Sites For Isomerization Research Articles

Proline Isomerization of Histone H3 Regulates Lysine Methylation and Gene Expression

The cis-trans isomerization of proline serves as a regulatory switch in signaling pathways. We identify the proline isomerase Fpr4, a member of the FK506 binding protein family in Saccharomyces cerevisiae, as an enzyme which binds the amino-terminal tail of histones H3 and H4 and catalyses the isomerization of H3 proline P30 and P38 in vitro. We show that P38 is necessary for methylation of K36 and that isomerization by Fpr4 inhibits the ability of Set2 to methylate H3 K36 in vitro. These results suggest that the conformational state of P38, controlled by Fpr4, is important for methylation of H3K36 by Set2. Consistent with such an antagonistic role, abrogation of Fpr4 catalytic activity in vivo results in increased levels of H3K36 methylation and delayed transcriptional induction kinetics of specific genes in yeast. These results identify proline isomerization as a novel noncovalent histone modification that regulates transcription and provides evidence for crosstalk between histone lysine methylation and proline isomerization.

Accurate Measurements of13C−13CJ-Couplings in the Rhodopsin Chromophore by Double-Quantum Solid-State NMR Spectroscopy

A new double-quantum solid-state NMR pulse sequence is presented and used to measure one-bond 13C-13C J-couplings in a set of 13C2-labeled rhodopsin isotopomers. The measured J-couplings reveal a perturbation of the electronic structure at the terminus of the conjugated chain but show no evidence for protein-induced electronic perturbation near the C11-C12 isomerization site. This work establishes NMR methodology for measuring accurate 1JCC values in noncrystalline macromolecules and shows that the measured J-couplings may reveal local electronic perturbations of mechanistic significance.

Pin1 in a mRNA Stability Complex

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that promotes eosinophil survival, maturation, and function. Factors, such as hyaluronic acid, that stimulate production of this cytokine are increased in asthmatic lung, as is the abundance of eosinophils. Eosinophils from asthmatic patients also exhibit elevated GM-CSF. Shen et al. show that Pin1, a peptidyl-prolyl isomerase specific for bonds between phosphorylated Ser-Pro or phosphorylated Thr-Pro, is part of a ribonucleoprotein complex that regulates the stability of GM-CSF mRNA. Pharmacological inhibition of Pin1 with juglone prevented increased mRNA abundance in and secretion of GM-CSF from isolated eosinophils in response to hyaluronic acid. The increase in GM-CSF mRNA was due to increased stability, not increased transcription. Pin1 coimmunoprecipitated with all four subunits of the AU-rich binding protein AUF1 (p45, p42, p40, and p37), of which p45 and p40 each have a potential Pin1 isomerization site. Hyaluronic acid triggered the dissociation of AUF1 from GM-CSF mRNA and the association of hnRNP C with GM-CSF mRNA, but only under conditions in which Pin1 was active. Hyaluronic acid stimulated the dephosphorylation of Pin1, which increased the activity of Pin1, and stimulated the phosphorylation of AUF1. In resting cells, the p37 subunit of AUF1 and Pin1 immunoprecipitated with the exosome-associated protein PM-Scl175, and hyaluronic acid decreased the association of p37 with the exosome. (The exosome is a complex associated with AU-rich mRNA decay.) Juglone blocked the prolonged survival observed for eosinophils isolated from allergen-challenged allergic donor patients, verifying the importance of Pin1 in vivo. The model for Pin1 in mRNA stability is as follows: In resting cells, Pin1 is inactive and the AUF1 complex recruits the exosome to decrease GM-CSF mRNA stability (see Anderson). Upon stimulation, Pin1 becomes dephosphorylated and AUF1 subunits are phosphorylated, leading to the release of the AUF1 complex from the mRNA and the binding of the hnRNP C, which promotes mRNA stability and allows increased production of the GM-CSF protein. Z.-J. Shen, S. Esnault, J. S. Malter, The peptidyl-prolyl isomerase Pin1 regulates the stability of granulocyte-macrophage colony-stimulating factor mRNA in activated eosinophils. Nat. Immunol. 6 , 1280-1287 (2005). [PubMed] P. Anderson, Pin1: A proline isomerase that makes you wheeze? Nat. Immunol. 6 , 1211-1212 (2005). [Online Journal]

Post-translational amino acid racemization in the frog skin peptide deltorphin I in the secretion granules of cutaneous serous glands

The dermal glands of the South American hylid frog Phyllomedusa bicolor synthesize and expel huge amounts of cationic, α-helical, 24- to 33-residue antimicrobial peptides, the dermaseptins B. These glands also produce a wide array of peptides that are similar to mammalian hormones and neuropeptides, including a heptapeptide opioid containing a d-amino acid, deltorphin I (Tyr– dAla–Phe–Asp–Val–Val–Gly NH 2). Its biological activity is due to the racemization of l-Ala2 to d-Ala. The dermaseptins B and deltorphins are all derived from a single family of precursor polypeptides that have an N-terminal preprosequence that is remarkably well conserved, although the progenitor sequences giving rise to mature opioid or antimicrobial peptides are markedly different. Monoclonal and polyclonal antibodies were used to examine the cellular and ultrastructural distributions of deltorphin I and dermaseptin B in the serous glands by immunofluoresence confocal microscopy and immunogold-electron microscopy. Preprodeltorphin I and preprodermaseptins B are sorted into the regulated pathway of secretion, where they are processed to give the mature products. Deltorphin I, [ l-Ala2]-deltorphin I and dermaseptin B are all stored together in secretion granules which accumulate in the cytoplasm of all serous glands. We conclude that the l- to d-amino acid isomerization of the deltorphin I occurs in the secretory granules as a post-translational event. Thus the specificity of isomerization depends on the presence of structural and/or conformational determinants in the peptide N-terminus surrounding the isomerization site.

Bond torsion affects the product distribution in the photoreaction of retinal model chromophores

Ab initio molecular dynamics (MD) calculations have been performed to study the photoisomerization of a 3-double-bond retinal model chromophore, the all-trans-4, 6-dimethylpenta-3, 5-dieniminium cation, and the possible influence of non-planar distortions on the product distribution. In total, 171 trajectories have been generated for four different conformations of the structure, a planar one and three in which the C4-C5 and the C5=C6 bonds were increasingly twisted out of plane. Starting geometries randomly distributed about the equilibrium geometry were generated by zero-point energy sampling; trajectories were calculated using CASSCF-BOMD methodology and were followed until the photoproduct and its configuration could be assigned. For the latter, two different approaches were applied, one involving the CASSCF configuration vectors, the other an analysis of the MD at the first possible hopping event. Isomerization was found to occur almost exclusively about the central C3=C4 double bond in the case of the planar model compound. Twisting the conjugated pi-system shifts the isomerization site from the central double bond to the terminal C5=C6 double bond. With both the C4-C5 and the C5=C6 bonds twisted by 20 degrees, about 35% of the trajectories lead to the configurationally inverted 5-cis product. The results are discussed with reference to the highly selective and efficient photo-induced isomerization of the retinal chromophore in rhodopsin.

Post-translational Amino Acid Isomerization: A FUNCTIONALLY IMPORTANT d-AMINO ACID IN AN EXCITATORY PEPTIDE

The post-translational modification of an L- to a D-amino acid has been documented in relatively few gene products, mostly in small peptides under 10 amino acids in length. In this report, we demonstrate that a 46-amino acid polypeptide toxin has one D-phenylalanine at position 44, and that the epimerization from an L-Phe to a D-Phe has a dramatic effect on the excitatory effects of the peptide. In one electrophysiological assay carried out, the D-Phe-containing peptide was extremely potent, whereas the unmodified polypeptide had no biological activity, demonstrating that the chirality of the post-translationally modified amino acid is functionally significant. The peptide toxin analyzed, r11a, belongs to the I-gene superfamily of conotoxins that has four disulfide cross-links. The D-Phe in r11a is at the third amino acid from the C terminus, the same relative position from the C-terminal end as the d-amino acid in omega-agatoxin TK from a spider, an unrelated peptide. Thus, although post-translational amino acid isomerization appears to have no strong specificity for the chemical nature of the amino acid side chain, the few peptides where this modification has been established suggest that there may be favored positions near the N or C terminus that are preferential sites for isomerization to a D-amino acid.

Enhanced diastereoselectivity via confinement: photoisomerization of 2,3-diphenylcyclopropane-1-carboxylic acid derivatives within zeolites.

From the perspective of asymmetric induction, the photochemistry of 24 chiral esters and amides of cis-2,3-diphenylcyclopropane-1-carboxylic acid from excited singlet and triplet states has been investigated within zeolites. The chiral auxiliaries placed at a remote location from the isomerization site functioned far better within a zeolite than in solution. Generally, chiral auxiliaries with an aromatic or a carbonyl substituent performed better than the ones containing only alkyl substituents. A model based on cation-binding-dependent flexibility of the chiral auxiliary accounts for the observed variation in de between aryl (and carbonyl) and alkyl chiral auxiliaries within zeolites. Cation-dependent diastereomer switch was also observed in select examples.

Sol–gel synthesis, characterization and catalytic properties of Fe–Ti mixed oxides

Fe 2O 3-TiO 2 solid acid catalysts have been prepared by a sol–gel route, characterized and tested in the isomerization of α-pinene oxide. The textural, morphological and microstructural characterization of the catalysts was carried out by BET surface area measurements, X-ray diffraction (XRD) and scanning electron microscopy with elemental X-ray analysis (SEM-EDX). Characterization data have shown that, on the pure TiO 2 catalyst , titania is mainly in the anatase phase. On the contrary the rutile phase and/or Fe 2TiO 5, amorphous iron–titanium mixed oxides and hematite were found on the Fe-promoted TiO 2 catalysts, depending on the Fe content. Temperature programmed desorption (TPD), microcalorimetry and FT-IR spectroscopy of adsorbed probe basic molecules (ammonia or pyridine) have shown that addition of iron to titania favours formation of new acid sites having a stronger Lewis character. Fe 2O 3-TiO 2 catalysts show a higher activity compared to pure titania in the isomerization of α-pinene oxide to campholenic aldehyde. The products distribution was also influenced by the composition of the catalysts. A correlation between the selectivity to campholenic aldehyde and the amount of Lewis acid sites on the catalysts was found. Based on the characterization and catalytic results, Fe–Ti–O mixed structures were proposed to be the active sites for the selective isomerization of α-pinene oxide to campholenic aldehyde.

On the Effect of the Strength of Acid Sites in Heterogeneous Catalysts on the Activity in the Skeletal Isomerization of n-Butane

The catalytic activity of three groups of acid catalysts different in the nature and strength of acid sites in the skeletal isomerization of n-butane was studied. It was found that the strength of the sites did not correlate with the rate of the reaction.

A method for the detection of asparagine deamidation and aspartate isomerization of proteins by MALDI/TOF-mass spectrometry using endoproteinase Asp-N.

A method was established for evaluating Asn deamidation and Asp isomerization/racemization. To detect the subtle changes in mass that accompany these chemical modifications, we used a combination of enzyme digestion by endoproteinase Asp-N, which selectively cleaves the N-terminus of L-alpha-Asp, and MALDI/TOF-mass spectrometry. To achieve better resolution, we employed digests of (15)N-labeled protein as an internal standard. To demonstrate the advantages of this method, we applied it to identify deamidated sites in mutant lysozymes in which the Asn residue is mutated to Asp. We also identified the deamidation or isomerization site of the lysozyme samples after incubating them under acidic or basic conditions.

H 2-reduced Pt/MoO 3 as a selective catalyst for heptane isomerization

H 2 reduction of Pt/MoO 3 was accompanied by an increase in the surface area. H 2-reduced Pt/MoO 3 exhibited the largest surface area of 250 m 2/g at reduction degrees of 60–70%. The surface area was enlarged only when Pt/MoO 3 was reduced after heating in a stream of H 2. The catalytic activity of H 2-reduced Pt/MoO 3 for heptane isomerization was also dependent on the extent of reduction. The highest isomerization activity appeared at a reduction degree of about 70%. Heptane was selectively isomerized on H 2-reduced Pt/MoO 3 with reduction degrees below 80%, and the high selectivity was preserved even at high conversion levels. The catalytic behaviors of H 2-reduced Pt/MoO 3 were strongly affected by heating atmosphere. Reduction after heating in H 2 provided a much more active and selective catalyst for heptane isomerization than reduction after heating in N 2. We suggest from the results of XRD measurements that reduction of Pt/MoO 3 through the formation of a H x MoO 3 phase can be an important step to enlarge the surface area and to generate the active sites for heptane isomerization.

Probing the Accessible Sites for n-Butene Skeletal Isomerization over Aged and Selective H-Ferrierite with d3-Acetonitrile

Aged H-ferrierite (H-FER) samples with different contents of deposits were prepared and studied under differential catalytic conditions in a tapered element oscillating microbalance. Subsequently, these samples were examined using infrared spectroscopy to determine the nature of carbonaceous deposits and probing the type and number of accessible sites with d 3-acetonitrile. From these results, for the first time, we have been able to calculate turnover frequencies (TOFs) for n-butene conversion and isobutene formation for both fresh and aged H-FER catalysts. It is observed that the deposition of carbonaceous species significantly lowers the number of accessible Brønsted sites. With short time on stream (TOS), cracking of the alkyl-aromatic deposits contributes to the overall isobutene production but simultaneously harms the selective catalytic action by inducing nonselective side reactions. With prolonged TOS these deposits become nonreactive and as a result the TOF drops, while isobutene is produced with high selectivity. It is demonstrated that at this stage no carbenium ions are detected, while Brønsted OH acid sites are still accessible for d 3-acetonitrile on the extensively aged and highly selective H-FER. This indicates that the latter sites are responsible for the selective catalytic conversion of n-butene into isobutene.

An FTIR study of the accessibility of the protonic sites of H-mordenites

The interaction of different probes with two H-MOR samples has been studied by IR. In the case of the sample with Si/Al = 10 acetonitrile perturbs all the hydroxy groups while 2,2-dimethylpropionitrile (pivalonitrile) perturbs only very few. Pyridine also perturbs all the hydroxy groups but only some of them protonate the pyridine, the others only H-bond to it. n-Hexane and 2,2-dimethylbutane give the same result, perturbing only some of the bridging hydroxy groups. The results are interpreted by assuming that no OHs are located in the 8-ring channels, and that the hydrocarbons cannot interact (due to the steric hindrance of the methyl group) with the OHs located in the side pockets. On the contrary, the flat molecule pyridine can enter slightly into the side pockets and H-bond with the OHs there. Pivalonitrile interacts only with the OHs which are well exposed in the main channels. It is concluded that the active sites for alkane isomerization are likely exclusively those that are well exposed in the main channels of H-MOR and that Al substitution in the T3 sites probably does not occur. The sample with Si/Al 45, taken as an example of a dealuminated sample, presents many less bridging OHs which are entirely available for interaction with even pivalonitrile.

WO x/ZrO 2 catalysts : Part 2. Isomerization of n-butane

The activity of WO x /ZrO 2 (ZW) catalysts for n-butane isomerization was investigated in the temperature range 423–673 K. Reference samples of crystalline WO 3 and ZrO 2 displayed low catalytic activity. When ZW catalysts were calcined at 873 or 1073 K, samples prepared from amorphous hydrous zirconia showed activity levels—both on areal rate and turnover frequency per total tungsten bases—that were 1 order of magnitude higher than those of the samples prepared from crystalline zirconia. The higher the calcination temperature in the range 873–1073 K the higher was the activity. The deactivation due to coking was fast, but treatment with flowing oxygen at 773 K completely restored the catalytic activity for several cycles. The activity decrease with time on stream was satisfactorily described up to 623 K by the empirical equation r= at − n . Active isomerization sites in the catalysts produced coke (average composition H/C=1.5). The presence of hydrogen in the reactant stream substantially decreased the deactivation rate, most probably by hindering the dehydrogenation reactions which ultimately lead to coke formation. Treating catalysts in hydrogen at 673 or at 443 K before the reaction shortened the induction period, suggesting that hydrogen adsorption had a role in generating hydroxyls active for the isomerization (Brønsted acid sites). The activity trend with surface density of tungsten indicated a polynuclear isomerization site.

The leucine zipper motif of the envelope glycoprotein ectodomain of human immunodeficiency virus type 1 contains conformationally flexible regions as revealed by NMR and circular dichroism studies in different media.

A 43-mer peptide derived from the coiled coil domain of the transmembrane glycoprotein, gp41, of human immunodeficiency virus type 1, was synthesized. Light scattering measurements suggested that the peptide molecules likely exist in the aqueous solution in trimeric form. Circular dichroism experiments showed a moderate helix population enhancement for the peptide in 80% methanol solution relative to helicity in sodium dodecyl sulfate micellar suspension. NMR spectroscopy indicated that the N-terminal section of the peptide was conformationally more sensitive to the medium. The conformationally labile regions contain residues implicated in gp41-gp120 association. Our data support the idea that the coiled coil region is responsible for oligomerization of the gp41 ectodomain and suggest a site of conformational isomerization following receptor binding-induced gp120 dissociation from gp41.

L to d Amino Acid Isomerization in a Peptide Hormone Is a Late Post-translational Event Occurring in Specialized Neurosecretory Cells

Modification of the chirality of a single amino acid residue within a peptide chain appears to be novel additional mechanism leading to structural and functional diversification of eukaryotic bioactive peptides. This phenomenon has been studied at the cellular level in a neuroendocrine organ which elaborates a mixture of diastereoisomers of a 72-residue neuropeptide, crustacean hyperglycemic hormone. For the first time, amino acid isomerization has been shown to occur in the perikarya of fully specialized neurosecretory cells, as a late step of the maturation of the hyperglycemic hormone precursor and after propeptide cleavage. The specificity and efficiency of this phenomenon indicates the existence of a new enzyme family involved in the biogenesis of peptide hormones.

Skeletal isomerization of n-butenes to isobutene over acid-treated natural clinoptilolite zeolites

The proton form of the natural clinoptilolite zeolite (HNZ) was modified by treatment with various acids. Effects of modification on catalytic performance were investigated for the skeletal isomerization of n-butenes to isobutene. Among HNZs treated with various acids, only those modified with boric acid (B-HNZ) showed an improved catalytic performance of skeletal isomerization in terms of activity and stability. Furthermore, B-HNZ exhibited higher isobutene selectivity compared to unmodified HNZ at the same conversion of n-butenes. The performance was comparable to that of a widely known ferrierite catalyst at the same reaction conditions. The main effect of boric acid treatment on HNZ was the selective increase in the number of acid sites of moderate strength required for efficient catalysts in skeletal isomerization without collapse of its framework. These acid sites of moderate strength were considered to be selective and stable reaction sites for skeletal isomerization of n-butenes. Coke deposition poisoned non-selective strong acid sites and imposed a spatial restriction on zeolite channels, leading to suppression of undesired dimerization reactions. Factors affecting selectivity to isobutene are discussed.

Analysis of isoaspartate in peptides by electrospray tandem mass spectrometry.

In view of the significance of Asn deamidation and Asp isomerization to isoAsp at certain sites for protein aging and turnover, it was desirable to challenge the extreme analytical power of electrospray tandem mass spectrometry (ESI-MS/MS) for the possibility of a site-specific detection of this posttranslational modification. For this purpose, synthetic L-Asp/L-isoAsp containing oligopeptide pairs were investigated by ESI-MS/MS and low-energy collision-induced dissociation (CID). Replacement of L-Asp by L-isoAsp resulted in the same kind of shifts for all 15 peptide pairs investigated: (1) the b/y intensity ratio of complementary b and y ions generated by cleavage of the (L-Asp/L-isoAsp)-X bond and of the X-(L-Asp/L-isoAsp) bond was decreased, and (2) the Asp immonium ion abundance at m/z 88 was also decreased. It is proposed that the isoAsp structure hampers the accepted mechanism of b-ion formation on both its N- and C-terminal side. The b/y ion intensity ratio and the relative immonium ion intensity vary considerably, depending on the peptide sequence, but the corresponding values are reproducible when recorded on the same instrument under identical instrumental settings. Thus, once the reference product ion spectra have been documented for a pair of synthetic peptides containing either L-Asp or L-isoAsp, these identify one or the other form. Characterization and relative quantification of L-Asp/L-isoAsp peptide mixtures are also possible as demonstrated for two sequences for which isoAsp formation has been described, namely myrG-D/isoD-AAAAK (deamidated peptide 1-7 of protein kinase A catalytic subunit) and VQ-D/isoD-GLR (deamidated peptide 41-46 of human procollagen alpha 1). Thus, the analytical procedures described may be helpful for the identification of suspected Asn deamidation and Asp isomerization sites in proteolytic digests of proteins.

Differentiating alpha- and beta-aspartic acids by electrospray ionization and low-energy tandem mass spectrometry.

Spectra obtained by low-energy electrospray ionization tandem mass spectrometry (ESI-MS/MS) of 34 peptides containing aspartic acids at position n were studied and unambiguously differentiated. beta-Aspartic acid yields an internal rearrangement similar to that of the C-terminal rearrangements of protonated and cationized peptides. As a result of this rearrangement, two different ions containing the N- and the C-terminal ends of the original peptide are formed, namely, the bn-1 + H2O and y"l - n + 1 - 46 ions, respectively, where e is the number of amino acid residues in the peptide. The structure suggested for the y"l - n + 1 - 46 ion is identical to that proposed for the vn ions observed upon high-energy collision-induced dissociation (CID) experiments. The intensity of these ions in the low-energy MS/MS spectra is greatly influenced by the presence and position of basic amino acids within the sequences. Peptides with a basic amino acid residue at position n - 1 with respect to the beta-aspartic acid yield very intense bn-1 + H2O ions, while the y"l - n + 1 - 46 ion was observed mostly in tryptic peptides. Comparison between the high- and low-energy MS/MS spectra of several isopeptides suggests that a metastable fragmentation process is the main contributor to this rearrangement, whereas for long peptides (40 AA) CID plays a more important role. We also found that alpha-aspartic acid containing peptides yield the normal immonium ion at 88 Da, while peptides containing beta-aspartic acid yield an ion at m/z 70, and a mechanism to explain this phenomenon is proposed. Derivatizing isopeptides to form quaternary amines, and performing MS/MS on the sodium adducts of isopeptides, both improve the relative intensity of the bn + 1 + H2O ions. Based on the above findings, it was possible to determine the isomerization sites of two aged recombinant growth proteins.

Stereoselective Photodimerization of (E)-Stilbenes in Crystalline γ-Cyclodextrin Inclusion Complexes

Solid-state irradiation of the crystalline inclusion complex of (E)-stilbene in gamma-cyclodextrin (gamma-CD) yields a single isomer of syn-tetraphenylcyclobutane stereoselectively in high yield. In contrast, the photodimerization of stilbene in solution is very inefficient and unselective, and no photodimer is observed even upon prolonged irradiation of pure crystals. The monosubstituted stilbenes form a pair of photodimers stereoselectively, viz. the syn head-to-head and syn head-to-tail isomers, in comparable yields. The photodimer yields of about 70% and the biphasic decay kinetics of the excited stilbene (as established by picosecond time-resolved diffuse-reflectance spectroscopy) indicate that the stilbene guests are located in at least two distinct sites in the gamma-CD crystal lattice, i.e., a dimerization site where excited stilbene is in close reach of another stilbene guest molecule and an isomerization site where excited stilbene does not find a close neighbor for dimerization and thus undergoes trans --> cis isomerization only.