Bochtler Lab.
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Max Planck-Institute of
Molecular Cell Biology and
Genetics
Dresden, Germany |
 
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International Institute of
Molecular and Cell Biology
Warsaw, Poland |
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Head:
Postdoctoral
fellows:
PhD students:
EU visiting expert:
Technician:
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Matthias Bochtler, PhD, Professor
Honorata Czapinska, PhD
Renata Filipek, PhD
Izabela Sabała, PhD
Monika Sokołowska, PhD
Roman Szczepanowski, PhD
Grzegorz Chojnowski, PhD
Patrycja Haniewicz, MSc
Henryk Korza, MSc
Marek Wojciechowski, MSc
Jean-Philippe Borges, PhD
Ewa Błażewicz |
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The MPI-PAN Junior Research group was set up jointly by the MPI-CBG Dresden and the Polish Academy of Sciences.
We use molecular biology, biochemistry, protein crystallography and NMR to elucidate protein structure and relate it to function. We focus mostly on peptidases and endonucleases, but have recently also started some “methods” oriented projects.
Peptidases
Our aim has been to find peptidases with novel folds and unusual active sites. I have focused on peptidases that cleave the non-ribosomally synthesized, mixed chirality peptides in peptidoglycan, the major component of bacterial cell walls. The elucidation of many peptidoglycan amidase structures by others and ourselves has allowed to ask questions such as: how promiscuous are protein folds? How many enzymes of different specificities belong to the same fold group? Conversely, how many different folds and active sites have evolved to catalyze the same reaction? Are folds specialized for chemical features of the substrate, e.g. are there specialized folds for D-D, L-D, or D-L linkages?
Some of the most promising candidates for novel peptidases are integral membrane proteins. We have no prior experience with membrane protein purification and crystallization, but have received a European Union Transfer of Knowledge grant to employ incoming researchers with membrane protein experience and to send group members abroad to learn new techniques. We currently have job openings in this area.
Protein degradation
In the area of protein degradation, a long-standing interest of the group is the structure of ubiquitin-activating enzyme, which is the most upstream component of the ubiquitin-proteasome pathway. The enzyme catalyzes the conversion of ubiquitin to the adenylate, and the subsequent conversion to an enzyme bound thioester. As we have been unable to crystallize the entire enzyme, we have instead studied fragments, either by crystallography or NMR.
Endonucleases
Many restriction endonucleases recognize palindromic or pseudopalindromic recognition sequences. Most of them catalyze “canonical” double strand breaks that result either in blunt ends or 4 nt 5’-overhangs. My focus has been on restriction endonucleases that generate other, unusual “footprints” in the DNA. It turns out that these enzymes are “exotic” and interesting also in other ways: Ecl18kI (/CCNGG) flips nucleotides to achieve specificity for its target sequence. In contrast to almost all other restriction endonucleases, MvaI (CC/WGG) and BcnI (CC/SGG) are monomeric and more similar to components of the cellular DNA repair machinery than to other restriction enzymes. We hope that this will be a more general theme, and currently study several other unusual enzymes, which we suspect to be adaptations of other components of the cellular DNA repair machinery. More recently, we have also started work on some eukaryotic enzymes that generate double strand breaks in DNA, but have no structures yet.
Methods development
In the “methods” area, we have looked for directly interpretable features in the diffraction pattern of 3D-crystals. Such features need to be distinguished from the largest random fluctuations. This has prompted us to study in detail the limiting distributions of the highest reflection intensity and of the highest local intensity average for crystals with randomly arranged atoms.
Recent publications - 2005-2007:
1. Sokolowska M, Kaus-Drobeka M, Czapinska H, Tamulaitis G, Szczepanowski RH, Urbanke C, Siksnys V, Bochtler M. Monomeric restriction endonuclease BcnI in the apo-form and in an asymmetric complex with target DNA. (in press). J Mol Biol.
2: Kaus-Drobek M, Czapinska H, Sokolowska M, Tamulaitis G, Szczepanowski RH, Urbanke C, Siksnys V, Bochtler M. Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetrically. Nucleic Acids Res. 2007; [Epub ahead of print]
3: Bochtler M, Chojnowski G. The highest reflection intensity in a resolution shell. Acta Crystallogr A. 2007; 63(Pt 2):146-55.
4: Firczuk M, Bochtler M. Mutational analysis of peptidoglycan amidase MepA. Biochemistry. 2007; 46(1):120-8.
5: Jaremko L, Jaremko M, Filipek R, Wojciechowski M, Szczepanowski RH, Bochtler M, Zhukov I. NMR assignment of a structurally uncharacterised fragment of recombinant mouse ubiquitin-activating enzyme. J Biomol NMR. 2006; [Epub ahead of print]
6: Bochtler M, Szczepanowski RH, Tamulaitis G, Grazulis S, Czapinska H, Manakova E, Siksnys V. Nucleotide flips determine the specificity of the Ecl18kI restriction endonuclease. EMBO J. 2006; 25(10):2219-29.
7: Firczuk M, Mucha A, Bochtler M. Crystal structures of active LytM. J Mol Biol. 2005; 354(3):578-90.
8: Grazulis S, Manakova E, Roessle M, Bochtler M, Tamulaitiene G, Huber R, Siksnys V. Structure of the metal-independent restriction enzyme BfiI reveals fusion of a specific DNA-binding domain with a nonspecific nuclease. Proc Natl Acad Sci U S A. 2005; 102(44):15797-802.
9: Odintsov SG, Sabala I, Bourenkov G, Rybin V, Bochtler M. Substrate access to the active sites in aminopeptidase T, a representative of a new metallopeptidase clan. J Mol Biol. 2005; 354(2):403-12.
10: Korza HJ, Bochtler M. Pseudomonas aeruginosa LD-carboxypeptidase, a serine peptidase with a Ser-His-Glu triad and a nucleophilic elbow. J Biol Chem. 2005; 280(49):40802-12.
11: Odintsov SG, Sabala I, Bourenkov G, Rybin V, Bochtler M. Staphylococcus aureus aminopeptidase S is a founding member of a new peptidase clan. J Biol Chem. 2005; 280(30):27792-9.
12: Dandanell G, Szczepanowski RH, Kierdaszuk B, Shugar D, Bochtler M. Escherichia coli purine nucleoside phosphorylase II, the product of the xapA gene. J Mol Biol. 2005; 348(1):113-25.
13: Azim MK, Goehring W, Song HK, Ramachandran R, Bochtler M, Goettig P. Characterization of the HslU chaperone affinity for HslV protease. Protein Sci. 2005; 14(5):1357-62.
14: Szczepanowski RH, Filipek R, Bochtler M. Crystal structure of a fragment of mouse ubiquitin-activating enzyme. J Biol Chem. 2005; 280(23):22006-11.
15: Groll M, Bochtler M, Brandstetter H, Clausen T, Huber R. Molecular machines for protein degradation. Chembiochem. 2005; 6(2):222-56. Review.
16: Filipek R, Potempa J, Bochtler M. A comparison of staphostatin B with standard mechanism serine protease inhibitors. J Biol Chem. 2005; 280(15):14669-74.
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