Biomedical subjects

G Folkers

Publications and source records attributed to G Folkers.

At least 37 records · Page 2Linked to original sources

PubMed2000

An analysis of the helix-to-strand transition between peptides with identical sequence.

An analysis of peptide segments with identical sequence but that differ significantly in structure was performed over non-redundant databases of protein structures. We focus on those peptides, which fold into an alpha-helix in one protein but a beta-strand in another. While the study shows that many such structurally ambivalent peptides contain amino acids with a strong helical preference collocated with amino acids with a strong strand preference, the results overwhelmingly indicate that the peptide's environment ultimately dictates its structure. Furthermore, the first naturally occurring structurally ambivalent nonapeptide from evolutionary unrelated proteins is described, highlighting the intrinsic plasticity of peptide sequences. We even find seven proteins that show structural ambivalence under different conditions. Finally, a computer algorithm has been implemented to identify regions in a given sequence where secondary structure prediction programs are likely to make serious mispredictions.

Algorithms↗

PubMed2000

Thermodynamic stability of HLA-B*2705. Peptide complexes. Effect of peptide and major histocompatibility complex protein mutations.

Designing synthetic vaccines from class I major histocompatibility complex (MHC)-binding antigenic peptides requires not only knowledge of the binding affinity of the designed peptide but also predicting the stability of the formed MHC-peptide complex. In order to better investigate structure-stability relationships, we have determined by circular dichroism spectroscopy the thermal stability of a class I MHC protein, HLA-B*2705, in complex with a set of 39 singly substituted peptide analogues. The influence of two anchoring side chains (P3 and P9) was studied by peptide mutation and appropriate site-directed mutagenesis of the HLA-B*2705 binding groove. The side chain at P9 is clearly the one that contributes the most to the thermal stability of the MHC-peptide complexes, as destabilization up to 25 degrees C are obtained after P9 mutation. Interestingly, structure-stability relationships do not fully mirror structure-binding relationships. As important as the C-terminal side chain are the terminal ammonium and carboxylate groups. Removal of a single H-bond between HLA-B27 and the terminal peptide moieties results in thermal destabilization up to 10 degrees C. Depending on the bound peptide and the location of the deleted H-bond, the decrease in the thermal stability of the corresponding complex is quantitatively different. The present study suggests that any peptidic amino acid at positions 3 and 9 promotes refolding of the B27-peptide complex. Once the complex is formed, the C-terminal side chain seems to play an important role for maintaining a stable complex.

Base Sequence↗

PubMed2000

Kinetics and crystal structure of the wild-type and the engineered Y101F mutant of Herpes simplex virus type 1 thymidine kinase interacting with (North)-methanocarba-thymidine.

Kinetic and crystallographic analyses of wild-type Herpes simplex virus type 1 thymidine kinase (TK(HSV1)) and its Y101F-mutant [TK(HSV1)(Y101F)] acting on the potent antiviral drug 2'-exo-methanocarba-thymidine (MCT) have been performed. The kinetic study reveals a 12-fold K(M) increase for thymidine processed with Y101F as compared to the wild-type TK(HSV1). Furthermore, MCT is a substrate for both wild-type and mutant TK(HSV1). Its binding affinity for TK(HSV1) and TK(HSV1)(Y101F), expressed as K(i), is 11 microM and 51 microM, respectively, whereas the K(i) for human cytosolic thymidine kinase is as high as 1.6 mM, rendering TK(HSV1) a selectivity filter for antiviral activity. Moreover, TK(HSV1)(Y101F) shows a decrease in the quotient of the catalytic efficiency (k(cat)/K(M)) of dT over MCT corresponding to an increased specificity for MCT when compared to the wild-type enzyme. Crystal structures of wild-type and mutant TK(HSV1) in complex with MCT have been determined to resolutions of 1.7 and 2.4 A, respectively. The thymine moiety of MCT binds like the base of dT while the conformationally restricted bicyclo[3.1.0]hexane, mimicking the sugar moiety, assumes a 2'-exo envelope conformation that is flatter than the one observed for the free compound. The hydrogen bond pattern around the sugar-like moiety differs from that of thymidine, revealing the importance of the rigid conformation of MCT with respect to hydrogen bonds. These findings make MCT a lead compound in the design of resistance-repellent drugs for antiviral therapy, and mutant Y101F, in combination with MCT, opens new possibilities for gene therapy.

Amino Acid Substitution↗

PubMed2000

Compulsory order of substrate binding to herpes simplex virus type 1 thymidine kinase. A calorimetric study.

Isothermal titration calorimetry has been used to investigate the thermodynamic parameters of the binding of thymidine (dT) and ATP to herpes simplex virus type 1 thymidine kinase (HSV1 TK). Binding follows a sequential pathway in which dT binds first and ATP second. The free enzyme does not bind ATP, whose binding site becomes only accessible in the HSV1 TK.dT complex. At pH 7.5 and 25 degrees C, the binding constants are 1.9 x 10(5) m(-1) for dT and 3.9 x 10(6) m(-1) for ATP binding to the binary HSV1 TK.dT complex. Binding of both substrates is enthalpy-driven and opposed by a large negative entropy change. The heat capacity change (DeltaCp) obtained from DeltaH in the range of 10-25 degrees C is -360 cal K(-1) mol(-1) for dT binding and -140 cal K(-1) mol(-1) for ATP binding. These large DeltaCp values are incompatible with a rigid body binding model in which the dT and ATP binding sites pre-exist in the free enzyme. Values of DeltaCp and TDeltaS strongly indicate large scale conformational adaptation of the active site in sequential substrate binding. The conformational changes seem to be more pronounced in dT binding than in the subsequent ATP binding. Considering the crystal structure of the ternary HSV1 TK.dT.ATP complex, a large movement in the dT binding domain and a smaller but substantial movement in the LID domain are proposed to take place when the enzyme changes from the substrate-free, presumably more open and less ordered conformation to the closed and compact conformation of the ternary enzyme-substrate complex.

Adenosine Triphosphate↗

PubMed2000

Highly purified recombinant varicella Zoster virus thymidine kinase is a homodimer.

Recombinant varicella zoster virus (VZV) thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli. The TK was expressed as a PreScission-cleavable fusion protein and purified by glutathione and ATP affinity chromatography, yielding homogeneous, highly pure VZV TK. The purified enzyme displays enzymatic activities with K(m) values of 0.3 +/- 0.06 microM for the natural substrate thymidine and 11.6 +/- 3.2 microM for ATP, indicating the biochemical equivalence with the viral VZV TK expressed in infected cells. Determinations of the native molecular weight by size exclusion chromatography and native polyacrylamide gel electrophoresis revealed that the pure enzyme is biologically active as a homodimer.

Blotting, Western↗

PubMed1999

Substrate diversity of herpes simplex virus thymidine kinase. Impact Of the kinematics of the enzyme.

Herpes simplex virus type 1 (HSV 1) thymidine kinase (TK) exhibits an extensive substrate diversity for nucleobases and sugar moieties, in contrast to other TKs. This substrate diversity is the crucial molecular basis of selective antiviral and suicide gene therapy. The mechanisms of substrate binding of HSV 1 TK were studied by means of site-directed mutagenesis combined with isothermal calorimetric measurements and guided by theoretical calculations and sequence comparison. The results show the link between the exceptionally broad substrate diversity of HSV 1 TK and the presence of structural features such as the residue triad His-58/Met-128/Tyr-172. The mutation of Met-128 into a Phe and the double mutant M128F/Y172F result in mutants that have lost their activity. However, by exchanging His to form the triple mutant H58L/M128F/Y172F, the enzyme regains activity. Strikingly, this triple mutant becomes resistant toward acyclovir. Furthermore, we give evidence for the importance of Glu-225 of the flexible LID region for the catalytic reaction. The data presented give new insights to understand mechanisms ruling substrate diversity and thus are crucial for both the development of new antiviral drugs and engineering of mutant TKs apt to accept novel substrate analogs for gene therapeutic approaches.

Amino Acid Sequence↗

PubMed1999

Nonapeptide analogues containing (R)-3-hydroxybutanoate and beta-homoalanine oligomers: synthesis and binding affinity to a class I major histocompatibility complex protein.

Crystal structures of antigenic peptides bound to class I MHC proteins suggest that chemical modifications of the central part of the bound peptide should not alter binding affinity to the MHC restriction protein but could perturb the T-cell response to the parent epitope. In our effort in designing nonpeptidic high-affinity ligands for class I MHC proteins, oligomers of (R)-3-hydroxybutanoate and(or) beta-homoalanine have been substituted for the central part of a HLA-B27-restricted T-cell epitope of viral origin. The affinity of six modified peptides to the B2705 allele was determined by an in vitro stabilization assay. Four out of the six designed analogues presented an affinity similar to that of the parent peptide. Two compounds, sharing the same stereochemistry (R,R,S,S) at the four stereogenic centers of the nonpeptidic spacer, bound to B2705 with a 5-6-fold decreased affinity. Although the chiral spacers do not strongly interact with the protein active site, there are configurations which are not accepted by the MHC binding groove, probably because of improper orientation of some lateral substituents in the bound state and different conformational behavior in the free state. However we demonstrate that beta-amino acids can be incorporated in the sequence of viral T-cell epitopes without impairing MHC binding. The presented structure-activity relationships open the door to the rational design of peptide-based vaccines and of nonnatural T-cell receptor antagonists aimed at blocking peptide-specific T-cell responses in MHC-associated autoimmune diseases.

3-Hydroxybutyric Acid↗

PubMed1999

Vaccines and their transfection potency.

Viral vaccines have been shown to contain residual host cell-DNA. There is no doubt about uptake and expression of foreign DNA in mammalian cells but the mechanism of transfection is not completely understood. It is suggested that DNA associates with several compounds and is transferred into the cell by endocytosis. In this study we estimate the potential of transfection of several original adjuvants by adding reporter plasmid DNA (pDNA) to vaccines. We used fibrosarcoma cells as an in vitro model and the results indicate that the cells are not able to express pDNA. Therefore we propose that adjuvants included in viral vaccines have no potential to transfect fibroblasts.

Aluminum Hydroxide↗

PubMed1999

Millisecond to microsecond time scale dynamics of the retinoid X and retinoic acid receptor DNA-binding domains and dimeric complex formation.

The all-trans retinoic acid and 9-cis retinoic acid receptors (RAR and RXR, respectively) belong to a family of ligand inducible transcription factors, which exert their effect via binding to hormone response elements. Both are members of the class II sub-family of nuclear receptors, which bind DNA as dimers, on tandem repeats of a hexamer motif separated by a variable spacer. The variability in spacer length and the head-to-tail organization of the hormone response elements result in different protein-protein interactions in each of the complexes. We show that the zinc-coordinating loop regions of RXR and RAR DNA-binding domains exhibit dynamics on the millisecond to microsecond time scale. The highly dynamic second zinc finger of RXR constitutes the primary protein-protein interface in many nuclear receptor assemblies on DNA. Dynamics is also observed in the first and second zinc fingers of RAR, which are implicated in dimeric interactions with RXR on response elements with spacers of 5 base pairs and 1 base pair, respectively. The striking correspondence between the regions that exhibit conformational exchange and the dimer interfaces of the proteins complexed with DNA suggests a functional role for the dynamics. The observed flexibility may allow the proteins to adapt to various partners and with different orientations upon assembly on DNA. Furthermore, the more extensive dynamics observed for RXR may reflect the greater ability of this protein to modulate its interaction surface since it participates in a wide variety of receptor complexes.

Amino Acid Sequence↗

PubMed1999

Peptides and polypeptides as modulators of the immune response: thymopentin--an example with unknown mode of action.

Peptides and polypeptides play a critical role in the immune system and are therefore predestined as a source for new approaches in immunotherapy. For example, antigenic peptides which can trigger a specific immunological response have been successfully used for vaccination. In contrast, cytokines have to be considered as rather non-specific immunomodulators. In addition, certain peptides with unknown mode of action have shown promising immunomodulating properties. An example is the pentapeptide thymopentin (TP5), which represents the active sequence of the originally described thymopoietin (TP). TP was recently identified as a fragment of the thymopoietins (TMPOs), a family of nuclear proteins. In vitro assays showed that TP5 affects the function of T cells and monocytes measured by enhanced cGMP level and the triggering of cellular signalling, respectively. In vivo studies demonstrated the capability of TP5 to improve an imbalanced immune system. TP5 exhibited important clinical features and further investigations on its mode of action are necessary to rationally create TP5 peptide analogs or peptidomimetics.

Adjuvants, Immunologic↗

PubMed1999

Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling.

The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in vitro with a technique called DNA family shuffling. A high-throughput robotic screen identified chimeras with an enhanced ability to phosphorylate zidovudine (AZT). Improved clones were combined, reshuffled, and screened on increasingly lower concentrations of AZT. After four rounds of shuffling and screening, two clones were isolated that sensitize Escherichia coli to 32-fold less AZT compared with HSV-1 TK and 16,000-fold less than HSV-2 TK. Both clones are hybrids derived from several crossover events between the two parental genes and carry several additional amino acid substitutions not found in either parent, including active site mutations. Kinetic measurements show that the chimeric enzymes had acquired reduced K(M) for AZT as well as decreased specificity for thymidine. In agreement with the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expense of thymidine. Despite the overall similarity of the two chimeric enzymes, each contains key contributions from different parents in positions influencing substrate affinity. Such mutants could be useful for anti-HIV gene therapy, and similar directed-evolution approaches could improve other enzyme-prodrug combinations.

Amino Acid Sequence↗

PubMed1999

Interaction of the recombinant herpes simplex virus type 1 thymidine kinase with thymidine and aciclovir: a kinetic study.

Herpes Simplex Virus type 1 thymidine kinase (HSV 1 TK) is a key target for antiviral therapy and it phosphorylates a broad spectrum of nucleosides and nucleotides. We report the results from kinetic and inhibition experiments with HSV 1 TK, and show that there is a preferred, but not exclusive, binding order of substrates, i.e. dT binds prior to ATP. Furthermore, the results provide new informations on the mechanism of binding suggesting that HSV1 TK undergoes conformational changes during the catalytic cycle.

Acyclovir↗

PubMed1999

Molecular cloning, genetic mapping, and developmental expression of bovine POU5F1.

We describe isolation and characterization of the bovine ortholog of POU5F1 (bPOU5F1) encoding octamer-binding transcription factor-4 (Oct-4). The organization of bPOU5F1 is similar to its human and murine orthologs, and it shares 90.6% and 81.7% overall identity at the protein level, respectively. Transient transfection of luciferase reporter constructs in murine P19 embryonal carcinoma cells demonstrated that bPOU5F1 has a functional promoter and contains two enhancer elements, of which one is repressed by retinoic acid. bPOU5F1 was mapped to the major histocompatibility complex on chromosome 23. bPOU5F1 mRNA was detected by nested reverse transcription-polymerase chain reaction in immature oocytes and in in vitro-produced preattachment-stage embryos. Oct-4 in oocytes and in vitro-produced preattachment-stage embryos was demonstrated by indirect immunofluorescence. Confocal laser scanning microscopy revealed Oct-4 in both the inner cell mass and trophoblast cells of the blastocyst until Day 10 of development. Immunofluorescence performed on the outgrowths formed at Day 13 postfertilization from in vitro-produced Day 8 blastocysts showed Oct-4 staining in all cells. This expression pattern suggests that bPOU5F1 acts early in bovine embryonic development but that its expression is not restricted to pluripotent cells of the blastocyst.

Amino Acid Sequence↗

PubMed1999

On the role of thymopoietins in cell proliferation. Immunochemical evidence for new members of the human thymopoietin family.

Thymopoietins (TMPOs) are a group of ubiquitously expressed nuclear proteins. They are suggested to play an important role in nuclear envelope organization and cell cycle control, as has been shown for lamina-associated polypeptides 2 alpha and beta, which are the rat homologs of human TMPOalpha and TMPObeta, respectively. The recent isolation and characterization of seven mouse TMPO mRNA transcripts named TMPO-alpha, beta, beta', gamma, epsilon delta and zeta, suggest that more than the three previously reported transcripts, alpha, beta, and gamma forms, may exist in humans. Here we report on the demonstration of putative human TMPOdelta and epsilon by immunoblotting of human cell lines using a newly prepared polyclonal antiserum against the common N-terminal region of TMPO. Furthermore, we prepared the first truly TMPO-beta-specific, affinity-purified polyclonal antiserum, using a part of the human analog of the beta-specific domain of mouse TMPO 220-259 for immunization. We showed that human TMPObeta is highly expressed in all cancerous cells tested, while hardly any cross-reactivities with other proteins could be detected. In contrast to the high expression of human TMPObeta in the cancer-derived neuroblastoma cell lines SK-N-MC and SMS-KAN, we found very low expression of human TMPObeta in low-proliferative nerve tissue. These data led us to the assumption that expression of TMPObeta may correlate with the occurrence of cancer, and therefore may serve as a new tumor marker, or even as a new target for cancer therapy.

Amino Acid Sequence↗

PubMed1998

Substituting nonpeptidic spacers for the T cell receptor-binding part of class I major histocompatibility complex-binding peptides.

X-ray diffraction studies as well as structure-activity relationships indicate that the central part of class I major histocompatibility complex (MHC)-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, three nonpeptidic spacers have been designed from the x-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the human leukocyte antigen-B*2705 protein was studied by an in vitro stabilization assay, and the thermal stability of all complexes was examined by circular dichroism spectroscopy. Depending on their chemical nature and length, the introduced spacers may be classified into two categories. Monofunctional spacers (11-amino undecanoate, (R)-3-hydroxybutyrate trimer) simply link two anchoring peptide positions (P3 and P9) but loosely contact the MHC binding groove and thus decrease more or less the affinity of the altered epitopes to human leukocyte antigen-B*2705. A bifunctional spacer ((R)-3-hydroxybutyrate tetramer) not only bridges the two distant anchoring amino acids but also strongly interacts with the binding cleft and leads to a 5-fold increase in binding to the MHC protein. To our knowledge, this is the first report of a nonpeptidic modification of T-cell receptor binding residues that significantly enhances the binding of altered peptide ligands to their host MHC protein. The presented modified ligands constitute interesting tools for perturbing the T cell response to the parent antigenic peptide.

Computer-Aided Design↗

PubMed1998

Drug resistance of herpes simplex virus type 1--structural considerations at the molecular level of the thymidine kinase.

Several drug-resistant strains of herpes simplex virus type 1 (HSV1) isolated in vivo or from tissue culture, have exhibited a mutated thymidine kinase (TK). Moreover, various site-directed-mutagenesis experiments conducted on HSV1 TK allowed the assignment of specific amino acid residues to specific functional properties. From this, a range of hypotheses was generated related to substrate binding of TK at the molecular level. A site-directed-mutagenesis study on Q125 was performed to clarify the contribution of this residue to the binding of thymidine or aciclovir beyond the hydrogen-bonding pattern observed in the crystal structure. While Q125L is only able to phosphorylate thymidine, Q125N accepts thymidine and aciclovir as substrates. Q125E shows no phosphorylation activity. Several mutations identified previously as relevant in drug resistance were studied in an attempt to further understand their role in these processes. Four amino acid positions are described (T63, A168, R176 and C336) that confer drug resistance when mutated; however, the molecular mechanisms are considerably different in each case. Analysis of the crystal structures and the molecular modeling presented in this paper suggest that T63 is essential for the binding of Mg2+ and thus the catalytic activity of the enzyme, while A168 limits steric accessibility and if mutated to a bulkier residue will exclude binding of larger substrate analogues. R176 appears to be essential for electrostatic balance within the active site, and C336, which is located at the surface of TK and directed toward the ATP-binding site, disrupts the three-dimensional structure of the whole active site by shifting the LID-domain. The present work contributes to a detailed understanding of nucleoside binding to TK, thereby facilitating the rational design of substrates for HSV1 TK and of drug-specific TK for gene therapy.

Acyclovir↗

PubMed1998

Density functional studies on herpes simplex virus type 1 thymidine kinase-substrate interactions: the role of Tyr-172 and Met-128 in thymine fixation.

The enzyme herpes simplex virus type 1 thymidine kinase (HSV1 TK) salvages thymidine into the DNA metabolism of the virus. In the active site, the thymine ring of the nucleoside binds in a pocket, formed by two residues, Tyr-172 and Met-128, in a sandwich-type orientation. To investigate the nature of the thymine-enzyme pocket interactions, we have carried out density functional theory calculations with gradient-corrected exchange-correlation functionals of models of the thymine-HSV1 TK adduct. Our calculations indicate that the role of Met-128 in the substrate fixation is purely steric and hydrophobic, while the substrate-Tyr-172 interaction is essentially electrostatic in nature. These findings are completely consistent with the available catalytic properties of mutants on the 128 position.

Adenosine Triphosphate↗

PubMed1998

A fast and inexpensive method for N-terminal fluorescein-labeling of peptides.

A new method has been developed to synthesize fluorescein labeled peptides, compounds of increasing importance in bioorganic chemistry, cell biology, pharmacology, drug targeting and medicinal chemistry. We show, that 4(5)-carboxyfluorescein is much more efficient than the hitherto predominantly utilized reagents 4(5)-carboxyfluorescein-N-succinimidylester and 4(5)-fluoresceinisothiocyanate.

Amino Acid Sequence↗