IMBB FORTH

Irákleion, Greece

IMBB FORTH

Irákleion, Greece
SEARCH FILTERS
Time filter
Source Type

Gessmann R.,IMBB FORTH | Axford D.,Diamond Light Source | Bruckner H.,Institute of Nutritional Science | Berg A.,Innovent Jena | Petratos K.,IMBB FORTH
Acta Crystallographica Section:F Structural Biology Communications | Year: 2017

Bergofungin is a peptide antibiotic that is produced by the ascomycetous fungus Emericellopsis donezkii HKI 0059 and belongs to peptaibol subfamily 2. The crystal structure of bergofungin A has been determined and refined to 0.84 Å resolution. This is the second crystal structure of a natural 15 residue peptaibol, after that of samarosporin I. The amino terminal phenylalanine residue in samarosporin I is exchanged to a valine residue in bergofungin A. According to agar diffusion tests, this results in a nearly inactive antibiotic peptide compared with the moderately active samarosporin I. Crystals were obtained from methanol solutions of purified bergofungin mixed with water. Although there are differences in the intramolecular hydrogen bonding scheme of samarosporin I, the overall folding is very similar for both peptaibols, namely 3 10 helical at the termini and α helical in the middle of the molecules. Bergofungin A and samarosporin I molecules are arranged in a similar way in both lattices. However, the packing of bergofungin A exhibits a second solvent channel along the twofold axis. This latter channel occurs in the vicinity of the N terminus, where the natural substitution resides. © 2017 International Union of Crystallography.


PubMed | University of Southern Denmark, IMBB FoRTH and Rega Institute for Medical Research
Type: | Journal: Methods in enzymology | Year: 2017

Protein folding is an intricate and precise process in living cells. Most exported proteins evade cytoplasmic folding, become targeted to the membrane, and then trafficked into/across membranes. Their targeting and translocation-competent states are nonnatively folded. However, once they reach the appropriate cellular compartment, they can fold to their native states. The nonnative states of preproteins remain structurally poorly characterized since increased disorder, protein sizes, aggregation propensity, and the observation timescale are often limiting factors for typical structural approaches such as X-ray crystallography and NMR. Here, we present an alternative approach for the in vitro analysis of nonfolded translocation-competent protein states and their comparison with their native states. We make use of hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS), a method based on differentiated isotope exchange rates in structured vs unstructured protein states/regions, and highly dynamic vs more rigid regions. We present a complete structural characterization pipeline, starting from the preparation of the polypeptides to data analysis and interpretation. Proteolysis and mass spectrometric conditions for the analysis of the labeled proteins are discussed, followed by the analysis and interpretation of HDX-MS data. We highlight the suitability of HDX-MS for identifying short structured regions within otherwise highly flexible protein states, as illustrated by an exported protein example, experimentally tested in our lab. Finally, we discuss statistical analysis in comparative HDX-MS. The protocol is applicable to any protein and protein size, exhibiting slow or fast loss of translocation competence. It could be easily adapted to more complex assemblies, such as the interaction of chaperones with nonnative protein states.


Gessmann R.,IMBB FORTH | Axford D.,Diamond Light Source | Evans G.,Diamond Light Source | Bruckner H.,Justus Liebig University | And 2 more authors.
Journal of Peptide Science | Year: 2012

The atomic resolution structures of samarosporin I have been determined at 100 and 293K. This is the first crystal structure of a natural 15-residue peptaibol. The amino acid sequence in samarosporin I is identical to emerimicin IV and stilbellin I. Samarosporin is a peptide antibiotic produced by the ascomycetous fungus Samarospora rostrup and belongs to peptaibol subfamily 2. The structures at both temperatures are very similar to each other adopting mainly a 310-helical and a minor fraction of α-helical conformation. The helices are significantly bent and packed in an antiparallel fashion in the centered monoclinic lattice leaving among them an approximately 10-Å channel extending along the crystallographic twofold axis. Only two ordered water molecules per peptide molecule were located in the channel. Comparisons have been carried out with crystal structures of subfamily 2 16-residue peptaibols antiamoebin and cephaibols. The repercussion of the structural analysis of samarosporin on membrane function is discussed. © 2012 European Peptide Society and John Wiley & Sons, Ltd.


Gessmann R.,IMBB FORTH | Axford D.,Diamond Light Source | Owen R.L.,Diamond Light Source | Bruckner H.,Justus Liebig University | And 2 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2012

The first crystal structure of a member of peptaibol antibiotic subfamily 4, trichovirin I-4A (14 residues), has been determined by direct methods and refined at atomic resolution. The monoclinic unit cell has two molecules in the asymmetric unit. Both molecules assume a 3 10 right-handed helical conformation and are significantly bent. The molecules pack loosely along the crystallographic twofold axis, forming two large tunnels between symmetry-related molecules in which no ordered solvent could be located. Carbonyl O atoms which are not involved in intramolecular hydrogen bonding participate in close van der Waals interactions with apolar groups. The necessary amphipathicity for biological activity of peptaibols is not realised in the crystal structure. Hence, a structural change of trichovirin to an α-helical conformation is proposed for membrane integration and efficient water/ion transportation across the lipid bilayer. © 2012 International Union of Crystallography.


Gessmann R.,IMBB FORTH | Bruckner H.,Justus Liebig University | Petratos K.,IMBB FORTH
Acta Crystallographica Section C: Structural Chemistry | Year: 2014

The title peptide, N-benzyloxycarbonyl-α-aminoisobutyryl-α- aminoisobutyryl-α-aminoisobutyryl-l-alanine tert-butyl ester or Z-Aib-Aib-Aib-l-Ala-OtBu (Aib is α-aminoisobutyric acid, Z is benzyloxycarbonyl and OtBu indicates the tert-butyl ester), C27H 42N4O7, is a left-handed helix with a right-handed conformation in the fourth residue, which is the only chiral residue. There are two 4→1 intramolecular hydrogen bonds in the structure. In the lattice, molecules are hydrogen bonded to form columns along the c axis. © 2014 International Union of Crystallography.


Gessmann R.,I.M.B.B. FO.R.T.H. | Kyvelidou C.,University of Crete | Papadovasilaki M.,I.M.B.B. FO.R.T.H. | Petratos K.,I.M.B.B. FO.R.T.H.
Biopolymers | Year: 2011

The Cu(II) center at the active site of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by Co(II) via denaturing of the protein, chelation and removal of copper by EDTA and refolding of the apo-protein, followed by addition of an aqueous solution of CoCl 2. Sitting drop vapour diffusion experiments produced green hexagonal crystals, which belong to space group P65, with unit cell dimensions a = b = 50.03, c = 98.80 Å Diffraction data, collected at 291 K on a copper rotating anode X-ray source, were phased by the anomalous signal of the cobalt atom. The structure was built automatically, fitted manually and subsequently refined to 1.86 Å resolution. The Co-substituted protein exhibits similar overall geometry to the native structure with copper. Cobalt binds more strongly to the axial Met86-Sδ and retains the tetrahedral arrangement with the four ligand atoms, His40-Nδ1, Cys78-SIγ, His81-Nδ1, and 86Met-Sδ, although the structure is less distorted than the native copper protein. The structure reported herein, is the first crystallographic structure of a Co(II)-substituted pseudoazurin. © 2010 Wiley Periodicals, Inc.


Kapellios E.A.,University of Crete | Karamanou S.,IMBB FORTH | Sardis M.F.,IMBB FORTH | Sardis M.F.,University of Crete | And 4 more authors.
Analytical and Bioanalytical Chemistry | Year: 2011

The determination of protein assembly size and relative molecular mass is currently of great importance in biochemical analysis. In particular, the technique of nanoelectrospray (nES) with a gas-phase electrophoretic mobility molecular analyzer (GEMMA) has received increased attention for such measurements. However, in order for the GEMMA technique to gain broader acceptance in protein analysis, it must be further evaluated and compared with other established bioanalytical techniques. In the present study, nES-GEMMA was evaluated for the analysis of a set of protein and protein complexes involved in the Sec and the bacterial type III secretion pathway of enteropathogenic Escherichia coli bacteria. The same set of proteins, isolated and purified using standard biochemical protocols, were also analyzed using multi-angle laser light scattering (MALLS) and quasi-elastic light scattering (QELS), following size exclusion chromatography. This allowed for direct comparisons between the three techniques. It was found that nES-GEMMA, in comparison to the more established MALLS and QELS techniques, offers several complementary advantages. It requires considerably less amount of material, i.e., nanogram vs. milligram amounts, and time per sample analysis, i.e., few minutes vs. tens of minutes. Whereas the determined size and relative molecular mass are similar between the compared methods, the electrophoretic diameters determined using nES-GEMMA seem to be systematically smaller compared to the hydrodynamic diameter derived by QELS. Some of the GEMMA technique disadvantages include its narrow dynamic range, limited by the fact that at elevated protein concentrations there is increased potential for the occurrence of nES-induced oligomers. Thus, it is preferred to analyze dilute protein solutions because non-specific oligomers are less likely to occur whereas biospecific oligomers remain detected. To further understand the formation of nES-oligomers, the effect of buffer concentration on their formation was evaluated. Also, nES-GEMMA is not compatible with all the buffers commonly used with MALLS and QELS. Overall, however, the nES-GEMMA technique shows promise as a high-throughput proteomics/protein structure tool. [Figure not available: see fulltext.] © 2011 Springer-Verlag.


PubMed | Justus Liebig University and IMBB FORTH
Type: Journal Article | Journal: Acta crystallographica. Section C, Structural chemistry | Year: 2015

Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (-aminoisobutyric acid) is severely restricted by the second methyl group attached to the C atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl--aminoisobutyrylglycyl--aminoisobutyric acid tert-butyl ester, C16H30N4O5, describes the first N-terminal-unprotected (Gly-Aib)n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N-H group of Aib4. This hydrogen bond is found in all tetrapeptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 ). Instead, every molecule is hydrogen bonded to six other symmetry-related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted molecule) and reverses the screw sense in the last two residues.


Gessmann R.,IMBB FORTH | Bruckner H.,Justus Liebig University | Petratos K.,IMBB FORTH
Acta Crystallographica Section C: Structural Chemistry | Year: 2015

Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α-aminoisobutyric acid) is severely restricted by the second methyl group attached to the Cα atom. Most of the crystal structures containing Aib are N-terminal protected. Deprotection of the N- or C-terminus of peptides may alter the hydrogen-bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl-α-aminoisobutyrylglycyl-α-aminoisobutyric acid tert-butyl ester, C16H30N4O5, describes the first N-terminal-unprotected (Gly-Aib) n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N - H group of Aib4. This hydrogen bond is found in all tetrapeptides and N-terminal-protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N⋯O = 5.88;A). Instead, every molecule is hydrogen bonded to six other symmetry-related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right-handed helical region (and the left-handed helical region for the inverted molecule) and reverses the screw sense in the last two residues. © 2015 International Union of Crystallography.


PubMed | Justus Liebig University and IMBB FORTH
Type: Journal Article | Journal: Acta crystallographica. Section C, Structural chemistry | Year: 2014

The title achiral peptide N-benzyloxycarbonyl--aminoisobutyryl--aminoisobutyryl--aminoisobutyrylglycine tert-butyl ester or Z-Aib-Aib-Aib-Gly-OtBu (Aib is -aminoisobutyric acid, Z is benzyloxycarbonyl, Gly is glycine and OtBu indicates the tert-butyl ester), C26H40N4O7, is partly hydrated (0.075H2O) and has two different conformations which together constitute the asymmetric unit. Both molecules form incipient 310-helices. They differ in the relative orientation of the N-terminal protection group and at the C-terminus. There are two 41 intramolecular hydrogen bonds.

Loading IMBB FORTH collaborators
Loading IMBB FORTH collaborators