Biomedical subjects

Y Engelborghs

Publications and source records attributed to Y Engelborghs.

At least 19 recordsLinked to original sources

PubMed2006

Stability analysis of the bacteriophage phiKMV lysin gp36C and its putative role during infection.

The kinetic, thermodynamic and structural stability of gp36C, the virion-associated peptidoglycan hydrolase domain of bacteriophage phiKMV, is analyzed. Recombinant gp36C is highly thermoresistant (k = 0.595 h(-1) at 95 degrees C), but not thermostable (T(m) = 50.2 degrees C, DeltaH(cal) = 6.86 x 10(4) cal mol(-1)). However, aggregation influences kinetic stability in an unusual manner since aggregation is more pronounced at 55 degrees C than at higher temperatures. Furthermore, gp36C reversibly unfolds in a two-state endothermic transition, and circular dichroism analysis shows that gp36C almost completely refolds after a 3-h heat treatment at 85 degrees C. These properties are in agreement with gp36C being part of the extensible tail which is ejected in an unfolded state during phage infection.

Amino Acid Sequence↗

PubMed2005

In vitro study of the pulmonary translocation of nanoparticles: a preliminary study.

Recent studies indicate that inhaled ultrafine particles can pass into the circulation. To study this translocation in an in vitro model three types of pulmonary epithelial cells were examined. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. TEER was too low in A549 cells. In these preliminary experiments, TEER values of 1007+/-300 and 348+/-62 Omega cm2 were reached for the Calu-3 cell line, using permeable membranes of 0.4 and 3 microm pore size, respectively. Growing primary rat type II pneumocytes on 0.4 microm pores, a TEER value of 241+/-90 Omega cm2 was reached on day 5; on 3 microm pores, no acceptable high TEER value was obtained. Translocation studies were done using 46 nm fluorescent polystyrene particles. When incubating polystyrene particles on membranes without a cellular monolayer, significant translocation was only observed using 3 microm pores: 67.5% and 52.7% for carboxyl- and amine-modified particles, respectively. Only the Calu-3 cell line was used in an initial experiment to investigate the translocation: on 0.4 microm pores no translocation was observed, on 3 microm pores approximately 6% translocation was observed both for carboxyl- and amine-modified particles.

Air Pollutants↗

PubMed2004

Novel inhibitors of HIV-1 integration.

Human immunodeficiency virus (HIV) is the etiological agent of the acquired immune deficiency syndrome (AIDS). The current strategy for the treatment of HIV infection is called Highly Active Antiretroviral Therapy (HAART) and is based on cocktails of drugs that are currently approved by the Food and Drug Administration. These drugs include compounds that target the viral entry step and the enzymes reverse transcriptase or protease. The introduction of HAART has dramatically changed the landscape of HIV disease. Death from AIDS-related diseases has been reduced significantly since HAART came into use. Nevertheless it is not clear how long clinical benefit will last taking into account the emergence of multiple drug-resistant viral strains. Addition of new anti-HIV drugs targeting other steps of the viral replication cycle may increase the potency of inhibition and delay resistance development. HIV integrase is an essential enzyme in the HIV life cycle and is an attractive target for new drug development. Despite years of intensive research, only two classes of compounds that inhibit integration have been identified until now, namely the diketo acids and the pyranodipyrimidines. In this review we will point to new potential antiviral targets related to retroviral integration that are amenable to drug development. We will describe the pitfalls of currently used integrase assays and propose new strategies and technologies for the discovery of HIV integration inhibitors. Furthermore, we will describe the two classes of integrase inhibitors and discuss their antiviral activity, molecular mechanism of anti-HIV action and the selection of HIV resistance against these drugs.

Animals↗

PubMed2003

Non-Gaussian statistics of the vibrational fluctuations of myoglobin.

Experiments on the dynamics of vibrational fluctuations in myoglobin revealed an interesting behavioral cross-over occurring in the range 180-200 K. In this temperature range the mean square displacement of atomic positions versus temperature sharply increases its slope, indicating the dissociation of CO from the heme group. In this paper we develop a theoretical model that provides a framework for the quantitative description of this phenomenon. The basis of our calculations is an assumption of an effective potential with multiple local minima. In particular, we consider a quartic potential in place of the simple quadratic. We then use non-Gaussian statistics to obtain a relationship between the mean square displacement and model parameters. We compare our model to published experimental data and show that it can describe the data set using physically meaningful parameters which are fitted to the experimental data. In the process we verify the Gaussian approximation's applicability only to the low-temperature régime. In the high-temperature limit, however, deviations from the Gaussian approximation are due to the double-well nature of our effective potential. We find that the published datasets showing the thermal transition display the qualitative trends predicted by appropriate algebraic approximations to our predicted myoglobin behavior.

Computer Simulation↗

PubMed2001

The analysis of time resolved protein fluorescence in multi-tryptophan proteins.

In the last decades, considerable progress has been made in the analysis of the fluorescence decay of proteins with more than one tryptophan. The construction of single tryptophan containing proteins has shown that the lifetimes of the wild type proteins are often the linear combinations of the family lifetimes of the contributing tryptophan residues. Additivity is not followed when energy transfer takes place among tryptophan residues or when the structure of the remaining protein is altered upon the modification. Progress has also been made in the interpretation of the value of the lifetime and the linkage with the immediate environment. Probably all the irreversible processes leading to return to the ground state have been catalogued and their rate constants are documented. Also, the process of electron transfer to the peptide carbonyl is becoming more and more documented and is linked to the rotameric state of tryptophan. Reversible excited state processes are also being considered, including reversible interconversions between rotamers. Interesting information about tryptophan and its environment comes also from anisotropy measurements for proteins in the native, the denatured and the molten globule states. Alterations of protein fluorescence due to the effects of ligand binding or side chain modifications can be analyzed via the ratio of the quantum yields of the modified protein and the reference state. Using the ratio of quantum yields and the (amplitude weighted) average lifetime, three factors can be identified: (1) a change in the apparent radiative rate constant reflecting either static quenching or an intrinsic change in the radiative properties; (2) a change in dynamic quenching; and (3) a change in the balance of the populations of the microstates or local static quenching.

Anisotropy↗

PubMed2001

Interactions between oligonucleotides and cationic polymers investigated by fluorescence correlation spectroscopy.

PURPOSE: To evaluate whether fluorescence correlation spectroscopy (FCS) can be used to characterize the complexation between oligonucleotides and cationic polymers. METHODS: The features of the complexes between rhodamine labeled oligonucleotides (Rh-ONs) and poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), poly(ethylene glycol)-poly(ethyleneimine) (pEG-pEI), and diaminobutane-dendrimer-(NH2)64 (DAB64) were characterized by light scattering, electrophoretic mobility, electrophoresis, and FCS. RESULTS: At low polymer/Rh-ON ratios, a decrease of the fluorescence of the Rh-ONs was observed on binding of the Rh-ONs to all cationic polymers. This was explained by the creation of "multimolecular complexes" in which the Rh-labels quench each other. The multimolecular complexes, which are highly fluorescent as they carry a number of Rh-ONs, resulted in high fluorescence peaks in the fluorescence fluctuation profile as measured by FCS. For pDMAEMA and DAB64, at higher polymer/Rh-ON ratios the fluorescence of the polyplexes increased, caused by the formation of "monomolecular complexes," which consist of only one Rh-ON per polymer. In the case of pEG-pEI, the fluorescence stayed constant when the polymer/Rh-ON ratio increased, so multimolecular polyplexes remained. FCS confirmed these results as the high fluorescence peaks disappeared in case of pDMAEMA/Rh-ON and DAB64/Rh-ON dispersions, but remained present for pEG-pEI/Rh-ON dispersions. CONCLUSIONS: FCS seems applicable for study of the interactions between ONs and different types of cationic polymers.

Cations↗

PubMed2001

Extending the capabilities of targeted molecular dynamics: simulation of a large conformational transition in plasminogen activator inhibitor 1.

Plasminogen activator inhibitor type 1 (PAI-1) is an inhibitor of plasminogen activators such as tissue-type plasminogen activator or urokinase-type plasminogen activator. For this molecule, different conformations are known. The inhibiting form that interacts with the proteinases is called the active form. The noninhibitory, noncleavable form is called the latent form. X-ray and modeling studies have revealed a large change in position of the reactive center loop (RCL), responsible for the interaction with the proteinases, that is inserted into a beta-sheet (s4A) in the latent form. The mechanism underlying this spontaneous conformational change (half-life = 2 h at 37 degrees C) is not known in detail. This investigation attempts to predict a transition path from the active to the latent structure at the atomic level, by using simulation techniques. Together with targeted molecular dynamics (TMD), a plausible assumption on a rigid body movement of the RCL was applied to define an initial guess for an intermediate. Different pathways were simulated, from the active to the intermediate, from the intermediate to the latent structure and vice versa under different conditions. Equilibrium simulations at different steps of the path also were performed. The results show that a continuous pathway from the active to the latent structure can be modeled. This study also shows that this approach may be applied in general to model large conformational changes in any kind of protein for which the initial and final three-dimensional structure is known.

Computer Simulation↗

PubMed2001

Membrane topology of the Streptomyces lividans type I signal peptidases.

Most bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins. For Streptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described. In this communication, we report the experimental determination of the membrane topology of these SPases. A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ have a predicted topology similar to that of SipY. These three S. lividans SPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast, S. lividans SipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria. Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo. Moreover, for the S. lividans SPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell. Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found.

Base Sequence↗

PubMed2001

Heuristic statistical analysis of fluorescence fluctuation data with bright spikes: application to ligand binding to the human serotonin receptor expressed in Escherichia coli cells.

A statistical method for the analysis of fluorescence fluctuation amplitudes including bright spikes is presented. This situation arises e. g. when fluorescent ligands interact with receptors carrying multiple binding sites. The technique gives information on the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscopy analysis, which cannot be applied in this situation. Two simple statistical tests are proposed that can discriminate between fluorescence intensities originating from free ligands or complexes. The performance of the two tests is evaluated and compared on mixtures of a fluorophore and fluorophore-coated beads that mimic the behaviour of multi-liganded complexes. An application to ligand binding to the serotonin receptor, expressed on Escherichia coli cells, is also provided. Specific binding of a fluorophore to this receptor, as well as competition with several ligands, is assessed.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2001

Fluorescence fluctuation analysis for the study of interactions between oligonucleotides and polycationic polymers.

The interactions between a cationic polymer, poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), and negatively charged rhodamine-labeled 25-mer phosphodiester oligonucleotides (Rh-ONs) were studied by fluorescence fluctuation spectroscopy and other techniques. The composition of the pDMAEMA/Rh-ON complexes was investigated as a function of the charge ratio (+/-) by increasing the pDMAEMA concentration and keeping the Rh-ON concentration constant. We applied two different methods for analyzing the fluorescence fluctuation profiles of the pDMAEMA/Rh-ON complexes, which depended on their composition. First, we analyzed the data with the photon counting histogram (PCH) technique, which determines the molecular brightness and the concentration of fluorophores (Chen et al, 1999). A particular challenge for the data analysis is the occurrence of sudden fluorescence bursts in the fluorescence fluctuation profiles, which are linked to the appearance of multimolecular complexes (i. e. when several Rh-ONs were present in one complex). A quantitative interpretation of the analysis for the complexes remains challenging and is connected to the rarity of the fluorescent bursts, which do not provide sufficient data statistics. To specifically address the problem of the fluorescent bursts we employed a method described by Van Craenenbroeck et al. (1999). This method, applicable only when data were integrated over much longer time bins, allowed us to estimate the number of fluorescence bursts which could be considered as a relative measure of the amount of multimolecular complexes present. When monomolecular complexes were formed, i. e. at high values of the charge ratio, highly intense fluorescence peaks were not present and the interpretation of the PCH analysis was more straightforward. The molecular brightness of the species (epsilon), as revealed from PCH analysis, was greater than epsilon for the free Rh-ONs, indicating that the Rh-ONs were attached to pDMAEMA chains.

Cations↗

PubMed2000

Kinetic analysis of novel multisubstrate analogue inhibitors of thymidine phosphorylase.

A kinetic analysis was performed for the novel 1-(8-phosphonooctyl)-6-amino-5-bromouracil and 1-(8-phosphonooctyl)-7-deazaxanthine inhibitors of Escherichia coli thymidine (dThd) phosphorylase (TPase). The structure of the compounds was rationally designed based on the available crystal structure coordinates of bacterial TPase. These inhibitors reversibly inhibited TPase. Kinetic analysis revealed that the compounds inhibited TPase in a purely competitive or mixed fashion not only when dThd, but also when inorganic phosphate (Pi), was used as the variable substrate. In contrast, the free bases 6-amino-5-bromouracil and 7-deazaxanthine behaved as non-competitive inhibitors of the enzyme in the presence of variable Pi concentrations while being competitive or mixed with respect to thymine as the natural substrate. Our kinetic data thus revealed that the novel 1-(8-phosphonooctyl)pyrimidine/purine derivatives are able to function as multisubstrate inhibitors of TPase, interfering at two different sites (dThd(Thy)- and phosphate-binding site) of the enzyme. To our knowledge, the described compounds represent the first type of such multisubstrate analogue inhibitors of TPase; they should be considered as lead compounds for the development of mechanistically novel type of TPase inhibitors.

Bromouracil↗

PubMed2000

Microenvironment of cysteine 242 in type-1 ribosome-inactivating protein from iris.

IRIP is a type-1 ribosome-inactivating protein isolated from the bulbs of Iris hollandica. It is one of the few type-1 RIPs that contain Cys residue(s) in their primary sequence. IRIP contains a single Cys residue at position 242. Although IRIP is thought to be a monomeric protein, SDS-PAGE indicates that part of the IRIP molecules can exist as disulphide bridge-linked dimers. Probing of the reactivity of the unique Cys residue by 5, 5'-dithiobis(2-nitrobenzoic acid) indicates that Cys(242) in IRIP is free but is only partially accessible to modifiers. Molecular modelling of IRIP is in agreement with this conclusion. Binding of the ligands adenine and poly(A) results in little or no effect on the conformation of Cys(242) in IRIP. Chemical modification of IRIP by a specific thiol modifier does not abolish the RNA N-glycosidase activity of IRIP, suggesting that Cys(242) is not critical for the enzymatic activity of IRIP. These results suggest that IRIP has the potential to be developed as a novel immunotoxin.

Amino Acid Sequence↗

PubMed2000

Molecular recognition of taxol by microtubules. Kinetics and thermodynamics of binding of fluorescent taxol derivatives to an exposed site.

We have determined the kinetic scheme and the reaction rates of binding to microtubules of two fluorescent taxoids, 7-O-[N-(4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-1) and 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-l-alanyl]Taxol (Flutax-2). Flutax-1 and Flutax-2 bind to microtubules with high affinity (K(a) approximately 10(7) m(-1), 37 degrees C). The binding mechanism consists of a fast bimolecular reaction followed by at least two monomolecular rearrangements, which were characterized with stopped-flow techniques. The kinetic constants of the bimolecular reaction were 6.10 +/- 0.22 x 10(5) m(-1) s(-1) and 13.8 +/- 1.8 x 10(5) m(-1) s(-1) at 37 degrees C, respectively. A second slow binding step has been measured employing the change of fluorescence anisotropy of the probe. The reversal of this reaction is the rate-limiting step of dissociation. A third step has been detected using small angle x-ray scattering and involves a 2-nm increase in the diameter of microtubules. It is suggested that the first step entails the binding of the Taxol moiety and the second a relative immobilization of the fluorescent probe. The equilibrium and some kinetic measurements required the use of stabilized cross-linked microtubules, which preserved taxoid binding. The results indicate that the Taxol binding site is directly accessible, in contrast with its location at lumen in the current model of microtubules. An alternative structural model is considered in which the binding site is located between protofilaments, accessible from the microtubule surface.

Animals↗

PubMed2000

Interaction between carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) and saturating concentrations of Calcofluor White. A fluorescence study.

Calcofluor White is a fluorescent probe that interacts with polysaccharides and is commonly used in clinical studies. Interaction between Calcofluor White and carbohydrate residues of alpha1-acid glycoprotein (orosomucoid) was previously followed by fluorescence titration of the Trp residues of the protein. A stoichiometry of one Calcofluor for one protein has been found [J.R. Albani and Y.D. Plancke, Carbohydr. Res., 318 (1999) 193-200]. Alpha1-acid glycoprotein contains 40% carbohydrate by weight and has up to 16 sialic acid residues. Since binding of Calcofluor to alpha1-acid glycoprotein occurs mainly on the carbohydrate residues, we studied in the present work the interaction between Calcofluor and the protein by following the fluorescence change of the fluorophore. In order to establish the role of the sialic acid residues in the interaction, the experiments were performed with the sialylated and asialylated protein. Interaction of Calcofluor with sialylated alpha1-acid glycoprotein induces a red shift of the emission maximum of the fluorophore from 438 to 450 nm at saturation (one Calcofluor for one sialic acid) and an increase in the fluorescence intensity. At saturation the fluorescence intensity increase levels off. Binding of Calcofluor to asialylated acid glycoprotein does not change the position of the emission maximum of the fluorophore and induces a decrease in its fluorescence intensity. Saturation occurs when 10 molecules of Calcofluor are bound to 1 mol of alpha1-acid glycoprotein. Since the protein contains five heteropolysaccharide groups, we have 2 mol of Calcofluor for each group. Addition of free sialic acid to Calcofluor induces a continuous decrease in the fluorescence intensity of the fluorophore but does not change the position of the emission maximum. Our results confirm the presence of a defined spatial conformation of the sialic acid residues, a conformation that disappears when they are free in solution. Dynamics studies on Calcofluor White and the carbohydrate residues of alpha1-acid glycoprotein are also performed at saturating concentrations of Calcofluor using the red-edge excitation spectra and steady-state anisotropy studies. The red-edge excitation spectra experiments show an important shift (13 nm) of the fluorescence emission maximum of the probe. This reveals that emission of Calcofluor occurs before relaxation of the surrounding carbohydrate residues occurs. Emission from a non-relaxed state means that the microenvironment of bound Calcofluor is rigid, inducing in this way the rigidity of the fluorophore itself, a result confirmed by anisotropy studies.

Benzenesulfonates↗

PubMed2000

Expression of chymotrypsin(ogen) in the thioredoxin reductase deficient mutant strain of Escherichia coli AD494(DE3) and purification via a fusion product with a hexahistidine-tail.

A reliable protocol was designed for fast expression and purification of recombinant chymotrypsin(ogen). The zymogen was overexpressed in soluble form as a (His)6-fusion construct in the cytoplasm of the thioredoxin reductase deficient Escherichia coli strain AD494(DE3). This allowed purification of chymotrypsinogen in a highly selective affinity chromatography capture step using a Ni-NTA column. After activation with enterokinase, the enzymatically active chymotrypsin was purified in a polishing step using a modified soybean trypsin inhibitor agarose column. This expression system and the use of affinity chromatography for capture and polishing, offers an easier and faster route to recombinant chymotrypsin(ogen) than the previously described use of Saccharomyces cerevisiae.

Base Sequence↗

PubMed2000

Purification and characterization of an 18-kd allergen of birch (Betula verrucosa) pollen: identification as a cyclophilin.

BACKGROUND: Five birch pollen allergens have been identified so far. In a previous study we detected new birch pollen allergens with an isoelectric point in the range 9.0 to 9.3, present only in extracts prepared at controlled basic pH. OBJECTIVE: The purpose of the current study was to purify and characterize those allergens. METHODS: The target allergens were purified by ion exchange and hydrophobic interaction chromatography. Analyses were carried out by SDS-PAGE, isoelectric focusing, immunoblotting, and amino acid sequencing. The in vivo reactivity of the allergens was evaluated by skin testing. RESULTS: An 18-kd protein, which we named Bet v 7, was purified. This 18-kd protein corresponded to 3 bands on isoelectric-focusing immunoblots that probably represent isoforms. On immunoblots up to 20.8% of birch pollen-allergic patients recognized those allergens. The clinical relevance of Bet v 7 was demonstrated by positive immediate-type skin testing on a patient allergic to birch pollen. Sequencing of an internal peptide yielded an amino acid sequence showing high homology with various plant cyclophilins. The rotamase activity of the protein, inhibited by cyclosporin A, further confirmed that Bet v 7 belongs to the group of cyclophilins. CONCLUSION: We have purified a novel allergen of birch pollen, Bet v 7, belonging to the cyclophilin family. Because cyclophilins are highly conserved proteins over the phylogeny, we may postulate that Bet v 7 is a member of a new family of panallergens.

Allergens↗

PubMed2000

A step toward the prediction of the fluorescence lifetimes of tryptophan residues in proteins based on structural and spectral data.

A method is presented that allows the calculation of the lifetimes of tryptophan residues on the basis of spectral and structural data. It is applied to two different proteins. The calcium binding protein from the sarcoplasm of the muscles of the sand worm Nereis diversicolor (NSCP) changes its conformation upon binding of Ca2+ or Mg2+. NSCP contains three tryptophan residues at position 4, 57, and 170, respectively. The fluorescence lifetimes of W57 are investigated in a mutant in which W4 and W170 have been replaced. The time resolved fluorescence properties of W57 are linked to its different microconformations, which were determined by a molecular dynamics simulation map. Together with the determination of the radiative rate constant from the wavelength of maximum intensity of the decay associated spectra, it was possible to determine an exponential relation between the nonradiative rate constant and the distance between the indole CE3 atom and the carbonyl carbon of the peptide bond reflecting a mechanism of electron transfer as the main determinant of the value for the nonradiative rate constant. This result allows the calculation of the fluorescence lifetimes from the protein structure and the spectra. This method was further tested for the tryptophan of Ha-ras p21 (W32) and for W43 of the Tet repressor, which resulted in acceptable values for the predicted lifetimes.

Animals↗

PubMed2000

Role of the switch II region in the conformational transition of activation of Ha-ras-p21.

The role of the switch II region in the conformational transition of activation of Ha-ras-p21 has been investigated by mutating residues predicted to act as hinges for the conformational transition of this loop (Ala59, Gly60, and Gly75) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997, Proteins 28:434-451), as well as mutating the catalytic residue Gln61. The proposed mutations of the hinge residues decrease the rate of the conformational transition of activation as measured by the binding of BeF3- to the GDP-p21 complex. Also, the thermodynamic parameters of the binding reaction are altered by a factor between three and five, depending on the temperature. (Due to changes in activation and reaction enthalpies, partially compensated by entropy changes.) The control mutation Q61H in which only the catalytic residue is changed has only a limited effect on the kinetic rate constants of the conformational transition and on the thermodynamic parameters of the reaction. The fact that mutations of the hinge residues of the switch II region affect both the binding of the phosphate analog and the conformational transition of activation indicates that the switch II is implicated both in the early and the late states of the transition.

Beryllium↗