A set of HA-detected experiments for measuring scalar and residual dipolar couplings

A new set of HCACO based three-dimensional NMR experiments for measuring residual dipolar couplings in proteins is presented. Using spin-state selection and editing in three dimensions, the experiments allow accurate measurement of intraresidual , and scalar and residual dipolar couplings of ¹⁵N/¹³C labeled proteins in D₂O and dilute liquid crystals with minimal spectral crowding. The presented experiments are especially suitable for small or medium sized proline-rich proteins, or proteins that require high pH solvent conditions, making ¹H^N detected experiments unattractive. In addition, the tetrahedral coordination of C is superior to the planar peptide bond for determination of local alignments in partially structured polypeptides. For the efficient use of spectrometer time and to avoid complications arising from the varying magnitude of the alignment tensor during relatively long experiments, the and couplings can also be measured simultaneously in an E.COSY like manner with high accuracy. The pulse sequences are balanced for cross-correlation effects and minimized for relaxation losses. The pulse sequences are tested with a sample of ¹⁵N/¹³C human ubiquitin. We find internuclear vector directions determined from the dipolar couplings to have an excellent correlation with those of ubiquitins refined solution structure.     

Measurement of eight scalar and dipolar couplings for methine–methylene pairs in proteins and nucleic acids

A new 3D, spin-state-selective coherence transfer NMR experiment is described that yields accurate measurements for eight scalar or dipolar couplings within a spin system composed of a methylene adjacent to a methine group. Implementations of the experiment have been optimized for proteins and for nucleic acids. The experiments are demonstrated for C–C moieties of the third IgG-binding domain from Streptococcal Protein G (GB3) and for C –C groups in a 24-nt RNA oligomer. Chemical shifts of C, C and H (respectively C , C and H ) are dispersed in the three orthogonal dimensions, and the absence of heteronuclear decoupling leads to distinct and well-resolved E.COSY multiplet patterns. In an isotropic sample, the E.COSY displacements correspond to ¹J_CH, ²J_CH2+²J_CH3, ²J_CH, ¹J_CH2+¹J_CH3, ¹J_CH2–²J_H2H3, ¹J_CH3–²J_H2H3, ³J_HH2 and ³J_HH3 for proteins, and ¹J , ²J J , ²J , ¹J +¹J , ¹J J , ¹J J , ³J and ³J in nucleic acids. The experiment, based on relaxation-optimized spectroscopy, yields best results when applied to residues where the methine–methylene group corresponds to a reasonably isolated spin system, as applies for C, F, Y, W, D, N and H residues in proteins, or the C –C groups in nucleic acids. Splittings can be measured under either isotropic or weakly aligned conditions, yielding valuable structural information both through the ³J couplings and the one-, two- and three-bond dipolar interactions. Dipolar couplings for 10 out of 13 sidechains in GB3 are found to be in excellent agreement with its X-ray structure, whereas one residue adopts a different backbone geometry, and two residues are subject to extensive ₁ rotamer averaging. The abundance of dipolar couplings can also yield stereospecific assignments of the non-equivalent methylene protons. For the RNA oligomer, dipolar data yielded stereospecific assignments for six out of the eight C H₂ groups in the loop region of the oligomer, in all cases confirmed by ¹J ^{1} $$" align="middle" border="0"> J , and H resonating downfield of H .Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-0175-z.     

Measurement of four-bond HN-Hα J-couplings in staphylococcal nuclease

Summary Quantitative J-correlation and triple-resonance ECOSY-type experiments are used to unambiguously establish the presence of four-bond sequential H^N-H J-couplings in the protein staphylococcal nuclease. Substantially negative values, ranging from -0.8 to -2.3 Hz, are observed when the angle is near +120°, and the following angle near +60°. For other conformations, the four-bond H^N-H J-couplings fall between -0.5 and +0.5 Hz.     

Simultaneous determination of Ψ and Φ angles in proteins from measurements of cross-correlated relaxation effects

A method is presented to determine both and backbone angles in proteins simultaneously. This is achieved by measuring the effect on two-spin coherences of cross-correlation between ¹⁵ N-¹H^N and ¹³ vectors. The cross-correlation rates are obtained by comparing two complementary three-dimensional experiments.     

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin -chain was established, completing the analysis of this hemoglobin. Comparisons with other avian -chains show variations smaller than those for the corresponding -chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform -chain, and a total of 35 positions are affected by differences among all avian -chains analyzed (versus 61 for the -chains). At three positions, the Psittacula -chain has residues unique to this species. Three ₁₁ contacts are modified, by substitutions at positions 51, 116, and 125.     

13C shieldings, 18O isotope effects on 13C shieldings, and 57Fe-13C spin couplings of the Fe-C-O unit in superstructured hemoprotein models: Comparison with hemoproteins, C-O vibrational frequencies, and X-ray structural data

¹³C NMR spectra of several carbon monoxide (99.7% ¹³C and 11.8% ¹⁸O enriched) hemoprotein models with varying polar and steric effects of the distal organic superstructure and constraints of the proximal side are reported. This enables the ⁵⁷Fe-¹³C(O) coupling constants ( ), ¹³C shieldings ((¹³C)), and ¹⁸O isotope effects on¹³ C shieldings (¹¹³C(¹⁸O/¹⁶O)) to be measured and hence comparisons with hemoproteins, C-O vibrational frequencies and X-ray structural data to be made. Negative polar interactions in the binding pocket and inhibition of Fe//CO back-donation or positive distal polar interactions with amide NH groups appear to have little direct effect on couplings. Similarly, the axial hindered base 1,2-dimethylimidazole has a minor effect on the values despite higher rates of CO desorption being observed for such complexes. On the contrary,¹³ C shieldings vary widely and an excellent correlation was found between the infrared C-O vibrational frequencies ((C-O)) and¹³ C shieldings and a reasonable correlation with¹⁸ O isotope effects on ¹³C shieldings. This suggests that (¹³C), (C-O) and^{1 13} C(¹⁸O/¹⁶O) are accurate monitors of the multiple mechanisms by which steric and electronic interactions are released in superstructured heme model compounds. The ¹³C shieldings of heme models cover a 4.0 ppm range which is extended to 7.0 ppm when several HbCO and MbCO species at different pH values are included. The latter were found to obey a similar linear (¹³ (¹³C) versus (C-O) relationship, which proves that both heme models and heme proteins are homogeneous from the structural and electronic viewpoint. Our results suggest that (C-O), (¹³C) and ¹¹³C(¹⁸O/¹⁶O) measurements reflect similar interaction which is primarily the modulation of back-bonding from the Fe d to the CO * orbital by the distal pocket polar interactions. The lack of correlation between^{1 13} C(¹⁸O/¹⁶O) and crystallographic CO bond lengths (r(C-O)) reflects significant uncertainties in the X-ray determination of the carbon and oxygen positions.     

Soret spectroscopic and molecular graphic analysis of human semi-beta-hemoglobin formation

The interaction of heme-free (^o) and heme-containing (^h) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at [5°C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 10⁷ M^–1 s^–1. This was 100-fold more rapid than the reported rate of combination of ^h and ^h proteins. This encounter-like rate of semi--hemoglobin (^oh) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB—GH helical entities. Molecular graphic analysis of modeled ^o protein superimposed over native ^h protein revealed an apo Mb-like structure with well-defined AB—GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the ₁₁ interface, was apparently responsible for the enhanced association properties of the ^o protein.     

Primary structure of alpha-globin chains from river buffalo (Bubalus bubalis L.) hemoglobins

Primary structure analysis of the four river buffalo -globin chains showed that haplotypes A and B differ from each other by a substitution at codon 64 that may encode Ala or Asn. The A haplotype encodes two -globin chains, ^I1 and ^II3, which differ at positions 129 and 131: ^I1 has 64 Ala, 129 Phe, 131 Asn; ^II3 has 64 Ala, 129 Leu, 131 Ser. The B haplotype encodes two -globin chains, ^I2 and ^II4, which differ at positions 10 and 11: ^I2 has 10 Ile, 11 Gln, 64 Asn; ^II4 has 10 Val, 11 Lys, 64 Asn. Apart from the Ala/Asn polymorphism at position 64, amino acid substitutions in allelic and nonallelic -globin chains seem to have arisen by single point mutations. Detection of electrophoretically silent mutations due to neutral amino acid substitutions and their influence on the isoelectric point are discussed. Furthermore, primary structures of river buffalo -globin chains are compared to other species of the Bovidae family to suggest evolutionary events that have characterized the amino acid substitutions of river buffalo hemoglobin.     

Mobility of NH bonds in DNA-binding protein HU of shape Bacillus stearothermophilus from reduced spectral density mapping analysis at multiple NMR fields

The dynamics of the backbone NH bonds of protein HU from Bacillus stearothermophilus (HUBst) have been characterized using measurements of cross-relaxation, longitudinal and transverse relaxation rates at 11.7, 14.1 and 17.6 T. Linear regression of the values with the squared Larmor frequency _N ² has revealed global exchange processes, which contributed on the order of 0.5–5.0 s^-1to the transverse relaxation rate. Subsequently, the experimental values were corrected for these exchange contributions. A reduced spectral density mapping procedure has been employed with the experimental relaxation rates and seven values of the spectral density function J() have been extracted. These spectral densities have been fitted within the framework of the model-free approach. The densities agree well with an axially symmetric rotational diffusion tensor with a diffusion anisotropy D_/D_ of 1.15, indicating that the flexible arms of HUBst do not significantly contribute to the rotational diffusion. The overall correlation time is 8.9 ± 0.6 ns/rad. The fast internal motions of most of the NH bonds in the core display order parameters ranging between 0.74 and 0.83 and internal correlation times between 1 and 20 ps. For the residues in the DNA-binding -arms, an extended version of the model function has been used. The slow internal motions show correlation times of 1–2 ns. The concomitant order parameters (0.3–0.6) are lower than those observed on the fast time scale, indicating that the flexibility of the -arms is mainly determined by the slower internal motions. A substantial decrease of the generalized order parameters in the -arms starting at residues Arg⁵⁵ and Ser⁷⁴, opposite on both strands of the -ribbon arms, has been explained as a hinge motion. A comparison of the order parameters for free and DNA-bound protein has demonstrated that the slow hinge motions largely disappear when HU binds DNA.     

Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins

Methods are described to correlate aromatic ¹H ²/¹³C ² or ¹H ¹/¹⁵N ¹ with aliphatic ¹³C chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [¹⁵N, ¹H]- and/or [¹³C, ¹H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with -carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [¹⁵N, ¹H]-TROSY-H(N)C^ar experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine -CH and tryptophan -NH groups.     

Effect of liquid culture on the growth and development of miniature rose (Rosa chinensis Jacq. ‘Minima’)

The growth of miniature rose (Rosa chinensis Jacq. Minima) shoots cultured on liquid medium was greater relative to those cultured on two-phase (solid + liquid) medium or solid medium alone. Shoot multiplication ratio (number of multiple shoots per explant per subculture) on liquid medium was higher with 17.8–26.6 M 6-benzyladenine at compared to that at 0–8.9 M. Shoots grown on 30 ml or more of liquid medium had a higher multiplication ratio than those grown on 10 or 20 ml. The growth and multiplication ratio increased when the culture period was extended from 3 to 6 weeks, although plantlets began to exhibit some chlorosis by the 6^th week. These conditions were maintained over four subcultures for cultivars Baby Katie, Lavender Jewel, Red Sunblaze and Royal Sunblaze, with no significant change in multiplication ratio over time.Abbreviations BA 6-benzyladenine - NAA 1-naphthaleneacetic acid     

Structural analyses of new tri- and tetrasaccharides produced from disaccharides by transglycosylation of purified Trichoderma viride β-glucosidase

A new -glucosidase was partially purified from Trichoderma viride cellulase. This -glucosidase catalyzed a transglycosylation reaction of cellobiose to give -D-Glc-(16)--D-Glc-(14)-D-Glc (1, yield: 18.8%) and -D-Glc-(16)--D-Glc-(16)--D-Glc-(14)-D-Glc (2, 3.7%), regioselectively. Furthermore, the enzyme regioselectively converted laminaribiose and gentiobiose into -D-Glc-(16)--D-Glc-(13)-D-Glc (3, 15.3%) and -D-Glc-(16)--D-Glc-(16)-D-Glc (4, 20.2%), respectively. The structures (1–4) of the products were determined by ¹H and ¹³C NMR spectroscopies. This high regio- and stereoselectively of the -glucosidase could be applied for oligosaccharide synthesis.     

A simple method to quantitatively measure polypeptide JHNHα coupling constants from TOCSY or NOESY spectra

A simple linear relationship between the J coupling constant and the linewidth (_1/2) of in-phase NMR peaks has been identified. This relationship permits the rapid and accurate determination of polypeptide J coupling constants from a simple inspection of amide cross peaks in homonuclear ¹H TOCSY or ¹H NOESY spectra. By using the appropriate set of processing parameters we show that J = 0.5(_1/2) – MW/5000 + 1.8 for TOCSY spectra and J = 0.6(_1/2) – MW/5000 – 0.9 for NOESY spectra, where _1/2 is the half-height linewidth in Hz and MW is the molecular weight of the protein in Da. The simplicity of this relationship, combined with the ease with which _1/2 measurements can be made, means that J coupling constants can now be rapidly determined (up to 100 measurements in less than 30 min) without the need for any complex curve-fitting algorithms. Tests on 11 different polypeptides involving more than 650 separate J measurements have shown that this method yields coupling constants with an rmsd error (relative to X-ray data) of less than 0.9 Hz. Furthermore, the correlation coefficient between the predicted NMR coupling constants and those derived from high-resolution X-ray crystal structures is typically better than 0.89. These simple linear relationships have been found to be valid for peptides as small as 1 kDa to proteins as large as 20 kDa. Despite the method's simplicity, these results are comparable to the accuracy and precision of the best techniques published to date.     

Two-dimensional1H NMR study of recombinant insect defensin A in water: Resonance assignments,secondary structure and global folding

Summary A 500 MHz 2D¹H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities,³J_NH-H coupling constants as well as¹H/²H exchange rates and /T temperature coefficients of NH protons strongly support the existence of an -helix (residues 14–24) and of an antiparallel -sheet (residues 27–40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from³J_NH-H coupling constants, (iii) 12 hydrogen bonds mostly deduced from¹H/²H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a -sheet is linked to an -helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.Abbreviations SCUBA Stimulated Cross peaks Under Bleached Alphas - MCD analysis Main Chain Directed analysis - CSH motif Cysteine Stabilized -Helix motif     

Laser kinetic spectroscopy studies of the bimolecular oxygenation of alpha- and beta-subunits within the R-state of human hemoglobin

Bimolecular oxygenation of tri-liganded R-state human hemoglobin (HbA) is described by bi-exponential kinetics with association rate constants k = 27.2 ± 1.3 (M·sec)^-1 and k = 62.9 ± 1.6 (M·sec)^-1. Both the observed processes have been assigned to the bimolecular oxygenation of - and -subunits of the native tetrameric protein by molecular oxygen. The quantum yields of photodissociation within the completely oxygenated R-state HbA are = 0.0120 ± 0.0017 and = 0.044 ± 0.005 for - and -subunits, respectively. The oxygenation reactions of isolated ^PCMB- and ^PCMB-hemoglobin chains are described by mono-exponential kinetics with the association rate constants k = 44 ± 2 (M·sec)^-1 and k = 51 ± 1 (M·sec)^-1, respectively. The quantum yields of photodissociation of isolated ^PCMB- and ^PCMB-chains (0.056 ± 0.006 and 0.065 ± 0.006, respectively) are greater than that observed for appropriate subunits within the R-state of oxygenated HbA.     

Expression of α and β tropomyosin subunits during early myogenesis in somites and limb buds of chick embryos

Summary The appearance of and subunits of skeletal tropomyosin in early myogenesis was studied histochemically using monoclonal antibody to tropomyosin and affinity-purified polyclonal antibody to tropomyosin. In muscle cells, in both somites and limb buds, the and subunits are simultaneously expressed and first appear in the somites at the 30–36 somites. The relatively greater amount of than tropomyosin found in early myogenesis is thus likely to result from a higher rate of tropomyosin synthesis.     

Ongoing ultradian activity rhythms in the common vole,Microtus arvalis,during deprivations of food,water and rest

Summary The timing mechanism underlying ultradian (2–3 h) activity patterns in the common vole, Microtus arvalis, was studied using behavioural deprivation experiments. These were aimed at distinguishing between a homeostatic control mechanism, in which the rhythmic behaviour itself is part of the causal loop, and a clock mechanism, independent of the behaviour.In 175 experiments, deprivation of food during 3 ultradian cycles in (subjective) daytime did not result in significant changes in the ultradian periodicity of attempts to obtain the food, compared with ad lib. access to food and water. A minor, but significant increase in ultradian activity time () occurred in the course of the deprivation, but this was compensated by a shorter ultradian rest (). These results were obtained both in intact animals (n = 24), which showed ultradian and circadian rhythmicity in behaviour, and in animals (n = 21) with electrolytic lesions aimed at the suprachiasmatic nuclei (SCN), which lacked the circadian modulation of behaviour. Simultaneous deprivation of water and food in 8 voles without circadian rhythmicity during 40 experiments also did not lead to any change in the ultradian periodicity of feeding attempts.Rest deprivation was studied in 5 SCN lesioned voles, by forcing running wheel activity to continue following spontaneous running. Thus, the experimental activity bout was artificially lengthened to 2–9 h in 67 experiments. The onset of the subsequent rest episodes occurred independent of the duration of the preceding . The duration of was dependent on the preceding, experimental in a periodic fashion. The interval experimental (=lengthened +following ) was equal to one, two or three times the control (obtained on nonexperimental days). This result fits the prediction of a clock model and is in conflict with a monotonicincrease of with , as expected in a homeostatic, restorative process.It is concluded that the ultradian timing of activity in the common vole can be explained neither by homeostatic hunger or thirst mechanisms nor by homeostatic rest/activity regulation. The results strongly suggest an independent clock system generating ultradian feeding rhythms in the common vole.Abbreviations DD continuous darkness - LD light-dark regime - LL continuous light - RCA retrochiasmatic area - ARC arcuate nucleus - SCN suprachiasmatic nuclei - ultradian period - ultradian activity time - ultradian rest time     

Larmor frequency selective model free analysis of protein NMR relaxation

Summary The Lipari-Szabo dynamical formalism is extended by setting the time constants of the Lorentzian terms to and . This analysis is compared to the earlier proposed three-parameter extended model free formalism with regard to the range of equivalence and the advantages of the simplified two-parameter (S _inff ^sup2 ,S _infH ^sup2 ) and (S _inff ^sup2 ,S _infN ^sup2 ) representations. Spectral density components are calculated and compared to those obtained from the spectral density analysis formalism. Protein relaxation data, commonly analyzed in terms of the two-parameter representation, may correspond to a dynamically heterogeneous behaviour that is more appropriately represented in terms of a fast limit order parameter and a second, lower frequency order parameter.     

Transformation of 16-dehydroprogesterone and 17α-hydroxyprogesterone by Mucor piriformis

Mucor piriformis was used to study the mode of transformation of 16-dehydroprogesterone (I, pregna-4, 16-diene-3, 20-dione) and 17-hydroxyprogesterone (II, 17-hydroxypregn-4-ene-3, 20-dione). Biotransformation products formed from I were 14-hydroxypregna-4, 16-diene-3, 20-dione (Ia), 7, 14-dihydroxypregna-4, 16-diene-3, 20-dione (Ib), 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Ic), and 3, 7, 14-trihydroxy-5-pregn-16-en-20-one (Id). Metabolites Ic and Id appear to be hitherto unknown. Time-course studies suggested that the transformation is initiated by hydroxylation at the 14-position (Ia) followed by hydroxylation at the 7-position (Ib). Microsomes (105,000 g sediment) prepared from 16-dehydroprogesterone-induced cells hydroxylate I to its 14-hydroxy derivative (Ia) in the presence of NADPH. Incubation of Ia with the organism resulted in the formation of Ib, Ic and Id. Biotransformation products formed from compound II were 17, 20-dihydroxypregn-4-en-3-one (IIa), 7, 17-dihydroxypregn-4-ene-3, 20-dione (IIb), 6, 17, 20-trihydroxypregn-4-en-3-one (IIc) and 11, 17, 20-trihydroxypregn-4-en-3-one (IId). Time-course studies indicated that IIa is the initial product formed, which is further hydroxylated either at the 6 or 11 position. Incubation of IIa with the organism resulted in the formation of IIc and IId. Reduction of the 4-en-3-one system and 20-keto group has not been observed before in organisms of the order Mucorales. In addition, M. piriformis has been shown to carry out hydroxylation at the C-6, C-7, C-11 and C-14 positions in the steroid molecules tested.     

Positive φ-angles in proteins by nuclear magnetic resonance spectroscopy

Summary Non-glycine residues with positive -angles have been identified in four proteins, barley serine proteinase inhibitor CI-2, bacterial ribonuclease (barnase) ofBacillus amyloliquefaciens, hen egg white lysozyme and a basic protein from barley seed (barwin) by use of nuclear magnetic resonance spectroscopy. By accurate measurements of the coupling constant and integration of the nuclear Overhauser H^N-H cross peak, positive -angles could be determined reliably to 60°±30°, in full agreement with the crystal structures for lysozyme, barnase and serine proteinase inhibitor CI-2. The work emphasizes that positive -angles can also occur in non-glycine residues and in the four proteins, positive -angles have been observed for the residue types aspartic acid, asparagine, arginine, serine, glutamine, histidine, tyrosine, tryptophan and phenylalanine. The measured coupling constants and the intensity of the intraresidue H^N-H NOEs agree well with the solution structures of three of the proteins, using the existing parametrization of the Karplus curve (Pardi, A., Billeter, M. and Wüthrich, K. (1984)J. Mol. Biol.,180, 741–751; Ludvigsen, S., Andersen, K.V. and Poulsen, F.M. (1991)J. Mol. Biol.,217, 731–736).