Pairwise NMR experiments for the determination of protein backbone dihedral angle Φ based on cross-correlated spin relaxation

Novel cross-correlated spin relaxation (CCR) experiments are described, which measure pairwise CCR rates for obtaining peptide dihedral angles Φ. The experiments utilize intra-HNCA type coherence transfer to refocus 2-bond coupling evolution and generate the or multiple quantum coherences which are required for measuring the desired CCR rates. The contribution from other coherences is also discussed and an appropriate setting of the evolution delays is presented. These CCR experiments were applied to ¹⁵N- and ¹³C-labeled human ubiquitin. The relevant CCR rates showed a high degree of correlation with the Φ angles observed in the X-ray structure. By utilizing these CCR experiments in combination with those previously established for obtaining dihedral angle Ψ, we can determine high resolution structures of peptides that bind weakly to large target molecules.     

Application of H(N)CA,CO-E.COSY experiments for calibrating the φ angular dependences of vicinal couplings J(C′i−1,Hi α), J(C′i−1,Ci β) and J(C′i−1,C′i) in proteins

A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly¹³C/¹⁵N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the³J(C_i–1,H_i ), ³J(C_i–1,C_i ) and³J(C_i–1,C_i)coupling constants in the indirectly detected ¹³Cdimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone -angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for³J(C_i–1,H_i ),³J(C_i–1,C_i ) and³J(C_i–1,C_i),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings.     

Measurement of two- and three-bond 13C−1H J couplings to the Cδ carbons of leucine residues in staphylococcal nuclease

Summary A new ¹H-detected 3D NMR experiment is described that permits quantitative measurement of two- and three-bond ¹³C–¹H couplings in proteins with selectively ¹³C-enriched methyl sites. The method is demonstrated for staphylococcal nuclease selectively [5,5 ¹³C]-labeled in all 11 leucine positions and ligated with thymidine 3,5-biphosphate and Ca²⁺. Two- and three-bond ¹³C methyl-proton couplings are reported and, together with the measured three-bond J_CC in uniformly ¹³C-enriched staphylococcal nuclease, the ²- and the stereospecific assignments of the C methyl group with respect to the prochiral -protons were determined. The same residues that were previously found to have high degrees of internal mobility on the basis of ¹³C relaxation times have measured coupling constants that are indicative of motional averaging.     

Combined use of 13C chemical shift and 1Hα−13Cα heteronuclear NOE data in monitoring a protein NMR structure refinement

Summary A large portion of the ¹³C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific ¹³C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting ¹H–¹³C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these ¹H–¹³C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC heteronuclear single-quantum coherence spectroscopy - PFG pulsed-field gradient - TOCSY ¹H-¹H total correlation spectroscopy - EGF epidermal growth factor - mEGF murine EGF - hEGF human EGF - hTGF human type- transforming growth factor - DIPSI spm-locking pulse sequence - NOE nuclear Overhauser effect - HNOE heteronuclear Overhauser effect     

Intraresidue 1H-15N-13C′ and 1Hα-13Cα-13C′ dipole-CSA relaxation interference as a source of constraints for structural refinement of metal-binding sites in zinc-finger proteins

¹H(i)-¹⁵N(i)-¹³C(i) dipole-chemical shift anisotropy (CSA) relaxation interference was quantified for the ¹³C,¹⁵N labeled zinc-finger protein qCRP2(LIM2). The cross-correlation rates obtained for residues located in the metal coordination sites of qCRP2(LIM2) show a high degree of correlation with the peptide plane torsion angles and taken from the solution structure. ¹H(i)-¹⁵N(i)-¹³C(i) as well as ¹³C(i)-¹H(i)-¹³C(i) dipole-CSA cross-correlation rates were subsequently used to improve the geometry of the metal binding site. The optimized dihedral angles of the two zinc-binding sites in qCRP2(LIM2) are in better agreement with values obtained from crystal structures of other zinc-finger proteins and thus establish the utility of this approach to improve the metal-binding site geometry of zinc-finger proteins studied by NMR spectroscopy in solution.     

Accurate measurements of the effects of deuteration at backbone amide positions on the chemical shifts of 15N, 13Cα, 13Cβ, 13CO and 1Hα nuclei in proteins

An approach towards accurate NMR measurements of deuterium isotope effects on the chemical shifts of all backbone nuclei in proteins (¹⁵N, ¹³C_α, ¹³CO, ¹H_α) and ¹³C_β nuclei arising from ¹H-to-D substitutions at amide nitrogen positions is described. Isolation of molecular species with a defined protonation/deuteration pattern at successive backbone nitrogen positions in the polypeptide chain allows quantifying all deuterium isotope shifts of these nuclei from the first to the fourth order. Some of the deuterium isotope shifts measured in the proteins ubiquitin and GB1 can be interpreted in terms of backbone geometry via empirical relationships describing their dependence on (φ; ψ) backbone dihedral angles. Because of their relatively large variability and notable dependence on the protein secondary structure, the two- and three-bond ¹³C_α isotope shifts, ²ΔC_α(N_iD) and ³ΔC_α(N_i+1D), and three-bond ¹³C_β isotope shifts, ³ΔC_β(N_iD), are useful reporters of the local geometry of the protein backbone.     

Changes in the suprageneric classification of Lasiocampidae (Lepidoptera) based on the nucleotide sequence of gene EF-1α

The nucleotide sequences of nuclear gene EF-1α were determined for 49 species of Lasiocampidae from Eurasia and Africa. Based on these data, the phylogeny of the family was reconstructed using the minimum evolution, maximum parsimony, maximum likelihood, and Bayesian inference methods. The molecular genetic research confirms the monophyly of Malacosominae which is treated as a separate subfamily. The genus Euthrix appears to be paraphyletic. The group of genera similar to Arguda, previously united with Odonestis in the tribe Odonestini, proved to be a separate lineage; contrary to the earlier assumption, the genera do not seem to be related. On the other hand, the genera Argonestis and Odonestis were found to be closely related and therefore were placed in the same tribe. The position of the genus Macrothylacia remains obscure. The genus Stoermeriana de Fr. et Witt is also para- or polyphyletic and consists of several independent lineages whose status remains to be determined. The new classification supports synonymization of Pinarinae with Lasiocampinae. The rank of subfamily is not supported for the Neotropical Macromphaliinae, which is downgraded to a tribe, Macromphaliini stat. n., within Poecilocampinae. The genus Hypopacha, previously considered within Macromphaliinae, is transferred to Poecilocampini; the close relation between Poecilocampini and Macromphaliini is additionally supported by the presence of a member of Poecilocampini in the New World. A new tribe, Argudini Zolotuhin trib. n., is established.     

Mapping of the spectral density function of a Cα−Hα bond vector from NMR relaxation rates of a 13C-labelled α-carbon in motilin

Summary The peptide hormone motilin was synthesised with a ¹³C-enriched -carbon in the leucine at position 10. In aqueous solution, six different relaxation rates were measured for the ¹³C–H fragment as a function of temperature and with and without the addition of 30% (v/v) of the cosolvent d ₂-1,1,1,3,3,3-hexafluoro-2-propanol (HFP). The relaxation rates were analysed employing the spectral density mapping technique introduced by Peng and Wagner [(1992) J. Magn. Reson., 98, 308–322] and using the model-free approach by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570]. The fit to various models of dynamics was also considered. Different procedures to evaluate the overall rotational correlation time were compared. A single exponential time correlation function was found to give a good fit to the measured spectral densities only for motilin in 30% (v/v) HFP at low temperatures, whereas at high temperatures in this solvent, and in D₂O at all temperatures, none of the considered models gave an acceptable fit. A new empirical spectral density function was tested and found to accurately fit the experimental spectral density mapping points. The application of spectral density mapping based on NMR relaxation data for specific ¹³C–¹H vector is shown to be a highly useful method to study biomolecular dynamics. Advantages are high sensitivity, high precision and uniform sampling of the spectral density function over the frequency range.Abbreviations CD circular dichroism - NOE nuclear Overhauser enhancement - NOESY two-dimensional NOE spectroscopy - INEPT insensitive nuclei enhanced by polarisation transfer - DANTE delays alternating with nutations for tailored excitation - WALTZ-16 wideband, alternating phase, low-power technique for zero residual splitting - FID Free induction decay - ppm parts per million - TSPA 3-trimethylsilyl-(3,3,2,2-d)-propionic acid - HFP d ₂-1,1,1,3,3,3-hexafluoro-2-propanol - CPMG Carr-Purcell-Meiboom-Gill - TFD time-resolved fluorescence depolarisation - CSA chemical shift anisotropy Partly presented at the symposium Dynamics and Function of Biomolecules, Szeged, Hungary, July 31–August 2, 1993.     

A New Experiment for the Measurement of nJ(C,P) Coupling Constants Including 3J(C4′i,Pi) and 3J(C4′i,Pi+1) in Oligonucleotides

A new experiment for the measurement of nJ(C,P) coupling constants along the phosphodiester backbone in RNA and DNA based on a quantitative-J HCP experiment is presented. In addition to coupling constants, in which a carbon atom couples to only one phosphorus atom, both the intraresidual 3J(C4i,Pi) and the sequential 3J(C4i,Pi+1) for the C4 resonances that couple to two phosphorus atoms can be obtained. Coupling constants obtained by this new method are compared to values obtained from the P-FIDS experiment. Together with 3J(H,P) coupling constants measured using the P-FIDS experiment, the backbone angles and can be determined.     

Measurement of homonuclear three-bond J(HNHα) coupling constants in unlabeled peptides complexed with labeled proteins: Application to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of hepatitis C virus (HCV)

A new isotope-filtered experiment has been designed to measure homonuclear three-bond J(H^NH) coupling constants of unlabeled peptides complexed with labeled proteins. The new experiment is based on the 3D HNHA pulse scheme, and belongs to the `quantitative J-correlation' type. It has been applied to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of human hepatitis C virus (HCV).     

Effects of 17 β-estradiol on the expression of interstitial collagenases-8 and −13 (MMP-8 and MMP-13) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in ovariectomized rat osteoblastic cells

Estrogen plays an important role in maintaining normal bone metabolism via the direct or indirect regulation of bone cells. Osteoblastic cells, as the target cells of estrogen, can secrete multiple matrix metalloproteinases (MMPs) that participate in bone remodeling. It has been demonstrated that bone loss induced by estrogen deficiency is closely related to the abnormal expression of multiple MMPs in osteoblastic cells. However, the regulating action of estrogen on the expression of interstitial collagenases MMP-8 and MMP-13 in osteoblastic cells in vivo remains unclear. We used an ovariectomized osteoporotic rat model to analyze the changes in the histomorphometric parameters of bone after and without treatment with 17-estradiol (E₂); We also used immunohistochemistry and in situ hybridization to observe changes in the expression of mRNA and the proteins MMP-8, MMP-13 and TIMP-1 in osteoblastic cells in rat proximal tibia. In this study, we found that in the ovariectomized rat the expression of MMP-13 mRNA and protein increased markedly, whereas the expression of MMP-8 and TIMP-1 mRNA and protein did not change significantly. Our analysis showed that the expression of MMP-13 protein was correlated positively to bone trabecular separation, osteoid surface area, and negatively to trabecular numbers and the percentage of trabecula bone volume/total tissue volume. Our results suggest that MMP-13 plays an important role in estrogen deficiency-induced bone loss, while estrogen can inhibit bone resorption and reduce bone turnover rate by down-regulating the expression of MMP-13 in osteoblastic cells.     

MQ-HCN-based pulse sequences for the measurement of 13C1′-1H1′, 13C1′-15N, 1H1′-15N, 13C1′-13C2′, 1H1′-13C2′, 13C6/8-1H6/8, 13C6/8-15N, 1H6/8-15N, 13C6-13C5, 1H6-13C5 dipolar couplings in 13C, 15N-labeled DNA (and RNA)

A suite of multiple quantum (MQ) HCN-based pulse sequences has been developed for the purpose of collecting dipolar coupling data in labeled nucleic acids. All the pulse sequences are based on the robust MQ-HCN experiment which has been utilized for assignment purposes in labeled nucleic acids for a number of years and provides much-needed resolution for the dipolar coupling measurements. We have attempted to collect multiple couplings centered on the 13C1' and 13C6/8 positions. Six pulse sequences are described, one each for measurement of one-bond 13C1'-1H1' and 13C6/8-1H6/8 couplings, one for measurement of one-bond 13C1'-15N and two-bond 1H1'-15N couplings, one for measurement of one-bond 13C6/8-15N and two-bond 1H6/8-15N couplings, one for measurement of one-bond 13C1'- 13C2' and two-bond 1H1'-13C2' couplings, and one for measurement of one-bond 13C6-13C5 and two-bond 1H6-13C5 couplings in the bases of C and T. These sequences are demonstrated for a labeled 18 bp DNA duplex in a 47 kDa ternary complex of DNA, CBFbeta, and the CBFalpha Runt domain, thus clearly demonstrating the robustness of the pulse sequences even for a very large complex.     

Assembly and Function of the Regulator of G protein Signaling 14 (RGS14)·H-Ras Signaling Complex in Live Cells Are Regulated by Gαi1 and Gαi-linked G Protein-coupled Receptors

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gα_i/o-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gα_i1/3-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gα_i1-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gα_i1-GDP but not active Gα_i1-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gα_i1 complex in brain lysates, and co-expression of RGS14 and Gα_i1 in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gα_i-linked α_2A-adrenergic receptor induces a conformational change in the Gα_i1·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gα_i1-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.     

G protein-coupled receptors and resistance to inhibitors of cholinesterase-8A (Ric-8A) both regulate the regulator of g protein signaling 14 RGS14·Gαi1 complex in live cells

Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional Gα(i/o) signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gα(i1/3)-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gα(i1)-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a Gα(i/o)-dependent complex with a G(i)-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the α(2A)-adrenergic receptor (α(2A)-AR) in a Gα(i/o)-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·α(2A)-AR complex occurs in the presence of Gα(i/o) but not Gα(s) or Gα(q). Unexpectedly, RGS14 does not dissociate from Gα(i1) in the presence of stimulated α(2A)-AR, suggesting preservation of RGS14·Gα(i1) complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gα(i1) complex and the Gα(i1)-dependent RGS14·α(2A)-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gα(i/o) and that these RGS14·Gα(i1)·GPCR complexes may be substrates for other signaling partners such as Ric-8A.     

Fractional <Superscript>13</Superscript>C enrichment of isolated carbons using [1-<Superscript>13</Superscript>C]- or [2-<Superscript>13</Superscript>C]-glucose facilitates the accurate measurement of dynamics at backbone C<Superscript>α</Superscript> and side-chain methyl positions in proteins

A simple labeling approach is presented based on protein expression in [1-¹³C]- or [2-¹³C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C^α sites, respectively. All of the methyl groups, with the exception of Thr and Ile(δ1) are produced with isolated ¹³C spins (i.e., no ¹³C–¹³C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-α sites are labeled without concomitant labeling at C^β positions for 17 of the common 20 amino acids and there are no cases for which ¹³C^α−¹³CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone ¹⁵N studies. The utility of the labeling is established by recording ¹³C R _1ρ and CPMG-based experiments on a number of different protein systems.     

Interleukin (IL)-32β-mediated CCAAT/Enhancer-binding Protein α (C/EBPα) Phosphorylation by Protein Kinase Cδ (PKCδ) Abrogates the Inhibitory Effect of C/EBPα on IL-10 Production

We previously reported that IL-32β promotes IL-10 production in myeloid cells. However, the underlying mechanism remains elusive. In this study, we demonstrated that IL-32β abrogated the inhibitory effect of CCAAT/enhancer-binding protein α (C/EBPα) on IL-10 expression in U937 cells. We observed that the phosphorylation of C/EBPα Ser-21 was inhibited by a PKCδ-specific inhibitor, rottlerin, or IL-32β knockdown by siRNA and that IL-32β shifted to the membrane from the cytosol upon phorbol 12-myristate 13-acetate treatment. We revealed that IL-32β suppressed the binding of C/EBPα to IL-10 promoter by using ChIP assay. These data suggest that PKCδ and IL-32β may modulate the effect of C/EBPα on IL-10 expression. We next demonstrated by immunoprecipitation that IL-32β interacted with PKCδ and C/EBPα, thereby mediating C/EBPα Ser-21 phosphorylation by PKCδ. We showed that IL-32β suppressed the inhibitory effect of C/EBPα on IL-10 promoter activity. However, the IL-10 promoter activity was reduced to the basal level by rottlerin treatment. When C/EBPα serine 21 was mutated to glycine (S21G), the inhibitory effect of C/EBPα S21G on IL-10 promoter activity was not modulated by IL-32β. Taken together, our results show that IL-32β-mediated C/EBPα Ser-21 phosphorylation by PKCδ suppressed C/EBPα binding to IL-10 promoter, which promoted IL-10 production in U937 cells.     

The effect of deleting residue C269 in the β12-β13 loop of protein phosphatase 2A (PP2A)catalytic subunit on the interaction between PP2A and metal ions, especially Mn(2+)

Protein phosphatase 2A (PP2A) is one of the most important Ser/Thr phosphatases in eukaryotic cells. The enzymatic core of PP2A (PP2A(D)) consists of a scaffold subunit (A subunit) and a catalytic subunit (C subunit). When residue Cys269 in the β12-β13 loop of the PP2A C subunit was deleted (ΔC269), the activity and the intrinsic fluorescence intensity of PP2A(D) decreased. Specify the effects of some metal ions on PP2A(D) were also changed. Mn(2+) in particular was an efficient activator of ΔC269 and altered the intrinsic fluorescence spectrum of ΔC269. Remarkably, after pre-treatment of ΔC269 with Mn(2+), the effects of other metal ions showed the same trends as they had on the WT. Molecular dynamics (MD) simulations showed that deletion of Cys269 decreased the polarity of the β12-β13 loop of PP2A Cα. We conclude that deletion of residue Cys269 alters the conformation and activity of PP2A(D) and influences the interaction between PP2A and various metal ions, notably Mn(2+).     

Two copies of the genes encoding the subunits of putative interleukin (IL)-4/IL-13 receptors,IL-4Rα, IL-13Rα1 and IL-13Rα2, have been identified in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) and have complex patterns of expression and modulation

Mammalian interleukin-4 (IL-4) and IL-13 are T helper type 2 (Th2) cytokines with pleiotropic functions in immunity. They signal through receptors containing IL-4Rα and IL-2Rγ or IL-13Rα1. In addition, a decoy receptor, IL-13Rα2, is known to exist and modulates the function of IL-13. The existence of fish orthologues to mammalian IL-4 and IL-13 is still under debate. However, the receptor chains have been predicted in zebrafish, and we have previously cloned IL-2Rγ and IL-13Rα2 in rainbow trout. In this study, we have cloned a further five novel trout IL-4/13 receptors. Thus, each of the IL-4Rα, IL-13Rα1 and IL-13Rα2 chains has two copies. The identities of the receptors is supported by homology analysis, characteristic domain structure, phylogenetic tree analysis and synteny analysis in zebrafish. However, the characteristic WSXWS motif of structural importance in mammalian type I cytokine receptors is missing in all fish IL-4Rα and IL-13Rα1 molecules. All the receptors have a characteristic domain structure that is similar to their mammalian counterparts except for IL-13Rα1b that has the N-terminal Ig domain missing. Since this Ig domain is a specific and critical binding unit for IL-13 but not for IL-4 signalling, its absence potentially converts the IL-13Rα1b into a receptor that can only signal via IL-4 ligation. The existence of duplicated receptor genes perhaps suggests that more ligands still remain to be discovered that will bind these receptors. The duplicated receptors are differentially expressed in most tissues and cell lines examined, and their expression can be modulated by LPS, polyIC and IFN-γ in cell lines. In contrast, the T-cell stimulant phytohaemagglutinin increased the expression of IL-4Rα1 and IL-4Rα2, but not IL-13Rα1/2, suggesting a role of an IL-4-like molecule in T-cell growth/activation in fish.