Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy

We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual ¹⁵N–T ₁ timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s⁻¹. Backbone amide ¹⁵N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41ε. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D₂O is employed as a solvent for sample preparation. Due to the intrinsically long ¹⁵N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.     

Bile acid amidoalcohols: simple organogelators

Simple bile acid amide synthesis of lithocholic and deoxycholic acids with 2-aminoethanol and 3-aminopropanol are reported. The structural properties of these amides were examined by NMR spectroscopic, ESI-TOF mass spectral, and X-ray crystallographic methods. The gelation properties of these amides in common organic solvents and in three different water solutions were also investigated using Tyndall effect, SEM, TEM, and optical microscopy. 2-Hydroxyethylamides were found to be effective gelators in chlorinated organic solvents and 3-hydroxypropylamides in aromatic solvents. Both derivatives thicken neutral and acidic water solutions.     

Hydration structure and dynamics of B- and Z-DNA in the presence of counterions via molecular dynamics simulations

Following our previous attempts at understanding the structural and dynamical properties of water and counterions hydrating nucleic acids, we have performed molecular dynamics simulations for B- and Z-DNA. In these simulations, the nucleic acids were held rigid. In the case of B-DNA, one turn of B-DNA double helix was considered in the presence of 1500 water molecules and 20 counterions (K⁺). The simulations were performed for 4.0 ps after equilibrating the system. For Z-DNA, we considered one turn of the double helix in the presence of 1851 water molecules and 24 counterions (K⁺). The simulations were carried out for 3.5 ps after equilibration. The average temperature of these simulations was ～ 360 K for Z-DNA and ～ 345 K for B-DNA. In these simulations the hydrogen atoms were explicitly taken into account. For both simulations, a fifth-order predictor-corrector was used for solving the translational equations of motion. The rotational motion of the water molecules was represented in terms of quaternion algebra and the rotational equations of motion were solved with a second-order quaternion method using a sixth-order predictor-corrector method. A time step of 0.5 · 10^?15 s was used in these simulations. The structural and the dynamical properties of water solvating the counterions, and the phosphate groups of the DNA, were computed to understand the hydration structure. Diffusion coefficients and velocity correlation functions were calculated for both ions and the water molecules. The velocity correlation functions for the ions exhibit a caged behavior. The dipole correlation functions for the water molecules indicate that the water molecules close to the helix retain the memory of their initial orientations for longer periods of time than those away from the helix. During the time period of our simulation (3–4 ps) the ion probability distributions show a well-defined pattern and suggest limited mobility for the ions, being close to the helix.     

Hydration of cis and trans N-methylformamide as revealed by the use of 17O-NMR,molecular mechanics,and ab initio calculations

The solvation of cis and trans N-methylformamide (NMF) by water was investigated using a combination of ¹⁷O-nmr spectroscopy, classical molecular mechanics [MM2(77) and MM2(87)] force field, and ab initio 4-31G* gradient optimization calculations. In dilute aqueous solution, the ¹⁷O-nmr spectra of NMF indicate strong shielding by 66.9 and 66.1 ppm for the cis and trans amide oxygens, respectively, compared to those values obtained in dilute toluene solution. This demonstrates that both isomers are equally solvated by molecules of water, which are further hydrogen bonded to molecules of water of the bulk solvent. Molecular mechanics simulations were carried out for cis and trans NMF in a cluster of water molecules. Radial distribution functions show structural contacts by several water molecules at the amide CO and NH group, which are significantly more pronounced with MM2 (87) calculations. Ab initio 4-31G* gradient optimization calculations on the supermolecule trans NMF-(H₂O)₃ indicates the presence of more than two hydrogen-bond contacts at the carbonyl oxygen. This is in agreement with MM2 calculations and provides further evidence for multiple acceptor properties of the amide oxygen and an out of the amide plane arrangement of the bound molecules of water. Comparison of the integration data to the first radial distribution function (rdf) minima shows that the local solvation of the CO and NH groups is very similar for both cis and trans isomers. The intermolecular geometric parameters of the supermolecule trans NMA–(H₂O)₃ and the first rdf maxima resulting from MM2 (87) and MM2 (77) calculations are compared with distribution of water molecules around the CO and NH groups of peptides and proteins resulting from x-ray and neutron diffraction experiments. The rdfs involving the methyl group of NMF demonstrate the nonrandom distribution of solvent sites with first maxima in reasonable agreement with distribution of water molecules around the apolar side chain of amino acid residues in proteins. © 1995 John Wiley & Sons, Inc.     

On the interaction of lithium salts with model amides

Results of a study of the interaction of alkali metal salts on model aliphatic amides are reported. Lithium salts appear to interact more strongly with amides than those of other alkali metals. Spectroscopic investigations suggest that Li⁺ ion binds to the amide group at the carbonyl oxygen, causing a change in the spectroscopic properties and the geometry of the amide. Such binding of ions to amide groups may be of importance when one studies the spectral and conformational changes of polypeptides and proteins in high salt media.     

Detection of dynamic water molecules in a microcrystalline sample of the SH3 domain of α-spectrin by MAS solid-state NMR

Water molecules are a major determinant of protein stability and are important for understanding protein–protein interactions. We present two experiments which allow to measure first the effective T₂ decay rate of individual amide proton, and second the magnetization build-up rates for a selective transfer from H₂O to H^N using spin diffusion as a mixing element. The experiments are demonstrated for a uniformly ²H, ¹⁵N labeled sample of a microcrystalline SH3 domain in which exchangeable deuterons were back-substituted with protons. In order to evaluate the NMR experimental data, as X-ray structure of the protein was determined using the same crystallization protocol as for the solid-state NMR sample. The NMR experimental data are correlated with the dipolar couplings calculated from H₂O–H^N distances which were extracted from the X-ray structure of the protein. We find that the H^N T₂ decay rates and H₂O–H^N build-up rates are sensitive to distance and dynamics of the detected water molecules with respect to the protein. We show that qualitative information about localization and dynamics of internal water molecules can be obtained in the solid-state by interpretation of the spin dynamics of a reporter amide proton.     

Calorimetric studies of the interactions of guanidinium hydrochloride and potassium iodide with model amides in aqueous solution.

The enthalpies of transfer, ΔH_tr, of a series of amides from water to aqueous solutions of either guanidinium hydrochloride (GuHCl) or potassium iodide were obtained from calorimetric measurements at 25°C. The amides were studied at molalities around 10^?2 m while salt molalities ranged from 0–10 m. The amides investigated were Ac-Gly-NHMe, Ac-Gly-Gly-NHMe, Ac-Ala-NHMe, and Ac-Leu-NHMe. Use of an additivity assumption allowed the calculation of group contributions to ΔH_tr in these two salt systems for the methyl group, leucyl side chain, and the peptide backbone unit. Values of the entropy of transfer were also obtained. The great ability of GuHCl to randomize protein structures appears to arise from effects on polar and nonpolar groups, which are characterized by enthalpies and entropies of transfer not substantially different from those with KI, a salt comprised of ions of comparable size and polarizability. The difference in the sign of the free energies of transfer of nonpolar groups from water to MX solutions, negative for GuHCl and positive for KI, is the result of these small differences in enthalpies and entropies of transfer. Variations in water structure produced by differences in ionic properties rather than a mode of action for GuHCl very different from that of other salts characterizes its superior denaturing ability.     

Drosophila melanogaster as a model system to study long-chain fatty acid amide metabolism

Long-chain fatty acid amides are cell-signaling lipids identified in mammals and, recently, in invertebrates, as well. Many details regarding fatty acid amide metabolism remain unclear. Herein, we demonstrate that Drosophila melanogaster is an excellent model system for the study long-chain fatty acid amide metabolism as we have quantified the endogenous levels of N-acylglycines, N-acyldopamines, N-acylethanolamines, and primary fatty acid amides by LC/QTOF-MS. Growth of D. melanogaster on media supplemented with [1-¹³C]-palmitate lead to a family of ¹³C-palmitate-labeled fatty acid amides in the fly heads. The [1-¹³C]-palmitate feeding studies provide insight into the biosynthesis of the fatty acid amides.     

Targeted Lipidomics in Drosophila melanogaster Identifies Novel 2-Monoacylglycerols and N-acyl Amides

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling.     

Fast hydrogen exchange affects 15N relaxation measurements in intrinsically disordered proteins

Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, ¹H–¹⁵N HSQC, is used to measure the ¹⁵N transverse relaxation rate (R₂), the measured R₂ rate is convoluted with the HX rate (k_HX) and has higher apparent R₂ values. Since the ¹⁵N R₂ measurement is important for analyzing protein backbone dynamics, the HX effect on the R₂ measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing ¹⁵N R ₂ ^CPMG experiments on α-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R ₂ ^CPMG can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D₂O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R ₂ ^CPMG values be obtained by methods described herein.     

Design,synthesis and SAR studies of 4-allyoxyaniline amides as potent 15-lipoxygensae inhibitors

A group of 4-allyloxyaniline amides 5a–o were designed, synthesized and evaluated as potential inhibitors of soybean 15-lipoxygenase (SLO) on the basis of eugenol and esteragol structures. Compound 5e showed the best IC₅₀ in SLO inhibition (IC₅₀ = 0.67 ± 0.06 μM). All compounds were docked in SLO active site retrieved from RCSB Protein Data Bank (PDB entry: 1IK3) and showed that allyloxy group of compounds is oriented towards the Fe³⁺-OH moiety in the active site of enzyme and fixed by hydrogen bonding with two conserved His⁵¹³ and Gln⁷¹⁶. It is resulted that molecular volume of the amide moiety would be a major factor in inhibitory potency variation of the synthetic amides, where the hydrogen bonding of the amide group could also involve in the activity of the inhibitors.     

The screening of some microorganisms for their ability to N-dealkylate drug molecules

Summary Selected microorganisms were screened for their ability to N-dealkylate drug molecules. The compounds studied enabled the investigation of N-alkyl groups in different chemical environments, including alkylaminoalkyl chains, saturated cyclic structures and in amide functions. Transformation products were extracted from the transformation mixtures, derivatised and analysed by gas liquid chromatography (GLC). For the purposes of screening, important transformation products were identified by comparison of their GLC retention data with similar derivatives of authentic standards. N-demethylation was effected by all test strains of the fungus Cunninghamella, except C. elegans, and also by three of the Streptomyces species tested. The Cunninghamella demonstrated the widest spectrum of transformation activity, N-demethylating the alkylaminoalkyl side chains of amitriptyline and chlorpromazine, the N-methylpiperidine function of codeine, and the cyclic amide function of diazepam. The N-demethylation of the latter substrate, which is only slightly water soluble, occurred in surprisingly high yield. There was no evidence that bulky groups such as N-dimethylallyl were celaved and selectivity for N-demethylation rather than O-demethylation was demonstrated when both N-and O-methyl functions were present in the same molecule. Comparisons are made with the mammalian metabolic pathways of the drug compounds studied.     

Lipase-catalyzed chemoselective aminolysis of various aminoalcohols with fatty acids

Candida antarctica lipase is well known to convert amines and alcohols into amides and esters. This report describes the development of a solvent-free enzymatic process for the production of fatty alkanolamides. The aminolysis of linoleyl ethyl ester with several aminoalcohols from C2 to C6 (linear or branched compounds), and the very high selectivity of amide compounds have been observed.Our investigation leads us to develop an original biotechnological process for the chemoselective synthesis of new active molecules for cutaneous application.     

南美天胡荽及其根际土壤水浸提液化感成分分析

为探讨南美天胡荽对其他植物种子萌发的影响以及筛选影响其他植物的主要化合物,该文采用种子萌发试验、气相色谱-质谱联用以及液相色谱-质谱联用的方法,分析了南美天胡荽不同溶剂浸提液对种子萌发的影响、南美天胡荽植株及其根际土壤浸提液成分。结果表明:(1)南美天胡荽不同溶剂浸提物均具有一定程度的抑制种子萌发作用。(2)气相色谱-质谱分析下,南美天胡荽植株水浸提液中共分离鉴定了35种化合物,其中,邻苯二甲酸二丁酯(15.2%)、10,15-十八烷二元酸(8.58%)、2,4-二叔丁基苯酚(6.81%)相对含量最高; 根际土壤水浸提液中共分离鉴定了17种化合物,其中,油酸酰胺(26.47%)、正二十七烷(9.63%)、十六酸乙酯(4.83%)相对含量最高。(3)液相色谱-质谱分析下,南美天胡荽植株水浸提液共分离鉴定了109种化合物,ESI⁺模式下,L-苯丙氨酸(3 483.99 ng·mg^-1)、木犀草素(2 306.64 ng·mg^-1)含量最多,ESI^-模式下,右旋奎宁酸(21 827.71 ng·mg^-1)、绿原酸(12 589.25 ng·mg^-1)含量最多; 根际土壤水浸提液中共分离鉴定了93种化合物,ESI⁺模式下,丁酸(7 660.53 ng·mg^-1)、棕榈酰胺(3 200.36 ng·mg^-1)含量最多,ESI^-模式下,正二十八酸(18 605.35 ng·mg^-1)、蔗糖(12 183.23 ng·mg^-1)含量最多。(4)南美天胡荽的潜在化感物质主要为脂肪酸类、酰胺类、酯类、芳香酸类化合物,而土壤中直接起化感作用的物质可能为丁酸、正二十八酸、羟基乙酸、油酸酰胺、棕榈酰胺、十六酸乙酯、苯甲酸,其中脂肪酸类化合物输入可能来源于南美天胡荽、土壤微生物和土壤动物,酰胺类、酯类、芳香类化合物则更可能来源于南美天胡荽植株。     

Influence of alkyl group on amide nitrogen atom on fluorescence quenching of tyrosine amide and N-acetyltyrosine amide

The steady-state and time-resolved fluorescence spectroscopy was applied to determine the influence of an alkyl substituent(s) (methyl or ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, or t-butyl) on amide nitrogen atom on photophysical properties of tyrosine and N-acetyltyrosine amides in water. Generally, the amide group strongly quenches the fluorescence of tyrosine, however, the size and number of substituents on amide nitrogen atom modify the quenching process only in small degree. The fluorescence intensity decays of all amides studied are bi-exponential. The contribution of both components (alphai) to the fluorescence decay undergoes irregular change. An introduction of alkyl substituent on amide nitrogen atom causes an increase of the fluorescence lifetime of tyrosine derivative compared to the unsubstituted amide for both N-acetyltyrosine and tyrosine with the protonated amino group. Calculated, basing on the fluorescence quantum yield (QY) and average lifetime, the radiative rate constants (kf) are similar, which indicates that the substituent(s) does not have substantial influence on radiative process of the deactivation of the excited state of the phenol chromophore for all compounds studied regardless the amino group status as well as the number and type of substituent (linear or branched). The comparison of the ground-state rotamer populations of tyrosine amides and N-acetyltyrosine amides with different alkyl substituent on amide nitrogen atom obtained from 1H NMR with the value of pre-exponential factors indicates that not the rotamer populations, but specific hydration of a whole molecule of the amino acid including chromophore and amino acid moiety, seems to be the main reason of the heterogenous fluorescence intensity decay of tyrosine derivatives.     

Phenolic Amides Are Potent Inhibitors of De Novo Nucleotide Biosynthesis

An outstanding challenge toward efficient production of biofuels and value-added chemicals from plant biomass is the impact that lignocellulose-derived inhibitors have on microbial fermentations. Elucidating the mechanisms that underlie their toxicity is critical for developing strategies to overcome them. Here, using Escherichia coli as a model system, we investigated the metabolic effects and toxicity mechanisms of feruloyl amide and coumaroyl amide, the predominant phenolic compounds in ammonia-pretreated biomass hydrolysates. Using metabolomics, isotope tracers, and biochemical assays, we showed that these two phenolic amides act as potent and fast-acting inhibitors of purine and pyrimidine biosynthetic pathways. Feruloyl or coumaroyl amide exposure leads to (i) a rapid buildup of 5-phosphoribosyl-1-pyrophosphate (PRPP), a key precursor in nucleotide biosynthesis, (ii) a rapid decrease in the levels of pyrimidine biosynthetic intermediates, and (iii) a long-term generalized decrease in nucleotide and deoxynucleotide levels. Tracer experiments using ¹³C-labeled sugars and [¹⁵N]ammonia demonstrated that carbon and nitrogen fluxes into nucleotides and deoxynucleotides are inhibited by these phenolic amides. We found that these effects are mediated via direct inhibition of glutamine amidotransferases that participate in nucleotide biosynthetic pathways. In particular, feruloyl amide is a competitive inhibitor of glutamine PRPP amidotransferase (PurF), which catalyzes the first committed step in de novo purine biosynthesis. Finally, external nucleoside supplementation prevents phenolic amide-mediated growth inhibition by allowing nucleotide biosynthesis via salvage pathways. The results presented here will help in the development of strategies to overcome toxicity of phenolic compounds and facilitate engineering of more efficient microbial producers of biofuels and chemicals.     

Binding of water to "types I and II" Cu2+ in proteins

Water proton spin-lattice relaxation times have been measured at 30MHz between 280 – 333 K in aqueous solutions of proteins containing Type I Cu²⁺ ions (azurin and umecyanin) and Type II Cu²⁺ ions (benzylamine oxidase and superoxide dismutase). These measurements show that Type II Cu²⁺ is accessible to exchangeable water molecules but Type I is not. This behaviour is consistent with the EPR and optical properties of these ions and their likely biochemical functions.     

Using Molecular Dynamics Simulations on Crambin to Evaluate the Suitability of Different Continuum Dielectric and Hydrogen Atom Models for Protein Simulations

Abstract

Molecular dynamics simulations of enzymes with enough explicit waters of solvation to realistically account for solute-solvent interactions can burden the computational resources required to perform the simulation by more than two orders of magnitude. Since enzyme simulations even with an implicit solvation model can be imposing for a supercomputer, it is important to assess the suitability of different continuum dielectric models for protein simulations. A series of 100-picosecond molecular dynamics simulations were performed on the X-ray crystal structure of the protein crambin to examine how well computed structures, obtained using seven continuum dielectric and two hydrogen atom models, agreed with the X-ray structure. The best level of agreement between computed and experimental structures was obtained using a constant dielectric of 2 and the all-hydrogen model. Continuum dielectric models of 1,1^*r, and 2^*r also led to computed structures in reasonably good agreement with the X-ray structure. In all cases, the all-hydrogen model gave better agreement than the united atom model, although, in one case, the difference was not significant. Dielectric models of 4, 80, and 4^*r with either hydrogen model yielded significantly poorer fits. It is especially noteworthy that the observed trends did not semiquantitatively converge until about 50 picoseconds into the simulations, suggesting that validation studies for protein calculations based on energy minimizations or short simulations should be viewed with caution.     

Binding of nonamer peptides to three HLA-B51 molecules which differ by a single amino acid substitution in the A-pocket

The interaction between 9-mer peptides and HLA-B51 molecules was investigated by quantitative peptide binding assay using RMA-S cell expressing human β2-microglobulin and HLA-B51 molecules. Of 147 chemically synthesized 9-mer peptides possessing two anchor residues corresponding to the motif of HLA-B^*5101 binding self-peptides, 27 paptides bound to HLA-B^*5101 molecules. Pro and Ala at position 2 as well as Ile at position 9 were confirmed to be main anchor residues, while Gly at position 2 as well as Val, Leu, and Met at position 9 were weak anchor residues for HLA-B^*5101. The A-pocket is suspected to have a critical role in peptide binding to MHC class I molecules because this pocket corresponds to the N-terminus of peptides and has a strong hydrogen bond formed by conserved Tyr residues. Further analysis of peptide binding to HLA-B^*5102 and B^*5103 molecules showed that a single amino acid substitution of Tyor for His at residue 171(B^*5102) and that of Gly for Trp at residue 167 (B^*5103) has a minimum effect in HLA-B51-peptide binding. Since previous studies showed that some HLA-B51 alloreactive CTL clones failed to kill the cells expressing HLA-B^*5102 or HLA-B^*5103, these results imply that the structural change of the A-pocket among HLA-B51 subtypes causes a critical conformational change of the epitope for TCR recognition rather than influences the interaction between peptides and MHC class I molecules.