Paramagnetic labelling of proteins and oligonucleotides for NMR

Paramagnetic effects offer a rich source of long-range structural restraints. Here we review current methods for site-specific tagging of proteins and oligonucleotides with paramagnetic molecules. The paramagnetic tags include nitroxide radicals and metal chelators. Particular emphasis is placed on tags suitable for site-specific and rigid attachment of lanthanide ions to macromolecules.     

Chemical Sensors for Solvent Vapors: Enthalpic and Entropic Contributions to Host-Guest Interactions

Chemical sensors, based on the mass-sensitive quartz micro balance (QMB) or the surface acoustic wave device (SAW), that are coated with thin cyclophane layers allow the detection of harmful organic vapors. The sensor signal of these supramolecular analyte-receptors can be predicted by a method that uses estimated free energies of the host-guest complex formation. From MM3 force field calculations, the reaction enthalpies (H° of host-guest interaction between the macrocycle and the analyte can be calculated, whereas the entropy changes are taken from condensation data. The validity of this condensation model is proven by an excellent linear correlation of the logarithm of the experimental equilibrium constant with the estimated Gibbs energy DG°. In this way promising sensor materials can be selected, even before they are synthesized.     

Capturing Conformational States in Proteins Using Sparse Paramagnetic NMR Data

Capturing conformational changes in proteins or protein-protein complexes is a challenge for both experimentalists and computational biologists. Solution nuclear magnetic resonance (NMR) is unique in that it permits structural studies of proteins under greatly varying conditions, and thus allows us to monitor induced structural changes. Paramagnetic effects are increasingly used to study protein structures as they give ready access to rich structural information of orientation and long-range distance restraints from the NMR signals of backbone amides, and reliable methods have become available to tag proteins with paramagnetic metal ions site-specifically and at multiple sites. In this study, we show how sparse pseudocontact shift (PCS) data can be used to computationally model conformational states in a protein system, by first identifying core structural elements that are not affected by the environmental change, and then computationally completing the remaining structure based on experimental restraints from PCS. The approach is demonstrated on a 27 kDa two-domain NS2B-NS3 protease system of the dengue virus serotype 2, for which distinct closed and open conformational states have been observed in crystal structures. By changing the input PCS data, the observed conformational states in the dengue virus protease are reproduced without modifying the computational procedure. This data driven Rosetta protocol enables identification of conformational states of a protein system, which are otherwise difficult to obtain either experimentally or computationally.     

Protein-Protein Interactions in Calcium Transport Regulation Probed by Saturation Transfer Electron Paramagnetic Resonance

We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) and its regulator phospholamban (PLB). SERCA is responsible for restoring calcium to the sarcoplasmic reticulum to allow muscle relaxation, whereas PLB inhibits cardiac SERCA unless phosphorylated at Ser¹⁶. To determine whether changes in protein association play essential roles in regulation, we detected the microsecond rotational diffusion of both proteins using saturation transfer EPR. Peptide synthesis was used to create a fully functional and monomeric PLB mutant with a spin label rigidly coupled to the backbone of the transmembrane helix, while SERCA was reacted with a Cys-specific spin label. Saturation transfer EPR revealed that sufficiently high lipid/protein ratios minimized self-association for both proteins. Under these dilute conditions, labeled PLB was substantially immobilized after co-reconstitution with unlabeled SERCA, reflecting their association to form the regulatory complex. Ser¹⁶ phosphorylation slightly increased this immobilization. Complementary measurements with labeled SERCA showed no change in mobility after co-reconstitution with unlabeled PLB, regardless of its phosphorylation state. We conclude that phosphorylating monomeric PLB can relieve SERCA inhibition without changes in the oligomeric states of these proteins, indicating a structural rearrangement within the heterodimeric regulatory complex.     

Host-Guest Interactions as a Model for the Solvent Influence on the Conformational Populations in [2.2.2.2]Paracyclophane

The interaction of a number of small organic molecules with the two lowest energy minimum conformations of [2.2.2.2]paracyclophane (4°-PCP), 1 and 2, are studied by MMP2(87) force field calculations. Formations of nesting as well as inclusion complexes were identified on the potential energy surface. With CH₂Cl₂ and CHCl₃ used as guests, the nesting complexes are lower in energy than the inclusion ones with both conformations 1 and 2. Furthermore, the nesting complexes with 2 are found to be more stable than the nesting ones with 1. Formation of the double nesting complexes of CH₂Cl₂ and CHCl₃ with 1 and 2 raise the difference in complexation energy in favour of 2. The preference of 4°-PCP for the form 2 in solution is explained based on the above analysis, although 2 is calculated to be 0.2 kcal·mol^–1 higher in steric energy than 1 in the gas phase.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089490050218     

Facilitated Protein Association via Engineered Target Search Pathways Visualized by Paramagnetic NMR Spectroscopy

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Paramagnetic NMR investigations of Co(II) and Ni(II) amicyanin

 The paramagnetic ¹H NMR spectra of the Co(II) and Ni(II) substituted forms of the type 1 blue copper protein (cupredoxin) amicyanin have been assigned. This is the first such analysis of a cupredoxin, which has a distorted tetrahedral active site with the ligands provided by two histidines, a cysteine and a methionine. The isotropic shifts of the resonances in these spectra are compared with those of Co(II) and Ni(II) azurin. A number of interesting similarities and differences are found. The coordination of the metal by the two equatorial histidine ligands is very similar in both proteins. The interaction between the introduced metal and the thiolate sulfur of the equatorial cysteine ligand is enhanced in the amicyanin derivatives. Resonances belonging to the weak axial methionine ligand exhibit much larger shifts in the amicyanin derivatives, indicative of shorter M(II)-S(Met) distances. The presence of shorter axial M(II)-S(Met) and equatorial M(II)-S(Cys) distances in both Co(II) and Ni(II) amicyanin is ascribed to the absence of a second axially interacting amino acid at the active site of this cupredoxin. Received: 2 February 1999 / Accepted: 19 May 1999     

Two-dimensional NMR Investigation of Solanascone

2-n-Heptyl-4-hydroxyquinoline-N-oxide (KF8940), isolated from Pseudomonas methanica, was a potent and selective inhibitor of the arachidonate 5-lipoxygenase of rat basophilic leukemia (RBL-1) cells. Kinetic analysis indicated that the inhibitory mode was non competitive. The Ki value was 3.5 × 10^?7 m. KF8940 also inhibited 12-lipoxygenase of bovine platelets in a non-competitive manner, but with a Ki value of 7 × 10^?5M. Ionophore A23187-stimulated SRS generation from rat peritoneal cells and antigen-stimulated SRS-A generation from sensitized rat lung were significantly inhibited by KF8940. KF8940 at a dose of 10 mg/kg (p.o.) suppressed the passive anaphylactic bronchoconstriction in guinea pigs.     

Prion Protein-Detergent Micelle Interactions Studied by NMR in Solution

Cellular prion proteins, PrP^C, carrying the amino acid substitutions P102L, P105L, or A117V, which confer increased susceptibility to human transmissible spongiform encephalopathies, are known to form structures that include transmembrane polypeptide segments. Herein, we investigated the interactions between dodecylphosphocholine micelles and the polypeptide fragments 90–231 of the recombinant mouse PrP variants carrying the amino acid replacements P102L, P105L, A117V, A113V/A115V/A118V, K110I/H111I, M129V, P105L/M129V, and A117V/M129V. Wild-type mPrP-(90–231) and mPrP[M129V]-(91–231) showed only weak interactions with dodecylphosphocholine micelles in aqueous solution at pH 7.0, whereas discrete interaction sites within the polypeptide segment 102–127 were identified for all other aforementioned mPrP variants by NMR chemical shift mapping. These model studies thus provide evidence that amino acid substitutions within the polypeptide segment 102–127 affect the interactions of PrP^C with membranous structures, which might in turn modulate the physiological function of the protein in health and disease.Transmissible spongiform encephalopathies (TSEs),² such as Creutzfeldt-Jakob disease and the Gerstmann-Sträussler-Scheinker syndrome in humans, are accompanied by the appearance in the brain of an aggregated “scrapie” isoform of the host-encoded prion protein, PrP^Sc (1–3). The cellular form, PrP^C, consists of an unstructured N-terminal “tail” of residues 23–125 and a globular domain of residues 126–231, and is attached by a C-terminal glycosylphosphatidylinositol (GPI) anchor to the outer plasma membrane. This structure ensures a role of membrane interactions in the physiological function of PrP^C and probably also in the disease-related events leading to TSEs. For example, transgenic mice expressing a prion protein variant lacking the GPI membrane anchor did not develop the typical clinical signs of TSE after inoculation with infectious brain homogenate, although significant amounts of PrP^Sc accumulated in the brain (4). This finding led to the conclusion that membrane-association of PrP^C is necessary for the development of a TSE. Independent evidence for the importance of membrane interactions for the onset of prion diseases was derived from cell-free conversion assays and cell culture experiments (5, 6).Data have also been presented that indicate that in addition to the normal form with the C terminus linked to a GPI anchor and the C-terminal domain located on the cell surface, PrP^C can adopt two different transmembrane topologies, ^CtmPrP and ^NtmPrP, which have the C-terminal polypeptide segment located in the lumen of the endoplasmic reticulum (^CtmPrP) or in the cytoplasm (^NtmPrP) (7–9). The population of the ^CtmPrP variant is <10% of the total wild-type prion protein present during cellular biosynthesis but is increased to 20–30% for the pathogenic mutations P102L, P105L, and A117V of human PrP and the designed variant mouse PrPs obtained with the amino acid exchanges A113V/A115V/A118V and K110I/H111I (10–13). The population of ^CtmPrP was further increased when an additional mutation, L9R, was present in the N-terminal signal sequence (14), so that ∼50% of the PrP was synthesized as the ^CtmPrP variant in granule neurons obtained from transgenic mice expressing a prion protein construct carrying the four amino acid replacements L9R, A113V, A115V, and A118V (15). Quite generally, an increase in the population of ^CtmPrP was also shown to be associated with severe neurodegeneration in transgenic mice, and it has been suggested that ^CtmPrP may be the proximate cause of neuronal death in certain prion disorders (10, 11, 15).In vitro studies on interactions of full-length and N-terminally truncated forms of recombinant PrP showed that acidic membranes caused the N-terminal part of the protein to become more structured, whereas the C-terminal domain was destabilized (16–19). Furthermore, zwitterionic gel-phase dipalmitoylphosphatidylcholine or raft-like membranes were shown to induce increased α-helical structure in recombinant Syrian hamster PrP-(90–231) at pH 7.0 (18, 19). Membrane interactions of polypeptides representing sequence motifs found in the prion protein have also been studied (20–23).In this report we describe investigations of PrP interactions with a membrane mimetic and focus on the mutations P102L, P105L, and A117V, which have been linked with familial Gerstmann-Sträussler-Scheinker syndrome in humans (2, 24, 25). Our interest in these variant proteins is related to open questions about the mechanisms by which pathogenic mutations predispose humans for prion diseases. We studied the interactions of a recombinant wild-type mouse prion protein fragment, mPrP-(90–231), and the variants mPrP[P102L]-(91–231), mPrP[P105L]-(91–231), mPrP[A117V]-(90–231), mPrP[A113V,A115V,A118V]-(90–231), and mPrP[K110I,H111I]-(90–231). For these studies, we used the N-terminally truncated protein composed of residues 90–231. This region contains the transmembrane segment, all known disease-associated point mutations, the entire polypeptide fragment with proteinase K-resistance in PrP^Sc, which is also sufficient to transmit disease (1, 25, 26). The amino acid substitutions in these variant PrPs are located either within a hydrophobic stretch of residues 112–127, which is highly conserved in mammalian PrPs (27, 28), or in the positively charged segment of residues 95–111 (Fig. 1, B and C). We also included the M129V polymorphism into this study, which was reported to have a significant influence on the susceptibility of humans to prion diseases and on the disease phenotype. For example, the mutations P105L and A117V are only pathogenic in the presence of valine at position 129 (2, 24). The zwitterionic detergent dodecylphosphocholine (DPC, Fig. 1A) was used as a biomembrane mimetic model system, and NMR spectroscopy was employed to screen for protein-detergent micelle interactions, and for the structural characterization of the various prion protein constructs interacting with the detergent micelles.Open in a separate windowFIGURE 1.Detergent and proteins used in this study. A, zwitterionic form of DPC. B, schematic diagram of the mPrP-(90–231) polypeptide indicating the locations of the regular secondary structures, i.e. three α-helices and two strands of an antiparallel β-sheet, a “positively charged cluster” (CC) of amino acid residues in positions 95–111, and a “hydrophobic polypeptide segment” (HPS) comprising residues 112–127. C, amino acid sequence alignment of residues 90–135 for wild-type mPrP-(90–231) and the protein variants studied in this paper, where for each variant mPrP the amino acid replacements are given and identical residues are indicated by dots; the numbering is according to Schätzl et al. (27).     

A Molecular Investigation of Genotype by Environment Interactions

The fitnesses conferred by seven lactose operons, which had been transduced into a common genetic background from natural isolates of Escherichia coli, were determined during competition for growth rate-limiting quantities of galactosyl-glycerol, a naturally occurring galactoside. The fitnesses of these same operons have been previously determined on lactose and three artificial galactosides, lactulose, methyl-galactoside and galactosyl-arabinose. Analysis suggests that although marked genotype by environment interactions occur, changes in the fitness rankings are rare. The relative activities of the β-galactosidases and the permeases were determined on galactosyl-glycerol, lactose, lactulose and methyl-galactoside. Both enzymes display considerable kinetic variation. The β-galactosidase alleles provide no evidence for genotype by environment interactions at the level of enzyme activity. The permease alleles display genotype by environment interactions with a few causing changes in activity rankings. The contributions to fitness made by the permeases and the β-galactosidases were partitioned using metabolic control analysis. Most of the genotype by environment interaction at the level of fitness is generated by changes in the distribution of control among steps in the pathway, particularly at the permease where large control coefficients ensure that its kinetic variation has marked fitness effects. Indeed, changes in activity rankings at the permease account for the few changes in fitness rankings. In contrast, the control coefficients of the β-galactosidase are sufficiently small that its kinetic variation is in, or close to, the neutral limit. The selection coefficients are larger on the artificial galactosides because the control coefficients of the permease and β-galactosidase are larger. The flux summation theorem requires that control coefficients associated with other steps in the pathway must be reduced, implying that the selection at these steps will be less intense on the artificial galactosides. This suggests that selection intensities need not be greater in novel environments.     

Homopolygalacturonan Molecular Size in Plant Cell Wall Matrices Via Paramagnetic Ion and Nitroxyl Amide Dipolar Spin-Spin Interactions

Mn²⁺, Cu²⁺, and nitroxyl amines have been shown to bond to plant homopolygalacturonan matrices in a spatially sequential fashion. As a consequence of this special form of cooperativity the lattice constant (κ), determined from Van Vleck's second moment relationship, approaches 1 only when the average number of dipolar interactions per spin approaches 1 (e.g., an array of dimers). Assuming that one paramagnetic ion or nitroxyl amide pair is bonded per polymer block within the matrix when κ = 1, the anionic ligand's average degree of polymerization ([unk]) can be estimated from the concentration of bonded paramagnetic dimers (e.g., [1/χ]_κ~1 = [unk]; χ is the mole fraction of bonded paramagnetic dimers). We have utilized this technique to estimate the average molecular size of homopolygalacturonan blocks in intact higher plant cortical cell walls ([unk] ~83), Nitella cell walls ([unk] ~27) and a commercially available galacturonic acid polymer ([unk] ~35). The [unk] determined from both the intact cortical cell wall lattice and the polygalacturonan were similar to literature values; these findings argue that the electron paramagnetic resonance, (EPR) dipolar spin-spin interaction technique reported herein is a valid approach for estimating molecular size in plant cell walls.     

Interactions between selected photosensitizers and model membranes: an NMR classification

Membrane interactions of porphyrinic photosensitizers (PSs) are known to play a crucial role for PS efficiency in photodynamic therapy (PDT). In the current paper, the interactions between 15 different porphyrinic PSs with various hydrophilic/lipophilic properties and phospholipid bilayers were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. PS-membrane interactions were deduced from analysis of the main DOPC 1H-NMR resonances (choline and lipid chain signals). Initial membrane adsorption of the PSs was indicated by induced changes to the DOPC choline signal, i.e. a split into inner and outer choline peaks. Based on this parameter, the PSs could be classified into two groups, Type-A PSs causing a split and the Type-B PSs causing no split. A further classification into two subgroups each, A1, A2 and B1, B2 was based on the observed time-dependent changes of the main DOPC NMR signals following initial PS adsorption. Four different time-correlated patterns were found indicating different levels and rates of PS penetration into the hydrophobic membrane interior. The type of interaction was mainly affected by the amphiphilicity and the overall lipophilicity of the applied PS structures. In conclusion, the NMR data provided valuable structural and dynamic insights into the PS-membrane interactions which allow deriving the structural constraints for high membrane affinity and high membrane penetration of a given PS.     

31P NMR Investigation of vanadium-treated chick muscle

The phosphorus NMR profile of normal and vanadium-treated chick muscle was obtained in vivo. The data show that the differentiation of breast and thigh muscles in terms of pH, lipid related metabolites, and bioenergetic parameters can be readily followed. Although the vanadium-treated chicks showed substantial retardation of growth, the only NMR parameter that was significanty affected by dietary vanadium was the pH of breast muscle, which was substantially more acidic in the vanadium-treated animals.     

Interactions of Lycopodium alkaloids with acetylcholinesterase investigated by 1H NMR relaxation rate

In order to further understand the interaction processes between the Lycopodium alkaloids and acetylcholinesterase, the binding properties of N-acetyl huperzine A (1), huperzine B (2) and huperzine F (3) with Torpediniforms Nacline acetylcholinesterase (TnAchE) were investigated by 1H NMR methods. The nonselective, selective and double-selective spin-lattice relaxation rates were acquired in the absence and presence of TnAchE at a ratio of [ligand]/[protein]=1:0.005. The selective relaxation rates show protons of 1-3 have dipole-dipole interaction with protons of TnAchE at the binding interface. The molecular rotational correlation time of bound ligands was calculated by double-selective relaxation rate at 298 K, which showed that 1-3 had high affinity with the protein. The results indicate that investigation of 1H NMR relaxation data is a useful method to locate the new Lycopodium alkaloids as AchE inhibitors.