Spatial structure of cro-repressor in a solution. II. Effect of ionized groups

The technique of 1H NMR spectroscopy and absorption UV spectroscopy were used to study the ionization of the tyrosine phenol cycles and the effect of ionizable groups on the chemical shifts of signals from protons in the side chains of several amino acid residues. The microenvironment of these residues was established by analysing the titration curves. The mutual orientation of two functionally important adjacent alpha-helical protein regions was determined in solution. The signals from methionine residues belonging to different regions of the secondary structure were assigned in the NMR spectrum. The results indicate that the spatial structure of the repressor is similar in solution an in the crystal. They confirm the model proposed for the cro repressor interaction with DNA and based on the data of X-ray diffraction analysis.     

Localization of hydrophobic ions in phospholipid bilayers using 1H nuclear Overhauser effect spectroscopy

The binding location for the hydrophobic ions tetraphenylphosphonium (TPP+) and tetraphenylboron (TPB-) was studied in sonicated phosphatidylcholine (PC) vesicles by measuring time-dependent and steady-state intermolecular 1H nuclear Overhauser effects (NOE's). Intermolecular cross-relaxation was also investigated by two-dimensional NOE spectroscopy. Information on the distance and order parameter dependence of the NOE's was obtained from a simple simulation of the NOE's in the alkyl chain region. Taken together, the NOE data and the simulation provide strong evidence that TPB- and TPP+, at low concentrations (less than or equal to 10 mol%), are localized in the alkyl chain region of the bilayer. At these lower concentrations of TPP+ or TPB-, no significant effect on lipid 13C T1 or T2 relaxation rates is detected. The proposed location is consistent with the expected free energy profiles for hydrophobic ions and with the carbonyl oxygens or interfacial water as the source of the membrane dipole potential. At higher ion/lipid ratios (greater than or equal to 20 mol%), TPB-/lipid NOE's increase. This results from a specific association of TPB- with the choline head group.     

Identification of localized redox states in plant-type two-iron ferredoxins using the nuclear Overhauser effect

The homonuclear Overhauser effect (NOE), in conjunction with nonselective spin-lattice relaxation measurements, has been employed to assign the contact-shifted resonances for the reduced form of two typical plant-type two-iron ferredoxins from the algae Spirulina platensis and Porphyra umbilicalis. These results demonstrate that the NOE should have broad general applicability for the assignments and electronic structural elucidation of diverse subclasses of paramagnetic iron-sulfur cluster proteins. NOE connectivities were detected only among sets of resonance exhibiting characteristically different deviations from Curie behavior, providing strong support for the applicability of the spin Hamiltonian formulation for the NMR properties of the antiferromagnetically coupled iron clusters [Dunham, W. R., Palmer, G., Sands, R. H., & Bearden, A. J. (1971) Biochim. Biophys. Acta 253, 373-384; Banci, L., Bertini, I., & Luchinat, C. (1989) Struct. Bonding (in press)]. The geminal beta-methylene protons for the two cysteines bound to the iron(II) center were clearly identified, as well as the C alpha H and one C beta H for each of the cysteines bound to the iron(III). The identification of the iron bound to cysteines 41 and 46 as the iron(II) in the reduced protein was effected on the basis of dipolar contacts between the bound cysteines, as predicted by crystal coordinates of S. platensis Fd [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. (Tokyo) 90, 1763-1773].(ABSTRACT TRUNCATED AT 250 WORDS)     

Local structure of cytochrome c from horse heart in solution. Conformational analysis using data of two-dimensional nuclear Overhauser effect spectroscopy]

Using the earlier suggested method the calculation of the backbone conformations of horse heart cytochrome c in oxidized (ferricytochrome c) and reduced (ferrocytochrome c) states has been performed by the two-dimensional nuclear Overhauser effect spectroscopy data. For both protein forms the secondary structure elements have been revealed and the conformations of the irregular polypeptide chain segments have been analysed. The similarity of the secondary structures of ferri- and ferrocytochrome c in solution was established from the comparison of their conformations. Small differences between the conformations of two molecule forms are shown to be localized within the polypeptide chain fragments situated in the spatial structure near the heme crevice. The comparison of the dihedral phi and psi angles in the calculated conformations of horse cytochrome C with the corresponding characteristics of X-ray structures of tuna ferri- and ferrocytochrome c made for the oxidized and reduced protein forms using the quantitative criteria testifies the similarity of their conformations in solution and crystal. In is shown that the conformational changes of the separate amino acid residues which take place as the result of the "solution-to-crystal" transition occur on the surface fragments of protein globule and do not lead to essential alterations of the secondary molecule structure.     

Conformation of oligonucleotides in solution from data of the nuclear Overhauser effect

In this review the results of studies of oligonucleotides conformation in solution by 1D and 2D NOE over the last four years are presented. Theoretical basis of 2D NOE and other 2D techniques are briefly considered. The few attempts of determination of oligonucleotides structures in solution on the half-quantitative level and the perspectives of using 2D NOE for quantitative structure resolution are discussed.     

The solution conformation of nicotinamide mononucleotide: a quantitative application of the nuclear Overhauser effect.

Torsion about the glycosidic linkage in nicotinamide mononucleotide has been investigated by quantitative application of the nuclear Overhauser effect. These measurements show that the syn (chi congruent to 20 degrees) and anti (chi congruent to 200 degrees) conformers of the title compound are isoenergetic, or nearly so, and interconverting rapidly. The syn/anti partition is not measurably affected by either changes in pH or temperature.     

Characterization of substance P-membrane interaction by transferred nuclear Overhauser effect

Substance P, one of the mammalian tachykinins, is known to interact strongly with lipid bilayers and this interaction may play a role in the receptor-peptide recognition process. The conformation of substance P bound to vesicles consisting of perdeuterated phosphatidylcholine has been investigated by means of two-dimensional transferred nuclear Overhauser (trNOE) spectroscopy. Nuclear magnetic resonance data analysis resulted in a unique conformational family characterized by a well-defined conformation of the last seven C-terminal amino acids, which consists of a sequence of nonstandard turns following each other in a helix-like manner. The absence of short- or medium-range trNOE in the N-terminal part indicates its structural flexibility.     

Investigation of the solution structure of a DNA octamer [d(GGAATTCC)]2 using two-dimensional nuclear Overhauser enhancement spectroscopy

Proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra in the pure absorption phase were obtained at 500 MHz for [d(GGAATTCC)]2 in aqueous solution at a series of mixing times. The experimental data were analyzed by comparison with theoretical spectra calculated using the complete 70 X 70 relaxation matrix including all proton dipole-dipole interactions and spin diffusion [Keepers, J. W. & James, T. L. (1984) J. Magn. Reson. 57, 404-426]. The theoretical spectra at each mixing time were calculated using two structures: a standard B-form DNA structure and an energy-minimized structure based on the similarity of the six internal residues of the title octamer with those of the dodecamer [d(CGCGAATTCGCG)]2, for which the crystal structure has been determined. Neither the standard B-form nor the energy-minimized structure will yield theoretical 2D NOE spectra which accurately reproduce all peak intensities in the experimental spectra. However, many features of the experimental spectra can be represented by both the B-form and the energy-minimized structure. Sequence-dependent structural characteristics are manifest in the 2D NOE spectra, in particular at the purine-pyrimidine junction as noted previously in the crystal structure. On the whole, the energy-minimized structure appears to yield theoretical 2D NOE spectra which mimic many, if not all, aspects of the experimental spectra. All 2D NOE data were consistent with nanosecond correction times as implied by proton spin-lattice relaxation time measurements. But better fits of some of the 2D NOE data using small variations in an effective isotropic correlation time suggest that there may be some local variations in mobility within the octamer duplex structure in solution.     

Acyl carrier protein from Escherichia coli I. Aspects of the solution structure as evidenced by proton nuclear Overhauser experiments at 500 MHz

The downfield aromatic (6-8 ppm) and upfield ring current shifted methyl regions (1-0 ppm) in the proton nuclear magnetic resonance spectrum of acyl carrier protein (ACP) from Escherichia coli have been examined at 500 MHz by using nuclear Overhauser methods. The data are analyzed in terms of the secondary structural model of Rock & Cronan (1979) [Rock, C. O., & Cronan, J. E., Jr. (1979) J. Biol. Chem. 254, 9778-9785], which suggests the existence of four alpha-helical segments joined by three beta-turns, and a short coil at the C terminus of the protein. Nuclear Overhauser effects among Tyr-71, Ile-69, Ile-72, and His-75 allow refinement of the secondary structure of the C terminus. Nuclear Overhauser effects among Tyr-71, Phe-28, and three Ile's also place stringent limitations on the folding of the four alpha-helices. These data allow the proposal of a tertiary structural model for ACP.     

Structural elucidation of a hydrophobic box in bovine alpha-lactalbumin by NMR: nuclear Overhauser effects

The proton nuclear Overhauser effects of bovine alpha-lactalbumin were studied at 200 MHz by irradiation of an upfield ring current shifted methylene at -2.45 ppm (assigned to Ile-95) and two aromatic protons, Tyr-103 (8.36 ppm) and Trp-60 (5.85 ppm). The experimental results were consistent with a putative three-dimensional alpha-lactalbumin model [Warne, P. K., Momany, F. A., Rumball, S. V., Tuttle, R. W., & Scheraga, H. A. (1974) Biochemistry 13, 768-782], which predicted the close proximity of Ile-95, Tyr-103, Trp-60, and Trp-104. Several of the assignments correlated with those previously made from chemically induced dynamic nuclear polarization experiments [Berliner, L. J., & Kaptein, R. (1981) Biochemistry 20, 799-807]. Subtle differences in the structure of this hydrophobic box region in alpha-lactalbumin were found between the Ca(II) and apo forms of the protein. The existence of this "hydrophobic box" in alpha-lactalbumin was strikingly similar to that in lysozyme, as verified in solution.     

Proton hyperfine resonance assignments in cyanide-ligated cytochrome c peroxidase using the nuclear Overhauser effect

The development of the proton nuclear Overhauser effect (NOE) for hyperfine shifted resonances of cyanide-ligated cytochrome c peroxidase (Saccharomyces cerevisiae) has been studied. In the pre-steady state regime, the major effects are due to primary NOEs to nearest neighbor protons. This has been used to advantage in making assignments of all of the remaining unassigned, resolved, downfield hyperfine shifted resonances. This work also determined the relative orientation of the heme pyrrole II substituents which is the cis configuration with the 4 alpha-vinyl proton pointing away from the 3CH3. In addition to heme protons, resonances of histidine 175, threonine 180, and histidine 52 have been assigned. These results indicate some structural rearrangement of the distal amino acids accompanying ligation.     

Conformation of the third domain of turkey ovomucoid in solution. Structural analysis by two-dimensional Overhauser nuclear effect spectroscopy]

The conformations of a polypeptide chain of turkey ovomucoid third domain and its modified form with split reactive site peptide bond Leu-18--Glu-19 have been determined by the literary two-dimensional nuclear Overhauser effect spectroscopy data using an earlier suggested method. It has been found that the polypeptide domain backbone contains an alpha-helical fragment (residues 32-47), five segments having extended conformation (1-5, 11-17, 19-25, 29-31, 48-50) and beta-turn type 1 (26-29). Segments 23-26, 28-31 and 50-51 form an antiparallel beta-structure. Conformational states of the residues entering irregular domain segments have been analysed. Splitting of the reactive site peptide bond Leu-18--Glu-19 is shown to cause insignificant changes in the conformations of a number of amino acid residues except for Val-6 and Asp-7 ones which undergo essential conformational alterations. The conformations of domain in solution and of japanese quail ovomucoid third domain in crystal have been compared. The root-mean-square deviations for phi and psi angles indicate their high similarity. The conformations of turkey ovomucoid third domain and proteinase inhibitor BUSI IIA in solution have been analysed. In spite of moderate (50%) homology of primary structures, some 75% of amino acid residues are shown to have close conformational phi and psi parameters.     

Conformational analysis of protein side chains using two-dimensional Overhauser nuclear effect spectroscopy]

The method has been proposed to determine the conformations of protein side-chains (dihedral angles chi 1) using two-dimensional nuclear Overhauser effect spectroscopy data. This method is grounded of the algorithm prepared on the basis of joint consideration of proton-proton distance dependences in dipeptide units of L-amino acid residues on the dihedral angles phi, psi and chi 1 with the accounting of the local sterical conditions of the polypeptide chain. The obtained results gave the possibility to bring the different regions of space (phi, psi) of amino acid residues into the line with the specific sets of nuclear Overhauser effect spectral parameters which unambiguously characterize in most cases the conformational states of their side-chains. The method efficiency was displayed on the test calculation with the utilization as the experimental data of the "model" nuclear Overhauser effect contacts derived from the X-ray atomic coordinates of the bovine pancreatic trypsin inhibitor molecule.     

500-MHz 1H NMR study of poly(dG).poly(dC) in solution using one-dimensional nuclear Overhauser effect

Secondary structures of poly(dG).poly(dC) and poly(dG).poly(dm5C) in solution are determined by nuclear Overhauser effect (NOE) measurements on GH8-deuterated and -nondeuterated DNAs with low presaturation pulse lengths (10-25 ms) and low-power and prolonged accumulations in the range of 50,000-72,000 scans. Under these conditions, the NOE difference spectra were free from diffusion. Primary NOEs between base protons GH8/CH6 and sugar protons H1', H2'/H2', and H3' suggest that in poly(dG).poly(dC) both guanine and cytosine nucleotides adopt a C3'-endo, low anti X = 200-220 degrees conformation. Computer modeling of the NOE data enable identification for the first time, in terms of the geometry of the nucleotide repeat, handedness, and helix geometry, of the structure of poly(dG).poly(dC) to be the A form, and the derived structure for the polymer duplex is very close to the single crystal structure of the double-helical d-GGGGCCCC [McCall, M., Brown, T., & Kennard, O. (1985) J. Mol. Biol. 183, 385-396]. Similar nuclear Overhauser effect data on poly(dG).poly(dm5C) revealed that G and m5C adopt a C2'endo, anti X = 240-260 degrees conformation, which indicates that this DNA exhibits the B form in solution. In summary, the results presented in this paper demonstrate that methylation of cytosines in poly(dG).poly(dC) causes A----B transition in the molecule.     

Interaction of troponin I and troponin C. Use of the two-dimensional nuclear magnetic resonance transferred nuclear Overhauser effect to determine the structure of the inhibitory troponin I peptide when bound to skeletal troponin C

We have used two-dimensional 1H nuclear magnetic resonance spectroscopy to determine the structure of the synthetic inhibitory peptide N alpha-acetyl TnI(104-115) amide bound to calcium-saturated skeletal troponin C (TnC). Conformational changes in the peptide induced by the formation of the troponin I (TnI) peptide-TnC complex were followed by the study of the transferred nuclear Overhauser effect, a technique that allows one to determine the structure of a ligand bound to a macromolecule. The structure of the bound TnI peptide reveals an amphiphilic alpha-helix, distorted around the two central proline residues. The central bend in the peptide functions to bring the residues on the hydrophobic face into closer proximity with each other, thereby forming a small hydrophobic pocket. The hydrophilic, basic residues extend off the opposite face of the peptide. Hydrophobic surfaces on TnC that become exposed upon binding of calcium are involved in the binding of the TnI peptide, but electrostatic interactions also contribute to the strength of the interaction. The role of amphiphilic helices in the targeting of calcium-binding proteins such as troponin C will be discussed.     

Detection of secondary structure elements in proteins by hydrophobic cluster analysis.

Hydrophobic cluster analysis (HCA) is a protein sequence comparison method based on alpha-helical representations of the sequences where the size, shape and orientation of the clusters of hydrophobic residues are primarily compared. The effectiveness of HCA has been suggested to originate from its potential ability to focus on the residues forming the hydrophobic core of globular proteins. We have addressed the robustness of the bidimensional representation used for HCA in its ability to detect the regular secondary structure elements of proteins. Various parameters have been studied such as those governing cluster size and limits, the hydrophobic residues constituting the clusters as well as the potential shift of the cluster positions with respect to the position of the regular secondary structure elements. The following results have been found to support the alpha-helical bidimensional representation used in HCA: (i) there is a positive correlation (clearly above background noise) between the hydrophobic clusters and the regular secondary structure elements in proteins; (ii) the hydrophobic clusters are centred on the regular secondary structure elements; (iii) the pitch of the helical representation which gives the best correspondence is that of an alpha-helix. The correspondence between hydrophobic clusters and regular secondary structure elements suggests a way to implement variable gap penalties during the automatic alignment of protein sequences.     

Proton hyperfine resonance assignments using the nuclear Overhauser effect for ferric forms of horse and tuna cytochrome c.

Proton hyperfine resonance assignments for cytochromes c from several species are currently being successfully pursued by several laboratories. These efforts focus mostly on the ferrous forms. In contrast to that work, we have pursued assignments of the proton hyperfine shifted resonances for horse and tuna ferricytochromes c. Our results indicate that assignments are nearly identical in those two proteins. Using the pre-steady state nuclear Overhauser effect, several additional assignments have been made for the tuna protein, whereas for the horse protein, the following protons have been assigned: heme 7, alpha CH2; heme 7, beta CH2; histidine 18, beta CH2 and alpha CH; and the methionine 80, beta CH2.     

Solution conformation of the biantennary N-linked oligosaccharide of human serotransferrin using H NMR nuclear Overhauser effect measurements

The conformation in solution of the biantennary complex type oligosaccharide unit derived from human serotransferrin has been investigated using ¹H—¹H Nuclear Overhauser Effect (NOE) measurements at 300 MHz. From quantitation of the NOE, the (1–3) antenna is shown to exist in a preferred solution conformation with respect to the mannosyl-chitobiose core. The flexibility of the (1–6) arm, together with the absence of NOE data between this arm and the core, indicates that, in contrast to the (1–3) arm the (1–6) arm has no preferred conformation with respect to the core.     

Two-dimensional nuclear Overhauser enhancement investigation of the solution structure and dynamics of the DNA octamer [d(GGTATACC)]2

The resonances of nearly all 70 of the non-exchangeable protons of the duplex [d(GGTATACC)]2 in aqueous solution are assigned by proton two-dimensional nuclear Overhauser enhancement (2D NOE) spectra obtained in pure absorption phase at 500 MHz. Experimental and theoretical 2D NOE spectra are compared at each mixing time (100, 175, 250 and 400 ms) using two B-DNA structures: a standard B-form and an energy-minimized form. The GG and CC ends of the octamer duplex are well represented by the regular B-DNA structure. But large discrepancies from these models are observed for the 'TATA' box. All 2D NOE data are consistent with nanosecond correlation times, as indicated by non-selective proton spin-lattice relaxation times, but small variations in the correlation time are observed, suggesting that there are some local differences in mobility within the octamer duplex structure in solution.