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Mann K.,Max Planck Institute For Biochemie | Edsinger E.,University of California at Berkeley | Edsinger E.,Okinawa Institute of Science and Technology
Proteome Science | Year: 2014

Background: Although the importance of proteins of the biomineral organic matrix and their posttranslational modifications for biomineralization is generally recognized, the number of published matrix proteomes is still small. This is mostly due to the lack of comprehensive sequence databases, usually derived from genomic sequencing projects. However, in-depth mass spectrometry-based proteomic analysis, which critically depends on high-quality sequence databases, is a very fast tool to identify candidates for functional biomineral matrix proteins and their posttranslational modifications. Identification of such candidate proteins is facilitated by at least approximate quantitation of the identified proteins, because the most abundant ones may also be the most interesting candidates for further functional analysis.Results: Re-quantification of previously identified Lottia shell matrix proteins using the intensity-based absolute quantification (iBAQ) method as implemented in the MaxQuant identification and quantitation software showed that only 57 of the 382 accepted identifications constituted 98% of the total identified matrix proteome. This group of proteins did not contain obvious intracellular proteins, such as cytoskeletal components or ribosomal proteins, invariably identified as minor components of high-throughput biomineral matrix proteomes. Fourteen of these major proteins were phosphorylated to a variable extent. All together we identified 52 phospho sites in 20 of the 382 accepted proteins with high confidence.Conclusions: We show that iBAQ quantitation may be a useful tool to narrow down the group of functional biomineral matrix protein candidates for further research in cell biology, genetics or materials research. Knowledge of posttranslational modifications in these major proteins could be a valuable addition to previously published proteomes. This is true especially for phosphorylation, because this modification was already shown to modify mineralization processes in some instances. © 2014 Mann and Edsinger; licensee BioMed Central Ltd.

Gerisch G.,Max Planck Institute For Biochemie
PMC Biophysics | Year: 2010

This report deals with actin waves that are spontaneously generated on the planar, substrate-attached surface of Dictyostelium cells. These waves have the following characteristics. (1) They are circular structures of varying shape, capable of changing the direction of propagation. (2) The waves propagate by treadmilling with a recovery of actin incorporation after photobleaching of less than 10 seconds. (3) The waves are associated with actin-binding proteins in an ordered 3-dimensional organization: with myosin-IB at the front and close to the membrane, the Arp2/3 complex throughout the wave, and coronin at the cytoplasmic face and back of the wave. Coronin is a marker of disassembling actin structures. (4) The waves separate two areas of the cell cortex that differ in actin structure and phosphoinositide composition of the membrane. The waves arise at the border of membrane areas rich in phosphatidylinositol (3,4,5) trisphosphate (PIP3). The inhibition of PIP3 synthesis reversibly inhibits wave formation. (5) The actin wave and PIP3 patterns resemble 2-dimensional projections of phagocytic cups, suggesting that they are involved in the scanning of surfaces for particles to be taken up. © 2010 Gerisch.

Gomis-Ruth F.X.,Molecular Biology Institute of Barcelona | Botelho T.O.,Molecular Biology Institute of Barcelona | Bode W.,Max Planck Institute For Biochemie
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2012

Visualization of three-dimensional structures is essential to the transmission of information to the general reader and the comparison of related structures. Therefore, it would be useful to provide a common framework. Based on the work of Schechter and Berger, and the finding that most peptidases bind their substrates in extended conformation, we suggest a "standard orientation" for the overall description of metallopeptidases (MPs) as done before for peptidases of other classes. This entails a frontal view of the horizontally-aligned active-site cleft. A substrate is bound N- to C-terminally from left (on the non-primed side of the cleft) to right (on the primed side), and the catalytic metal ion resides at the cleft bottom at roughly half width. This view enables us to see that most metalloendopeptidases are bifurcated into an upper and a lower sub-domain by the cleft, whose back is framed by a nearly horizontal "active-site helix." The latter comprises a short zinc-binding consensus sequence, either HEXXH or HXXEH, which provides two histidines to bind the single catalytic metal and the general-base/acid glutamate required for catalysis. In addition, an oblique "backing helix" is observed behind the active-site helix, and a mixed β-sheet of at least three strands is positioned in the upper sub-domain paralleling the cleft. The lowermost "upper-rim" strand of the sheet runs antiparallel to the substrate bound in the cleft and therefore contributes both to delimitating the cleft top and to binding of the substrate main-chain on its non-primed side through β-ribbon-like interactions. In contrast, in metalloexopeptidases, which chop off N- or C-terminal residues only, extensive binding on both sides of the cleft is not required and a different overall scaffold is generally observed. This consists of an αβα- sandwich, which is reminiscent of, but clearly distinct from, the archetypal α/β-hydrolase fold. Metalloexopeptidases have their active sites at the C-terminal end of a central, eight-stranded twisted β-sheet, and can contain one or two catalytic metal ions. As the zinc-binding site and the residues engaged in substrate binding and catalysis are mainly provided by loops connecting the β-sheet strands and the helices on either side, the respective standard orientations vary with respect to the position of the sheets. The standard orientation of eight prototypic MP structures is presented and discussed. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. © 2011 Elsevier B.V. All rights reserved.

Mann K.,Max Planck Institute For Biochemie | Wilt F.H.,University of California at Berkeley | Poustka A.J.,Max Planck Institute For Molekulare Genetik
Proteome Science | Year: 2010

Background: The sea urchin embryo has been an important model organism in developmental biology for more than a century. This is due to its relatively simple construction, translucent appearance, and the possibility to follow the fate of individual cells as development to the pluteus larva proceeds. Because the larvae contain tiny calcitic skeletal elements, the spicules, they are also important model organisms for biomineralization research. Similar to other biominerals the spicule contains an organic matrix, which is thought to play an important role in its formation. However, only few spicule matrix proteins were identified previously.Results: Using mass spectrometry-based methods we have identified 231 proteins in the matrix of the S. purpuratus spicule matrix. Approximately two thirds of the identified proteins are either known or predicted to be extracellular proteins or transmembrane proteins with large ectodomains. The ectodomains may have been solubilized by partial proteolysis and subsequently integrated into the growing spicule. The most abundant protein of the spicule matrix is SM50. SM50-related proteins, SM30-related proteins, MSP130 and related proteins, matrix metalloproteases and carbonic anhydrase are among the most abundant components.Conclusions: The spicule matrix is a relatively complex mixture of proteins not only containing matrix-specific proteins with a function in matrix assembly or mineralization, but also: 1) proteins possibly important for the formation of the continuous membrane delineating the mineralization space; 2) proteins for secretory processes delivering proteinaceous or non-proteinaceous precursors; 3) or proteins reflecting signaling events at the cell/matrix interface. Comparison of the proteomes of different skeletal matrices allows prediction of proteins of general importance for mineralization in sea urchins, such as SM50, SM30-E, SM29 or MSP130. The comparisons also help point out putative tissue-specific proteins, such as tooth phosphodontin or specific spicule matrix metalloproteases of the MMP18/19 group. Furthermore, the direct sequence analysis of peptides by MS/MS validates many predicted genes and confirms the existence of the corresponding proteins. © 2010 Mann et al; licensee BioMed Central Ltd.

Engelhardt H.,Max Planck Institute For Biochemie
Methods in Molecular Biology | Year: 2013

The ultrastructure of bacteria is only accessible by electron microscopy. Our insights into the architecture of cells and cellular compartments such as the envelope and appendages is thus dependent on the progress of preparative and imaging techniques in electron microscopy. Here, I give a short overview of the development and characteristics of methods applied for imaging (components of) the bacterial surface and refer to key investigations and exemplary results. In the beginning of electron microscopy, fixation of biological material and staining for contrast enhancement were the standard techniques. The results from freezeetching, metal shadowing and from ultrathin-sections of plastic-embedded material shaped our view of the cellular organization of bacteria. The introduction of cryo-preparations, keeping samples in their natural environment, and three-dimensional (3D) electron microscopy of isolated protein complexes and intact cells opened the door to a new dimension and has provided insight into the native structure of macromolecules and the in situ organization of cells at molecular resolution. Cryo-electron microscopy of single particles, together with other methods of structure determination, and cellular cryo-electron tomography will provide us with a quasi-atomic model of the bacterial cell surface in the years to come. © Springer Science+Business Media New York 2013.

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