A Biochemical Lanthanide-Encoding Approach Enables Quantitative Monitoring of the Bacterial Response to Vancomycin Treatment.

A pathogenic bacterium has its own mechanisms for not only pathogenic attack but also exogenous invasion defense, in which the bacterial cell wall is the front line of attack and defense. We developed a biochemical lanthanide-encoding approach to quantify the uncanonical d-amino acid (d-X) that was edited in a small proportion into the terminal acyl-d-Ala-d-X of nascent peptidoglycan UDP-MurNAc-pentapeptides in the bacterial cell wall. This approach overcomes the difficulties regarding quantification and accuracy issues encountered by the popular optical imaging and traditional high-performance liquid chromatography-based methods. Newly synthesized azide-d-Leu and ketone-d-Met were used together with alkynyl-d-Ala for their metabolic assembly and then bioorthogonally encoded by the correspondingly fabricated DBCO-DOTA-Gd, H2NO-DOTA-Eu, and azide-DOTA-Sm tags. This approach allows direct quantification of the d-X in situ in the cell wall using 158Gd, 153Eu, and 154Sm species-unspecific isotope dilution inductively coupled plasma mass spectrometry, avoiding any tedious and complex "cell-broken" pretreatment procedures that might induce racemization of the d-X. The obtained site-specific and accurate in situ information about the d-X enables quantitative monitoring of the bacterial response when Staphylococcus aureus meets vancomycin, showing that the amounts of azide-d-Leu and ketone-d-Met assembled are more important after determining the structure- and composition-dependent bacterial antibiotic resistance mechanisms. In addition, we found that the combined use of vancomycin and d-Ala restores the efficacy of vancomycin and might be a wise and simple way to combat vancomycin intermediate-resistant S. aureus.

Identification and quantification of leucine and isoleucine residues in peptides using photoexcited tryptophan.

The molecular recognition ability of tryptophan (Trp) for isomeric amino acids, such as leucine (Leu) and isoleucine (Ile), and isomeric amino acid-containing dipeptides, such as Leu-Gly, Ile-Gly, Gly-Leu, and Gly-Ile (where Gly denotes glycine), was investigated using a tandem mass spectrometer equipped with an electrospray ionization source and cold ion trap. The ultraviolet photodissociation spectra of the cold gas-phase clusters of Leu and Ile with Na+Trp in the wavelength range of 265-290 nm revealed that the relative intensities of Leu and Ile were only different in the wavelength range of 265-273 nm; however, no differences in the relative intensities were observed when the wavelength exceeded 274 nm. The molecular recognition ability of photoexcited Trp was used for the identification and quantification of Leu and Ile in dipeptides in solution. The mole fractions of Leu and Ile in dipeptides could be determined from the abundances observed in a single product ion spectrum of the cold gas-phase clusters of dipeptides with Na+Trp.

NMR-Based Serum Metabolomics Revealed Distinctive Metabolic Patterns in Reactive Arthritis Compared with Rheumatoid Arthritis.

Reactive arthritis (ReA) is a member of seronegative spondyloarthropathy (SSA), which involves an acute/subacute onset of asymmetrical lower limb joint inflammation weeks after a genitourinary/gastrointestinal infection. The diagnosis is clinical because it is difficult to culture the microbes from synovial fluid. Arthritis patients with a similar clinical picture but lapsed history of an immediate preceding infection that do not fulfill the diagnostic criteria of other members of SSA, such as ankylosing spondylitis, psoriatic arthritis, and arthritis associated with inflammatory bowel disease, are labeled as peripheral undifferentiated spondyloarthropathy (uSpA). Both ReA and uSpA patients show a strong association with class I major histocompatibility complex allele, HLA-B27, and a clear association with an infectious trigger; however, the disease mechanism is far from clear. Because the clinical picture is largely dominated by rheumatoid-arthritis (RA)-like features including elevated levels of inflammatory markers (such as ESR, CRP, etc.), these overlapping symptoms often confound the clinical diagnosis and represent a clinical dilemma, making treatment choice more generalized. Therefore, there is a compelling need to identify biomarkers that can support the diagnosis of ReA/uSpA. In the present study, we performed NMR-based serum metabolomics analysis and demonstrated that ReA/uSpA patients are clearly distinguishable from controls and further that these patients can also be distinguished from the RA patients based on the metabolic profiles, with high sensitivity and specificity. The discriminatory metabolites were further subjected to area under receiver operating characteristic curve analysis, which led to the identification of four metabolic entities (i.e., valine, leucine, arginine/lysine, and phenylalanine) that could differentiate ReA/uSpA from RA.

Capillary electrophoresis/visible-LED induced fluorescence of tryptophan: What's new?

Tryptophane (Trp) labelled by 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) is very difficult to identify using CE and fluorescence detection (480 nm). Why in this article some mass spectrometry experiments show that Trp is really labelled by CBQCA as Leucine (Leu)? If the maximum of UV absorption (λmax ) is the same between Leu-CBQCA and Trp-CBQCA, the molar extinction coefficient is around 2 fold higher for Trp-CBQCA. The fluorescence of the Leu-CBQCA derivative is 50 times more important than for Trp-CBQCA. The addition of 7.5 mM of ß-cyclodextrin (ß-CD) was found to be a good mean to improve 2.1 fold the sensitivity of the Trp-CBQCA fluorescence. Using a buffer containing SDS and ß-CD in CE, a LOD of 0.7 µM of L-Trp can be reached and the ratio of the intensities between Leu, Isoleucine, Valine, Trp is 100, 21, 15, 1. Negative ESI/ MS and MS/MS of the labeled amino acids show that a loss of the carboxylate function takes place. In the presence of two enantiomers of Trp-CBQCA, we have shown that this decarboxylation is not due to the derivatization process in the solution but rather occurs in the source of the mass spectrometer.

Linear Peptides Containing d-Leucine with Neuroprotective Activities from the Leech Whitmania pigra Whitman.

Three new linear peptides containing d-leucine, named whitmantides A-C (1-3), were isolated from the dried whole bodies of Whitmania pigra Whitman. Their structures with absolute configurations were elucidated by Edman degradation, mass spectrometry, Marfey's analysis, and solid-phase synthesis. It is the first time that peptides containing d-amino acid in leeches were discovered. Compounds 1-3 displayed neuroprotective activities against oxygen-glucose deprivation/reperfusion injury on Neuro-2a cells. In addition, ex vivo serum stability tests showed that 1-3 were resistant to protease degradation.

Investigation of the Complete Suite of the Leucine and Isoleucine Isomers: Toward Prediction of Ion Mobility Separation Capabilities.

In this study we investigated 11 isomers with the molecular formula C6H13NO2 (m/z 131) to ascertain the potential of utilizing drift tube ion mobility mass spectrometry to aid in the separation of isomeric mixtures. This study of small molecules provides a detailed examination of the application of uniform field ion mobility for a narrow scope of isomers with variations in both bond coordination and stereochemistry. For small molecules, it was observed that in general constitutional isomers are more readily separated by uniform field mobility in comparison to stereoisomers such as enantiomers or diastereomers. Diastereomers exhibited differences in their collision cross section (CCS), but were unresolvable in a mixture, whereas the enantiomers studied did not exhibit statistically different CCS values. A mathematical relationship relating the CCS to resolving power was developed in order to predict the required ion mobility resolving power needed to separate the various isomer classes. For the majority of isomers evaluated in this study, a uniform field-based resolving power of 100 was predicted to be sufficient to resolve over half (∼60%) of all hypothetical isomer pairs, including leucine and isoleucine, whereas their stereoisomers (d- and l-forms) are predicted to be significantly more challenging, if not impossible, to separate by conventional drift tube techniques.

Rapid Distinction of Leucine and Isoleucine in Monoclonal Antibodies Using Nanoflow LCMSn.

Monoclonal antibodies (mAbs) are large heterogeneous molecules that represent a growing class of therapeutics. De novo sequencing of mAbs becomes necessary when the original cell line or the cDNA is unavailable. An important feature in sequencing of mAbs is the discrimination of isobaric residues (Xle): leucine (Leu) and isoleucine (Ile). An incorrect identification of the Xle site, especially in the complementarity determining regions (CDRs), can result in the production of an antibody with severely compromised efficacy. Multistage fragmentation (MSn) in the mass spectrometer can provide sufficient evidence for Ile/Leu discrimination. However, most existing methods utilize direct infusion of purified peptides, demanding peptide enrichment which can be labor-intensive and requires large amount of material. Here we introduce an online nano-LCMSn method, which depending on the nature of the peptide, exploits either generation of a signature 69 Da ion from Ile or formation of unique w-ions employing MS3 (ETD-HCD) for rapid Ile/Leu distinction. This reliable and sensitive method utilizes the Orbitrap Fusion tribid mass spectrometer to rapidly assign multiple Xle residues in the CDRs of mAbs.

Mass Defect-Based N,N-Dimethyl Leucine Labels for Quantitative Proteomics and Amine Metabolomics of Pancreatic Cancer Cells.

Mass spectrometry-based stable isotope labeling has become a key technology for protein and small-molecule analyses. We developed a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nano reversed-phase liquid chromatography-tandem mass spectrometry (nanoRPLC-MS/MS), called mass defect-based N,N-dimethyl leucine (mdDiLeu) labeling. The duplex mdDiLeu reagents were custom-synthesized with a mass difference of 20.5 mDa, arising from the subtle variation in nuclear binding energy between the two DiLeu isotopologues. Optimal MS resolving powers were determined to be 240K for labeled peptides and 120K for labeled metabolites on the Orbitrap Fusion Lumos instrument. The mdDiLeu labeling does not suffer from precursor interference and dynamic range compression, providing excellent accuracy for MS1-centric quantification. Quantitative information is only revealed at high MS resolution without increasing spectrum complexity and overlapping isotope distribution. Chromatographic performance of polar metabolites was dramatically improved by mdDiLeu labeling with modified hydrophobicity, enhanced ionization efficiency, and picomole levels of detection limits. Paralleled proteomics and amine metabolomics analyses using mdDiLeu were systematically evaluated and then applied to pancreatic cancer cells.

Engineering a periplasmic binding protein for amino acid sensors with improved binding properties.

Periplasmic binding proteins (PBPs) are members of a widely distributed protein superfamily found in bacteria and archaea, and are involved in the cellular uptake of solutes. In this report, a leucine-binding PBP was engineered to detect l-Leu based on a fluorescence resonance energy transfer (FRET) change upon ligand binding. A fluorescent unnatural amino acid, l-(7-hydroxycoumarin-4-yl)ethylglycine (CouA), was genetically incorporated into the protein as a FRET donor, and a yellow fluorescent protein (YFP) was fused with its N-terminus as a FRET acceptor. When CouA was incorporated into position 178, the sensor protein showed a 2.5-fold increase in the FRET ratio. Protein engineering significantly improved its substrate specificity, showing minimal changes in the FRET ratio with the other 19 natural amino acids and d-Leu. Further modification increased the sensitivity of the sensor protein (14-fold) towards l-Leu, and it recognized l-Met as well with moderate binding affinity. Selected mutant sensors were used to measure concentrations of l-Leu in a biological sample (fetal bovine serum) and to determine the optical purity of Leu and Met. This FRET-based sensor design strategy allowed us to easily manipulate the natural receptor to improve its binding affinity and specificity and to recognize other natural molecules, which are not recognized by the wild-type receptor. The design strategy can be applied to other natural receptors, enabling engineering receptors that sense biochemically interesting molecules.

Exploration of doubtful cases of leucine and isoleucine discrimination in mass spectrometric peptide sequencing by electron-transfer and higher-energy collision dissociation-based method.

Electron-transfer dissociation (ETD) and electron-transfer and higher-energy collision dissociation (EThcD) spectra of short tryptic peptides with leucine/isoleucine residues in neighboring positions demonstrate intensive w-ions. On the contrary, u-ions possess very low intensities (if present at all). Therefore radical site migration is negligible in the applied conditions while ETD (EThcD) spectra allow for the reliable discrimination of the isomeric residues in the sequencing process. The presence of a fragment ion 43.055 mass units lower than z2-ion of peptides with IK sequence at their C-termini was shown to be a result of alternative fragmentation starting from the loss of propylammonium ion from the doubly protonated peptide molecule and formation of an oxazole fragment ion.

Distinguishing between Leucine and Isoleucine by Integrated LC-MS Analysis Using an Orbitrap Fusion Mass Spectrometer.

Despite the great success of mass spectrometry (MS) for de novo protein sequencing, Leu and Ile have been generally considered to be indistinguishable by MS because their molecular masses are exactly the same. Positioning of incorrect Leu/Ile residues in variable domains, especially in CDRs (complementarity determining regions) of an antibody, may result in substantial loss of antigen binding affinity and specificity of the antibody. Here, we describe an integrated LC-MS based strategy, encompassing a combination of HCD (high-energy collisional dissociation) multistage mass spectrometric analysis (HCD-MSn) and ETD (electron transfer dissociation)-HCD MS3 analysis using an Orbitrap Fusion mass spectrometer, to reliably identify Leu and Ile residues in proteins and peptides. The merits and limitations of this Leu/Ile discrimination approach are evaluated. Using the new approach, along with proposed decision-making guidelines we unambiguously identified every Leu/Ile residue in peptides containing up to five Leu/Ile residues and molecular masses up to 3000 Da. In addition, we have demonstrated, for the first time, that every Leu/Ile residue in the variable regions of a monoclonal antibody that could not be assigned by antibody germline sequence alignment could be correctly determined using this approach. Our results suggest that, by incorporating this approach into existing de novo antibody sequencing protocols, 100% of antibody amino acid sequences, including identity of Leu and Ile residues, can be accurately obtained solely by means of mass spectrometry. In principle, this integrated, online LC-MS approach for Leu/Ile assignment can be applied to de novo sequencing of any protein or peptide.

High-Speed Microdialysis-Capillary Electrophoresis Assays for Measuring Branched Chain Amino Acid Uptake in 3T3-L1 cells.

We have developed a high-throughput microdialysis-capillary electrophoresis (MD-CE) assay for monitoring branched chain amino acid (BCAA) uptake/release dynamics in 3T3-L1 cells. BCAAs (i.e., isoleucine, leucine, and valine) and their downstream metabolites (i.e., alanine, glutamine, and glutamate) are important indicators of adipocyte lipogenesis. To perform an analysis, amino acids were sampled using microdialysis, fluorescently labeled in an online reaction, separated using CE, and detected using laser-induced fluorescence (LIF) in a sheath flow cuvette. Separation conditions were optimized for the resolution of the BCAAs isoleucine, leucine, and valine, as well as 13 other amino acids, including ornithine, alanine, glutamine, and glutamate. CE separations were performed in <30 s, and the temporal resolution of the online MD-CE assay was <60 s. Limits of detection (LOD) were 400, 200, and 100 nM for isoleucine, leucine, and valine, respectively. MD-CE dramatically improved throughput in comparison to traditional offline CE methods, allowing 8 replicates of 15 samples (i.e., 120 analyses) to be assayed in <120 min. The MD-CE assay was used to assess the metabolism dynamics of 3T3-L1 cells over time, confirming the utility of the assay.

A Whole-Cell Surface Plasmon Resonance Sensor Based on a Leucine Auxotroph of Escherichia coli Displaying a Gold-Binding Protein: Usefulness for Diagnosis of Maple Syrup Urine Disease.

We developed a whole-cell surface plasmon resonance (SPR) sensor based on a leucine auxotroph of Escherichia coli displaying a gold-binding protein (GBP) in response to cell growth and applied this sensor to the diagnosis of maple syrup urine disease, which is represented by the elevated leucine level in blood. The leucine auxotroph was genetically engineered to grow displaying GBP in a proportion to the concentration of target amino acid leucine. The GBP expressed on the surface of the auxotrophs directly bound to the golden surface of an SPR chip without the need for any additional treatment or reagents, which consequently produced SPR signals used to determine leucine levels in a test sample. Gold nanoparticles (GNPs) were further applied to the SPR system, which significantly enhanced the signal intensity up to 10-fold by specifically binding to GBP expressed on the cell surface. Finally, the diagnostic utility of our system was demonstrated by its employment in reliably determining different statuses of maple syrup urine disease based on a known cutoff level of leucine. This new approach based on an amino acid-auxotrophic E. coli strain expressing a GBP that binds to an SPR sensor holds great promise for detection of other metabolic diseases of newborn babies including homocystinuria and phenylketonuria, which are also associated with abnormal levels of amino acids.

Leucine supplementation via drinking water reduces atherosclerotic lesions in apoE null mice.

AIM: Recent evidence suggests that the essential amino acid leucine may be involved in systemic cholesterol metabolism. In this study, we investigated the effects of leucine supplementation on the development of atherosclerosis in apoE null mice. METHODS: ApoE null mice were fed with chow supplemented with leucine (1.5% w/v) in drinking water for 8 week. Aortic atherosclerotic lesions were examined using Oil Red O staining. Plasma lipoprotein-cholesterol levels were measured with fast protein liquid chromatography. Hepatic gene expression was detected using real-time PCR and Western blot analyses. RESULTS: Leucine supplementation resulted in 57.6% reduction of aortic atherosclerotic lesion area in apoE null mice, accompanied by 41.2% decrease of serum LDL-C levels and 40.2% increase of serum HDL-C levels. The body weight, food intake and blood glucose level were not affected by leucine supplementation. Furthermore, leucine supplementation increased the expression of Abcg5 and Abcg8 (that were involved in hepatic cholesterol efflux) by 1.28- and 0.86-fold, respectively, and significantly increased their protein levels. Leucine supplementation also increased the expression of Srebf1, Scd1 and Pgc1b (that were involved in hepatic triglyceride metabolism) by 3.73-, 1.35- and 1.71-fold, respectively. Consequently, leucine supplementation resulted in 51.77% reduction of liver cholesterol content and 2.2-fold increase of liver triglyceride content. Additionally, leucine supplementation did not affect the serum levels of IL-6, IFN-γ, TNF-α, IL-10 and IL-12, but markedly decreased the serum level of MCP-1. CONCLUSION: Leucine supplementation effectively attenuates atherosclerosis in apoE null mice by improving the plasma lipid profile and reducing systemic inflammation.

A convenient and validated enantiomer separation of chiral aliphatic amines as nitrobenzoxadiazole derivatives on polysaccharide-derived chiral stationary phases under simultaneous ultraviolet and fluorescence detection.

A convenient method using a fluorogenic agent, 4-chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl), was developed for enantiomer separation of chiral aliphatic amines including amino alcohols by normal high-performance liquid chromatography. The enantiomer separation of chiral aliphatic amines as NBD derivatives was performed on six covalently bonded and four coated-type polysaccharide-derived chiral stationary phases (CSPs) under simultaneous ultraviolet (UV) and fluorescence detection (FLD). Among the covalently bonded CSPs, Chiralpak IE showed the best enantiomer separation for most analytes. The other CSPs also showed good enantioselectivity except for Chiralpak IB. On the other hand, Chiralpak AD-H and Amylose-1 generally exhibited better enantiomer separation of NBD derivatized chiral amines among the coated CSPs. The developed analytical technique was also applied to determine the optical purity of commercially available (R)- and (S)-leucinol; the impurity was found to be 0.06%. The developed method was validated and proved to be an accurate, precise, sensitive, and selective method suitable for separation of chiral aliphatic amines as NBD derivatives under simultaneous UV and FLD.

Structure-Based Assignment of Ile, Leu, and Val Methyl Groups in the Active and Inactive Forms of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 2.

Resonance assignments are the first step in most NMR studies of protein structure, function, and dynamics. Standard protein assignment methods employ through-bond backbone experiments on uniformly (13)C/(15)N-labeled proteins. For larger proteins, this through-bond assignment procedure often breaks down due to rapid relaxation and spectral overlap. The challenges involved in studies of larger proteins led to efficient methods for (13)C labeling of side chain methyl groups, which have favorable relaxation properties and high signal-to-noise. These methyls are often still assigned by linking them to the previously assigned backbone, thus limiting the applications for larger proteins. Here, a structure-based procedure is described for assignment of (13)C(1)H3-labeled methyls by comparing distance information obtained from three-dimensional methyl-methyl nuclear Overhauser effect (NOE) spectroscopy with the X-ray structure. The Ile, Leu, or Val (ILV) methyl type is determined by through-bond experiments, and the methyl-methyl NOE data are analyzed in combination with the known structure. A hierarchical approach was employed that maps the largest observed "NOE-methyl cluster" onto the structure. The combination of identification of ILV methyl type with mapping of the NOE-methyl clusters greatly simplifies the assignment process. This method was applied to the inactive and active forms of the 42-kDa ILV (13)C(1)H3-methyl labeled extracellular signal-regulated kinase 2 (ERK2), leading to assignment of 60% of the methyls, including 90% of Ile residues. A series of ILV to Ala mutants were analyzed, which helped confirm the assignments. These assignments were used to probe the local and long-range effects of ligand binding to inactive and active ERK2.

Metabolomics analysis and biomarker identification for brains of rats exposed subchronically to the mixtures of low-dose cadmium and chlorpyrifos.

Cadmium (Cd) and chlorpyrifos (CPF) are widespread harmful environmental pollutants with neurotoxicity to mammals. Although the exposure to Cd and CPF at the same time may pose a significant risk to human health, the subchronic combined neurotoxicity of these two chemicals at low levels in the brain is poorly understood. In this study, we treated rats with three doses (low, middle, and high) of Cd, CPF, or their mixture for 90 days. No obvious symptom was observed in the treated animals except those treated with high-dose CPF. Histological results showed that middle and high doses of the chemicals caused neuronal cell damage in brains. GC-MS-based metabonomics analysis revealed that energy and amino acid metabolism were disturbed in the brains of rats exposed to the two chemicals and their combinations even at low doses. We further identified the unique brain metabolite biomarkers for rats treated with Cd, CPF, or both. Two amino acids, tyrosine and l-leucine, were identified as the biomarkers for Cd and CPF treatment, respectively. In addition, a set of five unique biomarkers (1,2-propanediol-1-phosphate, d-gluconic acid, 9H-purine, serine, and 2-ketoisovaleric acid) was identified for the mixtures of Cd and CPF. Therefore, the metabolomics analysis is more sensitive than regular clinical observation and pathological examination for detecting the neurotoxicity of the individual and combined Cd and CPF at low levels. Overall, these results identified the unique biomarkers for Cd and CPF exposure, which provide new insights into the mechanism of their joint toxicity.

Method development for the determination of D- and L-isomers of leucine in human plasma by high-performance liquid chromatography tandem mass spectrometry and its application to animal plasma samples.

We developed a highly sensitive and specific high-performance liquid chromatography tandem mass spectrometry method with an electrospray ionization for the determination of D- and L-isomers of leucine in human plasma. Phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and quality control samples. The extraction of D- and L-leucine in plasma samples (100 µL) was performed using cationic exchange solid-phase extraction. The enantiomer separation of D- and L-leucine was successfully achieved without derivatization using a CHIRALPAK ZWIX(-) with an isocratic mobile phase comprised of methanol/acetonitrile/1 mol/L ammonium formate/formic acid (500:500:25:2, v/v/v/v) at a flow rate of 0.5 mL/min. In addition, the discrimination of DL-leucine from structural isomers DL-isoleucine and DL-allo-isoleucine was performed using the unique precursor and product ion pair transition of DL-leucine (m/z 132.1 > 43.0) and DL-leucine-d 7 (m/z 139.2 > 93.0) in positive electrospray ionization mode. The standard curves were linear throughout the calibration range from 0.001 to 1 µg/mL for D-leucine and from 1 to 1000 µg/mL for L-leucine, respectively, with acceptable intra- and inter-day precision and accuracy. The stability of D- and L-leucine in human plasma and solvents was confirmed. The endogenous level of D- and L-leucine in human plasma was 0.00197~0.00591 and 9.63~24.7 µg/mL, respectively. This method was also successfully applied to investigate the species difference in the ratios of D-leucine to total leucine from individual plasma concentrations in humans and various animals. The plasma D-leucine concentrations or their ratio to total leucine in rodents was much higher than that in humans.

Site reactivity in the free radicals induced damage to leucine residues: a theoretical study.

Several recent computational studies have tried to explain the observed selectivity in radical damage to proteins. In this work we use Density Functional Theory and Transition State Theory including tunnelling corrections, reaction path degeneracy, the effect of diffusion, and the role of free radicals to get further insights into this important topic. The reaction between a leucine derivative and free radicals of biological significance, in aqueous and lipid media, has been investigated. Both thermochemical and kinetic analyses, in both hydrophilic and hydrophobic environments, have been carried out. DPPH, ˙OOH, ˙OOCH3, ˙OOCH2Cl, ˙OOCHCl2 and ˙OOCHCH2 radicals do not react with the target molecule. The reactions are proposed to be kinetically controlled. The leucine gamma site was the most reactive for the reactions with ˙N3, ˙OOCCl3, ˙OCH3, ˙OCH2Cl, and ˙OCHCl2 radicals, with rate constants equal to 1.97 × 10(5), 3.24 × 10(4), 6.68 × 10(5), 5.98 × 10(6) and 8.87 × 10(8) M(-1) s(-1), respectively, in aqueous solution. The ˙Cl, ˙OH and ˙OCCl3 radicals react with leucine at the beta, gamma, and delta positions at rates close to the diffusion limit with the alpha position which is the slowest path and the most thermodynamically favored. The presented results confirm that the Bell-Evans-Polanyi principle does not apply for the reactions between amino acid residues and free radicals. Regarding the influence of the environment on the reactivity of the studied series of free radicals towards leucine residues, it is concluded that hydrophilic media slightly lower the reactivity of the studied radicals, compared to hydrophobic ones, albeit the trends in reactivity are very similar.

ValidNESs: a database of validated leucine-rich nuclear export signals.

ValidNESs (http://validness.ym.edu.tw/) is a new database for experimentally validated leucine-rich nuclear export signal (NES)-containing proteins. The therapeutic potential of the chromosomal region maintenance 1 (CRM1)-mediated nuclear export pathway and disease relevance of its cargo proteins has gained recognition in recent years. Unfortunately, only about one-third of known CRM1 cargo proteins are accessible in a single database since the last compilation in 2003. CRM1 cargo proteins are often recognized by a classical NES (leucine-rich NES), but this signal is notoriously difficult to predict from sequence alone. Fortunately, a recently developed prediction method, NESsential, is able to identify good candidates in some cases, enabling valuable hints to be gained by in silico prediction, but until now it has not been available through a web interface. We present ValidNESs, an integrated, up-to-date database holding 221 NES-containing proteins, combined with a web interface to prediction by NESsential.