Biomedical subjects

G Folkers

Publications and source records attributed to G Folkers.

At least 19 recordsLinked to original sources

PubMed2006

Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE.

The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation.

Amino Acid Sequence↗

PubMed2006

Application of high-throughput technologies to a structural proteomics-type analysis of Bacillus anthracis.

A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.

Bacillus anthracis↗

PubMed2003

Identification of steroid sulfate transport processes in the human mammary gland.

Circulating hormones and local biotransformation of steroid precursors are both sources of estrogen in human mammary tissue. Estrone-3-sulfate (E(1)S) is an important estrogenic form in premenopausal women, and dehydroepiandrosterone sulfate (DHEAS) constitutes a major adrenal precursor. Membrane transport systems that govern delivery of these anionic steroid conjugates to the mammary gland were investigated. RNA was screened by RT-PCR and Northern blotting for expression of organic anion transporting polypeptide (OATP) (solute carrier family 21A) and organic anion transporter (OAT) (solute carrier family 22A) gene families. OATP-B (SLC21A9) was the major carrier expressed; OATP-D (SLC21A11) and OATP-E (SLC21A12) were less abundant. In normal sections, OATP-B immunolocalized to the myoepithelium that surrounds the ductal epithelial cells. In invasive carcinoma, ductal epithelial cells were positive. OATP-B was characterized in stable transfected Chinese hamster ovary cells. E(1)S affinity constant (K(m)) [K(m) = 5 micro mol/liter, maximum velocity (V(max)) V(max) = 777 pmol/mg.min] and DHEAS (K(m) = 9 micro mol/liter, V(max) = 85 pmol/mg.min) were substrates. The prostaglandins (PG) A(1) and PGA(2) stimulated uptake of E(1)S and DHEAS by increasing V(max) 2-fold but not changing K(m). The effect of PGA was selectively blocked by the lipophilic thiol reagent N-ethylmaleimide but not by the hydrophilic acetamido-4'(iodoacetyl)aminostilbene-2,2'-disulfonic acid, suggesting an interaction between the electrophilic cyclopentenone ring and specific cysteine residues of OATP-B.

Algorithms↗

PubMed2003

Understanding the selectivity of fumagillin for the methionine aminopeptidase type II.

The aim of this study is to explain the selectivity of the antiangiogenic drug fumagillin for the eukaryotic enzyme methionine aminopeptidase type II (MetAP-II, EC 3.4.11.18) over the structurally very similar MetAP-I. A homology model for the human MetAP-I is constructed and molecular dynamics simulations are performed on this model with and without a docked fumagillin molecule. These simulations are compared with analogous simulations that were performed on the experimentally determined structure of the human MetAP-II enzyme. We observe an increased flexibility of the active site histidine that is covalently modified by fumagillin in the MetAP-I enzyme. The MetAP-I active site residues, particularly the fumagillin-binding histidine, have a lower probability to be in a conformation that is prone to react with the drug than their MetAP-II counterparts. This result offers an explanation for the selectivity of fumagillin for the eukaryotic MetAP-II enzyme.

Amino Acid Sequence↗

PubMed2002

Identification of a novel human organic anion transporting polypeptide as a high affinity thyroxine transporter.

Transport of various amphipathic organic compounds is mediated by organic anion transporting polypeptides (OATPs in humans, Oatps in rodents), which belong to the solute carrier family 21A (SLC21A/Slc21a). Several of these transporters exhibit a broad and overlapping substrate specificity and are expressed in a variety of different tissues. We have isolated and functionally characterized OATP-F (SLC21A14), a novel member of the OATP family. The cDNA (3059 bp) contains an open reading frame of 2136 bp encoding a protein of 712 amino acids. Its gene containing 15 exons is located on chromosome 12p12. OATP-F exhibits 47-48% amino acid identity with OATP-A, OATP-C, and OATP8, the genes of which are clustered on chromosome 12p12. OATP-F is predominantly expressed in multiple brain regions and Leydig cells of the testis. OATP-F mediates high affinity transport of T(4) and reverse T(3) with apparent K(m) values of approximately 90 nM and 128 nM, respectively. Substrates less well transported by OATP-F include T(3), bromosulfophthalein, estrone-3-sulfate, and estradiol-17beta-glucuronide. Furthermore, OATP-F-mediated T(4) uptake could be cis-inhibited by L-T(4) and D-T(4), but not by 3,5-diiodothyronine, indicating that T(4) transport is not stereospecific, but that 3',5'-iodination is important for efficient transport by OATP-F. Thus, in contrast to most other family members, OATP-F has a more selective substrate preference and may play an important role in the disposition of thyroid hormones in brain and testis.

Amino Acid Sequence↗

PubMed2001

Three-dimensional model of the cyclin-dependent kinase 1 (CDK1): Ab initio active site parameters for molecular dynamics studies of CDKS.

Cyclin-dependent kinase 1 (CDK1) is an interesting target for potential anticancer drugs, and its three-dimensional (3D) structure is presently unknown. The purpose of this work was to build a 3D model of CDK1, which could be used in drug design studies. The protein 3D structure was homology modeled, based on the known crystal structure of CDK2, and new nonbonded parameters for the Mg(2+) coordination complex were developed by means of ab initio quantum chemical calculations. These parameters were both obtained and validated using the CDK2 structure as reference, and then they were used for the refinement of the CDK1 model. The resulting CDK1 structure was satisfactory and stable at room temperature, as shown by the molecular dynamics simulations carried out over a 1-ns time interval on the entire protein. A number of representative kinases in the active and inactive form, including the inactive CDK1 modeled in this work, were compared. The results illustrate the conformational variability of the activation loop of the inactive form of the kinases and suggest a way for selectively targeting the single CDKs.

Amino Acid Sequence↗

PubMed2001

Folding and self-assembly of herpes simplex virus type 1 thymidine kinase.

Thymidine kinase from herpes simplex virus type 1 (HSV1 TK) has been postulated to be a homodimer throughout the X-ray crystallography literature. Our study shows that HSV1 TK exists as a monomer-dimer equilibrium mixture in dilute aqueous solutions. In the presence of 150 mM NaCl, the equilibrium is characterized by a dissociation constant of 2.4 microm; this constant was determined by analytical ultracentrifugation and gel filtration experiments. Dimerization seems to be unfavorable for enzymatic activity: dimers show inferior catalytic efficiency compared to the monomers. Moreover, soluble oligomers formed by self-assembly of TK in the absence of physiological salt concentrations are even enzymatically inactive. This study investigates enzymatic and structural relevance of the TK dimer in vitro. Dissociation of the dimers into monomers is not accompanied by large overall changes in secondary or tertiary structure as shown by thermal and urea-induced unfolding studies monitored by circular dichroism and fluorescence spectroscopy. A disulfide-bridge mutant TK (V119C) was designed bearing two cysteine residues at the dimer interface in order to crosslink the two subunits covalently. Under reducing conditions, the properties of V119C and wild-type HSV1 TK (wt HSV1 TK) were identical in terms of expression yield, denaturing SDS PAGE gel electrophoresis, enzyme kinetics, CD spectra and thermal stability. Crosslinked V119C (V119Cox) was found to have an increased thermal stability with a t(m) value of 59.1(+/-0.5) degrees C which is 16 deg. C higher than for the wild type protein. This is thought to be a consequence of the conformational restriction of the dimer interface. Furthermore, enzyme kinetic studies on V119Cox revealed a K(m) for thymidine of 0.2 microm corresponding to wt HSV1 TK, but a significantly higher K(m) for ATP. The present findings raise the question whether the monomer, not the dimer, might be the active species in vivo.

Adenosine Triphosphate↗

PubMed2001

Use of fluorescence polarization to monitor MHC-peptide interactions in solution.

We describe here fluorescence polarization-based methods to investigate class I MHC-peptide interactions in solution. Fluorescein-labelled peptides were used to determine MHC/peptide complex association and dissociation constants as well as the equilibrium binding constant (KD). Furthermore, we developed a competition assay for the determination of IC50 values of nonlabelled compounds. Both kinetic and equilibrium parameters are of prime importance for the development of immunomodulating compounds. The assays described here show a good reproducibility and require only picomolar amounts of labelled tracers. A high ratio between the experimental values obtained for bound and free labelled ligand as well as a low standard deviation, permits the detection of class I MHC ligands with low affinity. Fluorescence polarization allows the direct measurement of the ratio between free and bound labelled ligand in solution without any separation step. Thus, in combination with microtiter-plates, the time for analysis is significantly decreased to 10 s per sample. Our assays represent versatile tools for characterizing the binding of single ligands as well as for rapid screening of large numbers of compounds.

Fluorescence Polarization↗

PubMed2001

A spectrophotometric assay for quantitative determination of kcat of herpes simplex virus type 1 thymidine kinase substrates.

A simple method to determine the in vitro catalytic turnover constant of several substrates of herpes simplex virus type 1 thymidine kinase is presented in this study. The method is based on a continuous spectroscopic enzyme-coupled assay and allows one to monitor the herpes simplex virus type 1 thymidine kinase activity in the presence of unlabeled substrates. A clear correlation between the catalytic turnover constant and the rate of decrease in absorbance over time during the assay has been demonstrated. Exploiting this correlation, this method has been used to determine rapidly and precisely the catalytic turnover constant of antiviral lead compounds not readily available in the radioactive labeled form.

Animals↗

PubMed2001

Beta-amino acid scan of a class I major histocompatibility complex-restricted alloreactive T-cell epitope.

An HLA-B27-restricted self-octapeptide known to react with an alloreactive T-cell receptor has been modified by systematic substitution of a beta-amino acid for the natural alpha-amino acid residue, over the whole length of the parent epitope. All modified peptides were shown to bind to recombinant HLA-B*2705 and induce stable major histocompatibility complex-peptide complexes, but with some variation depending on the position of the beta-amino acid on the peptide sequence. Alteration of the natural peptide sequence at the two N-terminal positions (positions 1 and 2) decreases binding affinity and thermodynamic stability of the refolded complex, but all other positions (from position 3 to the C-terminal residue) were insensitive to the beta-amino acid substitution. All modified peptides were recognized by an alloreactive T-cell clone specific for the parent epitope with decreased efficiency, to an extent dependent of the position that was modified. Furthermore, the introduction of a single beta-amino acid at the first two positions of the modified peptide was shown to be sufficient to protect them against enzymatic cleavage. Thus, beta-amino acids represent new interesting templates for alteration of T-cell epitopes to design either synthetic vaccines of T-cell receptor antagonists.

Amino Acid Sequence↗

PubMed2001

The rational of catalytic activity of herpes simplex virus thymidine kinase. a combined biochemical and quantum chemical study.

Most antiherpes therapies exploit the large substrate acceptance of herpes simplex virus type 1 thymidine kinase (TK(HSV1)) relative to the human isoenzyme. The enzyme selectively phosphorylates nucleoside analogs that can either inhibit viral DNA polymerase or cause toxic effects when incorporated into viral DNA. To relate structural properties of TK(HSV1) ligands to their chemical reactivity we have carried out ab initio quantum chemistry calculations within the density functional theory framework in combination with biochemical studies. Calculations have focused on a set of ligands carrying a representative set of the large spectrum of sugar-mimicking moieties and for which structural information of the TK(HSV1)-ligand complex is available. The k(cat) values of these ligands have been measured under the same experimental conditions using an UV spectrophotometric assay. The calculations point to the crucial role of electric dipole moment of ligands and its interaction with the negatively charged residue Glu(225). A striking correlation is found between the energetics associated with this interaction and the k(cat) values measured under homogeneous conditions. This finding uncovers a fundamental aspect of the mechanism governing substrate diversity and catalytic turnover and thus represents a significant step toward the rational design of novel and powerful prodrugs for antiviral and TK(HSV1)-linked suicide gene therapies.

Antiviral Agents↗

PubMed2001

Mutation of Cys-67 alters the thermodynamic stability of the human leukocyte antigen HLA0-B*2705.

The B pocket of the class I major histocompatibility complex-encoded protein HLA-B*2705 has recently been suggested to be responsible for the misfolding of this HLA haplotype and thus to induce susceptibility to autoimmune inflammatory diseases. Four mutants of the B*2705 heavy chain were refolded in the presence of three control peptides. The monitoring of the thermal unfolding of the B*2705-peptide complexes by circular dichroism spectroscopy showed that all heterotrimeric mutants were markedly less stable than the corresponding complexes with the wild-type heavy chain. Among the four heavy chain mutations, the C67S change was investigated for unfolding and peptide binding properties because this position may mediate disulfide pair bridging and alter T-cell recognition of HLA-B*2705. Wild-type heterotrimers completely unfold in a single transition at mild acidic pH whereas increase of the pH to mild basic conditions induce only a partial biphasic unfolding. Cys-67 seems to play a crucial role in controlling the thermodynamic stability of the B*2705-peptide complexes as the C67S mutant unfolds faster and with a single transition, independent of pH. Fluorescence polarization and size exclusion chromatography of unfolding intermediates suggest that the peculiar unfolding of the B*2705 wild-type heavy chain cannot be explained by modified peptide binding properties but more likely by the formation of high molecular weight species.

Cysteine↗

PubMed2001

Cell fingerprinting: an approach to classifying cells according to mass profiles of digests of protein extracts.

We present a statistical framework for classifying cells according to the set of peptide masses obtained by mass spectrometric analysis of digestions of whole cell protein extracts. The digest is separated by high performance liquid chromatography (HPLC) coupled directly to a mass spectrometer either by an electrospray interface or by collection to a matrix-assisted laser desorption/ionization target plate. Here, the mass to charge ratio, intensity, and HPLC retention time of the peptides are measured. We have used defined bacterial strains to test this approach. For each bacterium, this process is repeated for extracts obtained at different points in the growth curve in order to try and define an invariant set of signals that uniquely identify the bacterium. This paper presents algorithms for the creation of this cell fingerprint database and develops a Bayesian classification scheme for deciding whether or not an unknown bacterium has a match in the database. Our initial testing based on a limited data set of three bacteria indicates that our approach is feasible. Via a jack-knife test, our Bayesian classification scheme correctly identified the bacterium in 67.8% of the cases.

Bacteria↗

PubMed2001

A rationally designed oligopeptide shows significant conformational changes upon binding to sulphate ions.

Oligopeptides that interact with oxoanions were developed by rational design methods. The substrate-binding site of the enzyme purine nucleoside phosphorylase served as a model for the design of the ionophores. The amino acids involved in the complexation of oxoanions were linked through flexible spacer residues. These spacers were chosen such that the relative orientation of the interacting amino acids was conserved. Several peptide sequences were preselected based on intermolecular H-bond frequencies. These frequencies were calculated from molecular dynamics trajectories of the corresponding peptide-anion complexes and used to score the binding properties of the peptides. The most promising peptides were prepared using solid phase peptide synthesis. Anion binding of the peptide ionophores was screened using circular dichroism (CD) and confirmed by NMR spectroscopy. CD measurements performed in methanol revealed a significant conformational change of a linear undecapeptide upon binding to sulphate ions. Two-dimensional-NMR experiments confirmed that a conformation with high helical content is formed in the presence of sulphate ions. These conformational changes induced by the anion stimulate the development of new transduction mechanisms in chemical sensors.

Amino Acid Sequence↗

PubMed2001

NMR-restrained docking of a peptidic inhibitor to the N-terminal domain of the phosphoenolpyruvate:sugar phosphotransferase enzyme I.

Starting from the NMR structure of the binary complex between the N-terminal domain of the unphosphorylated enzyme I (EIN) of the phosphoenolpyruvate:sugar phosphotransferase (PTS) and the histidine-containing phosphocarrier protein (HPr), a molecular model of the phosphorylated transition state of the related complex was established using constrained simulated annealing. The coordinates of the phosphorylated EIN enzyme were then used in a second step for flexible docking of a decapeptide inhibitor of EIN whose enzyme-bound conformation itself was determined by NMR using transferred nuclear Overhauser effects. Two phosphorylation models of the peptide inhibitor were investigated and shown to be both functional. Interestingly, one model is very similar to that of the complex between EIN and its natural substrate HPr. The present study demonstrates that NMR-guided flexible docking constitutes an interesting tool for docking highly flexible peptide ligands and facilitates the upcoming protein-based design of nonpeptide EIN inhibitors for discovering new antibiotics.

Amino Acid Sequence↗

PubMed2001

The effect of substrate binding on the conformation and structural stability of Herpes simplex virus type 1 thymidine kinase.

The structure of Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) is known at high resolution in complex with a series of ligands and exhibits important structural similarities to the nucleoside monophosphate (NMP) kinase family, which are known to show large conformational changes upon binding of substrates. The effect of substrate binding on the conformation and structural stability of TK(HSV1), measured by thermal denaturation experiments, far-UV circular dichroism (CD) and fluorescence is described, and the results indicate that the conformation of the ligand-free TK(HSV1) is less ordered and less stable compared to the ligated enzyme. Furthermore, two crystal structures of TK(HSV1) in complex with two new ligands, HPT and HMTT, refined to 2.2 A are presented. Although TK(HSV1):HPT does not exhibit any significant deviations from the model of TK(HSV1):dT, the TK(HSV1):HMTT complex displays a unique conformationally altered active site resulting in a lowered thermal stability of this complex. Moreover, we show that binding affinity and binding mode of the ligand correlate with thermal stability of the complex. We use this correlation to propose a method to estimate binding constants for new TK(HSV1)substrates using thermal denaturation measurements monitored by CD spectroscopy. The kinetic and structural results of both test substrates HPT and HMTT show that the CD thermal denaturation system is very sensitive to conformational changes caused by unusual binding of a substrate analog.

Antigens, Surface↗

PubMed2000

Protein-based virtual screening of chemical databases. 1. Evaluation of different docking/scoring combinations.

Three different database docking programs (Dock, FlexX, Gold) have been used in combination with seven scoring functions (Chemscore, Dock, FlexX, Fresno, Gold, Pmf, Score) to assess the accuracy of virtual screening methods against two protein targets (thymidine kinase, estrogen receptor) of known three-dimensional structure. For both targets, it was generally possible to discriminate about 7 out of 10 true hits from a random database of 990 ligands. The use of consensus lists common to two or three scoring functions clearly enhances hit rates among the top 5% scorers from 10% (single scoring) to 25-40% (double scoring) and up to 65-70% (triple scoring). However, in all tested cases, no clear relationships could be found between docking and ranking accuracies. Moreover, predicting the absolute binding free energy of true hits was not possible whatever docking accuracy was achieved and scoring function used. As the best docking/consensus scoring combination varies with the selected target and the physicochemistry of target-ligand interactions, we propose a two-step protocol for screening large databases: (i) screening of a reduced dataset containing a few known ligands for deriving the optimal docking/consensus scoring scheme, (ii) applying the latter parameters to the screening of the entire database.

Algorithms↗

PubMed2000

Nucleoside binding site of herpes simplex type 1 thymidine kinase analyzed by X-ray crystallography.

The crystal structures of the full-length Herpes simplex virus type 1 thymidine kinase in its unligated form and in a complex with an adenine analogue have been determined at 1.9 A resolution. The unligated enzyme contains four water molecules in the thymidine pocket and reveals a small induced fit on substrate binding. The structure of the ligated enzyme shows for the first time a bound adenine analogue after numerous complexes with thymine and guanine analogues have been reported. The adenine analogue constitutes a new lead compound for enzyme-prodrug gene therapy. In addition, the structure of mutant Q125N modifying the binding site of the natural substrate thymidine in complex with this substrate has been established at 2.5 A resolution. It reveals that neither the binding mode of thymidine nor the polypeptide backbone conformation is altered, except that the two major hydrogen bonds to thymidine are replaced by a single water-mediated hydrogen bond, which improves the relative acceptance of the prodrugs aciclovir and ganciclovir compared with the natural substrate. Accordingly, the mutant structure represents a first step toward improving the virus-directed enzyme-prodrug gene therapy by enzyme engineering.

Adenine↗