Two separate N-terminal fragments of the 470-amino-acidEscherichia coliDnaB helicase, comprising residues 1-142 and 1-161, were expressed inE. coli.The proteins were extracted in a soluble fraction, ...purified, and characterised physically. In contrast to the full-length protein, which is hexameric, both fragments exist as monomers in solution, as demonstrated by sedimentation equilibrium measurements. CD spectroscopy was used to confirm that the 161-residue fragment is highly structured (mostly α-helical) and undergoes reversible thermal denaturation. The structurally well-defined core of the N-terminal domain of the DnaB helicase is composed of residues 24 to 136, as determined by assignment of resonances from flexible residues in NMR spectra. The1H NMR signals of the flexible residues are located at random coil chemical shifts, and their linewidths are significantly narrower than those of the structured core, indicating complete disorder and increased mobility on the nanosecond time scale. The results support the idea of a flexible hinge region between the N- and C-terminal domains of the native hexameric DnaB protein.
The secondary structure of an N-terminally elongated Antennapedia (Antp) homeodomain (HD) polypeptide containing residues -14 to 67, where residues 1-60 constitute the HD, has been determined by NMR ...in solution. This polypeptide contains the conserved motif -Tyr-Pro-Trp-Met-(YPWM) at positions -9 to -6. Despite the hydrophobic nature of this tetrapeptide motif, the N-terminal arm consisting of residues -14 to 6 is flexibly disordered, and the well-defined part of the HD structure with residues 7-59 is indistinguishable from that of the shorter Antp HD polypeptide (where positions 0, 1, and 67 are methionine, arginine, and glycine, respectively). In vitro biochemical studies showed that the stability and specificity of the DNA binding previously observed for the shorter Antp HD polypeptide is preserved in the elongated polypeptide. These results strongly support the view that the HD is connected through a flexible linker to the main body in the Antp protein and that the minor groove contacts by the N-terminal arm (residues 1-6) in the Antp HD-DNA complex are an intrinsic feature of the DNA-binding interactions of the intact Antp protein.
The scalar coupling constants between protons, nitrogens, and phosphorus in the metal complex chloro(triphenylphosphine)bisbis(1-pyrazolyl)methaneruthenium(II) chloride, RuCl(PPh3)(BPM)2+Cl- (1), ...were measured with a set of specially adapted NMR experiments. The absolute sign of the coupling constants was determined by relating the signs of the measured couplings to that of a one-bond proton−carbon coupling constant. A complete set of coupling constants >|0.4| Hz was obtained with use of a single sample with 99% 15N-labeled bis-pyrazolyl ligands. The data show that the two-bond 15N−15N and 31P−15N couplings across the metal center are significantly larger, if the two metal-ligating atoms are trans rather than cis with respect to one another. Furthermore, all trans couplings 2 J PN and 2 J NN are positive, while the corresponding cis couplings are negative or too small to be measured. The conformation dependence of the scalar coupling constants supports the rapid structural characterization of catalytically active organometallic complexes by NMR spectroscopy. The proposed set of NMR experiments includes HSQC experiments with small flip angles, a quantitative long-range 15N−15N correlation experiment, and DQ/ZQ experiments for the determination of the sign and size of J NN and J PN coupling constants in linear spin systems.
During the reduction of ribonucleotides with
3Hformate by the class III anaerobic ribonucleotide reductase from
Escherichia coli
tritium appears in water and not in the product deoxyribonucleotide. ...In D
2O, deuterium replaces the OH-group at carbon-2′ with retention of configuration. In addition we find 1-2 % deuterium in the 3′-position demonstrating a small exchange of this hydrogen with the protons of water during catalysis. Class I and II enzymes catalyze identical reactions. Members of the three classes of reductases apparently use the same chemical mechanism in spite of having completely different protein structures.
The solution structure of a synthetic mutant type I antifreeze protein (AFP I) was determined in aqueous solution at pH 7.0 using nuclear magnetic resonance (NMR) spectroscopy. The mutations ...comprised the replacement of the four Thr residues by Val and the introduction of two additional Lys‐Glu salt bridges. The antifreeze activity of this mutant peptide, VVVV2KE, has been previously shown to be similar to that of the wild type protein, HPLC6 (defined here as TTTT). The solution structure reveals an α helix bent in the same direction as the more bent conformer of the published crystal structure of TTTT, while the side chain χ1 rotamers of VVVV2KE are similar to those of the straighter conformer in the crystal of TTTT. The Val side chains of VVVV2KE assume the same orientations as the Thr side chains of TTTT, confirming the conservative nature of this mutation. The combined data suggest that AFP I undergoes an equilibrium between straight and bent helices in solution, combined with independent equilibria between different side chain rotamers for some of the amino acid residues. The present study presents the first complete sequence‐specific resonance assignments and the first complete solution structure determination by NMR of any AFP I protein.
The three-dimensional backbone fold of a polypeptide fragment from the rat LFB1/HNF1 transcription factor was determined by nuclear magnetic resonance (NMR) spectroscopy in solution. This fragment ...contains an amino acid sequence that is similar to 22% homologous to the well known homeodomains, but which contains 81 amino acid residues as compared with 60 residues in 'typical' homeodomains. For the present studies we used a recombinant 99 amino acid polypeptide containing this sequence in positions 10-90, which was uniformly labelled with super(15)N and also doubly labelled with super(15)N and super(13)C. The NMR structure of this polypeptide contains three alpha -helices comprising the residues 18-29, 36-50 and 71-84, a loop formed by residues 30-35, and a long stretch of non-regular secondary structure linking the second and third helices. The relative location and orientation of the helices is very similar to that in the Antennapedia (Antp) homeodomain structure, despite the fact that helix II is elongated by about one turn. This confirms a recently advanced hypothesis based on sequence comparisons that this polypeptide segment of LFB1/HNF1 might represent a homeodomain-like structural element. The helix-turn-helix motif, which has been shown to comprise the DNA recognition helix in the Antp homeodomain, can readily be recognized in the LFB1/HNF1 homeodomain, in spite of an extensive modification of the primary structure. The two residues of the tight turn in the Antp homeodomain are replaced by a 23 residue linker region between the two helices in LFB1/HNF1, which bulges out from the rest of the molecule and thus enables the formation of a non-classical helix-turn-helix motif.
Recombinant flavodoxin from Escherichia coli was uniformly enriched with 15N and 13C isotopes and its oxidized form in aqueous solution investigated by three‐dimensional NMR spectroscopy. Nearly ...complete 1H, 15N and 13C resonance assignments were obtained. The secondary structure was determined from chemical shift, NOE and 3JHNHα coupling constant data. Like homologous long‐chain flavodoxins, E. coli flavodoxin contains a five‐stranded parallel β‐sheet and five helices. The β‐strands were found to comprise the residues 3–8, 29–34, 48–56, 80–89, 114–116 and 141–145. The helices comprise residues 12–25, 40–45, 62–73, 98–108 and 152–166. The FMN‐binding site was determined by intermolecular NOEs and low‐field shifted amide proton resonances induced by the phosphoester group of the cofactor. The data are in good agreement with a previously predicted model of E. coli flavodoxin Havel, T. F. (1993)Mol. Sim. 10, 175–210. The analysis of of water‐flavodoxin NOEs revealed the presence of two, possibly three, buried hydration water molecules which are located at sites, where homologous flavodoxins from Anacystis nidulans and Anabena 7120 contain conserved hydration water molecules. One of these water molecules mediates hydrogen bonds between the protein backbone and the ribityl chain of the FMN cofactor.
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