A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

High-resolution magic angle spinning 1H NMR analysis of whole cells of Thalassiosira pseudonana (Bacillariophyceae): Broad range analysis of metabolic composition and nutritional value

Chemical composition of the microalga Thalassiosira pseudonana Hasle & Heimdalwas studied with different proton nuclearmagnetic resonance (¹H NMR)techniques, and by comparing NMR spectrafrom extraction samples with a spectrumfrom a sample of whole cells we show thathigh-resolution magic angle spinning (HRMAS) ¹H NMR can be used for broadrange analysis of metabolic composition inmicroalgal whole cells. Signals fromimportant metabolites such aspolyunsaturated fatty acids (PUFAs)eicosapentaenoic (EPA) and docosahexaenoic(DHA) acids were seen in a ¹H NMRspectrum of lipophilic extract, andpossibly also signals from the carotenoidfucoxanthin. In a spectrum of hydrophilicextract we assigned signals to amino acidssuch as glutamine (Gln) and glutamic acid(Glu), carbohydrate and ATP. These findingswere compared to a spectrum of HR MAS¹H NMR analysis of whole cells, whereit was possible to find signals coincidentwith the different metabolites seen inspectra of the extraction samples. Sincethe position of resonance peaks in a NMRspectrum depends on the chemicalsurroundings of each atom at the time ofanalysis some peak shift differencesbetween extract and whole cell samplespectra may occur, but signal shifts werenot significantly different between theanalyses here. In addition, application ofHR MAS highly increased spectral resolutionin the complex whole cell sample. Wetherefore suggest that HR MAS ¹H NMRanalysis is a suitable analysis tool tostudy metabolic composition directly onwhole cells of microalgae, making itpossible to study a broad range ofmetabolites simultaneously without tediousextraction procedures.     

Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [N]H(4) Assimilation in Lemna minor L

Rapid, sensitive, and selective methods for the determination of the ¹⁵N abundance of amino acids in isotopic tracer experiments with plant tissues are described and discussed. Methodology has been directly tested in an analysis of the kinetics of [¹⁵N]H₄⁺ assimilation in Lemna minor L. The techniques utilize gas chromatography-mass spectrometry selected ion monitoring of major fragments containing the N moiety of N-heptafluorobutyryl isobutyl esters of amino acids. The ratio of selected ion pairs at the characteristic retention time of each amino acid derivative can be used to calculate ¹⁵N abundance with an accuracy of ±1 atom% excess ¹⁵N using samples containing as little as 30 picomoles of individual amino acids. Up to 11 individual amino acid derivatives can be selectively monitored in a single chromatogram of 30 minutes. It is suggested that these techniques will be useful in situations where the small quantities of N available for analysis have hitherto hindered the use of ¹⁵N-labeled precursors.     

The use of two-dimensional correlated spectroscopy to obtain new assignments in the high-resolution 1H nuclear magnetic resonance spectrum of the biantennary complex oligosaccharide isolated from human serum transferrin by hydrazinolysis

The asialo biantennary complex type oligosaccharide from human serum transffrrin was isolated by hydrazinolysis, a method which results in the quantitative release of the intact oligosaccharide free of all amino acids. The ¹H-NMR chemical shifts of the previously assigned anomeric and H-2 protons from the peripheral residues of the glycopeptide are identical to the corresponding values for the reduced oligosaccharide. The chemical shift of GlcNAc-1 H-1 proton in the reduced oligosaccharide was assigned by selective deuteration. Proton J connectivities were determined using two-dimensional ¹H-¹H correlated high resolution NMR spectroscopy. Twelve new assignments were made within the central envelope of the NMR spectrum and a further six were tentatively proposed. The ability to assign proton resonances in this way should allow further conformational studies of the oligosaccharide using nuclear Overhauser effects between the relevant assigned protons on different saccharide residues (Homans, S.W., Dwek, R.A., Fernandes, D.L. and Rademacher, T.W. (1982) FEBS Lett. 150, 503–506).     

Optimization of amino acid type-specific <Superscript>13</Superscript>C and <Superscript>15</Superscript>N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm

We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273–6279 (1982)), types of amino acids are labeled with ¹³C or/and ¹⁵N such that cross peaks between ¹³CO(i – 1) and ¹⁵NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with ¹³C and the second with ¹⁵N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B₂R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.     

A solid-state NMR study of the structure and dynamics of the myristoylated N-terminus of the guanylate cyclase-activating protein-2

Guanylate cyclase-activating protein-2 (GCAP-2) is a retinal Ca²⁺ sensor protein. It plays a central role in shaping the photoreceptor light response and in light adaptation through the Ca²⁺-dependent regulation of the transmembrane retinal guanylate cyclase (GC). GCAP-2 is N-terminally myristoylated and the full activation of the GC requires this lipid modification. The structural and functional role of the N-terminus and particularly of the myristoyl moiety is currently not well understood. In particular, detailed structural information on the myristoylated N-terminus in the presence of membranes was not available. Therefore, we studied the structure and dynamics of a 19 amino acid peptide representing the myristoylated N-terminus of GCAP-2 bound to lipid membranes by solid-state NMR. ¹³C isotropic chemical shifts revealed a random coiled secondary structure of the peptide. Peptide segments up to Ala₉ interact with the membrane surface. Order parameters for Cα and side chain carbons obtained from DIPSHIFT experiments are relatively low, suggesting high mobility of the membrane-associated peptide. Static ²H solid-state NMR measurements show that the myristoyl moiety is fully incorporated into the lipid membrane. The parameters of the myristoyl moiety and the DMPC host membrane are quite similar. Furthermore, dynamic parameters (obtained from ²H NMR relaxation rates) of the peptide's myristic acid chain are also comparable to those of the lipid chains of the host matrix. Therefore, the myristoyl moiety of the N-terminal peptide of GCAP-2 fills a similar conformational space as the surrounding phospholipid chains.     

Determination of argininosuccinate in normal blood serum and liver

Argininosuccinate has been determined in normal serum and liver extract of rats by chromatographic analysis on Dowex-1-acetate after a brief period of in vivo labeling with l-[U-¹⁴C] citrulline. The amounts found were within the range of other amino acid intermediates of the ornithine cycle, determined in the same samples. Both ¹⁴C and ninhydrin color were measured. Chromatography on Amberlite columns, subsequent to fractionation on Dowex, allowed determination of the other amino acids. Fractionation on Dowex-1-acetate provides a reliable and highly selective method for the analysis of minute amounts of argininosuccinate in biological samples containing other amino acids.     

An Economical Method for Production of 2H,13CH3-Threonine for Solution NMR Studies of Large Protein Complexes: Application to the 670 kDa Proteasome

NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby ¹³CH₃ methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[α-²H; β−²H;γ-¹³C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics. These reporters are particularly valuable, because unlike other methyl containing amino acids, Thr residues are localized predominantly to the surfaces of proteins, have unique hydrogen bonding capabilities, have a higher propensity to be found at protein nucleic acid interfaces and can play important roles in signaling pathways through phosphorylation. The utility of the labeling methodology is demonstrated with an application to the 670 kDa proteasome core particle, where high quality Thr ¹³C,¹H correlation spectra are obtained that could not be generated from samples prepared with commercially available U-[¹³C,¹H]-Thr.     

A quantitative study of organic phosphate-amine interaction by 31P nuclear magnetic resonance

Interactions between the phosphate group of 4-deoxypyridoxine 5′-phosphate and different protonated amines were quantitatively measured by means of {³¹P}-¹H nuclear magnetic double resonance technique combined with pD titration. An interaction of the phosphate group with added amine resulted in a measurable difference in the ³¹P chemical shift of these phosphate-containing samples with and without amine [Δδ(³¹P)]. Basic amino acids and biogenic amines had significant measurable Δδ(³¹P) values. No interactions were observed for acidic or neutral α, β and γ-amino acids.     

Rap1‐mediated nucleosome displacement can regulate gene expression in senescent cells without impacting the pace of senescence

Cell senescence is accompanied, and in part mediated, by changes in chromatin, including histone losses, but underlying mechanisms are not well understood. We reported previously that during yeast cell senescence driven by telomere shortening, the telomeric protein Rap1 plays a major role in reprogramming gene expression by relocalizing hundreds of new target genes (called NRTS, for n ew R ap1 t argets at s enescence) to the promoters. This leads to two types of histone loss: Rap1 lowers histone level globally by repressing histone gene expression, and it also causes local nucleosome displacement at the promoters of upregulated NRTS. Here, we present evidence of direct binding between Rap1 and histone H3/H4 heterotetramers, and map amino acids involved in the interaction within the Rap1 SANT domain to amino acids 392–394 (SHY). Introduction of a point mutation within the native RAP1 locus that converts these residues to alanines (RAP1^SHY), and thus disrupts Rap1‐H3/H4 interaction, does not interfere with Rap1 relocalization to NRTS at senescence, but prevents full nucleosome displacement and gene upregulation, indicating direct Rap1‐H3/H4 contacts are involved in nucleosome displacement. Consistent with this, the histone H3/H4 chaperone Asf1 is similarly unnecessary for Rap1 localization to NRTS but is required for full Rap1‐mediated nucleosome displacement and gene activation. Remarkably, RAP1^SHY does not affect the pace of senescence‐related cell cycle arrest, indicating that some changes in gene expression at senescence are not coupled to this arrest.     

Decreased labeling of amino acids by inhibition of the utilization of [3H,14C]glucose via the hexosemonophosphate shunt in rat brain in vivo

Treatment of rats with 6-aminonicotinamide showed a small but significant decrease in the labeling of amino acids in the brain after injection of [³H]acetate. The results of these experiments also gave evidence of the presence of [³H]glucose and [³H]lactate, and an increase in [³H]glucose content in the brain of 6-aminonicotinamide treated rats. To apportion the contribution of [³H]glucose formed by gluconeogenesis from [³H]acetate to the labeling of amino acids a method was formulated based on the measurement of radioactivity of amino acids, lactate and free sugars in brain after injection of [6-³H]glucose or [1-³H]glucose relative to that after co-injection of [U-¹⁴C]glucose or [2-¹⁴C]glucose. In contrast to the expected formation of [1, 6-³H]glucose by gluconeogenesis from [³H]acetate,³H-labeled glucose isolated from brain, blood and liver showed the presence of [6-³H]glucose only. The values corrected for the presence of [6-³H]glucose showed that treatment with 6-aminonicotinamide had no effect on the labeling of amino acids by oxidation of [³H]acetate. These findings indicated that a significant decrease in the labeling of amino acids from [U-¹⁴C]glucose reported previously and again confirmed using [1-³H], [6-³H], [2-¹⁴C] or [U-¹⁴C]glucose in the present investigation was not due to the inhibition of the activities of enzymes of the citric acid cycle. These results support the postulated role of the hexosemonophosphate shunt for the utilization of glucose in providing neurotransmitter amino acids glutamate and -aminobutyrate.Dedicated to Professor K. A. C. Elliott on his 80th birthday.     

Callyaerins A–F and H,new cytotoxic cyclic peptides from the Indonesian marine sponge Callyspongia aerizusa

Bioassay guided fractionation of the EtOAc fraction of the sponge Callyspongia aerizusa yielded seven new cytotoxic cyclic peptides callyaerins A–F (1–6) and H (8). Their structures were determined using extensive 1D (¹H, ¹³C and DEPT) and 2D (COSY, HMQC, HMBC, TOCSY, and ROESY) NMR and mass spectral (ESI and HRESI-TOF) data. All compounds were cyclic peptides containing ring systems of 5–9 amino acids and side chains of 2–5 amino acids in length. An unusual (Z)-2,3-diaminoacrylic acid unit provided the template for ring closure and afforded the linkage to the peptidic side chain which was always initiated with a proline moiety. All peptides contained three or more proline residues and the remaining residues were predominantly hydrophobic residues with all amino acids present in the l form. Callyaerins A–F (1–6) and H (8) showed biological activity in antibacterial assays and in various cytotoxicity assays employing different tumour cell-lines (L5178Y, HeLa, and PC12). Callyaerins E (5) and H (8) exhibited strong activity against the L5178Y cell line with ED₅₀ values of 0.39 and 0.48 μM, respectively. On the other hand, callyaerin A (1) showed strong inhibitory properties towards C. albicans.     

1H HRMAS-NMR based metabolic fingerprints for discrimination of cheeses based on sensory qualities

In this study, the ¹H HRMAS-NMR (High-resolution Magic Angle Spinning-Nuclear Magnetic Resonance) spectra of 52 cheese samples obtained from the South Korean dairy farms were evaluated for their metabolic profiling and intensities associating with the sensory qualities. The NMR profiles displayed a broad range of compounds comprising amino acids, carbohydrates, organic acids, and phospholipids. Afterwards, the cheese samples were categorized into three groups (more likeness - G1, moderate likeness - G2, less likeness - G3), in relating to their sensory scores. The NMR data of the samples were later investigated through multivariate statistical tools to define the variations in metabolic fingerprints of every cheese sample and their intensities hailing in individual sensory groups. The unsupervised PCA employing all cheese samples unveiled the uniqueness in metabolite profiles of the brown and cheddar type cheeses (outliers). Moreover, Gouda and other types of cheeses displayed samples positioning in respective of their metabolite profiles. The pairwise comparison of sensory groups in the supervised models perceived better separation in OPLS-DA than PLS-DA. The corresponding VIP (PLS-DA) and loading (OPLS-DA) plots revealed amino acids and organic acids (lactate, citrate) as significant variables. The discrimination of G 1 Gouda type of cheeses against G 2 and G 3 was highly associated with their citrate levels. Further investigation using heatmaps displayed clear differentiation between each sensory group in terms of the levels of amino acids, lactate, citrate, phospholipids, and glycerol, conveying these variations are likely due to proteolytic and metabolic processes in cheese ripening. This study concluded that ¹H HRMAS-NMR metabolite profile of the Korean cheeses is consistence with their sensory qualities. Further, the candidate metabolites identified in this study confers their potential application as biomarkers in cheese industries for faster and effective validation of sensory characteristics.     

A dansylation microassay for some amino acids in brain.

A method is described for the determination of γ-amino butyric acid, glycine, glutamine, glutamate, asparate, and taurine in small samples of brain tissue. This is an extension of a method based on the formation of derivatives with [³H]dansyl chloride (15) with the addition of ¹⁴C-labeled amino acids as internal standards to allow correction for incomplete and variable degrees of dansylation. An improved chromatographic separation of dansyl-taurine is employed. Estimates of the contents of these amino acids in rat cortical tissue are reported and compared with those obtained on an autoanalyser, and with those in the literature using autoanalytical and other methods.     

Inhibition of macromolecular synthesis in tumors by L-1-tosylamido-2-phenylethyl chloromethyl ketone.

L-1-tosylamido-2-phenylethyl chloromethyl ketone was observed to inhibit the incorporation of [³H] amino acids into protein and [³H] thymidine incorporation into DNA in Novikoff hepatoma ascites cells $\text{in vitro}$ Similar effects were seen with several Morris hepatomas and a transplanted colon tumor in rats, and were accompanied by decreased uptake of isotope into acid soluble tissue fractions. Under the same conditions, there was no significant inhibition in regenerating liver and there was an increased uptake of [³H] amino acids in the livers of normal and tumor bearing rats.     

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids

Summary Two-dimensional (2D)¹H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the¹H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D¹H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.     

Synthesis of Glucuronides of Fluoropyrimidine Drugs: N- and O-Glucuronides of 5-Fluorocytosine and 5-Fluorouracil

Abstract

The protected (4a-9a) and deprotected (4b-6b, 8b) glucuronides of 5-fluorocytosine and 5-fluorouracil were synthesized and characterized by mass spectrometry and ¹⁹F, ¹H and ¹³C NMR. The substitution position of the sugar moiety on the pyrimidine ring was determined from the ¹³C NMR chemical shift of the C1′ of the sugar. The α or β configuration of the glucuronide linkage was assigned on the basis of the value of the coupling constant between H1′ and H2′ of the sugar.     

Metabolomic profiling of Cheonggukjang during fermentation by 1H NMR spectrometry and principal components analysis

Metabolomic analysis of extracts of Cheonggukjang was carried out using ¹H nuclear magnetic resonance (NMR) spectrometry and principal components analysis (PCA). The major peaks in the ¹H NMR spectra of the 50% methanol fraction were assigned to isoleucine/leucine, lactate, alanine, acetic acid, citric acid, choline, fructose, sucrose, tyrosine, phenylalanine and formic acid. The first two principle components (PC1 and PC2) of the ¹H NMR spectra of the aqueous fraction allowed discrimination of Cheonggukjang extracts of samples obtained after different periods of fermentation. These two principal components cumulatively accounted for 98.5% of the total variation of all variables. The major peaks within the ¹H NMR spectra that contributed to discrimination of different samples were assigned to isoleucine/leucine, lactate, acetic acid, citric acid, choline, fructose, glucose and sucrose. This metabolomic analysis of samples of Cheonggukjang extract demonstrates that NMR and PCA can be used to obtain standard trajectory plots and related information for Cheonggukjang and other fermented foods.     

Terahertz time-domain spectroscopy of amino acids and polypeptides

Frequency-dependent absorption coefficients and refractive indices of amino acids (glycine and l-alanine) and polypeptides (polyglycine and poly-l-alanine) in the wavenumber region from 7 to 55 cm⁻¹ were measured by terahertz time-domain spectroscopy. A vibrational band was observed at 45.5 cm⁻¹ for polyglycine, which was assigned as an interchain mode. The reduced absorption cross sections of the amino acids and polypeptides show power-law behavior. The exponents are different between the monomers and polymers, and those of the two polypeptides suggest that the time dependences of the total dipole moments are similar in the timescale of subpico- to picoseconds.     

Equilibration of H labeling between body water and free amino acids: Enabling studies of proteome synthesis

Protein synthesis can be estimated by measuring the incorporation of a labeled amino acid into a proteolytic peptide. Although prelabeled amino acids are typically administered, recent studies have tested ²H₂O; the assumption is that there is rapid equilibration of ²H (in body water) with the carbon-bound hydrogens of amino acids before those amino acids are incorporated into a protein(s). We have determined the temporal changes in ²H labeling of body water and amino acids which should build confidence in ²H₂O-based studies of protein synthesis when one aims to measure the ²H labeling of proteolytic peptides.