Amplification of radiation damping in a 600-MHz NMR spectrometer: Application to the study of water-protein interactions

Summary A new application of a recently developed electronic radiation-damping (RD) control system is presented. It is possible to amplify radiation damping so as to make the water magnetization return back to its equilibrium direction in a time shorter than the characteristic RD time. Certain types of experiments involving radiation damping as a selective inversion pulse can be significantly improved by this new method. Moreover, amplification of RD is shown to improve water suppression and consequently the dynamics of 2D NOESY experiments on proteins.     

A joint 2D NMR and theoretical investigation of Ca2+ binding loops III and IV of calmodulin

A 2D NMR NOESY spectrum of integral CaM in water(148 residues) reveals a series of downfield-shifted crosspeaks stemming from the NH protons of the Ca2(+)-binding loops III and IV. Their attribution, with the help of already assigned proton resonances of isolated tryptic fragments, was complemented by means of energy-minimizations on the Ca2+ complexes of loops III and IV. From these calculations, a set of two alternative, related conformations was obtained for each loop. The first type of conformation provides a coordination pattern for Ca2+ that is similar to that found in loop EF of parvalbumin. The computed interproton distances in both loops are fully compatible with the inferences from the sets of NOESY cross-peaks. Evidence is also provided for interloop interactions.     

Nucleotide-binding domain 1 of cystic fibrosis transmembrane conductance regulator production of a suitable protein for structural studies.

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATP-binding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotide-binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR. Determination of the three dimensional structure of NBD1 is essential to better understand its structure-function relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant His-tagged NBD1, as a soluble, stable and isolated domain. The method avoids the use of renaturing processes or fusion constructs. ATPase activity assays show that the recombinant domain is functional. Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation DeltaF508, could differ from that of the nucleotide-binding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N-1H NMR spectra demonstrate that the domain is folded. The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1.     

1H-NMR investigation of yeast cytochrome c. Interaction with the corresponding specific reductase (L-lactate cytochrome)

1H-NMR spectroscopy has been used to study the modifications of certain characteristic resonances of the Hansenula anomala yeast cytochrome c on binding to its specific reductase (flavocytochrome b2) or to the isolated cytochrome domain obtained from the entire molecule. Normal titration curves are observed for the resonances at 37.8 ppm assigned to heme c methyl 8 and at 19.4 ppm, line of cytochrome b2 spectrum. In contrast, the shifts near 3.2 and 3.4 ppm for trimethyl-lysine resonances of this cytochrome c present abnormal titration curves, saturation being apparently reached at low molar (cytochrome b2)/(cytochrome c) ratio. An interpretation is proposed in terms of shifts due to local conformational transitions induced by reductase binding but not rapidly reversible upon dissociation.     

Alpha-cobratoxin: proton NMR assignments and solution structure.

The solution structure of alpha-cobratoxin, a neurotoxin purified from the venom of the snake Naja naja siamensis, at pH 3.2 is reported. Sequence-specific assignments of the NMR resonances was attained by a combination of a generalized main-chain-directed strategy and of the sequential method. The NMR data show the presence of a triple-stranded beta-sheet (residues 19-25, 36-41, and 52-57), a short helix, and turns. An extensive number of NOE cross peaks were identified in the NOESY NMR maps. These were applied as distance constraints in a molecular modeling protocol which includes distance geometry and dynamical simulated annealing calculations. A single family of structures is observed which fold in such a way that three major loops emerge from a globular head. The solution and crystal structures of alpha-cobratoxin are very similar. This is in clear contrast to results reported for alpha-bungarotoxin where significant differences exist.     

ASSOCIATION OF MURINE SPLENOCYTE CD3 COMPLEX TO THE CYTOSKELETON: ABSENCE OF MODULATION BY EXOGENOUS FATTY ACIDS

The cytoplasmic regions of the CD3 complex are presumably involved in signal transduction following ligand—receptor binding. We investigated the effects of incubating either stearic or oleic acid on the association of murine lymphocyte CD3 complex with the cytoskeleton. Both cytochalasin D, an inhibitor of microfilament formation, and W7, an inhibitor of calmodulin, inhibited capping of CD3. The association of CD3 with the cytoskeleton was confirmed by confocal laser scanning microscopy studies, which showed co-localization of the cross-linked CD3 receptors and the membrane attachment proteins ankyrin and fodrin. Although exogenous oleic acid increased plasma membrane fluidity, neither expression nor capping of CD3 receptors was increased. Nonetheless, oleic acid did increase uptake of tritiated thymidine after binding of anti-CD3 antibodies. Lymphoproliferation was progressively inhibited by both cytochalasin D and W7, confirming the importance of intact cytoskeleton for cellular activation.     

Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing

Single-cell sequencing is a powerful tool for delineating clonal relationship and identifying key driver genes for personalized cancer management. Here we performed single-cell sequencing analysis of a case of colon cancer. Population genetics analyses identified two independent clones in tumor cell population. The major tumor clone harbored APC and TP53 mutations as early oncogenic events, whereas the minor clone contained preponderant CDC27 and PABPC1 mutations. The absence of APC and TP53 mutations in the minor clone supports that these two clones were derived from two cellular origins. Examination of somatic mutation allele frequency spectra of additional 21 whole-tissue exome-sequenced cases revealed the heterogeneity of clonal origins in colon cancer. Next, we identified a mutated gene SLC12A5 that showed a high frequency of mutation at the single-cell level but exhibited low prevalence at the population level. Functional characterization of mutant SLC12A5 revealed its potential oncogenic effect in colon cancer. Our study provides the first exome-wide evidence at single-cell level supporting that colon cancer could be of a biclonal origin, and suggests that low-prevalence mutations in a cohort may also play important protumorigenic roles at the individual level.