvind M.
A1  - Seckler, Robert
T1  - Purification and characterization of a thermostable beta-galactosidase from kidney beans (Phaseolus vulgaris L.) cv. PDR14
N2  - Summary Using five different steps, ;-Galactosidase has been purified from kidney beans to apparent electrophoretic homogeniety with approximately 90-fold purificationwith a specific activity of 281 units mg;1 protein. A single bandwas observed in native PAGE. Activity staining of the native gel with 5-bromo4-chloro 3-indoxyl ;-D-galactopyranoside (X-Gal) at pH 4.0 also produceda single band. Analytical gel filtration in Superdex G-75 revealed the molecularmass of the native protein to be approximately 75 kD. 10 percnt; SDS-PAGE under reducingconditions showed two subunits of molecular masses, 45 and 30 kD, respectively.Hence, ;-galactosidase from kidney beans is a heterodimer. A typical proteinprofile with ;max at 280 nm was observed and A280/A260ratio was 1.52. The N-terminal sequence of the 45 kD band showed 86 percnt; sequencehomology with an Arabidopsis thaliana and 85 percnt; with Lycopersiconesculentum putative ;-galactosidase sequences. The Electrospray MassSpectrometric analysis of this band also revealed a peptide fragment that had90 percnt; sequence homology with an Arabidopsis thaliana putative ;- galactosidasesequence. The N-terminal sequencing of the 30 kD band as well as mass spectrometricanalysis both by MALDI- TOF and ES MS revealed certain sequences that matchedwith phytohemagglutinin of kidney beans. The optimum pH of the enzyme was 4.0and it hydrolysed o- and p-nitrophenyl ;-D galactopyranosidewith a Km value of 0.63 mmol/L and 0.74 mmol/L, respectively.The energy of activation calculated from the Arrhenius equation was 14.8 kcal/molenzyme site. The enzyme was found to be comparatively thermostable showing maximumactivity at 67 °C. Thermal denaturation of the enzyme at 65 °C obeyssingle exponential decay with first order-rate constant 0.105 min;1.Galactose, a hydrolytic product of this enzyme was a competitive inhibitor witha Ki of 2.7 mmol/L.
Y1  - 2003
SN  - 0176-1617
ER  - 
TY  - JOUR
A1  - Freiberg, Alexander
A1  - Morona, Renato
A1  - Van den Bosch, Luisa
A1  - Jung, Christiane
A1  - Behlke, Joachim
A1  - Carlin, Nung
A1  - Seckler, Robert
A1  - Baxa, Ulrich
T1  - The tailspike protein of Shigella phage Sf6 : a structural homolog of Salmonella phage P22 tailspike protein without sequence similarity in the beta-helix domain
N2  - Bacteriophage Sf6 tailspike protein is functionally equivalent to the well characterized tailspike ofSalmonella phage P22, mediating attachment of the viral particle to host cell-surface polysaccharide. However, there is significant sequence similarity between the two 70-kDa polypeptides only in the N-terminal putative capsid-binding domains. The major, central part of P22 tailspike protein, which forms a parallel ;-helix and is responsible for saccharide binding and hydrolysis, lacks detectable sequence homology to the Sf6 protein. After recombinant expression in Escherichia coli as a soluble protein, the Sf6 protein was purified to homogeneity. As shown by circular dichroism and Fourier transform infrared spectroscopy, the secondary structure contents of Sf6 and P22 tailspike proteins are very similar. Both tailspikes are thermostable homotrimers and resist denaturation by SDS at room temperature. The specific endorhamnosidase activities of Sf6 tailspike protein toward fluorescence-labeled dodeca-, deca-, and octasaccharide fragments of Shigella O-antigen suggest a similar active site topology of both proteins. Upon deletion of the N-terminal putative capsid-binding domain, the protein still forms a thermostable, SDS-resistant trimer that has been crystallized. The observations strongly suggest that the tailspike of phage Sf6 is a trimeric parallel ;-helix protein with high structural similarity to its functional homolog from phage P22.
Y1  - 2003
UR  - http://www.jbc.org/content/278/3/1542.full
SN  - 0021-9258
ER  - 
TY  - JOUR
A1  - Sunna, Anwar
A1  - Bergquist, Peter L.
T1  - A gene encoding a novel extremely thermostable 1,4-beta-xylanase isolated directly from an environmental DNA sample
N2  - Small-subunit (SSU) rRNA genes (rDNA) were amplified by PCR from a hot pool environmental DNA sample using Bacteria- or Archaea-specific rDNA primers. Unique rDNA types were identified by restriction fragment length polymorphism (RFLP) analysis and representative sequences were determined. Family 10 glycoside hydrolase consensus PCR primers were used to explore the occurrence and diversity of xylanase genes in the hot pool environmental DNA sample. Partial sequences for three different xylanases were obtained and genomic walking PCR (GWPCR), in combination with nested primer pairs, was used to obtained a unique 1,741-bp nucleotide sequence. Analysis of this sequence identified a putative XynA protein encoded by the xynA open reading frame. The single module novel xylanase shared sequence similarity to the family 10 glycoside hydrolases. The purified recombinant enzyme, XynA expressed in E. coli exhibited optimum activity at 100 degrees C and pH 6.0, and was extremely thermostable at 90 degrees C. The enzyme showed high specificity toward different xylans and xylooligosaccharides.
Y1  - 2003
ER  - 
TY  - JOUR
A1  - Müssig, Carsten
A1  - Altmann, Thomas
T1  - Genomic brassinosteroid effects
N2  - Detailed analysis of brassinosteroid (BR)-regulated genes can provide evidence of the molecular basis of BR effects. Classical techniques (such as subtractive cDNA cloning) as well as cDNA and oligonucleotide microarrays have been applied to identify genes which are upregulated or downregulated after BR treatment or are differently expressed in BR-deficient or -insensitive mutants compared with wild type plants. Genes encoding cell-wall-modifying enzymes, enzymes of the BR biosynthetic pathway, auxin response factors, and transcription factors are subject to BR regulation. Effects on several other metabolic pathways and interactions with other phytohormones have been reported as well, although some of these effects may depend on certain environmental conditions (for example, light/dark or stress), the developmental stage of the plants, and tissue types. The identification of components of the BR signal transduction pathway revealed different modes of transcriptional control in animals and plants. Steroid signaling in plants comprises the plasma membrane receptor kinases BRI1 and BAK1 and intracellular protein phosphorylations. Thus, BR signaling in plants is reminiscent of growth factor and TGF-beta signal transduction in animals. The phosphorylation cascade could be a basis of extensive signaling cross-talk and thereby explain the complexity of BR responses
Y1  - 2003
ER  - 
TY  - JOUR
A1  - Müssig, Carsten
A1  - Altmann, Thomas
T1  - Changes in gene expression in response to altered SHL transcript levels
N2  - The nuclear SHL protein is composed of a N-terminal BAH domain and a C-terminal PHD finger. Both domains are found in transcriptional regulators and chromatin-modifying proteins. Arabidopsis plants over-expressing SHL showed earlier flowering and senescence phenotype. To identify SHL regulated genes, expression profiles of 35S::SHL plants were established with Affymetrix ATH1 microarrays. About 130 genes showed reduced transcript levels, and about 45 genes showed increased transcript levels in 35S:: SHL plants. The up-regulated genes included AGL20 and AGL9, which most likely cause the early flowering phenotype of 35S:: SHL plants. Late-flowering SHL-antisense lines showed reduced AGL20 mRNA levels, suggesting that AGL20 gene expression depends on the SHL protein. The stronger expression of senescence- and defence-related genes (such as DIN2, DIN11 and PR-1) is in line with the early senescence phenotype of SHL-over- expressing plants. SHL-down-regulated genes included stress response genes and the PSR3.2 gene (encoding a beta- glucosidase). SHL over-expression did not alter the tissue specificity of PSR3.2 gene expression, but resulted in reduced transcript levels in both shoots and roots. Plants with glucocorticoid-inducible SHL over-expression were established and used for expression profiling as well. A subset of genes was identified, which showed consistent changes in the inducible system and in plants with constitutive SHL over-expression
Y1  - 2003
ER  - 
TY  - JOUR
A1  - Rahimi, G.
A1  - Isachenko, E.
A1  - Sauer, Heinrich
A1  - Isachenko, V.
A1  - Wartenberg, Maria
A1  - Hescheler, Jürgen
A1  - Mallmann, P.
A1  - Nawroth, F.
T1  - Effect of different vitrification protocols for human ovarian tissue on reactive oxygen species and apoptosis
Y1  - 2003
ER  - 
TY  - JOUR
A1  - Sachinidis, A.
A1  - Gissel, C.
A1  - Nierhoff, D.
A1  - Hippler-Altenburg, Rita
A1  - Sauer, Heinrich
A1  - Wartenberg, Maria
A1  - Hescheler, Jürgen
T1  - Identification of plateled-derived growth factor-BB as cardiogenisis-inducing factor in mouse embryonic stem cells under serum-free conditions
Y1  - 2003
ER  - 
TY  - JOUR
A1  - Klein, Florian
A1  - Feldhahn, Niklas
A1  - Lee, Sanggyu
A1  - Wang, Hui
A1  - Ciuffi, Fiammetta
A1  - von Elstermann, Mirko
A1  - Toribio, Maria L.
A1  - Sauer, Heinrich
A1  - Wartenberg, Maria
A1  - Barath, Varun Singh
A1  - Krönke, Martin
A1  - Wernet, Peter
A1  - Rowley, Janet D.
A1  - Müschen, Markus
T1  - T lymphoid differentiation in human bone marrow
Y1  - 2003
ER  -