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Erik Goormaghtigh

Publications and source records attributed to Erik Goormaghtigh.

25 records · Page 2Linked to original sources
PubMed2002

Insight into the factors influencing the backbone dynamics of three homologous proteins, dendrotoxins I and K, and BPTI: FTIR and time-resolved fluorescence investigations.

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, combined with hydrogen/deuterium exchange technique and time-resolved fluorescence spectroscopy, has been used to investigate the changes in structure and dynamics that underlie the thermodynamic stability differences observed for three closely homologous proteins: dendrotoxins I and K, and bovine pancreatic trypsin inhibitor (BPTI). The experiments were performed on proteins under their native state and a modified form, obtained by selective reduction of a disulfide bond at the surface of the molecule, increasing slightly the backbone flexibility without changing the average structure. The data confirmed the high local as well as global rigidity of BPTI. In protein K, the exchange process was slow during the first 2 h of exchange, presumably reflecting a compact three-dimensional conformation, and then increased rapidly, the internal amide protons of the beta-strands exchanging 10-fold faster than in BPTI or protein I. The most probable destabilizing element was identified as Pro32, in the core of the beta-sheet. Protein I was found to present a 10% more expanded volume than protein K or BPTI, and there is a possible correlation between the resulting increased flexibility of the molecule and the lower thermodynamic stability observed for this protein. Interestingly, the interior amide protons of the beta-sheet structure were found to be as protected against exchange in protein I as in BPTI, suggesting that, although globally more flexible than that of Toxin K or BPTI, the structure of Toxin I could be locally quite rigid. The structural factors suspected to be responsible for the differences in internal flexibility of the two toxins could play a significant role in determining their functional properties.

Amides↗
PubMed2002

Reconstitution of holin activity with a synthetic peptide containing the 1-32 sequence region of EJh, the EJ-1 phage holin.

Pneumococcal EJ-1 phage holin (EJh) is a hydrophobic polypeptide of 85 amino acid residues displaying lethal inner membrane disruption activity. To get an insight into holin structure and function, several peptides representing the different topological regions predicted by sequence analysis have been synthesized. Peptides were structurally characterized in both aqueous buffer and membrane environments, and their potential to induce membrane perturbation was determined. Among them, only the N-terminal predicted transmembrane helix increased the membrane permeability. This segment, only when flanked by the positive charged residues on its N-terminal side, which are present in the sequence of the full-length protein, folds into a major alpha-helix structure with a transmembrane preferential orientation. Fluorescein quenching experiments of N-terminal-labeled peptide evidenced the formation of oligomers of variable size depending on the peptideto-lipid molar ratio. The self-assembling tendency correlated with the formation of transmembrane pores that permit the release of encapsulated dextrans of various sizes. When analyzed by atomic force microscopy, peptide-induced membrane lesions are visualized as transbilayer holes. These findings are the first evidence for a lytic domain in holins and for the nature of membrane lesions caused by them.

Amino Acid Sequence↗
PubMed2002

A new experimental approach to detect long-range conformational changes transmitted between the membrane and cytosolic domains of LmrA, a bacterial multidrug transporter.

LmrA confers multidrug resistance to Lactococcus lactis by mediating the extrusion of antibiotics, out of the bacterial membrane, using the energy derived from ATP hydrolysis. Cooperation between the cytosolic and membrane-embedded domains plays a crucial role in regulating the transport ATPase cycle of this protein. In order to demonstrate the existence of a structural coupling required for the cross-talk between drug transport and ATP hydrolysis, we studied specifically the dynamic changes occurring in the membrane-embedded and cytosolic domains of LmrA by combining infrared linear dichroic spectrum measurements in the course of H/D exchange with Trp fluorescence quenching by a water-soluble attenuator. This new experimental approach, which is of general interest in the study of membrane proteins, detects long-range conformational changes, transmitted between the membrane-embedded and cytosolic regions of LmrA. On the one hand, nucleotide binding and hydrolysis in the cytosolic nucleotide binding domain cause a repacking of the transmembrane helices. On the other hand, drug binding to the transmembrane helices affects both the structure of the cytosolic regions and the ATPase activity of the nucleotide binding domain.

Adenosine Triphosphate↗
PubMed2002

Infrared spectroscopy as a tool for discrimination between sensitive and multiresistant K562 cells.

Fourier transform infrared spectroscopy was performed on human leukemic daunorubicin-sensitive K562 cells and their multiresistant counterpart derived by selection. Statistical analysis, including variable reduction and linear discriminant analysis was performed on sensitive and multiresistant cells spectra in order to establish a diagnostic tool for multiresistant pattern. For each of the two methods of data reduction tested [genetic algorithm or principal component analysis (PCA)] discrimination between the two cell lines was found to be possible. The best results, obtained with PCA-reduction, showed an accuracy of 93% on a distinct test set of spectra. These results demonstrate the efficiency of Fourier transform infrared spectroscopy for classification. Further analysis of the spectral differences indicated that discrimination between resistant and sensitive cells was based on variations in all cellular contents. Lipid and nucleic acid decreased, relatively, while the protein content increased.

Drug Resistance, Neoplasm↗
PubMed2002

Modeling the three-dimensional structure of H+-ATPase of Neurospora crassa.

Homology modeling in combination with transmembrane topology predictions are used to build the atomic model of Neurospora crassa plasma membrane H+-ATPase, using as template the 2.6 A crystal structure of rabbit sarcoplasmic reticulum Ca2+-ATPase [Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647-655]. Comparison of the two calcium-binding sites in the crystal structure of Ca2+-ATPase with the equivalent region in the H+-ATPase model shows that the latter is devoid of most of the negatively charged groups required to bind the cations, suggesting a different role for this region. Using the built model, a pathway for proton transport is then proposed from computed locations of internal polar cavities, large enough to contain at least one water molecule. As a control, the same approach is applied to the high-resolution crystal structure of halorhodopsin and the proton pump bacteriorhodopsin. This revealed a striking correspondence between the positions of internal polar cavities, those of crystallographic water molecules and, in the case of bacteriorhodopsin, the residues mediating proton translocation. In our H+-ATPase model, most of these cavities are in contact with residues previously shown to affect coupling of proton translocation to ATP hydrolysis. A string of six polar cavities identified in the cytoplasmic domain, the most accurate part of the model, suggests a proton entry path starting close to the phosphorylation site. Strikingly, members of the haloacid dehalogenase superfamily, which are close structural homologs of this domain but do not share the same function, display only one polar cavity in the vicinity of the conserved catalytic Asp residue.

Amino Acid Sequence↗
PubMedDate not supplied

Translocation of amino acyl residues from the membrane interface to the hydrophobic core: thermodynamic model and experimental analysis using ATR-FTIR spectroscopy.

The interactions of a series of histidine-containing peptides with biological model membranes have been investigated by attenuated total reflection Fourier transform infra red (ATR-FTIR) spectroscopy. Related peptides have previously been shown to exhibit antibiotic and DNA transfection activities. The 26-residue LAH4X4 peptides were designed in such a manner to form amphipathic helical structures in membrane environments. Four histidines and four variable amino acids X constitute one face of the helix whereas leucines and alanines characterize the opposite hydrophobic surface. The dichroic ratio of ATR-FTIR spectra has been used to follow the pH-dependent transition from in-plane to transmembrane alignments upon increase in pH. A theoretical model of the topological modulations is presented and the experimental transition curves analysed in order to reveal the Gibbs free energy of transition. The novel concept provides access to the free energy changes associated with the amino acids X incorporated into an extended alpha-helix and in the context of phospholipid bilayers. For the peptides of the series the Gibbs free energies associated with the transition from the membrane interface to the bilayer interior follow the sequence of amino acids: L<A approximately I<S approximately F<T approximately G<V approximately W<<Y.

Amino Acids↗