Biomedical subjects

Erik Goormaghtigh

Publications and source records attributed to Erik Goormaghtigh.

At least 19 recordsLinked to original sources

PubMed2006

Characteristics of fibers formed by cytochrome c and induced by anionic phospholipids.

Recent publications described the formation of millimeter-length fibers by diverse lipid-binding proteins (e.g., histone H1, cytochrome c, indolicidin, and endostatin) when they are mixed with 80:20 phosphatidylcholine/phosphatidylserine vesicles. Further, these fibers displayed amyloid characteristics when stained with Congo Red. In the study presented here, we found by FTIR the amide I absorption band to reveal significant variation in fibers formed by cytochrome c, with some consisting of cytochrome c in a nativelike conformation and some exhibiting strong amyloid (beta-sheet) characteristics. Protein structure also varied from amyloid to nearly native within single fibers. Fibers were frequently blue or bluish and sometimes iridescent, likely due to interference of light in the fibers. The amyloid-type amide I band was observed for blue fibers only. AFM shows that fibers consist of smaller 3-4 nm diameter fibers with 10 nm lateral spacing.

Amyloid↗

PubMed2006

Chemometric tools for classification and elucidation of protein secondary structure from infrared and circular dichroism spectroscopic measurements.

Protein classification and characterization often rely on the information contained in the protein secondary structure. Protein class assignment is usually based on X-ray diffraction measurements, which need the protein in a crystallized form, or on NMR spectra, to obtain the structure of a protein in solution. Simple spectroscopic techniques, such as circular dichroism (CD) and infrared (IR) spectroscopies, are also known to be related to protein secondary structure, but they have seldom been used for protein classification. To see the potential of CD, IR, and combined CD/IR measurements for protein classification, unsupervised pattern recognition methods, Principal Component Analysis (PCA) and cluster analysis, are proposed first to check for natural grouping tendencies of proteins according to their measured spectra. Partial Least Squares Discriminant Analysis (PLS-DA), a supervised pattern recognition method, is used afterwards to test the possibility to model explicitly each protein class and to test these models in class assignment of unknown proteins. Determination of the protein secondary structure, understood as the prediction of the abundance of the different secondary structure motifs in the biomolecule, was carried out with the local regression method interval Partial Least Squares (iPLS). CD, IR, and CD/IR measurements were correlated to the fraction of the motif to be predicted, determined from X-ray measurements. iPLS builds models extracting the spectral information most correlated to a specific secondary motif and avoids the use of irrelevant spectral regions. Spectral intervals chosen by iPLS models provide structural information which can be used to confirm previous biochemical assignments or identify new motif-related spectral features. The predictive ability of the models built with the selected spectral regions has a quality similar to previous classical approaches.

Circular Dichroism↗

PubMed2006

The papaya Kunitz-type trypsin inhibitor is a highly stable beta-sheet glycoprotein.

The papaya Kunitz-type trypsin inhibitor, a 24-kDa glycoprotein, was purified to homogeneity. The purified inhibitor stoichiometrically inhibits bovine trypsin in a 1:1 molar ratio. Circular dichroism and infrared spectroscopy analyses demonstrated that the inhibitor contains extensive beta-sheet structures. The inhibitor was found to retain its full inhibitory activity over a broad pH range (1.5-11.0) and temperature (up to 80 degrees C), besides being stable at very high concentrations of strong chemical denaturants (e.g., 5.5 M guanidine hydrochloride). The inhibitor retained its compact structure over the pH range analyzed as shown by 8-anilino-1-naphtalenesulfonic acid binding characteristics, excluding the formation of some relaxed or molten state. Exposure to 2.5 mM dithiothreitol for 120 min caused a 33% loss of the inhibitory activity, while a loss of 75% was obtained in the presence of 20 mM of dithiothreitol during the same time period. A complete loss of the inhibitory activity was observed after incubation with 50 mM dithiothreitol for 5 min. Incubation of the inhibitor with general proteases belonging to different families revealed its extraordinary resistance to proteolysis in comparison with the soybean trypsin inhibitor, the archetypal member of the Kunitz-type inhibitors family. The inhibitor also exhibited a remarkable resistance to proteolytic degradation against pepsin for at least a 24-h incubation period. Instead, the soybean inhibitor was completely degraded after 2 h incubation with this aspartic protease. All these data demonstrated the high stability of the papaya trypsin inhibitor.

Anilino Naphthalenesulfonates↗

PubMed2006

Conformational and orientational guidance of the analgesic dipeptide kyotorphin induced by lipidic membranes: putative correlation toward receptor docking.

The analgesic dipeptide kyotorphin (L-Tyr-L-Arg) and an acylated kyotorphin derivative were studied by a combination of theoretical (molecular dynamics simulation and quantum mechanics methods) and experimental (fluorescence and infrared spectroscopies) approaches both in solution and in model systems of membranes. At biological pH the peptides have a neutral net charge. Nevertheless, their phenolic rings interact with phospholipid molecules (partition coefficient varies from 6 x 10(2) to 2 x 10(4), depending on the lipid and pH used) despite being exposed to the aqueous bulk medium. The lowest energy transition dipole moment is displaced from the normal to the lipid bilayer by 20 degrees on average. The observed extensive interaction, pK(a), precise location, and well-defined orientation in membranes combined with the ability to discriminate rigid raftlike membrane domains suggest that kyotorphin meets the structural constraints needed for receptor-ligand interaction. The acylated kyotorphin derivative mimics kyotorphin properties and represents a promising way for entrapment in a drug carrier and transport across the blood-brain barrier.

Blood-Brain Barrier↗

PubMed2006

Evaluation of the information content in infrared spectra for protein secondary structure determination.

Fourier-transform infrared spectroscopy is a method of choice for the experimental determination of protein secondary structure. Numerous approaches have been developed during the past 15 years. A critical parameter that has not been taken into account systematically is the selection of the wavenumbers used for building the mathematical models used for structure prediction. The high quality of the current Fourier-transform infrared spectrometers makes the absorbance at every single wavenumber a valid and almost noiseless type of information. We address here the question of the amount of independent information present in the infrared spectra of proteins for the prediction of the different secondary structure contents. It appears that, at most, the absorbance at three distinct frequencies of the spectra contain all the nonredundant information that can be related to one secondary structure content. The ascending stepwise method proposed here identifies the relevance of each wavenumber of the infrared spectrum for the prediction of a given secondary structure and yields a particularly simple method for computing the secondary structure content. Using the 50-protein database built beforehand to contain as little fold redundancy as possible, the standard error of prediction in cross-validation is 5.5% for the alpha-helix, 6.6% for the beta-sheet, and 3.4% for the beta-turn.

Algorithms↗

PubMed2006

Interactions involved in the realignment of membrane-associated helices. An investigation using oriented solid-state NMR and attenuated total reflection Fourier transform infrared spectroscopies.

A series of histidine-containing peptides (LAH4X6) was designed to investigate the membrane interactions of selected side chains. To this purpose, their pH-dependent transitions from in-plane to transmembrane orientations were investigated by attenuated total reflection Fourier transform infrared and oriented solid-state NMR spectroscopies. Peptides of the same family have previously been shown to exhibit antibiotic and DNA transfection activities. Solution NMR spectroscopy indicates that these peptides form amphipathic helical structures in membrane environments, and the technique was also used to characterize the pK values of all histidines in the presence of detergent micelles. Whereas one face of the amphipathic helix is clearly hydrophobic, the opposite side is flanked by four histidines surrounding six leucine, alanine, glycine, tryptophan, or tyrosine residues, respectively. This diversity in peptide composition causes pronounced shifts in the midpoint pH of the in-plane to transmembrane helical transition, which is completely abolished for the peptides carrying the most hydrophilic amino acid residues. These properties open up a conceptually new approach to study in a quantitative manner the hydrophobic as well as specific interactions of amino acids in membranes. Notably, the resulting scale for whole residue transitions from the bilayer interface to the hydrophobic membrane interior is obtained from extended helical sequences in lipid bilayers.

Alanine↗

PubMed2005

Modulation of the membrane orientation and secondary structure of the C-terminal domains of Bak and Bcl-2 by lipids.

Infrared spectroscopy was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domains of the pro-apoptotic protein Bak (Bak-C) and the anti-apoptotic protein Bcl-2 (Bcl-2-C) when incorporated into different lipid vesicles. Whereas beta-pleated sheet was the predominant type of secondary structure of Bak-C in the absence of membranes, the same peptide adopted different structures depending on lipid composition when incorporated into membranes, with the predominance of the alpha-helical structure in the case of DMPC and other phospholipids, such as POPC and POPG. However, beta-pleated sheet was the predominant structure in other membranes containing phospholipids with longer fatty acyl chains and cholesterol, as well as in a mixture which imitates the composition of the outer mitochondrial membrane (OMM). Similarly, Bcl-2-C adopted a structure with a predominance of intermolecularly bound pleated beta-sheet in the absence of membranes, with alpha-helix as the main component in the presence of DMPC and POPG, but intermolecular beta-sheet in the presence of EYPC and cholesterol. Using ATR-IR, it was found that the orientation of the alpha-helical components of both domains was nearly perpendicular to the plane of the membrane in the presence of DMPC membranes, but not in EYPC or OMM membranes. (2)H NMR spectroscopy of DMPC-d(54) confirmed the transmembrane disposition of the domains, revealing that they broadened the phase transition temperature, although the order parameter of the C-D bonds was not affected, as might have been expected for intrinsic peptides. When all these results are taken together, it was concluded that the domains only form transmembrane helices in membranes of reduced thickness and that hydrophobic mismatching occurs in thicker membranes, as happens in the membrane imitating the composition of the OMM, where the peptides were partially located outside the membranes.

Humans↗

PubMed2005

Orientation and mode of lipid-binding interaction of human apolipoprotein E C-terminal domain.

ApoE (apolipoprotein E) is an anti-atherogenic lipid transport protein that plays an integral role in lipoprotein metabolism and cholesterol homoeostasis. Lipid association educes critical functional features of apoE, mediating reduction in plasma and cellular cholesterol levels. The 10-kDa CT (C-terminal) domain of apoE facilitates helix-helix interactions in lipid-free state to promote apoE self-association and helix-lipid interactions during binding with lipoproteins, although the mode of lipid-binding interaction is not well understood. We investigated the mode of lipid-binding interaction and orientation of apoE CT domain on reconstituted lipoproteins. Isolated recombinant human apoE CT domain (residues 201-299) possesses a strong ability to interact with phospholipid vesicles, yielding lipoprotein particles with an apparent molecular mass of approximately 600 kDa, while retaining the overall alpha-helical content. Electron microscopy and non-denaturing PAGE analysis of DMPC (dimyristoylphosphatidylcholine)--apoE CT domain lipoprotein complexes revealed discoidal complexes with a diameter of approx. 17 nm. Cross-linking apoE CT domain on discoidal particles yielded dimeric species as the major product. Attenuated total reflectance Fourier transform IR spectroscopy of phospholipid-apoE CT domain complexes reveals that the helical axis is oriented perpendicular to fatty acyl chains of the phospholipid. Fluorescence quenching analysis of DMPC-apoE CT domain discoidal complexes by spin-labelled stearic acid indicated a relatively superficial location of the native tryptophan residues with respect to the plane of the phospholipid bilayer. Taken together, we propose that apoE CT domain interacts with phospholipid vesicles, forming a long extended helix that circumscribes the discoidal bilayer lipoprotein complex.

Apolipoproteins E↗

PubMed2004

Conformational changes in gastric H+/K+-ATPase monitored by difference Fourier-transform infrared spectroscopy and hydrogen/deuterium exchange.

Gastric H+/K+-ATPase is a P-type ATPase responsible for acid secretion in the stomach. This protein adopts mainly two conformations called E1 and E2. Even though two high-resolution structures for a P-ATPase in these conformations are available, little structural information is available about the transition between these two conformations. In the present study, we used two experimental approaches to investigate the structural differences that occur when gastric ATPase is placed in the presence of various ligands and ligand combinations. We used attenuated total reflection-Fourier-transform IR experiments under a flowing buffer to modify the environment of the protein inside the measurement cell. The high accuracy of the results allowed us to demonstrate that the E1-E2 transition induces a net change in the secondary structure that concerns 10-15 amino acid residues of a total of 1324 in the proteins. The E2.K+ structure is characterized by a decreased beta-sheet content and an increase in the disordered structure content with respect to the E1 form of the enzyme. Modifications in the absorption of the side chain of amino acids are also suggested. By using hydrogen/deuterium-exchange kinetics, we show that tertiary-structure modifications occurred in the presence of the same ligands, but these changes involved several hundreds of residues. The present study suggests that conformational changes in the catalytic cycle imply secondary-structure rearrangements of small hinge regions that have an impact on large domain re-organizations.

Animals↗

PubMed2004

Filipin orientation revealed by linear dichroism. Implication for a model of action.

The organization of the polyene antibiotic filipin in membranes containing cholesterol is a controversial matter of debate. Two contradictory models exist, one suggesting a parallel and the other perpendicular organization of filipin with respect to the plane of the membrane. UV-vis linear dichroism, ATR-FTIR, and fluorescence anisotropy decay techniques were combined to study the orientation of filipin in model systems of membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC) with and without cholesterol. Filipin's orientation is determined by the presence/absence of cholesterol when it is inserted in gel crystalline phase model membranes. When cholesterol (33%) is present in DPPC bilayers, filipin stands perpendicular to the membrane surface as expected in "pore-forming" models. At variance, absence of cholesterol leaves filipin in an essentially random organization in the lipidic matrix. In liquid crystalline phase bilayers (POPC) filipin's orientation is perpendicular to the membrane surface even in absence of cholesterol. Thus filipin's activity/organization depends not only on cholesterol presence but also in the lipid phase domain it is inserted in. These findings were combined with spectroscopy and microscopy data in the literature, solving controversial matters of debate.

Anisotropy↗

PubMed2004

Evidence of an intramolecular interaction between the two domains of the BlaR1 penicillin receptor during the signal transduction.

The BlaR1 protein is a penicillin-sensory transducer involved in the induction of the Bacillus licheniformis beta-lactamase. The amino-terminal domain of the protein exhibits four transmembrane segments (TM1-TM4) that form a four-alpha-helix bundle embedded in the plasma bilayer. The carboxyl-terminal domain of 250 amino acids (BlaR-CTD) fused at the carboxyl end of TM4 possesses the amino acid sequence signature of penicillin-binding proteins. This membrane topology suggests that BlaR-CTD and the BlaR-amino-terminal domain are responsible for signal reception and signal transduction, respectively. With the use of phage display experiments, we highlight herein an interaction between BlaR-CTD and the extracellular, 63-amino acid L2 loop connecting TM2 and TM3. This interaction does not occur in the presence of penicillin. This result suggests that binding of the antibiotic to BlaR1 might entail the release of the interaction between L2 and BlaR-CTD, causing a motion of the alpha-helix bundle and transfer of the information to the cytoplasm of the cell. In addition, fluorescence spectroscopy, CD, and Fourier transform IR spectroscopy experiments indicate that in contrast to the behavior of the corresponding Staphylococcus aureus protein, the beta-lactam antibiotic does not induce a drastic conformational change in B. licheniformis BlaR-CTD.

Acylation↗

PubMed2004

The optimization of protein secondary structure determination with infrared and circular dichroism spectra.

We have used the circular dichroism and infrared spectra of a specially designed 50 protein database [Oberg, K.A., Ruysschaert, J.M. & Goormaghtigh, E. (2003) Protein Sci. 12, 2015-2031] in order to optimize the accuracy of spectroscopic protein secondary structure determination using multivariate statistical analysis methods. The results demonstrate that when the proteins are carefully selected for the diversity in their structure, no smaller subset of the database contains the necessary information to describe the entire set. One conclusion of the paper is therefore that large protein databases, observing stringent selection criteria, are necessary for the prediction of unknown proteins. A second important conclusion is that only the comparison of analyses run on circular dichroism and infrared spectra independently is able to identify failed solutions in the absence of known structure. Interestingly, it was also found in the course of this study that the amide II band has high information content and could be used alone for secondary structure prediction in place of amide I.

Algorithms↗

PubMed2004

Analysis of 1H/2H exchange kinetics using model infrared spectra.

This paper investigates the different approaches that best retrieve band shape parameters and kinetic time constants from series of protein Fourier transform infrared (FT-IR) spectra recorded in the course of 1H/2H exchange. In this first approach, synthetic spectra were used. It is shown that 1H/2H exchange kinetic measurements can help resolve spectral features otherwise hidden because of the overlap of various spectral contributions. We evaluated the efficiency of Fourier self-deconvolution, synchronous/asynchronous correlation, difference spectroscopy, principal component analysis, inverse Laplace transform, and determination of the underlying spectra by global analysis assuming first-order kinetics with either known or unknown time constants. It is demonstrated that some strategies allow the extraction of both the time dependence and the spectral shape of the underlying contributions.

Algorithms↗

PubMed2004

Evaluation of the ordering of membranes in multilayer stacks built on an ATR-FTIR germanium crystal with atomic force microscopy: the case of the H(+),K(+)-ATPase-containing gastric tubulovesicle membranes.

Polarized attenuated total reflection Fourier transform infrared spectra were recorded on multilayer stacks of native gastric tubulovesicle membranes. The spectral intensity and linear dichroism were measured for average thicknesses ranging between 0 and 100 bilayers. Atomic force microscopy was used to investigate the orientation of the membranes at the top of the stack. Height profiles were obtained along randomly drawn lines and slopes were computed over various distances. Orientation distribution functions were obtained from the slopes and decomposed into Legendre polynomials. It was found that the second Legendre polynomials coefficient characterizing the membrane orientation was always larger than 0.9. It could therefore be concluded that the membrane tilt does not significantly contribute to the infrared dichroism, even for the largest thicknesses tested.

Animals↗

PubMed2003

Phosphorylation-induced conformational changes of cystic fibrosis transmembrane conductance regulator monitored by attenuated total reflection-Fourier transform IR spectroscopy and fluorescence spectroscopy.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ABC protein superfamily. Phosphorylation of a regulatory domain of this protein is a prerequisite for activity. We analyzed the effect of protein kinase A (PKA) phosphorylation on the structure of purified and reconstituted CFTR protein. 1H/2H exchange monitored by attenuated total reflection Fourier transform IR spectroscopy demonstrates that CFTR is highly accessible to aqueous medium. Phosphorylation of the regulatory (R) domain by PKA further increases this accessibility. More specifically, fluorescence quenching of cytosolic tryptophan residues revealed that the accessibility of the cytoplasmic part of the protein is modified by phosphorylation. Moreover, the combination of polarized IR spectroscopy with 1H/2H exchange suggested an increase of the accessibility of the transmembrane domains of CFTR. This suggests that CFTR phosphorylation can induce a large conformational change that could correspond either to a displacement of the R domain or to long range conformational changes transmitted from the phosphorylation sites to the nucleotide binding domains and the transmembrane segments. Such structural changes may provide better access for the solutes to the nucleotide binding domains and the ion binding site.

Animals↗

PubMed2003

Characterization of a potent human interleukin-11 agonist.

Human interleukin-11 (hIL-11) is a multi-potential cytokine that is involved in numerous biological activities, such as haematopoiesis, osteoclastogenesis, neurogenesis and female fertility, and also displays anti-inflammatory properties. IL-11 is used clinically to treat chemotherapy-induced thrombocytopenia. Because of its broad spectrum of action, improved IL-11 agonists, as well as IL-11 antagonists, could be of interest for numerous clinical applications. IL-11 signalling is dependent on the formation of a tripartite ligand-receptor complex consisting of IL-11, the IL-11R (IL-11 receptor) alpha subunit (responsible for the specificity of the interaction) and gp130 (glycoprotein 130) receptor beta subunit (responsible for signal transduction). The interaction between IL-11 and IL-11Ralpha subunit occurs at its recently assigned site I. We have designed an IL-11 mutein whose hydrophobicity at site I has been increased. The mutein has been characterized in terms of structure, affinity, specificity and bioactivity. Electrophoretic analysis, gel filtration, IR spectroscopy and CD indicate that this new protein is more compact than wild-type IL-11. It binds to IL-11Ralpha with a three-fold-enhanced affinity, and retains the ability to recruit gp130 through site II. However, analysis of its biological activity revealed a complex pattern: although this mutein is 60-400-fold more active than wild-type IL-11 on the proliferation of 7TD1 murine hybridoma cell, it is less active than IL-11 on the proliferation of B9 cells, another murine hybridoma cell line. The results are interpreted on the basis of an IL-11 conformational change induced by the mutations, and the preferential use by the mutein of another unknown transducing receptor chain.

Animals↗

PubMed2003

Protein concentration is not an absolute prerequisite for the determination of secondary structure from circular dichroism spectra: a new scaling method.

We present here a simple and rapid method to extract good estimates of protein secondary structure content from circular dichroism (CD) spectra without any prior knowledge of the sample concentration. The method involves two steps: first, a single-wavelength normalization procedure and, second, the application for each secondary structure of a quadratic model based on one or two wavelength intensities. These quadratic models were derived by a cross-validation analysis of a new protein CD spectrum database. Tested on CD spectra of proteins at different concentrations, the normalization was shown to render the method virtually independent of the sample concentration. Further tests on CD spectra not recorded in our laboratory showed that our quadratic models are of general applicability. Even though the success of the present approach is less than that for currently available methods, its simplicity and the fact that the concentration is not needed may be very attractive for the study of small amounts of membrane proteins or peptides for which an accurate concentration determination might be very difficult or impossible to obtain.

Circular Dichroism↗

PubMed2003

Rationally selected basis proteins: a new approach to selecting proteins for spectroscopic secondary structure analysis.

Protein basis sets have been extensively used as reference data for the determination of protein structure with optical methods such as circular dichroism and infrared spectroscopies. We have taken a new approach to basis protein selection by utilizing three crystal structure classification databases: CATH, SCOP, and PDB_SELECT. Through the use of the information available in these and other online resources, we identified 115 commercially available proteins as potential basis set candidates. By carefully screening the quality of the crystal structures and commercial protein preparations, we obtained a final set of 50 rationally selected proteins (RaSP50) that has been optimized for use in spectroscopic protein structure determination studies. These proteins span the full range of known protein folds as well as alpha-helix and beta-sheet contents, and they represent a more comprehensive variety of fold types than any previous reference set. This report includes a detailed presentation of the reasoning behind the rational protein selection process, a description of the properties of the RaSP50 set, and a discussion of the types of structural and spectral variations that are represented in the set.

Circular Dichroism↗