Search

Vibrational circular dichroism (VCD) and IR absorption spectra are obtained in a chloroform solution for poly[γ-((R)-α-phenethyl)-L-glutamate] (PRPLG) and poly[γ-((S)-α-phenethyl)-L-glutamate] (PSPLG), whose only structural difference is an opposite chiral center in the side chain. Their characteristic amide A, I, and II bands show VCD patterns quite similar to those of poly[γ-benzyl-L-glutamate] (PBLG), indicating that the secondary structure of these polypeptides is a right-handed α-helix. The VCD spectra in the CH stretching region exhibit different patterns for PRPLG and PSPLG, reflecting the chirality difference in the side chains. This difference is interpreted on the basis of the additivity of optical activity contributions from the main chain conformation and the chirality difference in the side chains. The results indicate that a VCD difference spectrum of the CH stretching region is a useful diagnostic tool for elucidating local chirality differences. © 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 228–234, 2001

The C-terminal domain of the α-subunit of Escherichia coliRNA polymerase (αCTD) is responsible for transcriptional activation through interaction with both activator proteins and UP element DNA. Previously, we determined the solution structure of αCTD. Here, we investigated the interaction between αCTD and UP element DNA by NMR. DNA titration curves and intermolecular NOE measurements indicate that αCTD can bind to multiple sites on the UP element DNA. Unlike many transcription factors, αCTD does not have a strict base sequence requirement for binding. There is a good correlation between the strength of the interaction and the extent of intrinsic bending of the DNA oligomer estimated from the gel retardation assay. We propose that αCTD recognizes the backbone structure of DNA oligomers responsible for the intrinsic bending. Moreover, NMR studies and drug competition experiments indicated that αCTD interacts with the UP element on the minor groove side of the DNA. The C-terminal end of helix-1, the N-terminal end of helix-4, and the loop between helices 3 and 4 are used for the interaction. Based on these observations, we propose a model for the UP element-αCTD complex.

The C-terminal domain of the α subunit of the RNA polymerase (αCTD) from Escherichia coli(Ec) regulates transcription by interacting with many kinds of proteins and promoter upstream (UP) elements consisting of AT-rich sequences. However, it is unclear how this system is common in all eubacteria. We investigate the structure and properties of αCTD from an extremely thermophilic eubacterium, Thermus thermophilus(Tt). The solution structure of TtαCTD (85 amino acids) was determined by NMR, and the interaction betweenTtαCTD and DNA with different sequences was investigated by means of chemical shift perturbation experiments. The tertiary structure of TtαCTD is almost identical with that ofEcαCTD despite 32% sequence homology. However,TtαCTD interacts with the upstream region sequence of the promoter in the Tt16 S ribosomal protein operon rather than the EcUP element DNA. The upstream region sequence ofTtis composed of 25 base pairs with 40% AT, unlike theEcUP element with 80% AT. The DNA binding site inTtαCTD is located on the surface composed of helix 4 and the loop preceding helix 4. The electric charges on this surface are not remarkably localized like those of EcαCTD.

Caspase-activated DNase (CAD), which causes a genome fragmentation at the final stage of apoptosis, is a protein of about 40 kDa and exists as a complex form with the inhibitor ICAD in living cells. There is sequence homology of about 80 amino acid residues at the N termini of CAD and ICAD (called the CAD domain). Here, we report the three-dimensional structure of the CAD domain of CAD determined by multi-dimensional NMR spectroscopy and the property of CAD domains investigated by a surface plasmon resonance experiment. The CAD domain of CAD is an independently folded domain composed of one α-helix and five β-strands forming a single sheet. The overall structure is categorized in the ubiquitin superfold. This domain can bind strongly to the isolated CAD domain of ICAD (dissociation constant: 5.48(±0.003) × 10−8M). It suggests the function of the CAD domains in the CAD-ICAD system, that the protein-protein interaction through the CAD domains plays an important role in the inhibition of CAD DNase activity and in the correct folding of CAD. On the basis of structural comparison with other protein complexes containing the ubiquitin superfold, the interaction mode of the CAD domains is proposed.

Here we demonstrate the presence of the A′‐RNA conformation using the single crystal structure of a tridecamer: r(UGAGCUUCGGCUC). The average A′‐RNA conformation deduced from X‐ray fiber diffraction data had only been available previously, but now the presence of the A′‐RNA conformation has been found in a single crystal structure for the first time. Statistical analysis showed that the A′‐RNA conformation is distinguishable from the A‐RNA conformation in a plot of the major groove width against the base pair inclination angle. The major groove of the A′‐RNA conformation is wide enough to accommodate a protein or peptide while that of the A‐RNA conformation is too narrow to do so. The presence of the A′‐RNA conformation is significant for protein–RNA interaction.

AbstractThe specific interaction between λ phage Cro repressor and the DNA fragment bearing the consensus sequence of operators has been studied using nuclear magnetic resonance (NMR). Using both l5N- and 13C/15N- labeled λ-Cro in complex with unlabeled DNA, chemical shift assignments of the λ-Cro-DNA complex were obtained using heteronuclear NMR experiments. Inter-molecular contacts between the protein and DNA were identified using heteronuclear filtered NOESY experiments. The inter-molecular contacts were supplemented with intra-protein and intra-DNA NOE constraints to dock λ-Cro to the bent B-form DNA using restrained molecular dynamics. The structure of one of the subunits of λ-Cro in the complex is essentially the same as that of the unbound form. In the complex, inter-molecular NOEs were observed between the “helix-turn-helix” region comprising the α2 and α3 helices of the λ-Cro protein and the major groove of the DNA. The methyl group of Thrl7 forms a hydrophobic contact with the methyl group of the thymine at base pair 1 in the DNA, and Val25 and Ala29 make hydrophobic contacts with the methyl group of the thymine at base pair 5. The presence and the absence of these contacts can explain the difference in the affinity of λ-Cro to several variants of the operator sequence.

A new isotope labeling technique for peptide segments in a protein sample was recently established using the protein splicing element intein [Yamazaki et al. (1998) J. Am. Chem. Soc., 120, 5591–5592]. This method makes it possible to observe signals of a selected amino (N-) or carboxyl (C-) terminal region along a peptide chain. However, there is a problem with the yield of the segmentally labeled protein. In this paper, we report an increase in the yield of the protein that enables the production of sufficient amounts of segmentally 13C/15N-labeled protein samples. This was achieved by improvement of the expression level of the N-terminal fragment in cells and the efficiency of refolding into the active splicing conformation. The N-terminal fragment was expressed as a fused protein with the cellulose binding domain at its N-terminus, which was expressed as an insoluble peptide in cells and the expression level was increased. Incubation with 2.5 M urea and 50% glycerol increased the efficiency of the refolding greatly, thereby raising the final yields of the ligated proteins. The feasibility of application of the method to a high-molecular-weight protein was demonstrated by the results for a maltose binding protein consisting of 370 amino acids. All four examined joints in the maltose binding protein were successfully ligated to produce segmentally labeled protein samples.

Resonance Raman spectra have been obtained for Spinacea oleracea, Phytolacca americanaand Halobacterium halobiumferrodoxins. These spectra were very similar to each other in terms of both vibrational frequencies and relative intensities, strongly suggesting that their molecular structures of active centers are very similar. The resonance Raman spectra were, however, fairly different in the frequencies of Fe—S(Cys) stretching modes from the spectrum of beef adrenodoxin previously reported. Probably the nature of Fe—S(Cys)-bonding and/or the manner of cysteine ligation is somewhat different between adrenodoxin and the other [2Fe—2S] ferrodoxins.

Diastereoselective syntheses of [5'-²H]thymidine (6-T) [(5'R)/(5'S) = 31:69], N⁴-benzoyl-2'-deoxy[5'-²H]cytidine (6-C^Bz) [(5'R)/(5'S) = 27:76], N⁶-benzoyl-2'-deoxy[5'-²H]adenosine (6-A^Bz) [(5'R)/(5'S) = 39:61], and 2'-deoxy-N²-isobutyryl[5'-²H]guanosine (6-G^iBu) [(5'R)/(5'S) = 20:80] were attained by a radical reductive deuteration reaction of the corresponding 5'-O-acetyl-3'-O-TBDMS-2'-deoxy-5'-phenylselenoribonucleosides (4) with a Bu3Sn²H-Et3B system at <−70°C to give 5 (87–98% yields), followed by unmasking (66–79% yields); 4 were synthesized in three steps of reactions from the corresponding 2'-deoxy-3'-O-TBDMS-5'-O-Ms-ribonucleosides (1) (88–93% yields) by a known method. Chirality 9:435–442, 1997. © 1997 Wiley-Liss, Inc.

A photo‐CIDNP spectrum of RNase T1showed that 4 out of the total 9 tyrosines are accessible to the photosensitive dye, while none of the histidine and tryptophan residues are accessible. By comparison with the results of nitration of tyrosine side chains followed by peptide analysis, it can be concluded that Tyr 45 is mostly exposed on the surface of RNase T1. On addition of 2'‐GMP, the signal of Tyr 45 shifts upfield and is remarkably broadened, which suggests that the phenyl ring of Tyr 45 stacks on the guanine ring of 2'‐GMP. Similar phenomena were observed on addition of 3'‐GMP and 3'‐dGMP.

The¹H,¹⁵N and¹³C magnetic resonances of the λ-Cro repressor have been assigned almost completely, mainly through the use of heteronuclear multidimensional NMR methods. Inter-subunit NOEs were distinguished by means of heteronuclear spectral editing technique (¹³C double half filter technique). Based on the distance and dihedral angle constraints derived from the NMR data, the three-dimensional solution structure of the λ-Cro repressor in the dimeric form has been calculated by the simulated annealing method. The input for the structure calculations consisted of¹H-¹H distance constraints, of which 1536 were intra-subunit and 40 were inter-subunit, and dihedral angle, φ, constraints, which numbered 92. The average root-mean-square deviation (RMSD) for all backbone heavy- atoms of the 20 calculated structures for residues 3 to 59 of the total of 66 amino acid residues in both subunits was 1.57 Å, while the average RMSD for each subunit in the same residue range was 0.66 Å. The subunit is composed of three α-helices, residues 7 to 13, 16 to 23 and 27 to 36, and a three-stranded anti-parallel β-sheet composed of residues 3 to 6, 40 to 44 and 50 to 55. The solution structure of the subunit is essentially the same as that in the crystalline form, but the structure of the dimer form in solution differs from that of the dimer unit in the crystalline form. It is suggested that the solution dimer structure is distorted to fit the recognition helices in the major grooves of DNA on complex formation.

Nortriostin A, which is an analog of a cyclic octadepsipeptide antibiotic triostin A and contains one cystine and two valine residues substituted for the N,N′-dimethylcystine and N-methylvaline residues of the antibiotic, was synthesized with Z–d-Ser[Boc–Ala–Cys(MeBzl)–Val]–OH and Z–d-Ser[H–Ala–Cys(MeBzl)–Val]–OTce as key intermediates. The synthetic analog showed no antimicrobial activity at a concentration of 100 μg/ml and was found to exist as a single structure in solution as examined by 1H-NMR spectroscopy. The latter observation suggests that the conformer equilibrium known to occur with triostin A is a consequence of the presence of N-methyl peptide bonds in the antibiotic molecule.

The stereoselective deuterium labeling at the 5' methylene protons of the ribose ring recently developed by Kawashima et al. [1995, Tetrahedron Lett., 36, 6699–6700] enabled the assignment of pro-R and pro-S protons at the 5' position. The deuterium-labeled nucleotides, [(5'S)-2H]- and [(5'R)-2H]-diastereomers, in an approximate ratio of 2:1, were incorporated in the decamer 5'-d(GCATTAATGC)-3'. Thus, both pro-R and pro-S methylene proton signals without geminal coupling appeared in the NOESY and DQF-COSY spectra. Complete stereospecific assignments and simplified spin systems enabled the determination of 15 3J coupling constants between H4' and H5'/H5", and the unambiguous assignment of 135 NOESY cross peaks originating from H4'/H5'/H5" resonances.

Vibrational circular dichroism (VCD) and infrared absorption spectra in the amide A region of blocked oligopeptides containing proline have been obtained in apolar organic solvents in order to clarify the characteristic VCD for the intramolecularly hydrogen-bonded NH stretching involved in the turn structures of peptide chains as well as the intermolecularly hydrogen-bonded NH stretching in associated molecules. The γ-turn with the intramolecularly hydrogen-bonded C7conformation exhibits a characteristic positive VCD band at about 3330 cm−1in dilute solutions of Piv-Pro-NHMe and Ac-Pro-NHMe. The intramolecularly hydrogen-bonded NH stretching in the type II β-turn with C10conformation gives rise to a positive VCD band at 3345 cm−1for Piv-Pro-Gly-NHMe. The Gly NH group of Piv-Pro-Gly-OMe and Ac-Pro-Gly-OMe in dilute solutions assumes C7C5conformation stabilized by a bifurcated three-center intramolecular hydrogen bond and exhibits a positive VCD band near 3300 cm−1at a lower frequency than the intermolecularly hydrogen-bonded band. All the peptides studied give a characteristic negative—positive bisignate couplet from the high wavenumber side for the intermolecularly hydrogen-bonded NH stretching band at high concentrations.

The structure of DNA in a DNA–protein complex was studied by means of fluorescence resonance energy transfer (FRET) method. Oligonucleotide phosphorothioates were labeled with fluorescein and eosin to obtain a donor- and acceptor-labeled DNA. The formation of a complex of the DNA with PAP1(70), which is a DNA binding site fragment derived from transcription regulatory protein, PAP1, of fission yeast, was confirmed by gel retardation analysis and fluorescence measurements. FRET of the donor- and acceptor-labeled DNA with and without PAP1(70) indicated that the DNA in the complex was bent about 26° toward the protein-binding surface.

Classification of the Raman spectra of various hemoproteins including HbNO and HbCNC2H5suggests that there are two kinds of ferrous low spin states. They are specified by the binding nature of the sixth ligand (L) to heme iron. The Fe–L bond in HbNO and HbCNC2H5as well as in HbCO and HbO2is of π type while that in ferrous cytochromes b5and cis of σ type.

After the assignment of 1H NMR signals of aromatic side chains by means of specific deuteration, we analyzed the DNA binding site of GAL4 by measuring photo-CIDNP spectra. The results showed that Trp 36is involved in both the specific interaction with UAS Gand non-specific DNA binding. This residue is located inside the Cys-rich region, but outside the putative Zn-finger. The photo-CIDNP spectrum also showed that the side chains of Tyr 40and His 53are not exposed on the surface of the protein.

For structure analysis of peptides by multinuclear NMR, stable isotope-labeled samples are required. A direct over-expression system by E. colicells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells. We here developed an over-expression system by means of thioredoxin gene fusion system. The fused protein composed of thioredoxin and the objective peptide was expressed in E. coliand then the peptide part was released by enterokinase. This system was successfully applied for the production of 15N-labeled human adrenocorticotropic hormone fragment (ACTH-(1-24)) as needed for multinuclear NMR analysis.

The solution structure of the central domain of the human nucleotide excision repair protein XPA, which binds to damaged DNA and replication protein A (RPA), was determined by nuclear magnetic resonance (NMR) spectroscopy. The central domain consists of a zinc-containing subdomain and a C-terminal subdomain. The zinc-containing subdomain has a compact globular structure and is distinct from the zinc-fingers found in transcription factors. The C-terminal subdomain folds into a novel α/β structure with a positively charged superficial cleft. From the NMR spectra of the complexes, DNA and RPA binding surfaces are suggested.

Abstract: Summary-Proton NMR spectra of gustatory stimulated healthy human whole, parotid and submandibular and sublingual saliva were measured. The typical patterns of their spectra were obtained. Among these three kinds of fluids the parotid saliva which contains preferentially serous saliva presented a relatively well resolved spectrum with satisfactory signal to noise ratio in a given short time(30min.). For eight subjects of parotid saliva collected in the afternoon the marked increase in signal intensity in the methyl and methylene proton region of proteins(peptides)was observed as compared with those collected in the morning, reflecting the circadian rhythms in the protein concentration in saliva. On the other hand the methyl peak of lactic acid presented the opposite tendency, which might be also correlated with the circadian rhythms of the glucose metabolism of the parotid gland. The proton NMR spectra of three patients suspected of salivary gland disease were different from those of healthy subjects.

Crystals have been obtained of the DNA-binding domain of the yeast transcription factor PHO4 in complexes with several synthetic fragments of NDA with appropriate cognate sequences. Crystals suitable for X-ray diffraction studies were produced in the case of a complex of the protein with a 17 base-pair fragment of DNA from a solution in polyethylene glycol and calcium chloride. The crystals have the space group of P41212 or P43212 with unit cell dimensions a = b= 56·7 Å, c= 447·8 Å. The diffraction data at 3 Å resolution were collected using synchrotron radiation with a Weissenberg camera for macromolecular crystallography.

The dynamic properties of the C-terminal one-third of the α subunit of RNA polymerase were investigated. The intact α subunit exhibited almost the same NMR spectral pattern as the isolated C-terminal fragment, indicating that the C-terminal domain retains the same conformation as the isolated fragment, and that its motion is independent of that of the associated N-terminal domain. Analysis of the NMR dynamics data for the intact α subunit indicated that at least 13 residues between the N and C-terminal domains show distinctly higher motional flexibility than the structured parts. This flexible linker may endow the C-terminal domain with locational freedom in different kinds of initiation complex. The dynamics data also revealed that the residues in the contact site for DNA and transcription factors exhibited higher mobility than other secondary structural elements.

Vibrational modes for the resonance Raman lines of Ni(OEP) have been obtained on the basis of a normal coordinate analysis. The Raman line at 1383 cm−1, which corresponds to an oxidation-state marker band of hemoproteins, is mainly due to the breathing mode of the pyrrole rings. The vibrations of the sixteen-membered inner ring contribute to the Raman bands sensitive to the spin-states of the heme iron.

Natural abundance 15N nmr spectra of L-histidine, and τ- and π-methylhistidines were measured. The signal assignments to the τ- and π-nitrogen could be made by comparison of these spectra. From the pH titration of the 15N chemical shifts, it is directly proved that the τ-H tautomer is predominant in a basic solution.

The glycerol part of E.coliphospholipid was specifically deuterated by supplying 2H5-glycerol in growth media of glycerol-requiring auxotroph. The 2H-spectra of cardiolipin were obtained for dispersion of the purified deuterated cardiolipin mixed with non-deuterated other components. The structure of the whole polar region of cardiolipin in the lipid bilayer appeared to be dynamically symmetric, and quite rigid.

Natural abundance 15N nmr spectra of [Met5]-enkephalin and its constituent peptides were obtained. By comparison of these spectra five 15N resonance signals could be assigned to each amino acid residue. In the pH titration curves of the chemical shifts the effect of the protonation at the C-terminal Met carboxyl group was observed.

Intermolecular associations of the enantiomers of pantolactone in carbon tetrachloride solution have been studied by infrared and 1H-NMR spectroscopic methods. The spectroscopic data have been analyzed by the least squares method to obtain the association constants and thermodynamic parameters. It was found that pantolactone takes a cyclic dimer structure by dint of hydrogen bonding. The association constant for homochiral dimerization was obtained to be 8.9 mol-1dm3at 26°C and the enthalpy and entropy changes were -35 kJ mol-1and -97 J K-1mol-1, respectively. The association constant for heterochiral dimerization is just twice as large as that for homochiral dimerization, that is, no selective association exists between enantiomers.

A 31P NMR spectrum of chicken erythrocyte chromatins in vivo was obtained. The spectrum was characterized by examining the spectra of isolated nuclei and nondigested chromatins under various conditions. The results have shown that most chromatins in chicken erythrocyte nuclei assume the 30 nm filament structure as a second step of condensation of DNA in vivo.

The secondary structure elements of the DNA‐binding domain of mouse interferon regulatory factor 2 [IRF‐2(113)] were determined by heteronuclear multidimensional NMR spectroscopy. The sequential NOE connectivities, amide proton exchange rates, and 3JHNαcoupling constants indicated the presence of three α‐helical regions and four short β‐strands connected through relatively long loops. The long range NOEs indicated the four strands form an antiparallel β‐sheet and the three α‐helices form a bundle on the sheet. The arrangement of the secondary structure elements and the overall folding topology resemble those of the DNA binding domains of bacterial activator CAP, heat shock transcription factors, and fork‐head proteins, although there is no sequence homology among them.

The secondary structure elements of the DNA-binding domain of mouse interferon regulatory factor 2 [IRF-2(113)] were determined by heteronuclear multidimensional NMR spectroscopy. The sequential NOE connectivities, amide proton exchange rates, and 3J HNαcoupling constants indicated the presence of three α-helical regions and four short β-strands connected through relatively long loops. The long range NOEs indicated the four strands form an antiparallel β-sheet and the three α-helices form a bundle on the sheet. The arrangement of the secondary structure elements and the overall folding topology resemble those of the DNA binding domains of bacterial activator CAP, heat shock transcription factors, and fork-head proteins, although there is no sequence homology among them.

A photo-CIDNP spectrum of RNase T 1showed that 4 out of the total 9 tyrosines are accessible to the photosensitive dye, while none of the histidine and tryptophan residues are accessible. By comparison with the results of nitration of tyrosine side chains followed by peptide analysis, it can be concluded that Tyr 45 is mostly exposed on the surface of RNase T 1. On addition of 2'-GMP, the signal of Tyr 45 shifts upfield and is remarkably broadened, which suggests that the phenyl ring of Tyr 45 stacks on the guanine ring of 2'-GMP. Similar phenomena were observed on addition of 3'-GMP and 3'-dGMP.

Most of the 1H and 15N magnetic resonances of the 66 amino acid long POU homeodomain of mouse Oct-3 have been assigned by the combined use of the two-dimensional homonuclear, and two- and three-dimensional heteronuclear NMR methods. The sequential NOE connectivities and amide proton exchange measurements indicate the presence of three helical regions within the domain. The positions of the three helices correspond well to those of other homeodomains, the three-dimensional structures of which have already been determined. The present NMR study provides the first experimental evidence for the existence of a helix-turn-helix motif in the oct-3POU homeodomain.

Most of the 1H and 15N magnetic resonances of the 66 amino acid long POU homeodomain of mouse Oct‐3 have been assigned by the combined use of the two‐dimensional homonuclear, and two‐ and three‐dimensional heteronuclear NMR methods. The sequential NOE connectivities and amide proton exchange measurements indicate the presence of three helical regions within the domain. The positions of the three helices correspond well to those of other homeodomains, the three‐dimensional structures of which have already been determined. The present NMR study provides the first experimental evidence for the existence of a helix‐turn‐helix motif in the oct‐3POU homeodomain.

After the assignment of 1H NMR signals of aromatic side chains by means of specific deuteration, we analyzed the DNA binding site of GAL4 by measuring photo‐CIDNP spectra. The results showed that Trp36is involved in both the specific interaction with UASGand non‐specific DNA binding. This residue is located inside the Cys‐rich region, but outside the putative Zn‐finger. The photo‐CIDNP spectrum also showed that the side chains of Tyr40and His53are not exposed on the surface of the protein.

Diastereoselective syntheses of [5′‐2H]thymidine (6‐T) [(5′R)/(5′S) = 31:69], N4‐benzoyl‐2′‐deoxy[5′‐2H]cytidine (6‐CBz) [(5′R)/(5′S) = 27:76], N6‐benzoyl‐2′‐deoxy[5′‐2H]adenosine (6‐ABz[(5′R)/(5′S) = 39:61], and 2′‐deoxy‐N2‐isobutyryl[5′‐2H]guanosine (6‐GiBu) [(5′R)/(5′S) = 20:80] were attained by a radical reductive deuteration reaction of the corresponding 5′‐O‐acetyl‐3′‐O‐TBDMS‐2′‐deoxy‐5′‐phenylselenoribonucleosides (4) with a Bu3Sn2H‐Et3B system at <−70°C to give 5(87–98% yields), followed by unmasking (66–79% yields); 4were synthesized in three steps of reactions from the corresponding 2′‐deoxy‐3′‐O‐TBDMS‐5′‐O‐Ms‐ribonucleosides (1) (88–93% yields) by a known method. Chirality 9:435–442, 1997.© 1997 Wiley‐Liss, Inc.

Resonance Raman spectra have been obtained for Spinacea oleracea, Phytolacca americanaand Halobacterium halobiumferrodoxins. These spectra were very similar to each other in terms of both vibrational frequencies and relative intensities, strongly suggesting that their molecular structures of active centers are very similar. The resonance Raman spectra were, however, fairly different in the frequencies of Fe—S(Cys) stretching modes from the spectrum of beef adrenodoxin previously reported. Probably the nature of Fe—S(Cys)‐bonding and/or the manner of cysteine ligation is somewhat different between adrenodoxin and the other [2Fe—2S] ferrodoxins.

For structure analysis of peptides by multinuclear NMR, stable isotope‐labeled samples are required. A direct over‐expression system by E. colicells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells. We here developed an over‐expression system by means of thioredoxin gene fusion system. The fused protein composed of thioredoxin and the objective peptide was expressed in E. coliand then the peptide part was released by enterokinase. This system was successfully applied for the production of 15N‐labeled human adrenocorticotropic hormone fragment (ACTH‐(1‐24)) as needed for multinuclear NMR analysis.

A new four-dimensional pulse scheme is described for the main-chain assignment of proteins by means of the J connectivity of the amide proton and nitrogen resonances of adjacent residues. Since the new experiment, 4D CP-HN(COCA)NH, involves heteronuclear cross-polarization for magnetization transfer from 13C=O to 15N via 13Ca, a relatively strong WALTZ-16 decoupling rf field is applied to 13Ca during magnetization transfer. Consequently, 13Ca is effectively decoupled from its attached 2H in the case of deuterated proteins, in the absence of a decoupling rf field for 2H. This efficiently improves the sensitivity of the experiment through 13C line narrowing. The experiment was performed on a randomly 60% deuterated protein, and the sensitivity of the final 4D spectrum was found to be excellent.

The base proton (purine H8 and pyrimidine H6) resonances are key signals for the assignment of the proton resonances of DNA oligomers. They are classified into two groups, i.e., cytosine H6 signals, observed as doublets, and the other base proton signals, observed as singlets. Here we propose some experiments for distinguishing the cytosine H6 signals from the other base proton signals. Moreover, the ability of signal selection and the sensitivity as to signal detection were compared for all experiments, and the optimum conditions for spectral measurements were surveyed. Some of the experiments were employed as the NOESY detection pulse. Previously proposed experiments, such as HOENOE and HAL, were also used in the comparison.

The solution states of Aerosol OT were studied by 1H-NMR measurements. In water two hydrocarbon chains of Aerosol OT molecules in monomeric state aggregate with each other with hydrophobic interactions as in micellar state.

The vibrational circular dichroism of (1R,2R)-diaminocyclohexane complexes of Pd(II), Cu(II), Ni(II), and Co(III) in CD3CN are characterized by positive and negative bands for the antisymmetric and symmetric NH2stretching vibrations, respectively. This feature is attributable to the λ conformation of the chelate ring.

Proton magnetic resonance and Raman spectra indicate that the backbone of γ-aminobutyric acid is fairly flexible and rotational isomers are almost equally populated in aqueous solution. The population is not much affected by change of pH. On the other hand, in γ-amino-β-Hydroxybutyric acid the trans-transand gauche-transconformations of the backbone are equally populated in neutral solution, trans-transconformation being predominant in non-zwitterionic forms. The conformation in solution is discussed in relation to the structure of these molecules in crystal.

The vibrational circular dichroism associated with the OH stretching vibrations of methyl 3-hydroxybutyrate and methyl lactate has been measured in the CCl4solutions. The bisignate feature of VCD observed in methyl 3-hydroxybutyrate has been interpreted as the coexistence of rotational isomers in intramolecularly hydrogen bonded species.

Heat-stable enterotoxin (STp) produced by a porcine strain of enterotoxigenic Escherichia coliis a peptide of 18 amino acid residues. The full toxicity is generated by a core peptide of 13 amino acids from residue 5 to 17 (STp(5–17)). Detailed conformational analysis of STp(5–17) was performed by NMR spectroscopy and distance geometry calculations. The tertiary structure of STp(5–17) was found to have a right-handed spiral fold throughout the whole molecule which was stabilized by the network of disulfide linkages and hydrogen bonds. This folding pattern was demonstrated in both organic and aqueous solutions.

A 31P NMR spectrum of chicken erythrocyte chromatins in vivo was obtained. The spectrum was characterized by examining the spectra of isolated nuclei and nondigested chromatins under various conditions. The results have shown that most chromatins in chicken erythrocyte nuclei assume the 30 nm filament structure as a second step of condensation of DNA in vivo.

The four (2'S)-[2'-2H]-2'-deoxynucleosides (>90 atom % 2H), were synthesized from the corresponding ribonucleosides involving six steps of reactions, i.e., oxidation of their 2'-hydroxyl group, stereoselective reductive deuteration of the resulting 2'-ketonucleoside intermediates with NaB2H4 in EtOH-H2O or EtOH, triflation, bromination with LiBr, highly stereoselective Bu3SnH-Et3B reduction of the resulting bromide, and, finally, unmasking.