Interaction of Daunomycin with Dipalmitoylphosphatidylcholine Model Membranes. A 1H NMR Study

1H NMR parameters were obtained for daunomycin in water solution in the free state as well as in the presence of dipalmitoylphosphatidylcholine model membranes. Spin-lattice relaxation rates were measured under nonselective, single-selective, and double-selective irradiation modes, and 2D NOESY spectra were obtained at several values of the mixing time. Proton-proton distances were calculated and the motional correlation time was evaluated in both the free and bound states. NMR parameters were used to show that ring A and the glucosamine moiety of daunomycin strongly interact with the external surface of the bilayer, while the rest of the molecule penetrates the membrane without crossing it. The structures of both free and bound daunomycin were obtained and compared by using molecular modeling.     

Interaction of a type II myosin with biological membranes studied by 2H solid state NMR

Deuterium nuclear magnetic resonance spectroscopy (2H NMR) has been employed to investigate the interaction of lung type II myosin protein with neutral bilayers containing dimyristoylphosphatidylcholine (DMPC) as the only constituent and mixed bilayers containing the negatively charged lipid dimyristoylphosphatidylglycerol (DMPG). DMPC was deuterated at its headgroup by substituting the four protons at the alpha- and beta-positions (DMPC-d4) and the nine protons at the gamma-position (DMPC-d9). DMPG was perdeuterated at its headgroup (DMPG-d5). No changes were observed in the quadrupole splittings or spin-lattice relaxation times for the deuterated DMPC headgroup segments when increasing amounts of myosin were added to liposomes, made exclusively of DMPC-d9 or of DMPC-d4. However, upon the insertion of the negatively charged lipid DMPG at 1:1 molar ratio into the DMPC bilayers, myosin was found to interact electrostatically with the liposomes, thereby affecting significantly both the quadrupole splittings and spin-lattice relaxation rates of the alpha-, beta-, and gamma-deuterons in labeled DMPC. Monitoring DMPG-d5 in mixed DMPC/DMPG bilayers revealed a direct electrostatic interaction of DMPG with the protein, where positively charged lysine residues located at the tail domain of myosin provide the necessary sites for the interaction to occur. When ATP and Mg2+ were complexed to the head domain of myosin, a reduced interaction with the negatively charged bilayers was observed. The results clearly indicate that a type II myosin can interact with membranes without the need for a specific hydrophobic domain or an anchor in the protein molecule, provided that negatively charged lipids are present in the bilayer.     

Interaction of the second binding region of troponin I with the regulatory domain of skeletal muscle troponin C as determined by NMR spectroscopy

Two dimensional 1H,15N-heteronuclear single quantum correlation NMR was used to monitor the resonance frequency changes of the backbone amide groups belonging to the 15N-labeled regulatory domain of calcium saturated troponin C (N-TnC) upon addition of synthetic skeletal N-acetyl-troponin I 115-131-amide peptide (TnI115-131). Utilizing the change in amide chemical shifts, the dissociation constant for 1:1 binding of TnI115-131 to N-TnC in low salt and 100 mM KCl samples was determined to be 28 +/- 4 and 24 +/- 4 microM, respectively. The off rate of TnI115-131 was determined to be 300 s-1 from observed N-TnC backbone amide 1H,15N-heteronuclear single quantum correlation cross-peak line widths, which is on the order of the calcium off rates (Li, M. X., Gagné, S. M., Tsuda, S., Kay, C. M., Smillie, L. B., and Sykes, B. D. (1995) Biochemistry 34, 8330-8340), and agrees with kinetic expectations for biological regulation of muscle contraction. The TnI115-131 binding site on N-TnC was determined by mapping of chemical shift changes onto the N-TnC NMR structure and was demonstrated to be in the "hydrophobic pocket" (Gagné, S. M., Tsuda, S., Li, M. X., Smillie, L. B., and Sykes, B. D. (1995) Nat. Struct. Biol. 2, 784-789).     

Diagnosis of cancer in humans by 1H NMR of tissue biopsies

We describe methodology for the diagnosis of human cancer, at high levels of accuracy, sensitivity, and specificity, by 1H NMR of tissue biopsies. This method is made robust and accurate by careful specimen preparation, and by multivariate analysis of spectral data. Examples are presented for the diagnosis of cancer of the prostate gland and the ovary. The potential for use of these methods noninvasively, in vivo, is shown to be very positive.     

Interaction of histone H1 with cis-platinum modified DNA

Cis-diamminedichloroplatinum(II) (cis-DDP) is known as an effective anticancer drug. Its therapeutic effect is supposed to be a consequence of the covalent binding to DNA. A number of cellular proteins were found to bind selectively to DNA modified by cis-DDP (but not by its isomer trans-DDP). Here we present our observations on interaction of the linker histone H1 with cis- and trans-DDP modified DNA fragments. The results afford new experimental information about the preferential binding of histone H1 to cis-DDP-distorted DNAs versus trans-DDP modified ones.     

Delta-selective opioid peptides containing a single aromatic residue in the message domain: an NMR conformational analysis

The sequence of deltorphin I, a delta-selective opioid agonist, has been systematically modified by inserting conformationally constrained C alpha, alpha disubstituted apolar residues in the third position. As expected, substitution of Phe with Ac6c, Ac5c and Ac3c yields analogues with decreasing but sizeable affinity. Surprisingly, substitution with Aib yields an analogue with almost the same binding affinity of the parent compound but with a greatly increased selectivity. This is the first case of a potent and very selective opioid peptide containing a single aromatic residue in the message domain, that is, only Tyr1. Here we report a detailed conformational analysis of [Aib3]deltorphin I and [Ac6c3]deltorphin I in DMSO at room temperature and in a DMSO/water cryomixture at low temperature, based on NMR spectroscopy and energy calculations. The peptides are highly structured in both solvents, as indicated by the exceptional finding of a nearly zero temperature coefficient of Val5 NH resonance. NMR data cannot be explained on the basis of a single structure but it was possible to interpret all NMR data on the basis of a few structural families. The conformational averaging was analysed by means of an original computer program that yields qualitative and quantitative composition of the mixture. Comparison of the preferred solution conformation with two rigid delta-selective agonists shows that the shapes of [Aib3]deltorphin I and [Ac6c3]deltorphin I are consistent with those of rigid agonists and that the message domain of opioid peptides can be defined only in conformational terms.     

Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR

Raphanus sativus Antifungal Protein 1 (Rs-AFP1) is a 51 amino acid residue plant defensin isolated from radish (Raphanus sativus L.) seeds. The three-dimensional structure in aqueous solution has been determined from two-dimensional 1H NMR data recorded at 500 MHz using the DIANA/REDAC calculation protocols. Experimental constraints consisted of 787 interproton distances extracted from NOE cross-peaks, 89 torsional constraints from 106 vicinal interproton coupling constants and 32 stereospecific assignments of prochiral protons. Further refinement by simulated annealing resulted in a set of 20 structures having pairwise root-mean-square differences of 1.35(+/- 0.35) A over the backbone heavy atoms and 2.11(+/- 0.46) A over all heavy atoms. The molecule adopts a compact globular fold comprising an alpha-helix from Asn18 till Leu28 and a triple-stranded beta-sheet (beta 1 = Lys2-Arg6, beta 2 = His33-Tyr38 and beta 3 = His43-Pro50). The central strand of this beta-sheet is connected by two disulfide bridges (Cys21-Cys45 and Cys25-Cys47) to the alpha-helix. The connection between beta-strand 2 and 3 is formed by a type VIa beta-turn. Even the loop (Pro7 to Asn17) between beta-strand 1 and the alpha-helix is relatively well defined. The structure of Raphanus sativus Antifungal Protein 1 features all the characteristics of the "cysteine stabilized alpha beta motif". A comparison of the complete structure and of the regions important for interaction with the fungal receptor according to a mutational study, is made with the structure of gamma-thionin, a plant defensin that has no antifungal activity. It is concluded that this interaction is both electrostatic and specific, and some possible scenarios for the mode of action are given.     

Mechanism of action cryptophycin. Interaction with the Vinca alkaloid domain of tubulin

Cryptophycin is a potent antitumor agent that depletes microtubules in intact cells, including cells with the multidrug resistance phenotype. To determine the mechanism of action of cryptophycin, its effects on tubulin function in vitro were analyzed. Cryptophycin reduced the in vitro polymerization of bovine brain microtubules by 50% at a drug:tubulin ratio of 0.1. Cryptophycin did not alter the critical concentration of tubulin required for polymerization, but instead caused substoichiometric reductions in the amount of tubulin that was competent for assembly. Consistent with its persistent effects on intact cells, cryptophycin-treated microtubule protein remained polymerization-defective even after cryptophycin was reduced to sub-inhibitory concentrations. The effects of cryptophycin were not due to denaturation of tubulin and were associated with the accumulation of rings of microtubule protein. The site of cryptophycin interaction with tubulin was examined using functional and competitive binding assays. Cryptophycin blocked the formation of vinblastine-tubulin paracrystals in intact cells and suppressed vinblastine-induced tubulin aggregation in vitro. Cryptophycin inhibited the binding of [3H]vinblastine and the hydrolysis of [gamma32P]GTP by isolated tubulin, but did not block the binding of colchicine. These results indicate that cryptophycin disrupts the Vinca alkaloid site of tubulin; however, the molecular details of this interaction are distinct from those of other antimitotic drugs.     

A calculation strategy for the structure determination of symmetric dimers by 1H NMR

The structure determination of symmetric dimers by NMR is impeded by the ambiguity of inter- and intramonomer NOE crosspeaks. In this paper, a calculation strategy is presented that allows the calculation of dimer structures without resolving the ambiguity by additional experiments (like asymmetric labeling). The strategy employs a molecular dynamics-based simulated annealing approach to minimize a target function. The experimental part of the target function contains distance restraints that correctly describe the ambiguity of the NOE peaks, and a novel term that restrains the symmetry of the dimer without requiring the knowledge of the symmetry axis. The use of the method is illustrated by three examples, using experimentally obtained data and model data derived from a known structure. For the purpose of testing the method, it is assumed that every NOE crosspeak is ambiguous in all three cases. It is shown that the method is useful both in situations where the structure of a homologous protein is known and in ab initio structure determination. The method can be extended to higher order symmetric multimers.     

Anti-MUC1 antibodies react directly with MUC1 peptides presented by class I H2 and HLA molecules

Codon usage bias, the preferential use of particular codons within each codon family, is characteristic of synonymous base composition in many species, including Drosophila, yeast, and many bacteria. Preferential usage of particular codons in these species is maintained by natural selection acting largely at the level of translation. In Drosophila, as in bacteria, the rate of synonymous substitution per site is negatively correlated with the degree of codon usage bias, indicating stronger selection on codon usage in genes with high codon bias than in genes with low codon bias. Surprisingly, in these organisms, as well as in mammals, the rate of synonymous substitution is also positively correlated with the rate of nonsynonymous substitution. To investigate this correlation, we carried out a phylogenetic analysis of substitutions in 22 genes between two species of Drosophila, Drosophila pseudoobscura and D. subobscura, in codons that differ by one replacement and one synonymous change. We provide evidence for a relative excess of double substitutions in the same species lineage that cannot be explained by the simultaneous mutation of two adjacent bases. The synonymous changes in these codons also cannot be explained by a shift to a more preferred codon following a replacement substitution. We, therefore, interpret the excess of double codon substitutions within a lineage as being the result of relaxed constraints on both kinds of substitutions in particular codons.     

Solid-state NMR studies of the prion protein H1 fragment

Conformational changes in the prion protein (PrP) seem to be responsible for prion diseases. We have used conformation-dependent chemical-shift measurements and rotational-resonance distance measurements to analyze the conformation of solid-state peptides lacking long-range order, corresponding to a region of PrP designated H1. This region is predicted to undergo a transformation of secondary structure in generating the infectious form of the protein. Solid-state NMR spectra of specifically 13C-enriched samples of H1, residues 109-122 (MKHMAGAAAAGAVV) of Syrian hamster PrP, have been acquired under cross-polarization and magic-angle spinning conditions. Samples lyophilized from 50% acetonitrile/50% water show chemical shifts characteristic of a beta-sheet conformation in the region corresponding to residues 112-121, whereas samples lyophilized from hexafluoroisopropanol display shifts indicative of alpha-helical secondary structure in the region corresponding to residues 113-117. Complete conversion to the helical conformation was not observed and conversion from alpha-helix back to beta-sheet, as inferred from the solid-state NMR spectra, occurred when samples were exposed to water. Rotational-resonance experiments were performed on seven doubly 13C-labeled H1 samples dried from water. Measured distances suggest that the peptide is in an extended, possibly beta-strand, conformation. These results are consistent with the experimental observation that PrP can exist in different conformational states and with structural predictions based on biological data and theoretical modeling that suggest that H1 may play a key role in the conformational transition involved in the development of prion diseases.     

Studies of the internal flow process in polymers by 1H NMR microscopy at 500 MHz

Intrinsic subthreshold oscillations in the membrane potential are a common property of many neurons in the peripheral and central nervous system. When such oscillations are combined with noise, interesting signal encoding and neuromodulatory properties are obtained which allow, for example, sensitivity adjustment or differential encoding of stimuli. Here we demonstrate that a noisy Hodgkin/Huxley-model for subthreshold oscillations, when tuned to maximum sensitivity, can be significantly modulated by even minor physiological changes in the oscillation parameters amplitude or frequency. Given the ubiquity of subthreshold oscillating neurons, it can be assumed that these findings reflect principle encoding properties which are relevant for an understanding of sensitivity and neuromodulation in peripheral and central neurons.     

1H NMR determination of intracellular volume in cell suspensions

A 1H NMR method has been developed for determining the intracellular and extracellular volumes in a cell suspension. The method is quick, simple, and inexpensive. A comparison of the ratios of the water and Tris buffer resonances in a cell suspension and in a buffer solution gives the intracellular volume. The most important precaution to take is to ensure that coil loading is identical in both solutions and that the NMR signal is not saturating. The method was validated with a 20% polyethylene glycol solution. A comparison with radiolabel methods for volume determination found that the radiolabel probe of extracellular volume did not penetrate the cell wall water of Enterococcus faecalis, resulting in an overestimation of the intracellular volume, and that tritiated water probably exchanged with macromolecules, causing an underestimation of intracellular volume.     

Solution conformation of the synthetic bovine proenkephalin-A209-237 by 1H NMR spectroscopy

Proenkephalin-A has been described to generate enkephalins, opoid peptides, and several derived peptides, which display various biological effects, including antinociception and immunological enhancement. Recently, we have isolated from bovine chromaffin granules a new antibacterial peptide, named enkelytin, which corresponds to the bisphosphorylated form of PEAP209-237 (Goumon, Y., Strub, J. M., Moniatte, M., Nullans, G., Poteur, L., Hubert, P., Van Dorsselaer, A., Aunis, D., and Metz-Boutigue, M. H. (1996) Eur. J. Biochem. 235, 516-525). In this paper, the three-dimensional solution structure of synthetic PEAP209-237 was investigated by NMR. These studies indicate that this peptide, which is unstructured in water, folds into an alpha-helical structure in trifluoroethanol/water (1/1). NMR data revealed two possible three-dimensional models of PEAP209-237. In both models, the proline residue Pro-227 induces a 90 degrees hinge between two alpha-helical segments (Ser-215 to Ser-221 and Glu-228 to Arg-232) leading to an overall L-shaped structure for the molecule. The negative charge of PEAP209-237 and the low amphipathy of the two alpha-helical segments imply new mechanisms to explain the antibacterial activity of enkelytin.     

NMR analysis of interactions of a phosphatidylinositol 3'-kinase SH2 domain with phosphotyrosine peptides reveals interdependence of major binding sites

The interactions of the N-terminal src homology (SH2) domain (N-SH2) of the 85 kDa subunit of phosphatidylinositol 3'-kinase (PI-3K) with phosphotyrosine (ptyr) and a series of ptyr-containing peptides have been examined by NMR spectroscopy. HSQC (heteronuclear single-quantum coherence) NMR spectra of 15N-labeled SH2 were used to evaluate its interactions with ptyr-containing ligands. The ability of ligands to cause chemical shift changes was compared to their potency as competitors in in vitro binding experiments using polyoma virus middle T antigen (MT). The results suggest the interdependence of SH2 binding elements. Chemical shifts of residues involved in the ptyr binding were altered by variations of the sequence of the bound peptide, suggesting that the ptyr fit can be adjusted by the peptide sequence. Perturbations of chemical shifts of residues coordinating the methionine three residues C-terminal to the ptyr (the +3 residue) were affected by substitution in the binding peptide at +1 and vice versa. Such results show synergistic interplay between regions of the SH2 binding residues C-terminal to the ptyr.     

Effects of replacement of Lys25 with Gln on the conformation of ribonuclease T1: sequence-specific 1H NMR resonance assignments of Gln25 ribonuclease T1 by two-dimensional NMR spectroscopy

Two isozymes of ribonuclease (RNase) T1 exist in nature, i.e. Gln25 RNase T1 and Lys25 RNase T1. Gln25 RNase T1 is less stable than Lys25 RNase T1, although the enzymatic activity is not distinguishable between these two isozymes. To elucidate the effects of the replacement of Lys25 with Gln on the conformation and microenvironments of RNase T1 in detail, two-dimensional NMR spectra were measured, sequence-specific 1H NMR resonance assignments of Gln25 RNase T1 were performed, and then the determined parameters and microenvironments of Gln25 RNase T1 were compared with those of Lys25 isozyme [Hoffmann, E. and Rüterjans, H. (1988) Eur. J. Biochem. 177, 539-560]. The main chain protons were assigned for 101 out of the total of 104 amino acid residues. Secondary structure elements were identified from analysis of characteristic NOE patterns, interstrand NOE connectivities, and hydrogen-deuterium exchange rates of main chain amide protons. The results indicated that Gln25 RNase T1 contains a single alpha-helix and seven beta-strands. The secondary structure of Gln25 RNase T1 is, thus, essentially the same as that of Lys25 RNase T1. On the other hand, comparison of the conformation-dependent shifts of Gln25 RNase T1 with these of Lys25 RNase T1 showed that the replacement of Lys25 with Gln has significant effects on the C-terminal part of the alpha-helix region and the base-binding site. These results may indicate that the base-binding site is relatively flexible in the RNase T1 molecule. Among the residues of the C-terminal part of the alpha-helix region, the protons of Asp29 were most affected in terms of their chemical shifts, which may indicate that the side chain carboxylate anion of Asp29 is the counterpart of the electrostatic interaction of Lys25 in Lys25 RNase T1. The Gln25 of Gln25 RNase T1 may have little or no interaction with Asp29, and this may be the reason why Gln25 RNase T1 is less stable than the Lys25 isozyme.