The solution structure of ribonuclease HI (RNase HI) from Escherichia coli (E. coli), a protein of 155 residues, was determined. Three-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) was used to obtain 1,424 distance constraints between individually assigned polypeptide chain hydrogen atoms. Supplemental geometric constraints of 90phi angles and 12chi1 angles, and the distance constraints of 66 hydrogen bonds were experimentally derived. Using the DADAS90 program that calculates structures in dihedral angle space, 15 structures satisfying almost all constraints were obtained. The average root mean square deviation (RMSD) from the mean structure was 0.75 A for backbone atoms. The RMSD for backbone atoms between the representative NMR structure with the smallest constraint violation and crystal structures was within 1.2 A. Although the NMR and crystal structures thus resemble one another, a significant discrepancy was observed in a region termed 'basic protrusion.' The discrepancy observed in NMR experiments is explained by fluctuation in this region.

All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme that cleaves the RNA strand of a RNA/DNA hybrid, were pH-titrated, and their ionization constants (pKa) were determined from an analysis of the pH-dependent chemical shifts of the carboxyl carbon resonances obtained from 1H-13C heteronuclear two-dimensional NMR. The pKa values in the enzyme varied widely among individual residues, for example, in the unusual pKa values for two important catalytic residues, Asp10 (pKa 6.1) and Asp70 (pKa 2.6). Moreover, remarkable two-step titrations were observed for these carboxylates. The binding of Mg2+ ion to the enzyme, which is the cofactor necessary for catalytic activity, caused no significant change in the pKa values of the carboxyl groups, except for that of Asp10. The variations of the pKas that were dependent on the microenvironment in the protein were theoretically reproduced to compare with the experimental results by a numerical calculation, using a continuum electrostatic model. Most of the significant pKa decreases were brought about through strong electrostatic interactions with the neighboring basic amino acids, Arg or Lys. The pKa shifts and the two-step titrations of Asp10 and -70, which are close to each other, were interpreted to be due to the neighboring effect of two functional groups, as observed in the interacting titratable groups of a dicarboxyl compound or in the active site carboxylates of lysozyme and aspartic protease. The role of Asp10 in the catalytic action is either to be the proton donor to the RNA moiety or the binding partner of the Mg2+ ion cofactor. Asp70, on the other hand, is considered to be the proton acceptor from a water molecule.

Assignments of 1H, 15N, and 13C magnetic resonances for ribonuclease H from Escherichia coli have been completed using double- and triple-resonance 2D and 3D NMR experiments. These assignments include all types of 1H, 15N, and 13C nuclei detectable by NMR. The enzyme used, which cleaves the RNA moiety of an RNA-DNA duplex, consists of 155 amino acid residues and has 1962 nuclei (227 nitrogen, 762 carbons, and 973 protons) observable independently by NMR. Among those, 1868 nuclei (95%) have been assigned. Two methods, 3D HCH and 13C-13C-1H heteroSQC/homoSQC, were newly devised to complete the side chain assignments. These methods were used to elucidate the -CH2- and -C-CH-substructures. Triple-resonance experiments to detect other types of substructures, (e.g., -N-CH- and -C-NH-) were also applied. In total, 10 kinds of 3D NMR experiments were used to complete the assignments. The chemical shifts obtained through the assignments were analyzed in terms of the tertiary structure of the protein molecule. Among the 13C chemical shifts, larger secondary shifts (deviations from shifts at the random coil state) were observed for the C alpha, C beta, and C' nuclei, which reflect the local structures on the backbone, that is, the alpha-helix, beta-sheet, and left-handed helix, respectively.

A new strategy for the sequential assignment of backbone proton resonances in larger proteins involving a unique combination of four types of heteronuclear three-dimensional (3D) NMR spectroscopies is reported. This method relies on the uniform labeling of amide nitrogens with 15N and of alpha-carbons with 13C. Heteronuclear 1H-15N TOCSY-HMQC and NOESY-HMQC experiments can reveal connections between cross-peaks arising from the NHi-C alpha Hi-1 and NHi-C alpha Hi connectivities in the finger-print region in in general. They also specifically reveal the sequential amide-amide connectivities among the amide cross-peaks for the alpha-helices. Heteronuclear 1H-13C HMQC-TOCSY and HMQC-NOESY experiments can reveal connections between cross-peaks arising from the NHi-C alpha Hi and NHi+1-C alpha Hi connectivities in the finger-print region in general. The combination of the two sets of results reveals the complete unambiguous sequential connection of cross-peaks for the proton resonances in the peptide backbone. The application of the new strategy is reported for a protein, ribonuclease H, with a molecular weight of 17.6 kDa.

Intracellular K of the perfused rat mandibular salivary gland was measured by 39K NMR spectroscopy at 8.45 T. Multiple-quantum NMR arising from multiple-exponential decay was used to eliminate the resonance due to extracellular K in the perfused gland at 25 degrees C. The resonance due to intracellular K consisted of two Lorentzian signals stemming from the [spin 1/2 to -1/2] coherence (sharp resonance) and the [spin -1/2 to -3/2], [spin 3/2 to 1/2] coherences (broad resonance). The transverse relaxation time (T2) corresponding to the [spin 1/2 to -1/2] coherence was ca. 2.5 ms, and that corresponding to the [spin -1/2 to -3/2], [spin 3/2 to 1/2] coherences was ca. 0.4 ms. The relaxation time of the double-quantum coherence of rank 3 (originating from product operators like Ix2Iz) was determined to be ca. 0.2 ms. These results suggest the possibility of the presence of a single homogeneous population of intracellular K with a correlation time of ca. 2.5 x 10(-8) s and a quadrupolar coupling constant of ca. 1.4 MHz.

In this report, we focus on the spectral analysis problem and show that some extensions of the differential method (2) significantly enhance its application range and flexibility

The spatial and temporal pattern of oscillating temperatures on the cell surface of a plasmodial strand ofPhysarum polycephalum was measured with a sensitive thermal image camera. The longitudinal tension of the strand was studied simultaneously. In the absence of chemical stimulation, the phases of the temperature oscillation observed at various portions of the strand were entrained with almost coincidental phase. The temperature and tension oscillation were synchronized, although the phase difference between them was occasionally changed. With local chemical stimulation, the phase of the temperature oscillation advanced in the portion to which the plasmodium would be induced to migrate. The phases between temperature and tension oscillations then became constant. The mechanism by which the plasmodium processes local information of chemical stimulus to global information for the migration is discussed.

A new experiment is proposed for two-dimensional heteronuclear correlation spectroscopy. It involves the rapid creation of pure heteronuclear ZQC under cross-polarization, followed by an evolution period during which ZQC is converted to DQC. A read pulse is used to detect the signal. No phase cycling is required to determine the sign of ω1.

A new technique to obtain two-dimensional (2D) crystals of proteins is reported. A clean surface of mercury was utilized for monolayer formation and crystallization of the protein. Two different proteins, ferritin from a horse spleen and F1-ATPase from a thermophilic bacterium (TF1), were crystallized on the clean surface of mercury in an oxygen atmosphere. Two dimensional crystals were analyzed by electron crystallography. Ferritin was found to form a hexagonal lattice with unit cell parameters a = b = 12 nm and γ = 60°. The 2D crystal of ferritin at a 2.0 nm resolution in negative stain revealed a subunit formation with P3m1 symmetry. TF1 also formed a hexagonal lattice with unit cell parameters a = b = 10 nm and γ = 60°. The reconstructed image of TF1 at a 1.8 nm resolution showed a ring structure with six peripheral constituents and minor components located in the middle hole.

A general method of obtaining four-quadrant pure-phase spectra in two-dimensional NMR is described. Mixed-phase 2D spectra can be converted to pure-phase 2D spectra using the mathematical manipulation of time reversal in the t, dimension or frequency inversion in the m, direction. The characteristics of this method as compared with two previously proposed methods are discussed, focusing on the calculation time of a spectrum, memory demands, and sensitivity gain. The applications to COSY and NOESY methods are shown for a simple biomolecule.

A simple proton-selective α/β-HSQMBC experiment is proposed for the accurate measurement of long-range proton–carbon coupling constants (nJCH) in small molecules without need for an individualized and time-consuming post-processing fitting procedure. The method acquires two pure-phase In-phase (IP) and Anti-phase (AP) multiplets completely free of any phase distortion due to the absence of JHH evolution. Accurate nJCH values can be directly measured analyzing the relative displacement of the resulting IPAP cross-peaks. Discussion about signal intensity dependence and cross-talk is made for a range of experimental conditions. The robustness of the method is evaluated by comparing the nJCH value measured from the analysis of the three available IP, AP and IPAP multiplet patterns. Multiple-frequency and region-selective versions of the method can also be efficiently recorded provided that excited protons are not mutually coupled.

The pseudofilter is a new instrumental concept which fills a gap between real and digital filters. The idea was originally motivated by the desire to develop frequency filters in the direction of ω1(f1) in 2D NMR. The filter mechanism relies strictly on the accumulation of repeated coherent signals; therefore only the sensitivity gain results without reduction of noise. Applications are (1) a 2D filter to cut unnecessary time-domain signals, (2) a J filter in 13C-1H shift correlation, and (3) an ideal filter in 1 D and 2D NMR.