The near complete (>90%) NMR assignment of 15N, 13Cα, 13Cβ, and HN chemical shifts is presented for a 64 kDa trp repressor−operator complex consisting of two tandem dimers of 15N,13C,>90% 2H labeled trp repressor, unlabeled 22-base-pair DNA, and unlabeled corepressor, 5-methyltryptophan. The DNA sequence employed contains three copies of the palindromic sequence 5‘-CTAG-3‘, allowing two dimers of trp repressor to bind to each duplex operator DNA. Chemical shift data establish that each subunit within a given dimer in the complex is in a chemically distinct environment, and the pattern of chemical shift differences between subunits provides information regarding interdimer contacts. Because of the large size of the complex, a number of modifications were made to existing enhanced sensitivity triple-resonance correlation experiments which link 13Cβ, 15N, and HN chemical shifts; the pulse sequences which include these changes are presented. The experiments make use of constant-time chemical shift evolution of the carbon magnetization, resulting in significant improvements in spectral resolution compared to non-constant-time versions of the pulse schemes. An analysis of the utility of the enhanced sensitivity method for recording spectra of high molecular weight deuterated proteins indicates that this approach produces reasonable sensitivity gains for the 64 kDa trp repressor−operator complex studied here.

The solution structure of bovine lactoferricin (LfcinB) has been determined using 2D 1H NMR spectroscopy. LfcinB is a 25-residue antimicrobial peptide released by pepsin cleavage of lactoferrin, an 80 kDa iron-binding glycoprotein with many immunologically important functions. The NMR structure of LfcinB reveals a somewhat distorted antiparallel beta-sheet. This contrasts with the X-ray structure of bovine lactoferrin, in which residues 1-13 (of LfcinB) form an alpha-helix. Hence, this region of lactoferricin B appears able to adopt a helical or sheetlike conformation, similar to what has been proposed for the amyloidogenic prion proteins and Alzheimer's beta-peptides. LfcinB has an extended hydrophobic surface comprised of residues Phe1, Cys3, Trp6, Trp8, Pro16, Ile18, and Cys20. The side chains of these residues are well-defined in the NMR structure. Many hydrophilic and positively charged residues surround the hydrophobic surface, giving LfcinB an amphipathic character. LfcinB bears numerous similarities to a vast number of cationic peptides which exert their antimicrobial activities through membrane disruption. The structures of many of these peptides have been well characterized, and models of their membrane-permeabilizing mechanisms have been proposed. The NMR solution structure of LfcinB may be more relevant to membrane interaction than that suggested by the X-ray structure of intact lactoferrin. Based on the solution structure, it is now possible to propose potential mechanisms for the antimicrobial action of LfcinB.

High-field proton magic-angle sample-spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy is shown to yield high-resolution 'H spectra of smectic, nematic and hexagonal-I1 phase lipids, from which isotropic chemical shifts, order parameters and relaxation times (q, qp and T,) can be determined. Such experiments are possible because of the special form of the dipolar Hamiltonian in such systems. Resolution is about the same as that obtained with sonicated systems, using conventional NMR techniques. We also show that 13C MAS NMR spectra, of both fluid and solid phases, are even better resolved, and in some cases resonances can be observed in MAS NMR spectra which are not observable in sonicated systems. For example, essentially all of the carbon atoms in cholesterol (CHOL) can be readily detected and assigned in a lecithin-CHOL bilayer, using MAS, while few can be seen in sonicated bilayers. This leads directly to the observation of cholesterol in intact biological membranes, such as human myelin, where over 50 peaks can be observed, and ca. 40 of these resonances can be assigned to specific, single-carbon-atom sites in the membrane. In addition, a number of experiments with massively deuterated lipids are reported. Combination of cross-polarization techniques with MAS, and difference spectroscopy, leads to the observation of essentially pure sterol spectra (in the presence of lipid) and pure lipid spectra (in the presence of CHOL). Analysis of chemical-shift results indicates a substantial deshielding of chain carbon atom resonances caused by the presence of CHOL, due presumably to increased trans chain segments, an effect mirrored in variable temperature spectra of human myelin, and in goldfish myelin. Taken together, these results suggest a resurgence in NMR studies of membranes may soon occur.

The transcriptionally active fragment of the yeast RNA polymerase II transcription elongation factor, TFIIS, comprises a three-helix bundle and a zinc ribbon motif joined by a linker region. We have probed the function of this fragment of TFIIS using structureguided mutagenesis. The helix bundle domain binds RNA polymerase II with the same affinity as does the full-length TFIIS, and this interaction is mediated by a basic patch on the outer face of the third helix. TFIIS mutants that were unable to bind RNA polymerase II were inactive for transcription activity, confirming the central role of polymerase binding in the TFIIS mechanism of action. The linker and zinc ribbon regions play roles in promoting cleavage of the nascent transcript and read-through past the block to elongation. Mutation of three aromatic residues in the zinc ribbon domain (Phe 269 , Phe 296 , and Phe 308 ) impaired both transcript cleavage and read-through. Mutations introduced in the linker region between residues 240 and 245 and between 250 and 255 also severely impaired both transcript cleavage and read-through activities. Our analysis suggests that the linker region of TFIIS probably adopts a critical structure in the context of the elongation complex.Elongating RNA polymerase II stalls upon encountering blocks to elongation in vitro (1). In some cases, these transiently stalled polymerases convert to very stable arrested complexes. Arrested complexes are unable to resume transcription even after hours to weeks of incubation (2). The inability of such complexes to resume transcription results from a structural change in the stalled polymerase, which causes the active site to disengage from the 3Ј-end of the transcript (3). The general elongation factor TFIIS 1 reactivates arrested transcription complexes within minutes (4). The reactivation process involves a TFIIS-stimulated endonucleolytic cleavage of the transcript by the RNA polymerase II (5, 6), which relocates the polymerase active site to the new 3Ј-end of the RNA chain and allows for chain extension. The reactivation of stalled elongation complexes involves multiple steps, with the first being the interaction of TFIIS with RNA polymerase II. The TFIIS-binding domain on RNA polymerase II was identified by Friesen and colleagues (7), who discovered mutants in the largest subunit of RNA polymerase II, RPB1, that displayed the same phenotype as a strain deleted for the TFIIS gene (sensitivity to the drug 6-azauracil) and that also could be suppressed by overexpression of TFIIS. These mutants localized to a part of RPB1 between regions G and H, which are conserved from bacteria to man and are in close proximity to the RNA polymerase active site (8, 9). The genetic evidence for a TFIIS interacting domain was confirmed biochemically, when two of the mutant RNA polymerases were purified and shown directly to have 500-fold lower affinity for TFIIS compared with the wild-type polymerase (10).Transcript cleavage is the next essential step in the reactivation process. It is now clear that R...

TFIIS is a general transcription elongation factor that helps arrested RNA polymerase II elongation complexes resume transcription. We have previously shown that yeast TFIIS (yTFIIS) comprises three structural domains (I-III). The three-dimensional structures of domain II and part of domain III have been previously reported, but neither domain can autonomously stimulate transcription elongation. Here we report the NMR structural analysis of residues 131-309 of yTFIIS which retains full activity and contains all of domains II and III. We confirm that the structure of domain II in the context of fully active yTFIIS is the same as that determined previously for a shorter construct. We have determined the structure of the C-terminal zinc ribbon domain of active yTFIIS and shown that it is similar to that reported for a shorter construct of human TFIIS. The region linking domain II with the zinc ribbon of domain III appears to be conformationally flexible and does not adopt a single defined tertiary structure. NMR analysis of inactive mutants of yTFIIS support a role for the linker region in interactions with the transcription elongation complex.The rate of transcript elongation by RNA polymerase II is regulated by elongation factors, of which there are two classes with different mechanisms of action. One class, which includes elongin, ELL, pTEFb, and RAP74 stimulates the rate of nucleotide incorporation. The other class, includes TFIIS, its viral and archael sequence homologues, and the bacterial proteins, greA and greB. These proteins do not affect the rate of nucleotide incorporation, but rather stimulate an activity in RNA polymerase II that enables it to transcribe throught blocks to elongation such as DNA-binding proteins, DNA-binding drugs, or particular sequences of DNA that promote transcription arrest (1).Although TFIIS 1 and its bacterial homologues have very similar mechanisms of action, their primary sequences and three-dimensional structures are quite different. GreA and greB each comprise 160 amino acids that form two 80-residue structural domains. In greA, the N-terminal domain is composed of two extended, coiled antiparallel ␣-helices, and can be cross-linked to the nascent RNA when bound to RNA polymerase in a stalled elongation complex (2). The C-terminal domain of greA is globular and is composed of a ␤-sheet that cradles an ␣-helix. GreB is a sequence homologue of greA and likely has an identical tertiary structure. yTFIIS is composed of three domains (domains I, II, and III) as defined by limited proteolysis and structural studies using nuclear magnetic resonance (NMR) spectroscopy (3) (see Fig. 1). Domains II and III, which together extend from residues 131-309 in yeast, are sufficient for transcription activity. Domain II (residues 131-240 in yeast) contains a three-helix bundle (3) and domain III is a zinc ribbon, which contains a three-stranded ␤-sheet, stabilized by a tetrad of cysteine residues that chelate a zinc ion (4). Domain II is conserved in TFIIS homologues and is postulated to int...

Wideline 2H NMR of model membranes was used to consider the molecular consequences of factors often suggested as modulators of complex glycosphingolipid oligosaccharide arrangement and motional characteristics at cell surfaces. GM1, asialo-GM1, and globoside were studied as examples of plasma membrane recognition sites. The experimental approach involved substitution of deuterons (D) for protons at specific locations within the carbohydrate chains. Deuterated glycolipids were then dispersed at 7-10 mol% in unsonicated bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine. Factors tested for their significance to carbohydrate chain conformation and dynamics included glycolipid natural alkyl and acyl chain variability, membrane fluidity, and the presence of cholesterol and a charged sugar residue (neuraminic acid). Effects of Ca2+ and membrane-associated protein were briefly considered. Two distinct strategies were employed in substituting deuterons for selected protons of carbohydrate residues. Neither approach necessitated alteration of the glycolipid natural fatty acid composition. (i) Protons of the exocyclic hydroxymethyl group on the terminal Gal residue of GM1 and asialo-GM1, and on the terminal N-acetylgalactosamine (GalNAc) residue of globoside, were replaced with deuterium (producing -CDHOH) by an enzymatic oxidation/reduction cycle. This represents the first application of such an approach to deuteration of complex neutral glycolipids. Spectral results were compared to those obtained for the similarly-deuterated monoglycosyl lipid, galactosylceramide (GalCer), with natural fatty acid composition. Efficacy of this labeling method may in principle be influenced by structural variations within a given glycolipid family. Also, asymmetric rotation of the deuterated group made it less attractive than the second method for relating spectral features to receptor geometry. (ii) A general synthetic, nonenzymatic method was investigated for replacing amino sugar N-acetyl groups with deuterated acetate (-COCD3). The acetate group of the GalNAc residue of globoside, GM1, and asialo-GM1, as well as that on neuraminic acid in GM1, was replaced with -COCD3. This second method afforded better signal-to-noise--an important consideration for 2H NMR. The NMR technique employed had the potential for detecting changes of as little as 10% in oligosaccharide orientation or motional order. Each glycolipid demonstrated clear evidence of preferred average oligosaccharide conformations in all (fluid) membrane environments examined. The most striking observation was that, in fluid matrices, conformation and motional order of the complex oligosaccharide chains were only modestly influenced by factors tested, including natural variation in the glycolipid hydrocarbon chains, membrane fluidity, temperature, and the presence of cholesterol or the N-acetylneuraminic acid (NeuAc) residue on GM1.(ABSTRACT TRUNCATED AT 400 WORDS)

BackgroundIn water lily (Nymphaea) hybrid breeding, breeders often encounter non-viable seeds, which make it difficult to transfer desired or targeted genes of different Nymphaea germplasm. We found that pre-fertilization barriers were the main factor in the failure of the hybridization of Nymphaea. The mechanism of low compatibility between the pollen and stigma remains unclear; therefore, we studied the differences of stigma transcripts and proteomes at 0, 2, and 6 h after pollination (HAP). Moreover, some regulatory genes and functional proteins that may cause low pollen-pistil compatibility in Nymphaea were identified.ResultsRNA-seq was performed for three comparisons (2 vs 0 HAP, 6 vs 2 HAP, 6 vs 0 HAP), and the number of differentially expressed genes (DEGs) was 8789 (4680 were up-regulated), 6401 (3020 were up-regulated), and 11,284 (6148 were up-regulated), respectively. Using label-free analysis, 75 (2 vs 0 HAP) proteins (43 increased and 32 decreased), nine (6 vs 2 HAP) proteins (three increased and six decreased), and 90 (6 vs 0 HAP) proteins (52 increased and 38 decreased) were defined as differentially expressed proteins (DEPs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the DEGs and DEPs were mainly involved in cell wall organization or biogenesis, S-adenosylmethionine (SAM) metabolism, hydrogen peroxide decomposition and metabolism, reactive oxygen species (ROS) metabolism, secondary metabolism, secondary metabolite biosynthesis, and phenylpropanoid biosynthesis.ConclusionsOur transcriptomic and proteomic analysis highlighted specific genes, incuding those in ROS metabolism, biosynthesis of flavonoids, SAM metabolism, cell wall organization or biogenesis and phenylpropanoid biosynthesis that warrant further study in investigations of the pollen-stigma interaction of water lily. This study strengthens our understanding of the mechanism of low pollen-pistil compatibility in Nymphaea at the molecular level, and provides a theoretical basis for overcoming the pre-fertilization barriers in Nymphaea in the future.

Deuterium decoupled, triple resonance NMR spectroscopy was used to analyze complexes of 2H, 15N, 13C labelled intact and (des2-7) trp repressor (delta 2-7 trpR) from E. coli bound in tandem to an idealized 22 basepair trp operator DNA fragment and the corepressor 5-methyltryptophan. The DNA sequence used here binds two trpR dimers in tandem resulting in chemically nonequivalent environments for the two subunits of each dimer. Sequence- and subunit-specific NMR resonance assignments were made for backbone 1HN, 15N, 13c alpha positions in both forms of the protein and for 13 C beta in the intact repressor. The differences in backbone chemical shifts between the two subunits within each dimer of delta 2-7 trpR reflect dimer-dimer contacts involving the helix-turn-helix domains and N-terminal residues consistent with a previously determined crystal structure [Lawson and Carey (1993) Nature, 366, 178-182]. Comparison of the backbone chemical shifts of DNA-bound delta 2-7 trpR with those of DNA-bound intact trpR reveals significant changes for those residues involved in N-terminal-mediated interactions observed in the crystal structure. In addition, our solution NMR data contain three sets of resonances for residues 2-12 in intact trpR suggesting that the N-terminus has multiple conformations in the tandem complex. Analysis of C alpha chemical shifts using a chemical shift index (CSI) modified for deuterium isotope effects has allowed a comparison of the secondary structure of intact and delta 2-7 tprR. Overall these data demonstrate that NMR backbone chemical shift data can be readily used to study specific structural details of large protein complexes.