Synthesis of a Double Transmembrane Domain Fragment of Ste2p by Native Chemical Ligation

Native chemical ligation (NCL) approaches have been applied extensively to soluble proteins. Fewer successes have been achieved with membrane peptides. In this report, the synthesis and semisynthesis by NCL of peptides corresponding to 1.7 transmembrane domains of the α-factor receptor from Saccharomyces cerevisiae is described. Synthesis was achieved when the ligation point was approximately in the middle of the loop joining the two transmembrane regions. In contrast, little to no ligation was observed when the ligation point was at the putative membrane interface of the sixth transmembrane domain (TM6) and the third extracellular loop (EL3). Ligations of a chemically synthesized 22-residue thioester with a synthetic 29-residue N-Cys peptide and a biosynthetic 73-residue N-Cys peptide were successfully achieved in both trifluoroethanol/guanidinium hydrochloride (TFE/GnHCl) and sodium dodecyl sulfate (SDS) media when mercaptoethanesulfonic acid (MESNA) was used as a catalyst. The resulting 51-residue and 95-residue ligation products were purified by reversed phase HPLC and recovered on a mg scale. Both peptides were >95% pure as determined by HPLC and had the expected molecular weight as judged by mass spectrometry. Segmental labeling of the 95-residue fragment, in which the N-Cys portion was [¹⁵N] labeled, resulted in a peptide that gave an NMR spectrum which was comparable to that of the unligated 73-residue peptide alone. R B Merrifield personified the finest qualities of a human being. He was an outstanding individual who influenced the way research is conducted by tens of thousands of scientists. At the same time he was a warm, humble, sincere man who was extremely kind and generous. I (FN) personally saw his generosity during a seminar he invited me to give at Rockefeller University. He was already a Nobel laureate but he treated me as a colleague and the encouragement he offered concerning my research program was very important for my future in academia. It is an honor to be among the participants in a volume honoring his contributions to peptide science.     

Combined use of eBeam irradiation and the potential probiotic Lactobacillus rhamnosus Vahe for control of foodborne pathogen Klebsiella pneumoniae

The implementation of electron beam radiation coupled with the use of probiotics is one of the newest food processing technologies that may be used to ensure food safety and improve shelf life of food products. The purpose of this study was to evaluate the effect of 50–150-Gy electron beam irradiation on the antimicrobial activity of the putative probiotic strain Lactobacillus rhamnosus Vahe. Low-dose electron beam irradiation of lactobacilli cells was performed using the Advanced Research Electron Accelerator Laboratory’s electron accelerator, and the agar well diffusion method and Verhulst logistic function were used to evaluate the effect of radiation on anti–Klebsiella pneumoniae activity of the cell free supernatant of L. rhamnosus Vahe cells in vitro. Our results suggest that 50–150-Gy electron beam irradiation decreases the viability of the investigated lactobacilli, but does not significantly change the probiotic’s activity against K. pneumoniae. Results indicate that the combined use of irradiation and L. rhamnosus Vahe might be suggested for non-thermal food sterilizing technologies.     

Tertiary Structural Motif Sequence Statistics Enable Facile Prediction and Design of Peptides that Bind Anti-apoptotic Bfl-1 and Mcl-1

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Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves

Many factors that change the temperature position and interval of the DNA helix–coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, T_m, and temperature melting width, ΔT, of the entire DNA transition. Changes in T_m and ΔT can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining T_m and ΔT. Indeed, some of the methods give unreasonable “jagged” T_m and ΔT dependences on varying relative concentration of DNA chemical modifications (r_b), [Na⁺], and GC content. At the same time, T_m determined as the helix–coil transition average temperature, and ΔT, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of T_m and ΔT on r_b, [Na⁺], and GC content. For multi-peak DMCs, T_m value determined in this way is the closest to the thermodynamic melting temperature (the helix–coil transition enthalpy/entropy ratio).     

Low-Dose Electron-Beam Irradiation for the Improvement of Biofilm Formation by Probiotic Lactobacilli

The effects of 50–150 gray electron-beam irradiation on the biofilm-formation ability and cell surface hydrophobicity of the commercial strain, Lactobacillus acidophilus DDS®-1, from Lacto-G (a marketed synbiotic formulation) and the putative probiotic, L. rhamnosus Vahe, were evaluated. No significant changes in cell surface hydrophobicity were found after irradiation, while increases in biofilm-formation abilities were documented for both investigated microorganisms 0.22 ± 0.03 vs. 0.149 ± 0.02 (L. rhamnosus Vahe, 150 Gy) and 0.218 ± 0.021 vs. 0.17 ± 0.012 (L. acidophilus DDS®-1, 150 Gy). Given this, the use of electron-beam irradiation (50–100 Gy) for the treatment of L. rhamnosus Vahe and L. acidophilus DDS®-1 cells may be considered in product sterilization, quality improvement, and packaging practices.

     

Covalent binding of the organophosphorus agent FP-biotin to tyrosine in eight proteins that have no active site serine

Organophosphorus (OP) esters are known to bind covalently to the active site serine of enzymes in the serine hydrolase family. It was a surprise to find that proteins with no active site serine are also covalently modified by OP. The binding site in albumin, transferrin, and tubulin was identified as tyrosine. The goal of the present work was to determine whether binding to tyrosine is a general phenomenon. Fourteen proteins were treated with a biotin-tagged organophosphorus agent called FP-biotin. The proteins were digested with trypsin and the labeled peptides enriched by binding to monomeric avidin. Peptides were purified by HPLC and fragmented by collision induced dissociation in a tandem ion trap mass spectrometer. Eight proteins were labeled and six were not. Tyrosine was labeled in human alpha-2-glycoprotein 1 zinc-binding protein (Tyr 138, Tyr 174 and Tyr 181), human kinesin 3C motor domain (Tyr 145), human keratin 1 (Tyr 230), bovine actin (Tyr 55 and Tyr 200), murine ATP synthase beta (Tyr 431), murine adenine nucleotide translocase 1 (Tyr 81), bovine chymotrypsinogen (Tyr 201) and porcine pepsin (Tyr 310). Only 1–3 tyrosines per protein were modified, suggesting that the reactive tyrosine was activated by nearby residues that facilitated ionization of the hydroxyl group of tyrosine. These results suggest that OP binding to tyrosine is a general phenomenon. It is concluded that organophosphorus-reactive proteins include not only enzymes in the serine hydrolase family, but also proteins that have no active site serine. The recognition of a new OP-binding motif to tyrosine suggests new directions to search for mechanisms of long-term effects of OP exposure. Another application is in the search for biomarkers of organophosphorus agent exposure. Previous searches have been limited to serine hydrolases. Now proteins such as albumin and keratin can be considered.     

Quantification of Cardiovascular Biomarkers in Patient Plasma by Targeted Mass Spectrometry and Stable Isotope Dilution

Verification of candidate biomarkers requires specific assays to selectively detect and quantify target proteins in accessible biofluids. The primary objective of verification is to screen potential biomarkers to ensure that only the highest quality candidates from the discovery phase are taken forward into preclinical validation. Because antibody reagents for a clinical grade immunoassay often exist for a small number of candidates, alternative methodologies are required to credential new and unproven candidates in a statistically viable number of serum or plasma samples. Using multiple reaction monitoring coupled with stable isotope dilution MS, we developed quantitative, multiplexed assays in plasma for six proteins of clinical relevance to cardiac injury. The process described does not require antibodies for immunoaffinity enrichment of either proteins or peptides. Limits of detection and quantitation for each signature peptide used as surrogates for the target proteins were determined by the method of standard addition using synthetic peptides and plasma from a healthy donor. Limits of quantitation ranged from 2 to 15 ng/ml for most of the target proteins. Quantitative measurements were obtained for one to two signature peptides derived from each target protein, including low abundance protein markers of cardiac injury in the nanogram/milliliter range such as the cardiac troponins. Intra- and interassay coefficients of variation were predominantly <10 and 25%, respectively. The configured multiplex assay was then used to measure levels of these proteins across three time points in six patients undergoing alcohol septal ablation for hypertrophic obstructive cardiomyopathy. These results are the first demonstration of a multiplexed, MS-based assay for detection and quantification of changes in concentration of proteins associated with cardiac injury in the low nanogram/milliliter range. Our results also demonstrate that these assays retain the necessary precision, reproducibility, and sensitivity to be applied to novel and uncharacterized candidate biomarkers for verification of proteins in blood.Discovery of disease-specific biomarkers with diagnostic and prognostic utility has become an important challenge in clinical proteomics. In general, unbiased discovery experiments often result in the confident identification of thousands of proteins, hundreds of which may vary significantly between case and control samples in small discovery studies. However, because of the stochastic sampling of proteomes in discovery “omics” experiments, a large fraction of the protein biomarkers “discovered” in these experiments are false positives arising from biological or technical variability. Clearly discovery omics experiments do not lead to biomarkers of immediate clinical utility but rather produce candidates that must be qualified and verified in larger sample sets than were used for discovery (1).Traditional, clinical validation of biomarkers has relied primarily on immunoassays because of their specificity and sensitivity for the target analyte and high throughput capability. However, antibody reagents for a clinical grade immunoassay often only exist for a short list of candidates. The development of a reliable sandwich immunoassay for one target protein is expensive, has a long development time, and is dependent upon the generation of high quality protein antibodies. For the large majority of new, unproven candidate biomarkers, an intermediate verification technology is required that has shorter assay development time lines, lower assay cost, and effective multiplexing of dozens of candidates in low sample volumes. Ideally the approach should be capable of analyzing hundreds of samples of serum or plasma with good precision. The desired outcome of verification is a small number of highly credentialed candidates suitable for traditional preclinical and clinical validation studies.Multiple reaction monitoring (MRM)¹ coupled with stable isotope dilution (SID) MS has recently been shown to be well suited for direct quantification of proteins in plasma (2–4) and has emerged as the core technology for candidate biomarker verification. MRM assays can be highly multiplexed such that a moderate number of candidate proteins (in the range of 10–50) can be simultaneously targeted and measured in the statistically viable number of patient samples required for verification (hundreds of serum samples). However, sensitivity for unambiguous detection and quantification of proteins by MS-based assays is often constrained by sample complexity, particularly when the measurements are being made in complex fluids such as plasma.Many biomarkers of current clinical importance, such as prostate-specific antigen and the cardiac troponins, reside in the low nanogram/milliliter range in plasma and, until recently, have been inaccessible by non-antibody approaches. Our laboratory has recently shown for the first time that a combination of abundant protein depletion with limited fractionation at the peptide level prior to SID-MRM-MS provides robust limits of quantitation (LOQs) in the 1–20 ng/ml range with coefficient of variation (CV) of 10–20% at the LOQ for proteins in plasma (3).Here we demonstrate that this work flow can be extended to configure assays for a number of known markers of cardiovascular disease and, more importantly, can be deployed to measure their concentrations in clinical samples. We modeled a verification study comprising six patients undergoing alcohol septal ablation treatment for hypertrophic obstructive cardiomyopathy, a human model of “planned” myocardial infarction (PMI), and obtained targeted, quantitative measurements for moderate to low concentrations of cardiac biomarkers in plasma. This work provides additional evidence that MS-based assays can be configured and applied to verification of new protein targets for which high quality antibody reagents are not available.     

Identification of ligand binding regions of the Saccharomyces cerevisiae alpha-factor pheromone receptor by photoaffinity cross-linking

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), a photoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on a p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of alpha-factor to generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growth arrest assay) and binding affinities of all analogues for the alpha-factor receptor (Ste2p) were determined. Two of the analogues that were tested, Bpa(1) and Bpa(5), showed 3-4-fold lower affinity than the alpha-factor, whereas Bpa(3) and Bpa(13) had 7-12-fold lower affinities. Bpa(8) competed poorly with [(3)H]-alpha-factor for Ste2p. All of the analogues tested except Bpa(8) had detectable halos in the growth arrest assay, indicating that these analogues are alpha-factor agonists. Cross-linking studies demonstrated that [Bpa(1)]-alpha-factor, [Bpa(3)]-alpha-factor, [Bpa(5)]-alpha-factor, and [Bpa(13)]-alpha-factor were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa(1), Bpa(3), and Bpa(13) cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of the pheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between the position 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The results further define the sites of interaction between Ste2p and the alpha-factor, allowing refinement of a model for the pheromone bound to its receptor.