Gerd Folkers - Academia.edu (original) (raw)
Papers by Gerd Folkers
Journal of Molecular Graphics, 1985
ABSTRACT
Zweiter Teil der öffentlichen Vortragsreihe im Rahmen des im Oktober 2004 am Collegium Helveticum... more Zweiter Teil der öffentlichen Vortragsreihe im Rahmen des im Oktober 2004 am Collegium Helveticum etablierten Forschungsprojektes «Die Rolle der Emotion: ihr Anteil bei menschlichem Handeln und bei der Setzung sozialer Normen» GEFÜHLE ZEIGEN. MANIFESTATIONSFORMEN EMOTIONALER PROZESSE
Il Farmaco
In rational drug design the study of protein-ligand-interactions is one of the most important app... more In rational drug design the study of protein-ligand-interactions is one of the most important approaches to get knowledge of SAR. On this study N2-Phenylthioguanines were synthesized by Schiemann reaction from thioguanine followed by a substitution of the fluorine by aniline-derivatives. The activity of these HSV1 TK inhibitors was determined by kinetic measurements of thymidine phosphorylation. The N2-Phenylthioguanines gave the same activity as the oxoanalogues. Interaction energies between thymidine and HSV1 TK were measured by microcalorimetry. Results of the measurement showed negative delta G and delta H values which indicates that the binding of the natural substrate occurs spontaneously and is enthalpy driven.
Biochemical Pharmacology, 2014
Entropy (ΔS), enthalpy (ΔH) and heat capacity (ΔCp) changes attending the oxytocin interaction wi... more Entropy (ΔS), enthalpy (ΔH) and heat capacity (ΔCp) changes attending the oxytocin interaction with its two binding sites on myometrial cell membranes in sheep were derived from the temperature dependence of Kd values. The high affinity oxytocin site (Kd on the order of 10(-9)mol l(-1), 25 °C), ascribed to the oxytocin receptor (OXTR), is entropy-driven in the temperature range 0-37 °C. Enthalpy component prevails as a driving force in the binding to the low affinity site (Kd ≈ 10(-7)) within the higher temperature range. ΔCp values in both cases do not differ significantly from zero but become highly relevant in the presence of a GTP analog (10(-4)M GTP-γS). Under these conditions, ΔCp in the low site interaction becomes negative and ΔS is shifted toward negative values (enthalpy drift); ΔCp of the high affinity site rises to a high positive value and the interaction is even more strongly entropy driven. Atosiban, a competitive antagonist of oxytocin at OXTR displays a single significant binding site on myometrial cells (Kd about 10(-7)mol l(-1)). Thermodynamic profiles of atosiban and the low affinity oxytocin site show conspicuous similarities, indicating that the inhibitor is bound to the low affinity site, and not, with a lower affinity, to the putative receptor protein. It is suggested that the interaction of oxytocin with its responding system on myometrial membranes follows in two distinct steps that are likely to be associated with several independent binding domains in the GPCR receptor.
Journal of Peptide Science
Quantitative Structure-activity Relationships, 1999
European Journal of Biochemistry, 1996
A new approach is reported that includes multiple-peptide synthesis and CD spectroscopy of overla... more A new approach is reported that includes multiple-peptide synthesis and CD spectroscopy of overlapping peptides to evaluate the secondary structure of the vaccinia-virus thymidine kinase (TK). We divided the sequence of the vaccinia-virus TK into 82 peptides of IS residues that overlapped by 13 rcsidues and covered the complete sequence of vaccinia-virus TK. All peptides were synthesized by solid-phase multiple-peptide synthesis by means of the Fmoclfert-butyl strategy. Subsequently, the secondary structure of each peptide was studied by means of CD spectroscopy in a mixture of 30% trifluoroethanol and sodium phosphate, pH 7. Secondary-structure evaluation led to determination of a vaccinia-virus-TK secondary-structure pattern.
Quantitative Structure-Activity Relationships, 1998
Based on the structures of known ligand molecules, a pseudoreceptor modeling concept developed at... more Based on the structures of known ligand molecules, a pseudoreceptor modeling concept developed at our laboratory allows the construction of a peptidic binding-site model for a structurally uncharacterized bioregulator. Such a three-dimensional receptor surrogate validated using an external ...
Clinical Pharmacology & Therapeutics, 2005
Background: Renal failure (RF) decreases the hepatic elimination of substrates of basolateral Org... more Background: Renal failure (RF) decreases the hepatic elimination of substrates of basolateral Organic Anion Transporting Polypeptides (Oatp) and canalicular Multidrug Resistance Protein 2 (Mrp2), e.g. bromosulfophthalein, cerivastatin, and pravastatin. Therefore we investigated, the expression of these hepatocellular transporters in RF.Methods: Male SD rats (120–140g) underwent 2-step 5/6-nephrectomy (6, N) or sham operation (8, C), were pair fed and killed after
Quantitative Structure-Activity Relationships, 2002
ABSTRACT
Journal of computer-aided molecular design, 2001
Starting from the NMR structure of the binary complex between the N-terminal domain of the unphos... more 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 ...
The Journal of biological chemistry, Jan 15, 2001
Most antiherpes therapies exploit the large substrate acceptance of herpes simplex virus type 1 t... more 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 neg...
Nature biotechnology, 1999
The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in ... more 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 ...
The Journal of biological chemistry, Jan 24, 1998
X-ray diffraction studies as well as structure-activity relationships indicate that the central p... more 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 le...
Journal of computer-aided molecular design, 1997
Starting from the X-ray structure of a class I major histocompatibility complex (MHC)-encoded pro... more Starting from the X-ray structure of a class I major histocompatibility complex (MHC)-encoded protein (HLA-B*2705), a naturally presented self-nonapeptide and two synthetic analogues were simulated in the binding groove of two human leukocyte antigen (HLA) alleles (B*2703 and B*2705) differing in a single amino acid residue. After 200 ps molecular dynamics simulations of the solvated HLA-peptide pairs, some molecular properties of the complexes (distances between ligand and protein center of masses, atomic fluctuations, buried versus accessible surface areas, hydrogen-bond frequencies) allow a clear discrimination of potent from weak MHC binders. The binding specificity of the three nonapeptides for the two HLA alleles could be explained by the disruption of one hydrogen-bonding network in the binding pocket of the HLA-B*2705 protein where the single mutation occurs. Rearrangements of interactions in the B pocket, which binds the side chain of peptide residue 2, and a weakening of i...
Journal of computer-aided molecular design, 1997
A cannabinoid pseudoreceptor model for the CB1-receptor has been constructed for 31 cannabinoids ... more A cannabinoid pseudoreceptor model for the CB1-receptor has been constructed for 31 cannabinoids using the molecular modelling software YAK. Additionally, two CoMFA studies were performed on these ligands, the first of which was conducted prior to the building of the pseudoreceptor. Its pharmacophore is identical with the initial superposition of ligands used for pseudoreceptor construction. In contrast, the ligand alignment for the second CoMFA study was taken directly from the final cannabinoid pseudoreceptor model. This altered alignment gives markedly improved cross-validated r2 values as compared to those obtained from the original alignment with r2 cross values of 0.79 and 0.63, respectively, for five components. However, the pharmacophore alignment has the better predictive ability. Both the CoMFA and pseudoreceptor methods predict the free energy of binding of test ligands well.
Quantitative Structure-Activity Relationships, 1995
ABSTRACT
ChemInform, 1998
--(SEEBACH, D.; POENARU, S.; FOLKERS, G.; ROGNAN, D.; Helv. Chim.
International journal of peptide and protein research, 1990
The preferred solution conformation of the PRP-hexapeptide (Tyr-Val-Pro-Leu-Phe-Pro) and of some ... more The preferred solution conformation of the PRP-hexapeptide (Tyr-Val-Pro-Leu-Phe-Pro) and of some of its structural analogues was investigated by NMR-spectroscopy, spectrofluorimetry and computer simulation technic. It was found that the preferred conformation is characterized by cis'-conformation of Pro3 and the gamma-turn on the Leu4-residue: for Val2 and Phe5 a beta-structure seems to be privileged. In such a conformation Val2 and Leu4 residues occupy exactly the same positions in space as residues i and i + 3 in an alpha-helix. It suggests that the PRP-hexapeptide can interact with receptor protein inducing or stabilizing its helical conformation by "knobs into holes" packing.
Thymidine kinase from herpes simplex virus type 1 (HSV1 TK) has been postulated to be a homodimer... more 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.
Journal of Molecular Graphics, 1985
ABSTRACT
Zweiter Teil der öffentlichen Vortragsreihe im Rahmen des im Oktober 2004 am Collegium Helveticum... more Zweiter Teil der öffentlichen Vortragsreihe im Rahmen des im Oktober 2004 am Collegium Helveticum etablierten Forschungsprojektes «Die Rolle der Emotion: ihr Anteil bei menschlichem Handeln und bei der Setzung sozialer Normen» GEFÜHLE ZEIGEN. MANIFESTATIONSFORMEN EMOTIONALER PROZESSE
Il Farmaco
In rational drug design the study of protein-ligand-interactions is one of the most important app... more In rational drug design the study of protein-ligand-interactions is one of the most important approaches to get knowledge of SAR. On this study N2-Phenylthioguanines were synthesized by Schiemann reaction from thioguanine followed by a substitution of the fluorine by aniline-derivatives. The activity of these HSV1 TK inhibitors was determined by kinetic measurements of thymidine phosphorylation. The N2-Phenylthioguanines gave the same activity as the oxoanalogues. Interaction energies between thymidine and HSV1 TK were measured by microcalorimetry. Results of the measurement showed negative delta G and delta H values which indicates that the binding of the natural substrate occurs spontaneously and is enthalpy driven.
Biochemical Pharmacology, 2014
Entropy (ΔS), enthalpy (ΔH) and heat capacity (ΔCp) changes attending the oxytocin interaction wi... more Entropy (ΔS), enthalpy (ΔH) and heat capacity (ΔCp) changes attending the oxytocin interaction with its two binding sites on myometrial cell membranes in sheep were derived from the temperature dependence of Kd values. The high affinity oxytocin site (Kd on the order of 10(-9)mol l(-1), 25 °C), ascribed to the oxytocin receptor (OXTR), is entropy-driven in the temperature range 0-37 °C. Enthalpy component prevails as a driving force in the binding to the low affinity site (Kd ≈ 10(-7)) within the higher temperature range. ΔCp values in both cases do not differ significantly from zero but become highly relevant in the presence of a GTP analog (10(-4)M GTP-γS). Under these conditions, ΔCp in the low site interaction becomes negative and ΔS is shifted toward negative values (enthalpy drift); ΔCp of the high affinity site rises to a high positive value and the interaction is even more strongly entropy driven. Atosiban, a competitive antagonist of oxytocin at OXTR displays a single significant binding site on myometrial cells (Kd about 10(-7)mol l(-1)). Thermodynamic profiles of atosiban and the low affinity oxytocin site show conspicuous similarities, indicating that the inhibitor is bound to the low affinity site, and not, with a lower affinity, to the putative receptor protein. It is suggested that the interaction of oxytocin with its responding system on myometrial membranes follows in two distinct steps that are likely to be associated with several independent binding domains in the GPCR receptor.
Journal of Peptide Science
Quantitative Structure-activity Relationships, 1999
European Journal of Biochemistry, 1996
A new approach is reported that includes multiple-peptide synthesis and CD spectroscopy of overla... more A new approach is reported that includes multiple-peptide synthesis and CD spectroscopy of overlapping peptides to evaluate the secondary structure of the vaccinia-virus thymidine kinase (TK). We divided the sequence of the vaccinia-virus TK into 82 peptides of IS residues that overlapped by 13 rcsidues and covered the complete sequence of vaccinia-virus TK. All peptides were synthesized by solid-phase multiple-peptide synthesis by means of the Fmoclfert-butyl strategy. Subsequently, the secondary structure of each peptide was studied by means of CD spectroscopy in a mixture of 30% trifluoroethanol and sodium phosphate, pH 7. Secondary-structure evaluation led to determination of a vaccinia-virus-TK secondary-structure pattern.
Quantitative Structure-Activity Relationships, 1998
Based on the structures of known ligand molecules, a pseudoreceptor modeling concept developed at... more Based on the structures of known ligand molecules, a pseudoreceptor modeling concept developed at our laboratory allows the construction of a peptidic binding-site model for a structurally uncharacterized bioregulator. Such a three-dimensional receptor surrogate validated using an external ...
Clinical Pharmacology & Therapeutics, 2005
Background: Renal failure (RF) decreases the hepatic elimination of substrates of basolateral Org... more Background: Renal failure (RF) decreases the hepatic elimination of substrates of basolateral Organic Anion Transporting Polypeptides (Oatp) and canalicular Multidrug Resistance Protein 2 (Mrp2), e.g. bromosulfophthalein, cerivastatin, and pravastatin. Therefore we investigated, the expression of these hepatocellular transporters in RF.Methods: Male SD rats (120–140g) underwent 2-step 5/6-nephrectomy (6, N) or sham operation (8, C), were pair fed and killed after
Quantitative Structure-Activity Relationships, 2002
ABSTRACT
Journal of computer-aided molecular design, 2001
Starting from the NMR structure of the binary complex between the N-terminal domain of the unphos... more 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 ...
The Journal of biological chemistry, Jan 15, 2001
Most antiherpes therapies exploit the large substrate acceptance of herpes simplex virus type 1 t... more 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 neg...
Nature biotechnology, 1999
The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in ... more 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 ...
The Journal of biological chemistry, Jan 24, 1998
X-ray diffraction studies as well as structure-activity relationships indicate that the central p... more 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 le...
Journal of computer-aided molecular design, 1997
Starting from the X-ray structure of a class I major histocompatibility complex (MHC)-encoded pro... more Starting from the X-ray structure of a class I major histocompatibility complex (MHC)-encoded protein (HLA-B*2705), a naturally presented self-nonapeptide and two synthetic analogues were simulated in the binding groove of two human leukocyte antigen (HLA) alleles (B*2703 and B*2705) differing in a single amino acid residue. After 200 ps molecular dynamics simulations of the solvated HLA-peptide pairs, some molecular properties of the complexes (distances between ligand and protein center of masses, atomic fluctuations, buried versus accessible surface areas, hydrogen-bond frequencies) allow a clear discrimination of potent from weak MHC binders. The binding specificity of the three nonapeptides for the two HLA alleles could be explained by the disruption of one hydrogen-bonding network in the binding pocket of the HLA-B*2705 protein where the single mutation occurs. Rearrangements of interactions in the B pocket, which binds the side chain of peptide residue 2, and a weakening of i...
Journal of computer-aided molecular design, 1997
A cannabinoid pseudoreceptor model for the CB1-receptor has been constructed for 31 cannabinoids ... more A cannabinoid pseudoreceptor model for the CB1-receptor has been constructed for 31 cannabinoids using the molecular modelling software YAK. Additionally, two CoMFA studies were performed on these ligands, the first of which was conducted prior to the building of the pseudoreceptor. Its pharmacophore is identical with the initial superposition of ligands used for pseudoreceptor construction. In contrast, the ligand alignment for the second CoMFA study was taken directly from the final cannabinoid pseudoreceptor model. This altered alignment gives markedly improved cross-validated r2 values as compared to those obtained from the original alignment with r2 cross values of 0.79 and 0.63, respectively, for five components. However, the pharmacophore alignment has the better predictive ability. Both the CoMFA and pseudoreceptor methods predict the free energy of binding of test ligands well.
Quantitative Structure-Activity Relationships, 1995
ABSTRACT
ChemInform, 1998
--(SEEBACH, D.; POENARU, S.; FOLKERS, G.; ROGNAN, D.; Helv. Chim.
International journal of peptide and protein research, 1990
The preferred solution conformation of the PRP-hexapeptide (Tyr-Val-Pro-Leu-Phe-Pro) and of some ... more The preferred solution conformation of the PRP-hexapeptide (Tyr-Val-Pro-Leu-Phe-Pro) and of some of its structural analogues was investigated by NMR-spectroscopy, spectrofluorimetry and computer simulation technic. It was found that the preferred conformation is characterized by cis'-conformation of Pro3 and the gamma-turn on the Leu4-residue: for Val2 and Phe5 a beta-structure seems to be privileged. In such a conformation Val2 and Leu4 residues occupy exactly the same positions in space as residues i and i + 3 in an alpha-helix. It suggests that the PRP-hexapeptide can interact with receptor protein inducing or stabilizing its helical conformation by "knobs into holes" packing.
Thymidine kinase from herpes simplex virus type 1 (HSV1 TK) has been postulated to be a homodimer... more 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.