The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic gr... more The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic growth of Salmonella bacteriophage P22. The solution structure of its C-terminal tetramerization domain, which holds together the two dimeric DNA-binding domains, has been determined by NMR spectroscopy. This structure reveals an assembly of four alpha-helical subunits, consisting of a dimer of two antiparallel coiled coils with a unique right-handed twist. The superhelical winding is considerably stronger and the interhelical separation closer than those found in the well-known left-handed coiled coils in fibrous proteins and leucine zippers. An unusual asymmetry arises between the two monomers that comprise one right-handed coiled coil. A difference in the packing to the adjacent monomer of the other coiled coil occurs with an offset of two helical turns. The two asymmetric monomers within each coiled coil interconvert on a time scale of seconds. Both with respect to symmetry and handedn...
Human liver fatty acid-binding protein (L-FABP) has been efficiently expressed in Escherichia col... more Human liver fatty acid-binding protein (L-FABP) has been efficiently expressed in Escherichia coli. The cDNA encoding human liver FABP was under the control of T7 RNA polymerase promoter in the expression vector pET-3b. Expression required overnight induction with isopropyl beta-D-thiogalactopyranoside in the presence of the bacterial RNA polymerase inhibitor, rifampicin. The protein could be purified by (NH4)2SO4 fractionation, anion-exchange and gel filtration chromatography, and was recognized by anti-(human L-FABP) antiserum. The binding characteristics of delipidated recombinant human L-FABP and muscle FABP (M-FABP) for fatty acids of different chain length and saturation grade, and for various hydrophobic ligands, were determined by radiochemical analysis and also by fluorescence for L-FABP. The apparent binding affinity of the ligands was calculated by using displacement curves of oleic acid and dansylamino-undecanoic acid (DAUDA). L-FABP showed a preference for the binding o...
Received March 2 N a y 13, 1993) -EJB 93 0314/2 Scrutiny of NOE data available for the protein en... more Received March 2 N a y 13, 1993) -EJB 93 0314/2 Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a P-sheet structure which is partly five stranded. The DNA-binding domain of IKe GVP was investigated using a spin-labeled deoxytrinucleotide. The paramagnetic-relaxation effects observed in the 'H-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two-dimensional difference spectroscopy. In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the structure of the protein. They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage MI 3 (M13 GVP), for which similar spectral perturbations are induced by such a spin-labeled oligonucleotide. Binding studies with negatively charged lanthanide-I ,4,7,10tetraazacyclodecanetrayl-1,4,7,1O-tetrakis(methylene)tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M13 GVP monomer, which coincides with the single-stranded DNA-binding domain as mapped with the aid of spin-labeled oligonucleotide fragments. The results show that singlestranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP. These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues. At pH 5, a second Gd(DOTP)'--binding site becomes apparent. The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrophobic residues in the cores of the M13 and IKe dimers.
The solution structure of the hyperstable MYL mutant (R31M/E36Y/R40L) of the Arc repressor of bac... more The solution structure of the hyperstable MYL mutant (R31M/E36Y/R40L) of the Arc repressor of bacteriophage P22 was determined by NMR spectroscopy and compared to that of the wild-type Arc repressor. A backbone rmsd versus the average of 0.37 A was obtained for the well-defined core region. For both Arc-MYL and the wild-type Arc repressor, evidence for a fast equilibrium between a packed ("in") conformation and an extended ("out") conformation of the side chain of Phe 10 was found. In the MYL mutant, the "out" conformation is more highly populated than in the wild-type Arc repressor. The Phe 10 is situated in the DNA-binding beta-sheet of the Arc dimer. While its "in" conformation appears to be the most stable, the "out" conformation is known to be present in the operator-bound form of Arc, where the Phe 10 ring contacts the phosphate backbone [Raumann, B. E., et al. (1994) Nature 367, 754-757]. As well as DNA binding, denaturation by urea and high temperatures induces the functionally active "out" conformation. With a repacking of the hydrophobic core, this characterizes a premelting transition of the Arc repressor. The dynamical properties of the Arc-MYL and the wild-type Arc repressor were further characterized by 15N relaxation and hydrogen-deuterium exchange experiments. The increased main chain mobility at the DNA binding site compared to that of the core of the protein as well as the reorientation of the side chain of Phe 10 is suggested to play an important role in specific DNA binding.
The subject RNA models the binding site for the coat protein of the R17 virus, as well as the rib... more The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the 1H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5'/H5" for RNA plus H2'/H2" for DNA), as well as for H5/H6, for H3'/H4' in sugars with substantial populations of the N-pucker, for H1'/H2' for S-puckered sugars, and usually for H2'/H3'. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectr...
The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic gr... more The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic growth of Salmonella bacteriophage P22. The solution structure of its C-terminal tetramerization domain, which holds together the two dimeric DNA-binding domains, has been determined by NMR spectroscopy. This structure reveals an assembly of four alpha-helical subunits, consisting of a dimer of two antiparallel coiled coils with a unique right-handed twist. The superhelical winding is considerably stronger and the interhelical separation closer than those found in the well-known left-handed coiled coils in fibrous proteins and leucine zippers. An unusual asymmetry arises between the two monomers that comprise one right-handed coiled coil. A difference in the packing to the adjacent monomer of the other coiled coil occurs with an offset of two helical turns. The two asymmetric monomers within each coiled coil interconvert on a time scale of seconds. Both with respect to symmetry and handedn...
Human liver fatty acid-binding protein (L-FABP) has been efficiently expressed in Escherichia col... more Human liver fatty acid-binding protein (L-FABP) has been efficiently expressed in Escherichia coli. The cDNA encoding human liver FABP was under the control of T7 RNA polymerase promoter in the expression vector pET-3b. Expression required overnight induction with isopropyl beta-D-thiogalactopyranoside in the presence of the bacterial RNA polymerase inhibitor, rifampicin. The protein could be purified by (NH4)2SO4 fractionation, anion-exchange and gel filtration chromatography, and was recognized by anti-(human L-FABP) antiserum. The binding characteristics of delipidated recombinant human L-FABP and muscle FABP (M-FABP) for fatty acids of different chain length and saturation grade, and for various hydrophobic ligands, were determined by radiochemical analysis and also by fluorescence for L-FABP. The apparent binding affinity of the ligands was calculated by using displacement curves of oleic acid and dansylamino-undecanoic acid (DAUDA). L-FABP showed a preference for the binding o...
Received March 2 N a y 13, 1993) -EJB 93 0314/2 Scrutiny of NOE data available for the protein en... more Received March 2 N a y 13, 1993) -EJB 93 0314/2 Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a P-sheet structure which is partly five stranded. The DNA-binding domain of IKe GVP was investigated using a spin-labeled deoxytrinucleotide. The paramagnetic-relaxation effects observed in the 'H-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two-dimensional difference spectroscopy. In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the structure of the protein. They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage MI 3 (M13 GVP), for which similar spectral perturbations are induced by such a spin-labeled oligonucleotide. Binding studies with negatively charged lanthanide-I ,4,7,10tetraazacyclodecanetrayl-1,4,7,1O-tetrakis(methylene)tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M13 GVP monomer, which coincides with the single-stranded DNA-binding domain as mapped with the aid of spin-labeled oligonucleotide fragments. The results show that singlestranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP. These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues. At pH 5, a second Gd(DOTP)'--binding site becomes apparent. The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrophobic residues in the cores of the M13 and IKe dimers.
The solution structure of the hyperstable MYL mutant (R31M/E36Y/R40L) of the Arc repressor of bac... more The solution structure of the hyperstable MYL mutant (R31M/E36Y/R40L) of the Arc repressor of bacteriophage P22 was determined by NMR spectroscopy and compared to that of the wild-type Arc repressor. A backbone rmsd versus the average of 0.37 A was obtained for the well-defined core region. For both Arc-MYL and the wild-type Arc repressor, evidence for a fast equilibrium between a packed ("in") conformation and an extended ("out") conformation of the side chain of Phe 10 was found. In the MYL mutant, the "out" conformation is more highly populated than in the wild-type Arc repressor. The Phe 10 is situated in the DNA-binding beta-sheet of the Arc dimer. While its "in" conformation appears to be the most stable, the "out" conformation is known to be present in the operator-bound form of Arc, where the Phe 10 ring contacts the phosphate backbone [Raumann, B. E., et al. (1994) Nature 367, 754-757]. As well as DNA binding, denaturation by urea and high temperatures induces the functionally active "out" conformation. With a repacking of the hydrophobic core, this characterizes a premelting transition of the Arc repressor. The dynamical properties of the Arc-MYL and the wild-type Arc repressor were further characterized by 15N relaxation and hydrogen-deuterium exchange experiments. The increased main chain mobility at the DNA binding site compared to that of the core of the protein as well as the reorientation of the side chain of Phe 10 is suggested to play an important role in specific DNA binding.
The subject RNA models the binding site for the coat protein of the R17 virus, as well as the rib... more The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the 1H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5'/H5" for RNA plus H2'/H2" for DNA), as well as for H5/H6, for H3'/H4' in sugars with substantial populations of the N-pucker, for H1'/H2' for S-puckered sugars, and usually for H2'/H3'. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectr...
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