Science.gov

Sample records for 15n labeled proteins

  1. An economical method for (15)N/(13)C isotopic labeling of proteins expressed in Pichia pastoris.

    PubMed

    Rodriguez, E; Krishna, N R

    2001-07-01

    We report a new and cost-effective approach to prepare (15)N/(13)C labeled proteins for NMR using the Pichia pastoris expression system. Four protocols (P1 to P4) were defined and compared using recombinant Ovine interferon-tau (rOvIFN-tau). Our results demonstrate that in order to get full incorporation of (15)N and (13)C, the isotopes are not totally required during the initial growth phase of P. pastoris culture. The addition of small amounts of (15)N and (13)C compounds 6 h prior to the methanol induction phase is sufficient to obtain 99% incorporation of heavy isotopes into the protein. Our optimized protocol P4 is two-thirds less costly than the classical method using (15)N and (13)C isotopes during the entire growth phase.

  2. Stereospecific assignments of glycine in proteins by stereospecific deuteration and {sup 15}N labeling

    SciTech Connect

    Hansen, A.P.; Curley, R.W. Jr.; Panigot, M.J.; Fesik, S.W.

    1994-12-01

    Stereospecific assignments are important for accurately determining the three-dimensional structures of proteins through the use of multidimensional NMR techniques. It is especially important to stereospecifically assign the glycine {alpha}-protons in proteins because of the potential for different backbone conformations of this residue. These stereospecific assignments are critical for interpreting the {sup 3}J{sub NH,{alpha}H} coupling constants and NOEs involving the glycine {alpha}-protons that determine the conformation of this part of the protein. However, it is often difficult to unambiguously obtain the stereospecific assignments for glycine residues by using only NOE data. In this poster, we present a method for unambiguous, stereospecific assignment of the {alpha}-protons of glycine residues. This method involves synthesis of stereo-specifically deuterated and {sup 15}N-labeled Gly using a slightly modified procedure originally described by Woodard and coworkers for the stereoselective deuteration of glycine. The stereospecifically deuterated and {sup 15}N-labeled Gy has been incorporated into recombinant proteins expressed in both bacterial systems (FKBP) and mammalian cells (u-PA). Two- and three-dimensional isotope-filtered and isotope-edited NMR experiments were used to obtain the stereospecific assignments of the glycine {alpha}-protons for these proteins.

  3. Automated Protein Turnover Calculations from 15N Partial Metabolic Labeling LC/MS Shotgun Proteomics Data

    PubMed Central

    Lyon, David; Castillejo, Maria Angeles; Staudinger, Christiana; Weckwerth, Wolfram; Wienkoop, Stefanie; Egelhofer, Volker

    2014-01-01

    Protein turnover is a well-controlled process in which polypeptides are constantly being degraded and subsequently replaced with newly synthesized copies. Extraction of composite spectral envelopes from complex LC/MS shotgun proteomics data can be a challenging task, due to the inherent complexity of biological samples. With partial metabolic labeling experiments this complexity increases as a result of the emergence of additional isotopic peaks. Automated spectral extraction and subsequent protein turnover calculations enable the analysis of gigabytes of data within minutes, a prerequisite for systems biology high throughput studies. Here we present a fully automated method for protein turnover calculations from shotgun proteomics data. The approach enables the analysis of complex shotgun LC/MS 15N partial metabolic labeling experiments. Spectral envelopes of 1419 peptides can be extracted within an hour. The method quantifies turnover by calculating the Relative Isotope Abundance (RIA), which is defined as the ratio between the intensity sum of all heavy (15N) to the intensity sum of all light (14N) and heavy peaks. To facilitate this process, we have developed a computer program based on our method, which is freely available to download at http://promex.pph.univie.ac.at/protover. PMID:24736476

  4. A spectral correlation function for efficient sequential NMR assignments of uniformly (15)N-labeled proteins.

    PubMed

    Bartels, C; Wüthrich, K

    1994-11-01

    A new computer-based approach is described for efficient sequence-specific assignment of uniformly (15)N-labeled proteins. For this purpose three-dimensional (15)N-correlated [(1)H, (1)H]-NOESY spectra are divided up into two-dimensional (1)H-(1)H strips which extend over the entire spectral width along one dimension and have a width of ca. 100 Hz, centered about the amide proton chemical shifts along the other dimension. A spectral correlation function enables sorting of these strips according to proximity of the corresponding residues in the amino acid sequence. Thereby, starting from a given strip in the spectrum, the probability of its corresponding to the C-terminal neighboring residue is calculated for all other strips from the similarity of their peak patterns with a pattern predicted for the sequentially adjoining residue, as manifested in the scalar product of the vectors representing the predicted and measured peak patterns. Tests with five different proteins containing both α-helices and β-sheets, and ranging in size from 58 to 165 amino acid residues show that the discrimination achieved between the sequentially neighboring residue and all other residues compares well with that obtained with an unguided interactive search of pairs of sequentially neighboring strips, with important savings in the time needed for complete analysis of 3D (15)N-correlated [(1)H, (1)H]-NOESY spectra. The integration of this routine into the program package XEASY ensures that remaining ambiguities can be resolved by visual inspection of the strips, combined with reference to the amino acid sequence and information on spin-system types obtained from additional NMR spectra.

  5. Cosine Modulated HSQC: A Rapid Determination of 3JHNHα Scalar Couplings in 15N-labeled Proteins

    NASA Astrophysics Data System (ADS)

    Petit, Audrey; Vincent, Sébastien J. F.; Zwahlen, Catherine

    2002-06-01

    A two-dimensional HSQC-based NMR method, 15N-COSMO-HSQC, is presented for the rapid determination of homonuclear 3JHNHα couplings in 15N-labeled proteins in solution. Scalar couplings are extracted by comparing the intensity of two separate datasets recorded with and without decoupling of the 3JHNHα during a preparation period. The scalar couplings are introduced through a cosine modulation of the peak intensities. The experiment relies on a BIRD sandwich to selectively invert all amide protons H N and is very simple to implement. 3JHNHα couplings were determined using both the 15N-COSMO-HSQC and quantitative- J on 15N-labeled chemokine RANTES. The two experiments show well-correlated values.

  6. Triple resonance experiments for aligned sample solid-state NMR of 13C and 15N labeled proteins

    PubMed Central

    Sinha, Neeraj; Grant, Christopher V.; Park, Sang Ho; Brown, Jonathan Miles; Opella, Stanley J.

    2013-01-01

    Initial steps in the development of a suite of triple-resonance 1H/13C/15N solid-state NMR experiments applicable to aligned samples of 13C and 15N labeled proteins are described. The experiments take advantage of the opportunities for 13C detection without the need for homonuclear 13C/13C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are ~20% randomly labeled with 13C in all backbone and side chain carbon sites and ~100% uniformly 15N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are 13C labeled at only the α-carbon and 15N labeled at the amide nitrogen of a few residues. The requirement for homonuclear 13C/13C decoupling while detecting 13C signals is avoided in the first case because of the low probability of any two 13C nuclei being bonded to each other; in the second case, the labeled 13Cα sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the 13C chemical shift and 1H–13C and 15N–13C heteronuclear dipolar coupling frequencies associated with the 13Cα and 13C′ backbone sites, which provide orientation constraints complementary to those derived from the 15N labeled amide backbone sites. 13C/13C spin-exchange experiments identify proximate carbon sites. The ability to measure 13C–15N dipolar coupling frequencies and correlate 13C and 15N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the 13C chemical shift and 1H–13C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach. PMID:17293139

  7. Ner protein of phage Mu: Assignments using {sup 13}C/{sup 15}N-labeled protein

    SciTech Connect

    Strzelecka, T.; Gronenborn, A.M.; Clore, G.M.

    1994-12-01

    The Ner protein is a small (74-amino acid) DNA-binding protein that regulates a switch between the lysogenic and lytic stages of phage Mu. It inhibits expression of the C repressor gene and down-regulates its own expression. Two-dimensional NMR experiments on uniformly {sup 15}N-labeled protein provided most of the backbone and some of the sidechain proton assignments. The secondary structure determination using two-dimensional NOESY experiments showed that Ner consists of five {alpha}-helices. However, because most of the sidechain protons could not be assigned, the full structure was not determined. Using uniformly {sup 13}C/{sup 15}N-labeled Ner and a set of three-dimensional experiments, we were able to assign all of the backbone and 98% of the sidechain protons. In particular, the CBCANH and CBCA(CO)NH experiments were used to sequentially assign the C{alpha} and C{beta} resonances; the HCCH-CTOCSY and HCCH-COSY were used to assign sidechain carbon and proton resonances.

  8. Mapping membrane protein backbone dynamics: a comparison of site-directed spin labeling with NMR 15N-relaxation measurements.

    PubMed

    Lo, Ryan H; Kroncke, Brett M; Solomon, Tsega L; Columbus, Linda

    2014-10-01

    The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR (15)N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R1 relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.

  9. Production, Purification, and Characterization of 15N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements

    PubMed Central

    Jaruga, Pawel; Nelson, Bryant C.; Lowenthal, Mark S.; Jemth, Ann-Sofie; Loseva, Olga; Coskun, Erdem; Helleday, Thomas

    2016-01-01

    Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA–protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length 15N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of 15N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented. PMID:26791985

  10. Metabolic labeling with stable isotope nitrogen (15N) to follow amino acid and protein turnover of three plastid proteins in Chlamydomonas reinhardtii

    PubMed Central

    2014-01-01

    Background The length of time that a protein remains available to perform its function is significantly influenced by its turnover rate. Knowing the turnover rate of proteins involved in different processes is important to determining how long a function might progress even when the stimulus has been removed and no further synthesis of the particular proteins occurs. In this article, we describe the use of 15N-metabolic labeling coupled to GC-MS to follow the turnover of free amino acids and LC-MS/MS to identify and LC-MS to follow the turnover of specific proteins in Chlamydomonas reinhardtii. Results To achieve the metabolic labeling, the growth medium was formulated with standard Tris acetate phosphate medium (TAP) in which14NH4Cl was replaced with 15NH415NO3 and (14NH4)6Mo7O24.4H2O was replaced with Na2MoO4.2H2O. This medium designated 15N-TAP allowed CC-125 algal cells to grow normally. Mass isotopic distribution revealed successful 15N incorporation into 13 amino acids with approximately 98% labeling efficiency. Tryptic digestion of the 55 kDa SDS-PAGE bands from 14N- and 15N-labeled crude algal protein extracts followed by LC-MS/MS resulted in the identification of 27 proteins. Of these, five displayed peptide sequence confidence levels greater than 95% and protein sequence coverage greater than 25%. These proteins were the RuBisCo large subunit, ATP synthase CF1 alpha and beta subunits, the mitochondrial protein (F1F0 ATP synthase) and the cytosolic protein (S-adenosyl homocysteine hydroxylase). These proteins were present in both labeled and unlabeled samples. Once the newly synthesized 15N-labeled free amino acids and proteins obtained maximum incorporation of the 15N-label, turnover rates were determined after transfer of cells into 14N-TAP medium. The t½ values were determined for the three plastid proteins (RuBisCo, ATP synthase CF1 alpha and beta) by following the reduction of the 15N-fractional abundance over time. Conclusion We describe a more

  11. Determination of Multimodal Isotopic Distributions: The Case of a (15)N Labeled Protein Produced into Hairy Roots.

    PubMed

    Trouillard, Romain; Hubert-Roux, Marie; Tognetti, Vincent; Guilhaudis, Laure; Plasson, Carole; Menu-Bouaouiche, Laurence; Coquet, Laurent; Guerineau, François; Hardouin, Julie; Ele Ekouna, Jean-Pierre; Cosette, Pascal; Lerouge, Patrice; Boitel-Conti, Michèle; Afonso, Carlos; Ségalas-Milazzo, Isabelle

    2015-06-16

    Isotopic labeling is widely used in various fields like proteomics, metabolomics, fluxomics, as well as in NMR structural studies, but it requires an efficient determination of the isotopic enrichment. Mass spectrometry is the method of choice for such analysis. However, when complex expression systems like hairy roots are used for production, multiple populations of labeled proteins may be obtained. If the isotopic incorporation determination is actually well-known for unimodal distributions, the multimodal distributions have scarcely been investigated. Actually, only a few approaches allow the determination of the different labeled population proportions from multimodal distributions. Furthermore, they cannot be used when the number of the populations and their respective isotope ratios are unknown. The present study implements a new strategy to measure the (15)N labeled populations inside a multimodal distribution knowing only the peptide sequence and peak intensities from mass spectrometry analyses. Noteworthy, it could be applied to other elements, like carbon and hydrogen, and extended to a larger range of biomolecules.

  12. Interresidue carbonyl-carbonyl polarization transfer experiments in uniformly 13C, 15N-labeled peptides and proteins

    NASA Astrophysics Data System (ADS)

    Janik, Rafal; Ritz, Emily; Gravelle, Andrew; Shi, Lichi; Peng, Xiaohu; Ladizhansky, Vladimir

    2010-03-01

    In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl-carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly 13C, 15N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory. At spinning frequencies used in this study, 22 kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600 MHz. To match HORROR condition over all powder orientations, variable amplitude radio frequency (RF) fields are required, and efficient direct transfers on the order of 20-30% can be straightforwardly established. Two- and three-dimensional chemical shift correlation experiments establishing long-range interresidue connectivities (e.g., (N[i]-CO[i - 2])) are demonstrated on the model peptide N-acetyl-valine-leucine, and on the third immunoglobulin binding domain of protein G. Possible future developments are discussed.

  13. Interresidue carbonyl-carbonyl polarization transfer experiments in uniformly 13C,15N-labeled peptides and proteins.

    PubMed

    Janik, Rafal; Ritz, Emily; Gravelle, Andrew; Shi, Lichi; Peng, Xiaohu; Ladizhansky, Vladimir

    2010-03-01

    In this work, we demonstrate that Homonuclear Rotary Resonance Recoupling (HORROR) can be used to reintroduce carbonyl-carbonyl interresidue dipolar interactions and to achieve efficient polarization transfer between carbonyl atoms in uniformly (13)C,(15)N-labeled peptides and proteins. We show that the HORROR condition is anisotropically broadened and overall shifted to higher radio frequency intensities because of the CSA effects. These effects are analyzed theoretically using Average Hamiltonian Theory. At spinning frequencies used in this study, 22kHz, this broadening is experimentally found to be on the order of a kilohertz at a proton field of 600MHz. To match HORROR condition over all powder orientations, variable amplitude radio frequency (RF) fields are required, and efficient direct transfers on the order of 20-30% can be straightforwardly established. Two- and three-dimensional chemical shift correlation experiments establishing long-range interresidue connectivities (e.g., (N[i]-CO[i-2])) are demonstrated on the model peptide N-acetyl-valine-leucine, and on the third immunoglobulin binding domain of protein G. Possible future developments are discussed. PMID:20060344

  14. Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots*

    PubMed Central

    Li, Lei; Nelson, Clark J.; Solheim, Cory; Whelan, James; Millar, A. Harvey

    2012-01-01

    The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a 15N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (KS) and degradation (KD) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify KS and KD for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. KS and KD correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive 15N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability. PMID:22215636

  15. hNCOcanH pulse sequence and a robust protocol for rapid and unambiguous assignment of backbone ((1)H(N), (15)N and (13)C') resonances in (15)N/(13)C-labeled proteins.

    PubMed

    Kumar, Dinesh; Hosur, Ramakrishna V

    2011-09-01

    A three-dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in (15)N/(13)C-labeled proteins. The experiment has been derived by a simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147]; t2 evolution is used to frequency label (13)C' rather than (15)N (similar trick has also been used in the design of hNCAnH pulse sequence from hNcaNH [Frueh et al., JACS, 131 (2009) 12880-12881]). The modification results in a spectrum equivalent to HNCO, but in addition to inter-residue correlation peaks (i.e. Hi , Ci-1), the spectrum also contains additional intra-residue correlation peaks (i.e. Hi-1 , Ci-1) in the direct proton dimension which has maximum resolution. This is the main strength of the experiment and thus, even a small difference in amide (1) H chemical shifts (5-6 Hz) can be used for establishing a sequential connectivity. This experiment in combination with the HNN experiment described previously [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] leads to a more robust assignment protocol for backbone resonances ((1) H(N) , (15)N) than could be derived from the combination of HNN and HN(C)N experiments [Bhavesh et al., Biochemistry, 40 (2001) 14727-14735]. Further, this new protocol enables assignment of (13)C' resonances as well. We believe that the experiment and the protocol presented here will be of immense value for structural-and functional-proteomics research by NMR. Performance of this experiment has been demonstrated using (13)C/(15)N labeled ubiquitin.

  16. hNCOcanH pulse sequence and a robust protocol for rapid and unambiguous assignment of backbone ((1)H(N), (15)N and (13)C') resonances in (15)N/(13)C-labeled proteins.

    PubMed

    Kumar, Dinesh; Hosur, Ramakrishna V

    2011-09-01

    A three-dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in (15)N/(13)C-labeled proteins. The experiment has been derived by a simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147]; t2 evolution is used to frequency label (13)C' rather than (15)N (similar trick has also been used in the design of hNCAnH pulse sequence from hNcaNH [Frueh et al., JACS, 131 (2009) 12880-12881]). The modification results in a spectrum equivalent to HNCO, but in addition to inter-residue correlation peaks (i.e. Hi , Ci-1), the spectrum also contains additional intra-residue correlation peaks (i.e. Hi-1 , Ci-1) in the direct proton dimension which has maximum resolution. This is the main strength of the experiment and thus, even a small difference in amide (1) H chemical shifts (5-6 Hz) can be used for establishing a sequential connectivity. This experiment in combination with the HNN experiment described previously [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] leads to a more robust assignment protocol for backbone resonances ((1) H(N) , (15)N) than could be derived from the combination of HNN and HN(C)N experiments [Bhavesh et al., Biochemistry, 40 (2001) 14727-14735]. Further, this new protocol enables assignment of (13)C' resonances as well. We believe that the experiment and the protocol presented here will be of immense value for structural-and functional-proteomics research by NMR. Performance of this experiment has been demonstrated using (13)C/(15)N labeled ubiquitin. PMID:21818779

  17. A new strategy for sequential assignment of intrinsically unstructured proteins based on 15N single isotope labelling

    NASA Astrophysics Data System (ADS)

    Lopez, Juan; Ahuja, Puneet; Gerard, Melanie; Wieruszeski, Jean-Michel; Lippens, Guy

    2013-11-01

    We describe a new efficient strategy for the sequential assignment of amide resonances of a conventional 15N-1H HSQC spectrum of intrinsically unfolded proteins, based on composite NOESY-TOCSY and TOCSY-NOESY mixing times. These composite mixing times lead to a Hα-proton mediated unidirectional transfer of amide to amide proton. We have implemented the composite mixing times in an HSQC-NOESY-HSQC manner to obtain directional connectivity between amides of neighbouring residues. We experimentally determine the optimal mixing times for both transfer schemes, and demonstrate its use in the assignment for both a fragment of the neuronal tau protein and for α-synuclein.

  18. Uniform {sup 15}N- and {sup 15}N/{sup 13}C-labeling of proteins in mammalian cells and solution structure of the amino terminal fragment of u-PA

    SciTech Connect

    Hansen, A.P.; Petros, A.M.; Meadows, R.P.; Mazar, A.P.; Nettesheim, D.G.; Pederson, T.M.; Fesik, S.W.

    1994-12-01

    Urokinase-type plasminogen activator (u-PA) is a 54-kDa glycoprotein that catalyzes the conversion of plasminogen to plasmin, a broad-specificity protease responsible for the degradation of fibrin clots and extracellular matrix components. The u-PA protein consists of three individual modules: a growth factor domain (GFD), a kringle, and a serine protease domain. The amino terminal fragment (ATF) includes the GFD-responsible for u-PA binding to its receptor-and the kringle domains. This protein was expressed and uniformly {sup 15}N-and {sup 15}N/{sup 13}C-labeled in mammalian cells by methods that will be described. In addition, we present the three-dimensional structure of ATF that was derived from 1299 NOE-derived distance restraints along with the {phi} angle and hydrogen bonding restraints. Although the individual domains in the structures were highly converged, the two domains are structurally independent. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor domain of u-PA and other homologous proteins were observed in the region that has been implicated in binding the urokinase receptor. These results may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.

  19. An intraresidual i(HCA)CO(CA)NH experiment for the assignment of main-chain resonances in 15N, 13C labeled proteins.

    PubMed

    Mäntylahti, Sampo; Tossavainen, Helena; Hellman, Maarit; Permi, Perttu

    2009-11-01

    An improved pulse sequence, intraresidual i(HCA)CO(CA)NH, is described for establishing solely (13)C'(i), (15)N(i), (1)HN(i) connectivities in uniformly 15N/13C-labeled proteins. In comparison to the "out-and-back" style intra-HN(CA)CO experiment, the new pulse sequence offers at least two-fold higher experimental resolution in the (13)C' dimension and on average 1.6 times higher sensitivity especially for residues in alpha-helices. Performance of the new experiment was tested on a small globular protein ubiquitin and an intrinsically unfolded 110-residue cancer/testis antigen CT16/PAGE5. Use of intraresidual i(HCA)CO(CA)NH experiment in combination with the established HNCO experiment was crucial for the assignment of highly disordered CT16. PMID:19768387

  20. HCN, A Triple-Resonance NMR Technique for Selective Observation of Histidine and Tryptophan Side Chains in 13C/ 15N-Labeled Proteins

    NASA Astrophysics Data System (ADS)

    Sudmeier, James L.; Ash, Elissa L.; Günther, Ulrich L.; Luo, Xuelian; Bullock, Peter A.; Bachovchin, William W.

    1996-12-01

    HCN, a new 3D NMR technique for stepwise coherence transfer from1H to13C to15N and reverse through direct spin couplings1JCHand1JCN, is presented as a method for detection and assignment of histidine and tryptophan side-chain1H,13C, and15N resonances in uniformly13C/15N-labeled proteins. Product-operator calculations of cross-peak volumes vs adjustable delay τ3were employed for determination of optimal τ3. For the phosphatidylinositol 3-kinase (PI3K SH3 domain, MW = 9.6 kD) at pH 6, H(C)N, the1H/15N projection, produced observable cross peaks within 20 min. and was completely selective for the single tryptophan and single histidine. The 3D HCN experiment yielded well-defined cross peaks in 20 h for the13C/15N-labeled origin-specific DNA binding domain from simian virus 40 T-antigen (T-ag-OBD131-259, MW = 15.4 kD) at pH 5.5. Resonances from all six histidines in T-ag-OBD were observed, and 11 of the 121H and13C chemical shifts and 10 of the 1215N chemical shifts were determined. The13C dimension proved essential in assignment of the multiply overlapping1H and15N resonances. From the spectra recorded at a single pH, three of the imidazoles were essentially neutral and the other three were partially protonated (22-37%). HCN yielded strong cross peaks after 18 h on a 2.0 mMsample of phenylmethanesulfonyl fluoride (PMSF)-inhibited α-lytic protease (MW = 19.8 kD) at pH 4.4. No spectra have been obtained, however, of native or boronic acid-inhibited α-lytic protease after 18 h at various temperatures ranging from 5 to 55°C, probably due to efficient relaxation of active-site imidazole1H and/or15N nuclei.

  1. Restraints on backbone conformations in solid state NMR studies of uniformly labeled proteins from quantitative amide 15N–15N and carbonyl 13C–13C dipolar recoupling data

    PubMed Central

    Hu, Kan-Nian; Qiang, Wei; Bermejo, Guillermo A.; Schwieters, Charles D.; Tycko, Robert

    2013-01-01

    Recent structural studies of uniformly 15N, 13C-labeled proteins by solid state nuclear magnetic resonance (NMR) rely principally on two sources of structural restraints: (i) restraints on backbone conformation from isotropic 15N and 13C chemical shifts, based on empirical correlations between chemical shifts and backbone torsion angles; (ii) restraints on inter-residue proximities from qualitative measurements of internuclear dipole–dipole couplings, detected as the presence or absence of inter-residue crosspeaks in multidimensional spectra. We show that site-specific dipole–dipole couplings among 15N-labeled backbone amide sites and among 13C-labeled backbone carbonyl sites can be measured quantitatively in uniformly-labeled proteins, using dipolar recoupling techniques that we call 15N-BARE and 13C-BARE (BAckbone REcoupling), and that the resulting data represent a new source of restraints on backbone conformation. 15N-BARE and 13C-BARE data can be incorporated into structural modeling calculations as potential energy surfaces, which are derived from comparisons between experimental 15N and 13C signal decay curves, extracted from crosspeak intensities in series of two-dimensional spectra, with numerical simulations of the 15N-BARE and 13C-BARE measurements. We demonstrate this approach through experiments on microcrystalline, uniformly 15N, 13C-labeled protein GB1. Results for GB1 show that 15N-BARE and 13C-BARE restraints are complementary to restraints from chemical shifts and inter-residue crosspeaks, improving both the precision and the accuracy of calculated structures. PMID:22449573

  2. Three-Dimensional Solid-State NMR Spectroscopy Is Essential for Resolution of Resonances from In-Plane Residues in Uniformly 15N-Labeled Helical Membrane Proteins in Oriented Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Marassi, Francesca M.; Ma, Che; Gesell, Jennifer J.; Opella, Stanley J.

    2000-05-01

    Uniformly 15N-labeled samples of membrane proteins with helices aligned parallel to the membrane surface give two-dimensional PISEMA spectra that are highly overlapped due to limited dispersions of 1H-15N dipolar coupling and 15N chemical shift frequencies. However, resolution is greatly improved in three-dimensional 1H chemical shift/1H-15N dipolar coupling/15N chemical shift correlation spectra. The 23-residue antibiotic peptide magainin and a 54-residue polypeptide corresponding to the cytoplasmic domain of the HIV-1 accessory protein Vpu are used as examples. Both polypeptides consist almost entirely of α-helices, with their axes aligned parallel to the membrane surface. The measurement of three orientationally dependent frequencies for Val17 of magainin enabled the three-dimensional orientation of this helical peptide to be determined in the lipid bilayer.

  3. Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded {sup 15}N/{sup 19}F-labeled unnatural amino acid

    SciTech Connect

    Shi, Pan; Xi, Zhaoyong; Wang, Hu; Shi, Chaowei; Xiong, Ying; Tian, Changlin

    2010-11-19

    Research highlights: {yields} Chemical synthesis of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine. {yields} Site-specific incorporation of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine to SH3. {yields} Site-specific backbone and side chain chemical shift and relaxation analysis. {yields} Different internal motions at different sites of SH3 domain upon ligand binding. -- Abstract: SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for {sup 15}N/{sup 19}F-trifluoromethyl-phenylalanine ({sup 15}N/{sup 19}F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.

  4. 15N chemical shift tensors and conformation of solid polypeptides containing 15N-labeled glycine residue by 15N NMR

    NASA Astrophysics Data System (ADS)

    Shoji, Akira; Ozaki, Takuo; Fujito, Teruaki; Deguchi, Kenzo; Ando, Isao; Magoshi, Jun

    1998-01-01

    The correlation between the isotropic 15N chemical shift ( δiso) and 15N chemical shift tensor components ( δ11, δ22 and δ33) and the main-chain conformation such as the polyglycine I (PGI: β-sheet), II (PGII: 3 1-helix), α-helix and β-sheet forms of solid polypeptides [Gly∗,X] n consisting of 15N-labeled glycine (Gly∗) and other amino acids (X: natural abundance of 15N) has been studied by solid-state 15N NMR method. A series of polypeptides [Gly∗,X] n (X = glycine, L-alanine, L-leucine, L-valine, L-isoleucine, β-benzyl L-aspartate, γ-benzyl L-glutamate, ɛ-carbobenzoxy L-lysine, and sarcosine) were synthesized by the α-amino acid N-carboxy anhydride (NCA) method. Conformations of these polypeptides in the solid state were characterized on the basis of conformation-dependent 13C chemical shifts in the 13C cross-polarization-magic angle spinning (CP-MAS) NMR spectra and by the characteristic bands in the IR and far-IR spectra. The δiso, δ11, δ22 and δ33 of the polypetides were determined from the 15N CP-MAS and 15N CP-static (powder pattern) spectra. It was found that the δiso, δ11, δ22 and δ33 in the PGI form (δ 83.5, 185, 40.7 and 25 ppm, resp.) are upfield from those in the PGII form (88.5, 194, 42.1 and 29 ppm, resp.), which were reproduced by the calculated 15N shielding constants using the finite perturbation theory (FPT)-INDO method. It was also found that the δ22 of the Gly∗ of [Gly∗,X] n is closely related to the main-chain conformation and the neighboring amino acid sequence, although the δiso is almost independent of the glycine content and conformation. Consequently, the δ22 value of Gly∗ containing copolypeptides is useful for the structural (main-chain conformation and neighboring amino acid sequence) analysis in the solid state by 15N NMR, if the 15N-labeled copolypeptide or natural protein can be provided. In addition, it is shown that the δiso of the glycine residue is useful for the conformational study of some

  5. Refining cotton-wick method for 15N plant labelling.

    NASA Astrophysics Data System (ADS)

    Fustec, Joëlle; Mahieu, Stéphanie

    2010-05-01

    The symbiosis Fabaceae/Rhizobiaceae plays a critical role in the nitrogen cycle. It gives the plant the ability to fix high amounts of atmospheric N. A part of this N can be transferred to the soil via rhizodeposition. The contribution of Fabaceae to the soil N pool is difficult to measure, since it is necessary for assessing N benefits for other crops, for soil biological activity, and for reducing water pollution in sustainable agriculture (Fustec, 2009). The aim of this study was to test and improve the reliability of the 15N cotton-wick method for measuring the soil N derived from plant rhizodeposition (Mahieu et al., 2007). The effects of the concentration of the 15N-urea labelling solution and of the feeding frequency (continuous or pulses) on the assessment of nitrogen rhizodeposition were studied in two greenhouse experiments using the field pea (Pisum sativum L.) and the non-nodulating isoline P2. The plant parts and the soil were prepared for 15N:14N measurements for assessing N rhizodeposition (Mahieu et al., 2009). The fraction of plants' belowground nitrogen allocated to rhizodeposition in both Frisson pea and P2 was 20 to more than 50% higher when plants were labelled continuously than when they were labelled using fortnightly pulses. Our results suggested that when 15N root enrichment was high, nitrogen rhizodeposition was underestimated only for plants that were 15N-fed by fortnightly pulses, and not in plants 15N-fed continuously. This phenomenon was especially observed for plants relying on symbiotic N fixation for N acquisition; it may be linked to the concentration of the labelling solution. In conclusion, N rhizodeposition assessment was strongly influenced by the 15N-feeding frequency and the concentration of the labelling solution. The estimation of N rhizodeposition was more reliable when plants were labelled continuously with a dilute solution of 15N urea. Fustec et al. 2009. Agron. Sustain. Dev., DOI 10.1051/agro/2009003, in press. Mahieu

  6. In vivo uniform (15)N-isotope labelling of plants: using the greenhouse for structural proteomics.

    PubMed

    Ippel, Johannes H; Pouvreau, Laurice; Kroef, Toos; Gruppen, Harry; Versteeg, Geurt; van den Putten, Peter; Struik, Paul C; van Mierlo, Carlo P M

    2004-01-01

    Isotope labelling of proteins is important for progress in the field of structural proteomics. It enables the utilisation of the power of nuclear magnetic resonance spectroscopy (NMR) for the characterisation of the three-dimensional structures and corresponding dynamical features of proteins. The usual approach to obtain isotopically labelled protein molecules is by expressing the corresponding gene in bacterial or yeast host organisms, which grow on isotope-enriched media. This method has several drawbacks. Here, we demonstrate that it is possible to fully label a plant with (15)N-isotopes. The advantage of in vivo labelling of higher organisms is that all constituting proteins are labelled and become available as functional, post-translationally modified, correctly folded proteins. A hydroponics set-up was used to create the first example of a uniformly (15)N-labelled (> 98%) plant species, the potato plant (Solanum tuberosum L., cv. Elkana). Two plants were grown at low costs using potassium-[(15)N]-nitrate as the sole nitrogen source. At harvest time, a total of 3.6 kg of potato tubers and 1.6 kg of foliage, stolons and roots were collected, all of which were fully (15)N-labelled. Gram quantities of soluble (15)N-labelled proteins (composed mainly of the glycoprotein patatin and Kunitz-type protease inhibitors) were isolated from the tubers. NMR results on the complete proteome of potato sap and on an isolated protease inhibitor illustrate the success of the labelling procedure. The presented method of isotope labelling is easily modified to label other plants. Its envisioned impact in the field of structural proteomics of plants is discussed.

  7. Synthesis and NMR of {sup 15}N-labeled DNA fragments

    SciTech Connect

    Jones, R.A.

    1994-12-01

    DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

  8. hnCOcaNH and hncoCANH pulse sequences for rapid and unambiguous backbone assignment in (13C, 15N) labeled proteins.

    PubMed

    Kumar, Dinesh; Reddy, Jithender G; Hosur, Ramakrishna V

    2010-09-01

    Time-saving in data acquisition is a major thrust of NMR pulse sequence development in the context of structural proteomics research. The conventional HNCA and HN(CA)CO pulse sequences, routinely used for sequential backbone assignment, have the limitation that they cannot distinguish inter- and intra-residue correlations. In order to remove this ambiguity, one has to record HNCO and HN(CO)CA or sequential HNCA experiments which provide unambiguous information of sequential correlations. However, this almost doubles the experimental time. Besides, they require repeated scanning through the (15)N planes to search for the matching peaks along the carbon dimension. In this background, we present here two pulse sequences, termed as hncoCANH and hnCOcaNH that lead to spectra equivalent to HNCA and HN(CA)CO spectra, respectively, but with direct distinction of inter- and intra-residue peaks; these occur with opposite signs in the new experiments. The two pulse sequences have been derived by simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] to frequency-label (13)C(alpha) or (13)C' instead of (15)N during the t(1) period. Like HN(C)N, these spectra also exhibit special patterns of self and sequential peaks around glycines and prolines, which enable direct identification of certain triplets of residues and thus provide internal checks during the sequential assignment walk. The spectra enable rapid and unambiguous assignment of H(N), (15)N and (13)C(alpha) (or (13)C') in a single experiment, and thus would be of great value in high-throughput structural proteomics. PMID:20643567

  9. Affordable uniform isotope labeling with (2)H, (13)C and (15)N in insect cells.

    PubMed

    Sitarska, Agnieszka; Skora, Lukasz; Klopp, Julia; Roest, Susan; Fernández, César; Shrestha, Binesh; Gossert, Alvar D

    2015-06-01

    For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80% can be achieved for (15)N and (13)C with yields comparable to expression in full media. For (2)H,(15)N and (2)H,(13)C,(15)N labeling, incorporation is only slightly lower with 75 and 73%, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins. PMID:25929326

  10. Biosynthetic uniform 13C,15N-labelling of zervamicin IIB. Complete 13C and 15N NMR assignment.

    PubMed

    Ovchinnikova, Tatyana V; Shenkarev, Zakhar O; Yakimenko, Zoya A; Svishcheva, Natalia V; Tagaev, Andrey A; Skladnev, Dmitry A; Arseniev, Alexander S

    2003-01-01

    Zervamicin IIB is a member of the alpha-aminoisobutyric acid containing peptaibol antibiotics. A new procedure for the biosynthetic preparation of the uniformly 13C- and 15N-enriched peptaibol is described This compound was isolated from the biomass of the fungus-producer Emericellopsis salmosynnemata strain 336 IMI 58330 obtained upon cultivation in the totally 13C, 15N-labelled complete medium. To prepare such a medium the autolysed biomass and the exopolysaccharides of the obligate methylotrophic bacterium Methylobacillus flagellatus KT were used. This microorganism was grown in totally 13C, 15N-labelled minimal medium containing 13C-methanol and 15N-ammonium chloride as the only carbon and nitrogen sources. Preliminary NMR spectroscopic analysis indicated a high extent of isotope incorporation (> 90%) and led to the complete 13C- and 15N-NMR assignment including the stereospecific assignment of Aib residues methyl groups. The observed pattern of the structurally important secondary chemical shifts of 1H(alpha), 13C=O and 13C(alpha) agrees well with the previously determined structure of zervamicin IIB in methanol solution. PMID:14658801

  11. The “Speedy” Synthesis of Atom-Specific 15N Imino/Amido-Labeled RNA

    PubMed Central

    Kreutz, Christoph; Micura, Ronald

    2016-01-01

    Although numerous reports on the synthesis of atom-specific 15N-labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid-phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of 15N(1) adenosine and 15N(1) guanosine amidites, which are the much needed counterparts of the more straightforward-to-achieve 15N(3) uridine and 15N(3) cytidine amidites in order to tap full potential of 1H/15N/15N-COSY experiments for directly monitoring individual Watson–Crick base pairs in RNA. Demonstrated for two preQ1 riboswitch systems, we exemplify a versatile concept for individual base-pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered. PMID:26237536

  12. Preparation of 13C/15N-labeled oligomers using the polymerase chain reaction

    DOEpatents

    Chen, Xian; Gupta, Goutam; Bradbury, E. Morton

    2001-01-01

    Preparation of .sup.13 C/.sup.15 N-labeled DNA oligomers using the polymerase chain reaction (PCR). A PCR based method for uniform (.sup.13 C/.sup.15 N)-labeling of DNA duplexes is described. Multiple copies of a blunt-ended duplex are cloned into a plasmid, each copy containing the sequence of interest and restriction Hinc II sequences at both the 5' and 3' ends. PCR using bi-directional primers and uniformly .sup.13 C/.sup.15 N-labeled dNTP precursors generates labeled DNA duplexes containing multiple copies of the sequence of interest. Twenty-four cycles of PCR, followed by restriction and purification, gave the uniformly .sup.13 C/.sup.15 N-labeled duplex sequence with a 30% yield. Such labeled duplexes find significant applications in multinuclear magnetic resonance spectroscopy.

  13. Measurement of 1J(Ni,Calpha(i)), 1J(Ni,C'i-1), 2J(Ni,Calpha(i-1)), 2J(H(N)i,C'i-1) and 2J(H(N)i,Calpha(i)) values in 13C/15N-labeled proteins.

    PubMed

    Mukherjee, Sulakshana; Mustafi, Sourajit M; Atreya, H S; Chary, K V R

    2005-04-01

    Use of partial or selective (13)C/(15)N labeling of specific amino acid residues in a given protein to measure the values of (1)J((15)N(i),(13)C(alpha) (i)), (2)J((1)H(N),(13)C(alpha) (i)), (2)J((15)N(i),(13)C(alpha) (i-1)), (1)J((15)N(i),(13)C'(i-1)) and (2)J((1)H(N),(13)C'(i-1)) is described. This was achieved by recording a sensitivity-enhanced 2D [(15)N-(1)H] HSQC experiment, without mixing the spin states of C(alpha) and C' during the course of entire experiment.

  14. Fungal Functioning In A Pine Forest: Evidence From A 15N-Labeled Global Change Experiment

    NASA Astrophysics Data System (ADS)

    Hobbie, E. A.; Hofmockel, K.; van Diepen, L.

    2012-12-01

    In this study, we used tracer 15N labeling and a concurrent six-year nitrogen fertilization experiment in a Pinus taeda Free Air CO2 Enrichment (FACE) experiment to investigate the functioning of saprotrophic and ectomycorrhizal fungi in nitrogen cycling. Ectomycorrhizal fungi with hydrophobic ectomycorrhizae acquired nitrogen from deeper in the soil profile than taxa with hydrophilic ectomycorrhizae, whereas saprotrophic fungi acquired nitrogen primarily from relatively recent surface litter. By combining natural abundance and tracer measurements, we estimated the 15N enrichment of fungi relative to source nitrogen at between 4‰ and 9‰, depending on genus, with some genera sampling pools more 15N-enriched in the labeling experiment than could be measured by bulk analyses. Thus, biologically relevant nitrogen fractions may correspond poorly to what can be extracted chemically. The multiple 15N labels in this FACE experiment proved useful for tracking nitrogen into different fungal taxa from potential nitrogen sources.

  15. Numerical evaluation of subsoil diffusion of (15) N labelled denitrification products during employment of the (15) N gas flux method in the field

    NASA Astrophysics Data System (ADS)

    Well, Reinhard; Buchen, Caroline; Lewicka-Szczebak, Dominika; Ruoss, Nicolas

    2016-04-01

    Common methods for measuring soil denitrification in situ include monitoring the accumulation of 15N labelled N2 and N2O evolved from 15N labelled soil nitrate pool in soil surface chambers. Gas diffusion is considered to be the main accumulation process. Because accumulation of the gases decreases concentration gradients between soil and chamber over time, gas production rates are underestimated if calculated from chamber concentrations. Moreover, concentration gradients to the non-labelled subsoil exist, inevitably causing downward diffusion of 15N labelled denitrification products. A numerical model for simulating gas diffusion in soil was used in order to determine the significance of this source of error. Results show that subsoil diffusion of 15N labelled N2 and N2O - and thus potential underestimation of denitrification derived from chamber fluxes - increases with cover closure time as well as with increasing diffusivity. Simulations based on the range of typical gas diffusivities of unsaturated soils show that the fraction of subsoil diffusion after chamber closure for 1 hour is always significant with values up to >30 % of total production of 15N labelled N2 and N2O. Field experiments for measuring denitrification with the 15N gas flux method were conducted. The ability of the model to predict the time pattern of gas accumulation was evaluated by comparing measured 15N2 concentrations and simulated values.

  16. Sub-cellular localisation of a 15N-labelled peptide vector using NanoSIMS imaging

    NASA Astrophysics Data System (ADS)

    Römer, Winfried; Wu, Ting-Di; Duchambon, Patricia; Amessou, Mohamed; Carrez, Danièle; Johannes, Ludger; Guerquin-Kern, Jean-Luc

    2006-07-01

    Dynamic SIMS imaging is proposed to map sub-cellular distributions of isotopically labelled, exogenous compounds. NanoSIMS imaging allows the characterisation of the intracellular transport pathways of exogenous molecules, including peptide vectors employed in innovative therapies, using stable isotopes as molecular markers to detect the compound of interest. Shiga toxin B-subunit (STxB) was chosen as a representative peptide vector. The recombinant protein ( 15N-STxB) was synthesised in Escherichia coli using 15NH 4Cl as sole nitrogen source resulting in 15N enrichment in the molecule. Using the NanoSIMS 50 ion microprobe (Cameca), different ion species ( 12C 14N -, 12C 15N -, 31P -) originating from the same sputtered micro volume were simultaneously detected. High mass resolving power enabled the discrimination of 12C 15N - from its polyatomic isobars of mass 27. We imaged the membrane binding and internalisation of 15N-STxB in HeLa cells at spatial resolutions of less than 100 nm. Thus, the use of rare stable isotopes like 15N with dynamic SIMS imaging permits sub-cellular detection of isotopically labelled, exogenous molecules and imaging of their transport pathways at high mass and spatial resolution. Application of stable isotopes as markers can replace the large and chemically complex tags used for fluorescence microscopy, without altering the chemical and physical properties of the molecule.

  17. Identification of the magnesium-histidine stretching vibration of the bacteriochlorophyll cofactors in photosynthetic reaction centers via {sup 15}N-labeling of the histidines

    SciTech Connect

    Czarnecki, K.; Bocian, D.F.; Chynwat, V.; Erickson, J.P.; Frank, H.A.

    1997-03-12

    In this communication, we report low-frequency, near-infrared-exciation RR spectra of bacterial RCs in which the histidine residues of the protein are selectively labeled with {sup 15}N. For practical reasons, the studies were conducted by comparing the vibrational signatures of RCs in which {sup 15}N was universally incorporated (all cofactors and all protein residues) (designated all-{sup 15}N RCs) with those in which [{sup 14}N]histidine was introduced as a reverse label (disignated {sup 14}N-His RCs) into the all {sup 15}N-labeled RCs. The studies of the histidine-labeled RCs reveal that the vibrational characteristics of the BChl core are far more complicated than originally anticipated. These results have clear implications for the photoexcitation dynamics of the BChls in RCs and may also have significant consequences for the dynamics of exogenous ligand binding to heme-based oxygen carriers. 12 refs., 2 figs.

  18. Compound-specific 15N analysis of amino acids in 15N tracer experiments provide an estimate of newly synthesised soil protein from inorganic and organic substrates

    NASA Astrophysics Data System (ADS)

    Charteris, Alice; Michaelides, Katerina; Evershed, Richard

    2015-04-01

    Organic N concentrations far exceed those of inorganic N in most soils and despite much investigation, the composition and cycling of this complex pool of SOM remains poorly understood. A particular problem has been separating more recalcitrant soil organic N from that actively cycling through the soil system; an important consideration in N cycling studies and for the soil's nutrient supplying capacity. The use of 15N-labelled substrates as stable isotope tracers has contributed much to our understanding of the soil system, but the complexity and heterogeneity of soil organic N prevents thorough compound-specific 15N analyses of organic N compounds and makes it difficult to examine any 15N-labelled organic products in any detail. As a result, a significant proportion of previous work has either simply assumed that since the majority of soil N is organic, all of the 15N retained in the soil is organic N (e.g. Sebilo et al., 2013) or subtracted 15N-labelled inorganic compounds from bulk values (e.g. Pilbeam et al., 1997). While the latter approach is more accurate, these methods only provide an estimate of the bulk 15N value of an extremely complex and non-uniformly labelled organic pool. A more detailed approach has been to use microbial biomass extraction (Brookes et al., 1985) and subsequent N isotopic analysis to determine the 15N value of biomass-N, representing the fraction of 15N assimilated by microbes or the 15N cycling through the 'living' or 'active' portion of soil organic N. However, this extraction method can only generate estimates and some lack of confidence in its validity and reliability remains. Here, we present an alternative technique to obtain a measure of the assimilation of an applied 15N substrate by the soil microbial biomass and an estimate of the newly synthesized soil protein, which is representative of the magnitude of the active soil microbial biomass. The technique uses a stable isotope tracer and compound-specific 15N analysis, but

  19. Expression, purification, and mass spectrometric analysis of 15N, 13C-labeled RGD-hirudin, expressed in Pichia pastoris, for NMR studies.

    PubMed

    Huang, Yinong; Zhang, Yanling; Wu, Yi; Wang, Jue; Liu, Xingang; Dai, Linsen; Wang, Longsheng; Yu, Min; Mo, Wei

    2012-01-01

    A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed (15)N, (13)C-labeled RGD-hirudin in Pichia pastoris in a fermenter. The protein was subsequently purified to yield sufficient quantities for structural and functional studies. The purified protein was characterized by HPLC and MALDI-TOF mass spectroscopy. Analysis revealed that the protein was pure and uniformly labeled with (15)N and (13)C. A bioassay showed that the anti-thrombin activity and the anti-platelet aggregation ability of the labeled protein were the same as those of unlabeled RGD-hirudin. Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone (15)N, (13)C and (1)H resonance assignments of the r-RGD-Hirudin. The (15)N-(1)H HSQC spectrum of uniformly (15)N, (13)C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the (15)N-(l)H correlation spectrum, and by selected planes of the CBCA(CO)NH, CBCANH, and HNCO experiments. These results provide a basis for further studies on the structure-function relationship of RGD-hirudin with thrombin and platelets. PMID:22879918

  20. Differential growth of the fungus Absidia cylindrospora on 13C/15N-labelled media.

    PubMed

    Crotty, F V; Blackshaw, R P; Murray, P J

    2011-06-15

    Many studies utilise enrichment of stable isotopes as tracers to follow the interactions occurring within soil food webs and methods have been developed to enrich bacteria, soil fauna and plant litter, Here for the first time we attempt to enrich a soil fungus to 99 atom% with (13)C and (15)N stable isotopes. In this study our objectives were to (a) assess whether the saprotrophic zygomycete fungus Absidia cylindrospora could grow on a medium enriched to 99 atom% with (13)C-glucose and (15)N-ammonium chloride, (b) to determine the level of enrichment obtained, and (c) to examine the change in growth rate of this fungus while it was growing on the dually enriched medium. To achieve this, the fungus was grown on agar enriched with (13)C and (15)N to 99 atom% and its growth rate monitored. The results showed that A. cylindrospora would grow on the highly labelled growth medium, but that its rate of growth was affected compared with the rate on either natural abundance media or media highly enriched with a single isotope ((13)C or (15)N). The implications of these results is that although the fungus is able to utilise these heavier isotopes, the biochemical processes involved in growth are affected, and consideration should be given to these differences when using stable isotope tracers in, for example, soil food web studies.

  1. Measurement of multiple psi torsion angles in uniformly 13C,15N-labeled alpha-spectrin SH3 domain using 3D 15N-13C-13C-15N MAS dipolar-chemical shift correlation spectroscopy.

    PubMed

    Ladizhansky, Vladimir; Jaroniec, Christopher P; Diehl, Annette; Oschkinat, Hartmut; Griffin, Robert G

    2003-06-01

    We demonstrate the simultaneous measurement of several backbone torsion angles psi in the uniformly (13)C,(15)N-labeled alpha-Spectrin SH3 domain using two different 3D 15N-13C-13C-15N dipolar-chemical shift magic-angle spinning (MAS) NMR experiments. The first NCCN experiment utilizes double quantum (DQ) spectroscopy combined with the INADEQUATE type 13C-13C chemical shift correlation. The decay of the DQ coherences formed between 13C'(i) and 13C(alphai) spin pairs is determined by the "correlated" dipolar field due to 15N(i)-13C(alphai) and 13C'(i)-15N(i+1) dipolar couplings and is particularly sensitive to variations of the torsion angle in the regime |psi| > 140 degrees. However, the ability of this experiment to constrain multiple psi-torsion angles is limited by the resolution of the 13C(alpha)-(13)CO correlation spectrum. This problem is partially addressed in the second approach described here, which is an NCOCA NCCN experiment. In this case the resolution is enhanced by the superior spectral dispersion of the 15N resonances present in the 15N(i+1)-13C(alphai) part of the NCOCA chemical shift correlation spectrum. For the case of the 62-residue alpha-spectrin SH3 domain, we determined 13 psi angle constraints with the INADEQUATE NCCN experiment and 22 psi constraints were measured in the NCOCA NCCN experiment.

  2. Development and application of 15N-tracer substances for measuring the whole-body protein turnover rates in the human, especially in neonates: a review.

    PubMed

    Wutzke, Klaus D

    2012-06-01

    Our research group of the Children's Hospital of the University of Rostock (Rostock group) has long-time experience in (15)N-labelling and in using yeast protein and its hydrolysates for tracer kinetic studies to evaluate parameters of the whole-body protein metabolism in premature infants. The particular advantage of applying an economically convenient, highly (15)N-enriched, and completely labelled yeast protein for evaluating protein turnover rates is the fact that the (15)N dose is spread among all proteinogenic amino acids. The absorption has been improved by hydrolysing [(15)N]yeast protein with thermitase into a mixture of amino acids, dipeptides and tripeptides so that faecal analysis becomes unnecessary when determining turnover rates. The review shows that, in contrast to the application of single (15)N-labelled amino acids with resulting overestimation of protein turnover rates, the (15)N-labelled yeast protein thermitase hydrolysate represents the amino acid metabolism more closely without causing amino acid imbalances. The (15)N-labelled yeast protein thermitase hydrolysate leads to the estimation of reliable protein turnover rates, particularly in premature infants.

  3. A facile method for expression and purification of (15)N isotope-labeled human Alzheimer's β-amyloid peptides from E. coli for NMR-based structural analysis.

    PubMed

    Sharma, Sudhir C; Armand, Tara; Ball, K Aurelia; Chen, Anna; Pelton, Jeffrey G; Wemmer, David E; Head-Gordon, Teresa

    2015-12-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized β-amyloid peptides with Aβ42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aβ42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aβ42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled Aβ42 for structural studies by NMR. The protocol involves utilization of an auto induction system with (15)N isotope labeled medium, for high-level expression of Aβ42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled Aβ42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aβ42 peptide (Garai et al., 2009). We obtain a final yield of ∼ 6 mg/L culture for (15)N isotope-labeled Aβ42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric Aβ42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with (15)N and (13)C in Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants.

  4. An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts.

    PubMed

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan

    2015-07-01

    Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein (15)N and (13)C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor.

  5. Worms from venus and mars: proteomics profiling of sexual differences in Caenorhabditis elegans using in vivo 15N isotope labeling.

    PubMed

    Tops, Bastiaan B J; Gauci, Sharon; Heck, Albert J R; Krijgsveld, Jeroen

    2010-01-01

    Hermaphrodites of the nematode Caenorhabditis elegans produce both sperm and oocytes in the same germline. To investigate the process underlying spermatogenesis and oogenesis separately, we used a quantitative proteomics approach applied to two mutant worm lines (fem-3(q20) and fem-1(hc17)) developing only male and female germlines, respectively. We used stable isotopic labeling of whole animals by feeding them either (14)N or (15)N labeled Escherichia coli. This way, we could confidently identify and quantify 1040 proteins in two independent experiments. Of these, approximately 400 proteins showed significant differential expression between female-like and male-like animals. As expected, proteins linked to oogenesis were found to be highly upregulated in the feminized worms, whereas proteins involved in spermatogenesis were found to be highly upregulated in the masculinized worms. This was complemented by many proteins strongly enriched in either mutant. Although the function of the majority of these proteins is unknown, their expression profile indicates that they have an as yet unrecognized role in the development and/or function of the female- and male germline in C. elegans. We show that members of several protein complexes as well as functionally similar proteins show comparable abundance ratios, indicating coregulation of protein expression. Additional analysis comparing our protein data to a previously published microarray data set shows that mRNA and protein expression are poorly correlating. We provide one of the first examples of a large-scale quantitative proteomics experiment in C. elegans and show the potential and feasibility of an approach enabling system-wide accurate quantitative proteomics experiments in this model organism. PMID:19916504

  6. Tracking the incorporation of 15N from labeled beech litter into mineral-organic associations

    NASA Astrophysics Data System (ADS)

    Kleber, M.; Hatton, P.; Derrien, D.; Lajtha, K.; Zeller, B.

    2008-12-01

    Nitrogen containing organic compounds are thought to have a role in the complex web of processes that control the turnover time of soil organic matter. The sequential density fractionation technique is increasingly used for the purpose of investigating the association of organic materials with the mineral matrix. Organic materials in the denser fractions (>2.0 kg L-1) typically show 13C NMR signals indicative of carbohydrate and aliphatic structures, an absence of lignin and tannin structures and a narrow C:N ratio, suggesting a microbial origin of organic matter in these fractions. Here we take advantage of a labeling experiment conducted at two different sites in Germany and in France to investigate the incorporation of organic nitrogen into physical fractions of increasing density, representing a proximity gradient to mineral surfaces. 15N labeled beech litter was applied to two acidic forest topsoils 8 and 12 years ago. Although there are differences in the distribution patterns between the two soils, and the majority of the organic nitrogen was recovered in fractions representing organic matter of plant origin and not bound to the mineral matrix, our data clearly show that after a decade, significant amounts of the nitrogen had been incorporated in mineral-organic fractions of supposedly slow turnover. It remains to be shown to which extent the N in the densest fractions was incorporated by soil microbiota and associated with mineral surfaces in organic form or adsorbed to mineral surfaces in inorganic form (NH4+).

  7. 1H, 13C, and 15N resonance assignments of murine amelogenin, an enamel biomineralization protein.

    SciTech Connect

    Buchko, Garry W.; Bekhazi, Jacky G.; Cort, John R.; Valentine, Nancy B.; Snead, Malcolm L.; Shaw, Wendy J.

    2008-06-01

    Amelogenin is the predominant matrix protein in developing dental enamel. Making extensive use of residue-specific 15N-labeled amino acids samples, the majority of the main and side chain resonances for murine amelogenin were assigned in 2% aqueous acetic acid at pH 3.0. This research was performed at Pacific Northwest National Laboratory, operated by Battelle for the US-DOE. A large part of this research was performed at the W.R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by U.S. Department of Energy’s Office of Biological and Environmental Research (BER) program located at Pacific Northwest National Laboratory (PNNL).

  8. Comparison of three 15N methods to correct for microbial contamination when assessing in situ protein degradability of fresh forages.

    PubMed

    Kamoun, M; Ammar, H; Théwis, A; Beckers, Y; France, J; López, S

    2014-11-01

    The use of stable (15)N as a marker to determine microbial contamination in nylon bag incubation residues to estimate protein degradability was investigated. Three methods using (15)N were compared: (15)N-labeled forage (dilution method, LF), (15)N enrichment of rumen solids-associated bacteria (SAB), and (15)N enrichment of rumen liquid-associated bacteria (LAB). Herbage from forages differing in protein and fiber contents (early-cut Italian ryegrass, late-cut Italian ryegrass, and red clover) were freeze-dried and ground and then incubated in situ in the rumen of 3 steers for 3, 6, 12, 24, and 48 h using the nylon bag technique. The (15)N-labeled forages were obtained by fertilizing the plots where herbage was grown with (15)NH4 (15)NO3. Unlabeled forages (obtained from plots fertilized with NH4NO3) were incubated at the same time that ((15)NH4)2SO4 was continuously infused into the rumen of the steers, and then pellets of labeled SAB and LAB were isolated by differential centrifugation of samples of ruminal contents. The proportion of bacterial N in the incubation residues increased from 0.09 and 0.45 g bacterial N/g total N at 3 h of incubation to 0.37 and 0.85 g bacterial N/g total N at 48 h of incubation for early-cut and late-cut ryegrass, respectively. There were differences (P < 0.001) between uncorrected N degradability values and those corrected for microbial contamination with all of the methods. Apparent N degradability of the low-N, high-fiber forage (late-cut ryegrass) was 0.51, whereas the corrected values were 0.85, 0.84, and 0.77 for the LF, SAB, and LAB methods, respectively. With early-cut ryegrass and red clover, the differences between uncorrected and corrected values ranged between 6% and 13%, with small differences among the labeling methods. Generally, methods using labeled forage or labeled SAB and LAB provided similar corrected degradability values. The accuracy in estimating the extent of degradation of protein in the rumen from in situ

  9. Synthesis of 13C and 15N labeled 2,4-dinitroanisole.

    PubMed

    Jagadish, Bhumasamudram; Field, Jim A; Chorover, Jon; Sierra-Alvarez, Reyes; Abrell, Leif; Mash, Eugene A

    2014-05-30

    Syntheses of [(13)C6]-2,4-dinitroanisole (ring-(13)C6) from [(13)C6]-anisole (ring-(13)C6) and [(15)N2]-2,4-dinitroanisole from anisole using in situ generated acetyl nitrate and [(15)N]-acetyl nitrate, respectively, are described. Treatment of [(13)C6]-anisole (ring-(13)C6) with acetyl nitrate generated in 100% HNO3 gave [(13)C6]-2,4-dinitroanisole (ring-(13)C6) in 83% yield. Treatment of anisole with [(15)N]-acetyl nitrate generated in 10 N [(15)N]-HNO3 gave [(15)N2 ]-2,4-dinitroanisole in 44% yield after two cycles of nitration. Byproducts in the latter reaction included [(15)N]-2-nitroanisole and [(15)N]-4-nitroanisole.

  10. Paramagnetic Inversion of the Sign of the Interference Contribution to the Transverse Relaxation of the Imido Protons of the Coordinated Imidazoles in the Uniformly 15N-Labeled Cytochrome c3

    NASA Astrophysics Data System (ADS)

    Ohmura, Tomoaki; Harada, Erisa; Fujiwara, Toshimichi; Kawai, Gota; Watanabe, Kimitsuna; Akutsu, Hideo

    1998-04-01

    In the spectrum of uniformly15N-labeled cytochromec3, the relative linewidths of the doublet peaks of the15N-coupled imido proton of the coordinated imidazole group were reversed on oxidation. This inversion was explained by the interference relaxation process between the electron-proton dipolar and15N-1H dipolar interactions. The inversion can be used to assign the imido protons of the coordinated imidazole groups in heme proteins.

  11. Novel labeling technique illustrates transfer of 15N2 from Sphagnum moss to vascular plants via diazotrophic nitrogen fixation

    NASA Astrophysics Data System (ADS)

    Thorp, N. R.; Vile, M. A.; Wieder, R.

    2013-12-01

    We used 15N2 gas to trace nitrogen (N) from biological N2-fixation to vascular plant uptake in an Alberta bog in order to determine if neighboring bog plants acquire recently fixed N from diazotrophs associating with Sphagnum mosses. Recent evidence indicates high rates of N2-fixation in Sphagnum mosses of Alberta bogs (Vile et al. 2013). Our previous work has shown that mosses can assimilate fixed N from associated diazotrophs as evidenced by the high N content of mosses despite minimal inputs from atmospheric deposition, retranslocation, and N mineralization. Therefore, the potential exists for vascular plants to obtain N from ';leaky' tissues of live mosses, however, this phenomenon has not been tested previously. Here we document the potential for relatively rapid transfer to vascular plants of N fixed by Sphagnum moss-associated diazotrophs. We utilized the novel approach of incubating mosses in 15N2 to allow the process of diazotrophic N2-fixation to mechanistically provide the 15N label, which is subsequently transferred to Sphagnum mosses. The potential for vascular bog natives to tap this N was assessed by planting the vascular plants in the labeled moss. Sphagnum mosses (upper 3 cm of live plants) were incubated in the presence of 98 atom % 15N2 gas for 48 hours. Two vascular plants common to Alberta bogs; Picea mariana and Vaccinium oxycoccus were then placed in the labeled mosses, where the mosses served as the substrate. Tissue samples from these plants were collected at three time points during the incubation; prior to 15N2 exposure (to determine natural abundance 15N), and at one and two months after 15N2 exposure. Roots and leaves were separated and run separately on a mass spectrometer to determine 15N concentrations. Sphagnum moss capitula obtained N from N2-fixation (δ15N of -2.43 × 0.40, 122.76 × 23.78, 224.92 × 68.37, 143.74 × 54.38 prior to, immediately after, and at 1 and 2 months after exposure to 15N2, respectively). Nitrogen was

  12. A new approach for the comparative analysis of multiprotein complexes based on 15N metabolic labeling and quantitative mass spectrometry.

    PubMed

    Trompelt, Kerstin; Steinbeck, Janina; Terashima, Mia; Hippler, Michael

    2014-01-01

    The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling ((14)N/(15)N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions. PMID:24686495

  13. Production of 15N-Labelled Liquid Organic Fertilisers Based on Manure and Crop Residue for Use in Fertigation Studies.

    PubMed

    Martínez-Alcántara, Belén; Martínez-Cuenca, Mary-Rus; Fernández, Carlos; Legaz, Francisco; Quiñones, Ana

    2016-01-01

    Large quantities of crop residue and animal manure from agricultural and livestock activities are annually produced worldwide. With proper management, these residues are potentially valuable sources of plant nutrients, mainly N. Recycling such subproducts in sustainably-based agricultural systems can minimise the use of mineral fertilisers, and hence reduce the potential risk of surface and groundwater pollution. Therefore, the purpose of this study was to obtain (small scale) two liquid labelled-organic fertilisers, an animal- and a vegetal-based organic (AO and VO, respectively) fertiliser, to be used as organic N sources in subsequent fertigation studies. Forage maize (Zea mays L.) grown under 15N-labelled fertiliser supply was used as raw material for VO fertiliser production, and also as 15N-labelled sheep feed to obtain 15N-labelled manure. The labelled faeces fraction was used as raw material for the AO fertiliser. The VO fertiliser was obtained after an acidic and an enzyme-driven hydrolysis. The AO fertiliser was obtained after acidic hydrolysis. The VO liquid fertiliser presented an N concentration of 330 mg·L-1, 85% of total N was organic, while ammonium and nitrate N accounted for 55% and 45% of the mineral nitrogen fraction, respectively. This fertiliser also exhibited high K, Ca and S concentrations and notable values for the remaining macro- and micronutrients. The AO liquid fertiliser had a similar total N concentration (496 mg·L-1, 82% of total N in an organic form) to that of VO, but its mineral N fraction significantly differed, which came in a predominantly (95%) ammonia form. It also had a high content of N, P, K and other macronutrients, and sufficient Fe, Zn, Mn, Cu and B levels, which suggests its suitability as a potential fertiliser. The percentage of 15N enrichment in both VO and AO liquid fertilisers exceeded 2% 15N atom excess, which enabled their use in subsequent assays run to assess nitrogen uptake efficiency.

  14. Water proton spin saturation affects measured protein backbone 15 N spin relaxation rates

    NASA Astrophysics Data System (ADS)

    Chen, Kang; Tjandra, Nico

    2011-12-01

    Protein backbone 15N NMR spin relaxation rates are useful in characterizing the protein dynamics and structures. To observe the protein nuclear-spin resonances a pulse sequence has to include a water suppression scheme. There are two commonly employed methods, saturating or dephasing the water spins with pulse field gradients and keeping them unperturbed with flip-back pulses. Here different water suppression methods were incorporated into pulse sequences to measure 15N longitudinal T1 and transversal rotating-frame T1ρ spin relaxation. Unexpectedly the 15N T1 relaxation time constants varied significantly with the choice of water suppression method. For a 25-kDa Escherichiacoli. glutamine binding protein (GlnBP) the T1 values acquired with the pulse sequence containing a water dephasing gradient are on average 20% longer than the ones obtained using a pulse sequence containing the water flip-back pulse. In contrast the two T1ρ data sets are correlated without an apparent offset. The average T1 difference was reduced to 12% when the experimental recycle delay was doubled, while the average T1 values from the flip-back measurements were nearly unchanged. Analysis of spectral signal to noise ratios ( s/ n) showed the apparent slower 15N relaxation obtained with the water dephasing experiment originated from the differences in 1H N recovery for each relaxation time point. This in turn offset signal reduction from 15N relaxation decay. The artifact becomes noticeable when the measured 15N relaxation time constant is comparable to recycle delay, e.g., the 15N T1 of medium to large proteins. The 15N relaxation rates measured with either water suppression schemes yield reasonable fits to the structure. However, data from the saturated scheme results in significantly lower Model-Free order parameters (< S2> = 0.81) than the non-saturated ones (< S2> = 0.88), indicating such order parameters may be previously underestimated.

  15. Nitrogen mineralization from selected /sup 15/N-labelled crop residues and humus as affected by inorganic nitrogen

    SciTech Connect

    Santos, J.A.

    1987-01-01

    The use of cover crops or crop residues as a source of N to succeeding crops has become a matter of increasing importance for economic and environmental reason. Greenhouse and field studies were conducted to determine the N contribution of four /sup 15/N labelled crop residues, rye (Secale cereale L.), wheat (Triticum aestivum L.), crimson clover (Trifolium encarnatum L.), and hairy vetch (Vicia sativa L.), to successive crops and to evaluate the effect of different organic (ON) and inorganic N (IN) combinations on mineralization of the above residues. Total /sup 15/N recovery from the residues ranged from 51% to 85% and 4% to 74% for the greenhouse and field studies, respectively.

  16. Comparison of five soil organic matter decomposition models using data from a 14C and 15N labeling field experiment

    NASA Astrophysics Data System (ADS)

    Pansu, Marc; Bottner, Pierre; Sarmiento, Lina; Metselaar, Klaas

    2004-12-01

    Five alternatives of the previously published MOMOS model (MOMOS-2 to -6) are tested to predict the dynamics of carbon (C) and nitrogen (N) in soil during the decomposition of plant necromass. 14C and 15N labeled wheat straw was incubated over 2 years in fallow soils of the high Andean Paramo of Venezuela. The following data were collected: soil moisture, total 14C and 15N and microbial biomass (MB)-14C and -15N, daily rainfall, air temperature and total radiation. Daily soil moisture was predicted using the SAHEL model. MOMOS-2 to -4 (type 1 models) use kinetic constants and flow partitioning parameters. MOMOS-2 can be simplified to MOMOS-3 and further to MOMOS-4, with no significant changes in the prediction accuracy and robustness for total-14C and -15N as well as for MB-14C and -15N. MOMOS-5 (type 2 models) uses only kinetic constants: three MB-inputs (from labile and stable plant material and from humified compounds) and two MB-outputs (mortality and respiration constants). MOMOS-5 did not significantly change the total-14C and -15N predictions but markedly improved the predictive quality and robustness of MB-14C and -15N predictions (with a dynamic different from the predictions by other models). Thus MOMOS-5 is proposed as an accurate and ecologically consistent description of decomposition processes. MOMOS-6 extends MOMOS-5 by including a stable humus compartment for long-term simulations of soil native C and N. The improvement of the predictions is not significant for this 2-year experiment, but MOMOS-6 enables prediction of a sequestration in the stable humus compartment of 2% of the initially added 14C and 5.4% of the added 15N.

  17. Variability of the 15N Chemical Shielding Tensors in the B3 Domain of Protein G from 15N Relaxation Measurements at Several Fields

    PubMed Central

    Hall, Jennifer B.; Fushman, David

    2008-01-01

    We applied a combination of 15N relaxation and CSA/dipolar cross-correlation measurements at five magnetic fields (9.4, 11.7, 14.1, 16.4, and 18.8 Tesla) to determine the 15N chemical shielding tensors for backbone amides in protein G in solution. The data were analyzed using various model-independent approaches and those based on Lipari-Szabo approximation, all of them yielding similar results. The results indicate a range of site-specific values of the anisotropy (CSA) and orientation of the 15N chemical shielding tensor, similar to those in ubiquitin. Assuming a Gaussian distribution of the 15N CSA values, the mean anisotropy is -173.9 to -177.2 ppm (for 1.02-Å NH-bond length) and the site-so-site CSA variability is ±17.6 to ±21.4 ppm, depending on the method used. This CSA variability is significantly larger than derived previously for ribonuclease H or recently, using “meta-analysis” for ubiquitin. Standard interpretation of 15N relaxation studies of backbone dynamics in proteins involves an a priori assumption of a uniform 15N CSA. We show that this assumption leads to a significant discrepancy between the order parameters obtained at different fields. Using the site-specific CSAs obtained from our study removes this discrepancy and allows simultaneous fit of relaxation data at all five fields to Lipari-Szabo spectral densities. These findings emphasize the necessity of taking into account the variability of 15N CSA for accurate analysis of protein dynamics from 15N relaxation measurements. PMID:16771499

  18. Synthesis and biosynthesis of {sup 13}C-, {sup 15}N-labeled deoxynucleosides useful for biomolecular structural determinations

    SciTech Connect

    Ashburn, D.A.; Garcia, K.; Hanners, J.L.; Silks, L.A. III; Unkefer, C.J.

    1994-12-01

    Currently, there is a great emphasis on elucidating the structure, function, and dynamics of DNA. Much of the research involved in this study uses nuclear magnetic resonance (NMR) spectroscopy. Effective use of NMR spectroscopy for DNA molecules with mw > 10,000 requires stable isotope enrichment. We present strategies for site-specific isotopic labeling of the purine bases adenosine and guanosine and the biosynthesis of (U-{sup 13}C, {sup 15}N) DNA from methylotropic bacteria. With commercially available 6-chloropurine, an effective two-step route leads to 2{prime}-deoxy-(amino-{sup 15}N)adenosine (dA). The resulting d(amino-{sup 15}N)A is used in a series of reactions to synthesize 2{prime}-deoxy-(2-{sup 13}C,1,amino-{sup 15}N{sub 2})guanosine or any combination thereof. An improved biosynthesis of labeled DNA has been accomplished using Methylobacterium extorquens AS1. Each liter of growth medium contains 4 g of methanol to yield 1 g of lyophilized cells. As much as 200 mg of RNA per liter of culture has been obtained. We are currently developing large-scale isolation protocols. General synthetic pathways to oligomeric DNA will be presented.

  19. Exploring the nitrogen ingestion of aphids--a new method using electrical penetration graph and (15)N labelling.

    PubMed

    Kuhlmann, Franziska; Opitz, Sebastian E W; Inselsbacher, Erich; Ganeteg, Ulrika; Näsholm, Torgny; Ninkovic, Velemir

    2013-01-01

    Studying plant-aphid interactions is challenging as aphid feeding is a complex process hidden in the plant tissue. Here we propose a combination of two well established methods to study nutrient acquisition by aphids focusing on the uptake of isotopically labelled nitrogen ((15)N). We combined the Electrical Penetration Graph (EPG) technique that allows detailed recording of aphid feeding behaviour and stable isotope ratio mass spectrometry (IRMS) to precisely measure the uptake of nitrogen. Bird cherry-oat aphids Rhopalosiphum padi L. (Hemiptera, Aphididae) fed for 24 h on barley plants (Hordeum vulgare L., cultivar Lina, Poaceae) that were cultivated with a (15)N enriched nutrient solution. The time aphids fed in the phloem was strongly positive correlated with their (15)N uptake. All other single behavioural phases were not correlated with (15)N enrichment in the aphids, which corroborates their classification as non-feeding EPG phases. In addition, phloem-feeding and (15)N enrichment of aphids was divided into two groups. One group spent only short time in the phloem phase and was unsuccessful in nitrogen acquisition, while the other group displayed longer phloem-feeding phases and was successful in nitrogen acquisition. This suggests that several factors such as the right feeding site, time span of feeding and individual conditions play a role for the aphids to acquire nutrients successfully. The power of this combination of methods for studying plant-aphid interactions is discussed.

  20. High Resolution 13C MRI With Hyperpolarized Urea: In Vivo T2 Mapping and 15N Labeling Effects

    PubMed Central

    Reed, Galen D.; von Morze, Cornelius; Bok, Robert; Koelsch, Bertram L.; Van Criekinge, Mark; Smith, Kenneth J.; Shang, Hong; Larson, Peder E. Z.; Kurhanewicz, John; Vigneron, Daniel B.

    2014-01-01

    13C steady state free precession (SSFP) magnetic resonance imaging and effective spin-spin relaxation time (T2) mapping were performed using hyperpolarized [13C] urea and [13C, 15N2] urea injected intravenously in rats. 15N labeling gave large T2 increases both in solution and in vivo due to the elimination of a strong scalar relaxation pathway. The T2 increase was pronounced in the kidney, with [13C, 15N2] urea giving T2 values of 6.3±1.3 s in the cortex and medulla, and 11±2 s in the renal pelvis. The measured T2 in the aorta was 1.3±0.3 s. [13C] urea showed shortened T2 values in the kidney of 0.23±0.03 s compared to 0.28±0.03 s measured in the aorta. The enhanced T2 of [13C, 15N2] urea was utilized to generate large signal enhancement by SSFP acquisitions with flip angles approaching the fully refocused regime. Projection images at 0.94 mm in-plane resolution were acquired with both urea isotopes, with [13C, 15N2] urea giving a greater than four-fold increase in signal-to-noise ratio [13C] over urea. PMID:24235273

  1. Quantitative analysis of 15N labeled positional isomers of glutamine and citrulline via electrospray ionization tandem mass spectrometry of their dansyl derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The enteral metabolism of glutamine and citrulline are intertwined because, while glutamine is one of the main fuel sources for the enterocyte, citrulline is one of its products. It has been shown that the administration of 15N labeled glutamine results in the incorporation of the 15N label into cit...

  2. Site-specific φ- and ψ-torsion angle determination in a uniformly/extensively 13C- and 15N-labeled peptide

    NASA Astrophysics Data System (ADS)

    Wi, Sungsool; Spano, Justin

    2011-10-01

    A solid-state rotational-echo double resonance (REDOR) NMR method was introduced to identify the ϕ- and ψ-torsion angle from a 1H- 15N or 1H- 13C' spin system of alanine-like residues in a selectively, uniformly, or extensively 15N-/ 13C-labeled peptide. When a C α( i) or a 15N peak is site-specifically obtainable in the NMR spectrum of a uniformly 15N/ 13C-labeled sample system, the ψ- or ϕ-torsion angle specified by the conformational structure of peptide geometry involving 15N( i)- 1H αi - 15N( i + 1) or 13C'( i - 1)- 1H Ni- 13C'( i) spin system can be identified based on 13C α- or 15N-detected 1H α- 15N or 1H N- 13C REDOR experiment. This method will conveniently be utilized to identify major secondary motifs, such as α-helix, β-sheet, and β-turn, from a uniformly 15N-/ 13C-labled peptide sample system. When tested on a 13C-/ 15N-labeled model system of a three amino acid peptide Gly-[U- 13C, 15N]Ala-[U- 13C, 15N]Leu, the ψ-angle of alanine obtained experimentally, ψ = -40 ± 30°, agreed reasonably well with the X-ray determined angle, ψ = -39°.

  3. Design and operation of a continuous 13C and 15N labeling chamber for uniform or differential, metabolic and structural, plant isotope labeling.

    PubMed

    Soong, Jennifer L; Reuss, Dan; Pinney, Colin; Boyack, Ty; Haddix, Michelle L; Stewart, Catherine E; Cotrufo, M Francesca

    2014-01-16

    Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as (13)C with (15)N, (18)O or (2)H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation(1-4). From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage(5-7). The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing (13)C and (15)N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components. Here, we present the construction and operation of a continuous (13)C and (15)N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%(13)C and 6.7 atom%(15)N uniform plant label, or material that is differentially labeled by up to 1.29 atom%(13)C and 0.56 atom%(15)N in its metabolic and structural components (hot water extractable and hot water

  4. Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

    PubMed Central

    Soong, Jennifer L; Reuss, Dan; Pinney, Colin; Boyack, Ty; Haddix, Michelle L; Stewart, Catherine E; Cotrufo, M. Francesca

    2014-01-01

    Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or 2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components. Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components

  5. Production of 15N-Labelled Liquid Organic Fertilisers Based on Manure and Crop Residue for Use in Fertigation Studies.

    PubMed

    Martínez-Alcántara, Belén; Martínez-Cuenca, Mary-Rus; Fernández, Carlos; Legaz, Francisco; Quiñones, Ana

    2016-01-01

    Large quantities of crop residue and animal manure from agricultural and livestock activities are annually produced worldwide. With proper management, these residues are potentially valuable sources of plant nutrients, mainly N. Recycling such subproducts in sustainably-based agricultural systems can minimise the use of mineral fertilisers, and hence reduce the potential risk of surface and groundwater pollution. Therefore, the purpose of this study was to obtain (small scale) two liquid labelled-organic fertilisers, an animal- and a vegetal-based organic (AO and VO, respectively) fertiliser, to be used as organic N sources in subsequent fertigation studies. Forage maize (Zea mays L.) grown under 15N-labelled fertiliser supply was used as raw material for VO fertiliser production, and also as 15N-labelled sheep feed to obtain 15N-labelled manure. The labelled faeces fraction was used as raw material for the AO fertiliser. The VO fertiliser was obtained after an acidic and an enzyme-driven hydrolysis. The AO fertiliser was obtained after acidic hydrolysis. The VO liquid fertiliser presented an N concentration of 330 mg·L-1, 85% of total N was organic, while ammonium and nitrate N accounted for 55% and 45% of the mineral nitrogen fraction, respectively. This fertiliser also exhibited high K, Ca and S concentrations and notable values for the remaining macro- and micronutrients. The AO liquid fertiliser had a similar total N concentration (496 mg·L-1, 82% of total N in an organic form) to that of VO, but its mineral N fraction significantly differed, which came in a predominantly (95%) ammonia form. It also had a high content of N, P, K and other macronutrients, and sufficient Fe, Zn, Mn, Cu and B levels, which suggests its suitability as a potential fertiliser. The percentage of 15N enrichment in both VO and AO liquid fertilisers exceeded 2% 15N atom excess, which enabled their use in subsequent assays run to assess nitrogen uptake efficiency. PMID:26982183

  6. Production of 15N-Labelled Liquid Organic Fertilisers Based on Manure and Crop Residue for Use in Fertigation Studies

    PubMed Central

    Martínez-Alcántara, Belén; Martínez-Cuenca, Mary-Rus; Fernández, Carlos; Legaz, Francisco; Quiñones, Ana

    2016-01-01

    Large quantities of crop residue and animal manure from agricultural and livestock activities are annually produced worldwide. With proper management, these residues are potentially valuable sources of plant nutrients, mainly N. Recycling such subproducts in sustainably-based agricultural systems can minimise the use of mineral fertilisers, and hence reduce the potential risk of surface and groundwater pollution. Therefore, the purpose of this study was to obtain (small scale) two liquid labelled-organic fertilisers, an animal- and a vegetal-based organic (AO and VO, respectively) fertiliser, to be used as organic N sources in subsequent fertigation studies. Forage maize (Zea mays L.) grown under 15N-labelled fertiliser supply was used as raw material for VO fertiliser production, and also as 15N-labelled sheep feed to obtain 15N-labelled manure. The labelled faeces fraction was used as raw material for the AO fertiliser. The VO fertiliser was obtained after an acidic and an enzyme-driven hydrolysis. The AO fertiliser was obtained after acidic hydrolysis. The VO liquid fertiliser presented an N concentration of 330 mg·L-1, 85% of total N was organic, while ammonium and nitrate N accounted for 55% and 45% of the mineral nitrogen fraction, respectively. This fertiliser also exhibited high K, Ca and S concentrations and notable values for the remaining macro- and micronutrients. The AO liquid fertiliser had a similar total N concentration (496 mg·L-1, 82% of total N in an organic form) to that of VO, but its mineral N fraction significantly differed, which came in a predominantly (95%) ammonia form. It also had a high content of N, P, K and other macronutrients, and sufficient Fe, Zn, Mn, Cu and B levels, which suggests its suitability as a potential fertiliser. The percentage of 15N enrichment in both VO and AO liquid fertilisers exceeded 2% 15N atom excess, which enabled their use in subsequent assays run to assess nitrogen uptake efficiency. PMID:26982183

  7. Heavy labeling of recombinant proteins.

    PubMed

    Rodriguez, Eric

    2007-01-01

    Because of the cost of isotopic chemicals and heterologous proteins to produce, an economical 15N/13C isotopic labeling method is critically needed. Four protocols have been tested for the expression of Ovine interferon-tau in Pichia pastoris. 13C-glucose in place of 13C-glycerol as well as the need for 15N/13C-sources were evaluated during the growth phase. Sequential addition of 15NH4Cl and 13C-methanol were also evaluated at different ratio. Our results demonstrate that 15N/13C isotopes are not required throughout the initial growth period but are necessary at low concentration a few hours prior to the methanol induction period. We have evaluated the cost of the use of isotopes 15NH4Cl, 13C-glucose and 13C-methanol in our optimised P4 protocol conditions. The cost was one-third that of the standard method using 15NH4Cl and 13C-glucose throughout the entire growth period and was even lower using 13C-glycerol.

  8. Non-homogeneity of isotopic labelling in 15N gas flux studies: theory, some observations and possible lessons

    NASA Astrophysics Data System (ADS)

    Well, Reinhard; Buchen, Caroline; Deppe, Marianna; Eschenbach, Wolfram; Gattinger, Andreas; Giesemann, Anette; Krause, Hans-Martin; Lewicka-Szczebak, Dominika

    2015-04-01

    Quantifying dinitrogen (N2) and nitrous oxide (N2O) fluxes from different soil N pools and processes can be accomplished using the 15N tracer technique but this is subject to four different sources of bias (i. - iv.). This approach includes 15N labelling of selected N pools in soil and subsequent isotope analysis of all relevant N pools as well as of gas samples from enclosures, i.e. mixtures of soil-derived and atmospheric N2 and N2O. Depending on the processes of interest, there may be 15N labelling of one or several N pools, were several labelling treatment are needed in the latter case (e.g. Müller et al., 2004). Measuring pool-derived N2 or N2O has been shown to include two calculation problems, (i.) arising from multiple pools (e.g. Arah, 1992) and (ii.) dealing with the non-random distribution of N2 and N2O mole masses (Hauck et al., 1958). Non-randomness can be solved if m/z 28, 29 and 30 are correctly analysed and the 15N enrichment of one (to distinguish two pools, i.e. soil and atmosphere) or two pools (in case of three pools) is known (Spott & Stange, 2008). Moreover (iii.), NO3- pools generating N2 and N2O via denitrification can be identical or different, e.g. if N2O evolved from higher enriched NO3- in deeper soil was more reduced to N2 compared to N2O evolved from N2O from shallow soil with lower enrichment, or vice versa. Apportioning N2O fluxes to NH4+ (nitrification and/or nitrifier denitrification) and NO3- (denitrification) is often conducted by NO3-labeling, measuring δ15N of emitted N2O and applying mixing equations were the measured 15N enrichment of NH4+and NO3-pool is used. However, this assumes that the average 15N enrichment of NH4+and NO3-in the soil is identical to the enrichment in the active soil domain producing N2 and/or N2O. Violation of this precondition must lead to bias in source apportionment (iv.), but to our knowledge this has not been investigated until now. Here we present conceptual models and model calculations

  9. Nitrogen and Carbon Flows Estimated by 15N and 13C Pulse-Chase Labeling during Regrowth of Alfalfa.

    PubMed Central

    Avice, J. C.; Ourry, A.; Lemaire, G.; Boucaud, J.

    1996-01-01

    The flow of 15N and 13C from storage compounds in organs remaining after defoliation (sources) to regrowing tissue (sinks), and 13C losses through root or shoot respiration were assessed by pulse-chase labeling during regrowth of alfalfa (Medicago sativa L.) following shoot removal. A total of 73% of labeled C and 34% of labeled N were mobilized in source organs within 30 d. Although all of the 15N from source organs was recovered in the regrowing tissue, much of the 13C was lost, mainly as CO2 respired from the root (61%) or shoot (8%), and was found to a lesser extent in sink tissue (5%). After 3, 10, or 30 d of regrowth, 87, 66, and 52% of shoot N, respectively, was derived from source tissue storage compounds; the rest resulted from translocation of fixed N2. Overall results suggest that most shoot C was linked to photosynthetic activity rather than being derived from mobilization of stored C in source organs. Furthermore, isotopic analysis of different chemical fractions of plant tissue suggests that between 14 and 58% of the shoot C derived from source tissues was linked to the mobilization of N compounds, not carbohydrates. PMID:12226391

  10. Millipedes and earthworms increase the decomposition rate of 15N-labelled winter rape litter in an arable field.

    PubMed

    Martens, H; Alphei, J; Schaefer, M; Scheu, S

    2001-01-01

    Effects of millipedes and earthworms on the decomposition of 15N-labelled litter of winter oilseed rape were investigated in a microcosm field experiment over a period of 264 days on an oat field near Göttingen managed by integrated farming. A total of 32 microcosms were filled with defaunated soil. 15N-labelled rape litter was placed either on top of the soil or buried into the soil simulating mulching and ploughing, respectively. To the microcosms nine adult individuals of Blaniulus guttulatus (Diplopoda) and two of Aporrectodea caliginosa (Lumbricidae) were added separately or in combination. In general, the presence of the animals accelerated the decomposition rate of the litter material. The effects were most pronounced in the presence of Aporrectodea caliginosa. The total amount of nitrate, ammonium and the amount of 35N leached from the microcosms was increased in the presence of earthworms or of both earthworms and millipedes. Both species proved to be important members of the detritus food web of the agricultural system studied.

  11. High-throughput backbone resonance assignment of small 13C, 15N-labeled proteins by a triple-resonance experiment with four sequential connectivity pathways using chemical shift-dependent, apparent 1J ( 1H, 13C): HNCACB codedHAHB

    NASA Astrophysics Data System (ADS)

    Pegan, Scott; Kwiatkowski, Witek; Choe, Senyon; Riek, Roland

    2003-12-01

    The proposed three-dimensional triple-resonance experiment HNCACB codedHAHB correlates sequential 15N, 1H moieties via the chemical shifts of 13C α, 13C β, 1H α, and 1H β. The four sequential correlation pathways are achieved by the incorporation of the concept of chemical shift-coding [J. Biomol. NMR 25 (2003) 281] to the TROSY-HNCACB experiment. The monitored 1H α and 1H β chemical shifts are then coded in the line shape of the cross-peaks of 13C α, 13C β along the 13C dimension through an apparent residual scalar coupling, the size of which depends on the attached hydrogen chemical shift. The information of four sequential correlation pathways enables a rapid backbone assignment. The HNCACB codedHAHB experiment was applied to ˜85% labeled 13C, 15N-labeled amino-terminal fragment of Vaccinia virus DNA topoisomerase I comprising residues 1-77. After one day of measurement on a Bruker Avance 700 MHz spectrometer and 8 h of manual analysis of the spectrum 93% of the backbone assignment was achieved.

  12. Choice of dietary protein of vegetarians and omnivores is reflected in their hair protein 13C and 15N abundance.

    PubMed

    Petzke, Klaus J; Boeing, Heiner; Metges, Cornelia C

    2005-01-01

    Stable isotopic (15N, 13C) composition of tissues depends on isotopic pattern of food sources. We investigated whether the isotopic compositions of human hair protein and amino acids reflect the habitual dietary protein intake. Hair samples were analyzed from 100 omnivores (selected randomly out of the 1987-1988 German nutrition survey VERA), and from 15 ovo-lacto-vegetarians (OLV), and from 6 vegans recruited separately. Hair bulk and amino acid specific isotopic compositions were analyzed by isotope-ratio mass spectrometry (EA/IRMS and GC/C/IRMS, respectively) and the results were correlated with data of the 7 day dietary records. Hair bulk 15N and 13C abundances clearly reflect the particular eating habits. Vegans can be distinguished from OLV and both are significantly distinct from omnivores in both 15N and 13C abundances. 15N and 13C abundances rose with a higher proportion of animal to total protein intake (PAPI). Individual proportions of animal protein consumption (IPAP) were calculated using isotopic abundances and a linear regression model using animal protein consumption data of vegans (PAPI = 0) and omnivores (mean PAPI = 0.639). IPAP values positively correlated with the intake of protein, meat, meat products, and animal protein. Distinct patterns for hair amino acid specific 15N and 13C abundances were measured but with lower resolution between food preference groups compared with bulk values. In conclusion, hair 13C and 15N values both reflected the extent of animal protein consumption. Bulk isotopic abundance of hair can be tested for future use in the validation of dietary assessment methods. PMID:15880664

  13. Choice of dietary protein of vegetarians and omnivores is reflected in their hair protein 13C and 15N abundance.

    PubMed

    Petzke, Klaus J; Boeing, Heiner; Metges, Cornelia C

    2005-01-01

    Stable isotopic (15N, 13C) composition of tissues depends on isotopic pattern of food sources. We investigated whether the isotopic compositions of human hair protein and amino acids reflect the habitual dietary protein intake. Hair samples were analyzed from 100 omnivores (selected randomly out of the 1987-1988 German nutrition survey VERA), and from 15 ovo-lacto-vegetarians (OLV), and from 6 vegans recruited separately. Hair bulk and amino acid specific isotopic compositions were analyzed by isotope-ratio mass spectrometry (EA/IRMS and GC/C/IRMS, respectively) and the results were correlated with data of the 7 day dietary records. Hair bulk 15N and 13C abundances clearly reflect the particular eating habits. Vegans can be distinguished from OLV and both are significantly distinct from omnivores in both 15N and 13C abundances. 15N and 13C abundances rose with a higher proportion of animal to total protein intake (PAPI). Individual proportions of animal protein consumption (IPAP) were calculated using isotopic abundances and a linear regression model using animal protein consumption data of vegans (PAPI = 0) and omnivores (mean PAPI = 0.639). IPAP values positively correlated with the intake of protein, meat, meat products, and animal protein. Distinct patterns for hair amino acid specific 15N and 13C abundances were measured but with lower resolution between food preference groups compared with bulk values. In conclusion, hair 13C and 15N values both reflected the extent of animal protein consumption. Bulk isotopic abundance of hair can be tested for future use in the validation of dietary assessment methods.

  14. Partitioning Residue-derived and Residue-induced Emissions of N2O Using 15N-labelled Crop Residues

    NASA Astrophysics Data System (ADS)

    Farrell, R. E.; Carverhill, J.; Lemke, R.; Knight, J. D.

    2014-12-01

    Estimates of N2O emissions in Canada indicate that 17% of all agriculture-based emissions are associated with the decomposition of crop residues. However, research specific to the western Canadian prairies (including Saskatchewan) has shown that the N2O emission factor for N sources in this region typically ranges between 0.2 and 0.6%, which is well below the current IPCC default emission factor of 1.0%. Thus, it stands to reason that emissions from crop residues should also be lower than those calculated using the current IPCC emission factor. Current data indicates that residue decomposition, N mineralization and N2O production are affected by a number of factors such as C:N ratio and chemical composition of the residue, soil type, and soil water content; thus, a bench-scale incubation study was conducted to examine the effects of soil type and water content on N2O emissions associated with the decomposition of different crop residues. The study was carried out using soils from the Black, Dark Brown, Brown, and Gray soil zones and was conducted at both 50% and 70% water-filled pore space (WFPS); the soils were amended with 15N-labeled residues of wheat, pea, canola, and flax, or with an equivalent amount of 15N-labeled urea; 15N2O production was monitored using a Picarro G5101-i isotopic N2O analyzer. Crop residue additions to the soils resulted in both direct and indirect emissions of N2O, with residue derived emissions (RDE; measured as 15N2O) generally exceeding residue-induced emissions (RIE) at 50% WFPS—with RDEs ranging from 42% to 88% (mean = 58%) of the total N2O. Conversely, at 70% WFPS, RDEs were generally lower than RIEs—ranging from 21% to 83% (mean = 48%). Whereas both water content and soil type had an impact on N2O production, there was a clear and consistent trend in the emission factors for the residues; i.e., emissions were always greatest for the canola residue and lowest for the wheat residue and urea fertilizer; and intermediate for pea

  15. Backbone 1H, 13C, and 15N NMR assignments for the Cyanothece 51142 protein cce_0567: a protein associated with nitrogen fixation in the DUF683 family

    SciTech Connect

    Buchko, Garry W.; Sofia, Heidi J.

    2008-06-01

    The recently sequenced genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 (contig 83.1_1_243_746) contains the sequence for an hypothetical protein that falls into the DUF683 family. As observed for the other 54 DUF683 proteins currently listed in the GenBank database, this 78-residue (9.0 kDa) protein in Cyanothece is also found in a nitrogen fixation gene cluster suggesting that it is involved in the process. To date no structural information exists for any of the proteins in the DUF683 family. In an effort to elucidate the biochemical role DUF683 may play in nitrogen fixation and to obtain structural information for a member of the DUF683 protein family, a construct containing DUF683 from Cyanothece 51142 was generated, expressed, purified, and the solution properties characterized. A total rotational correlation time (tc) of 17.1 ns was estimated by nuclear magnetic resonance (NMR) spectroscopy suggesting a molecular weight of ~ 40 kDa, an observation dictating that DUF683 is a tetramer in solution. Using triple-labeled (2H, 13C, 15N) and residue-specific 15N-labeled amino acids (L, K, V, and E/Q) samples, most of the backbone and side chain resonances for DUF683 were assigned. The 13C alpha chemical shifts and NOESY NMR data indicate that the protein is helical from K18-E75.

  16. Utilizing the charge field effect on amide (15)N chemical shifts for protein structure validation.

    PubMed

    Bader, Reto

    2009-01-01

    Of all the nuclei in proteins, the nuclear magnetic resonance (NMR) chemical shifts of nitrogen are the theoretically least well understood. In this study, quantum chemical methods are used in combination with polarizable-continuum models in order to show that consideration of the effective electric field, including charge screening due to solvation, improves considerably the consistencies of statistical relationships between experimental and computed amide (15)N shifts between various sets of charged and uncharged oligopeptides and small organic molecules. A single conversion scheme between shielding parameters from first principles using density functional theory (DFT) and experimental shifts is derived that holds for all classes of compounds examined here. This relationship is then used to test the accuracy of such (15)N chemical shift predictions in the cyclic decapeptide antibiotic gramicidin S (GS). GS has previously been studied in great detail, both by NMR and X-ray crystallography. It adopts a well-defined backbone conformation, and hence, only a few discrete side chain conformational states need to be considered. Moreover, a charge-relay effect of the two cationic ornithine side chains to the protein backbone has been described earlier by NMR spectroscopy. Here, DFT-derived backbone amide nitrogen chemical shifts were calculated for multiple conformations of GS. Overall, the structural dynamics of GS is revisited in view of chemical shift behavior along with energetic considerations. Together, the study demonstrates proof of concept that (15)N chemical shift information is particularly useful in the analysis and validation of protein conformational states in a charged environment.

  17. Measurement and interpretation of 15N-1H residual dipolar couplings in larger proteins.

    PubMed

    Bhattacharya, Akash; Revington, Matthew; Zuiderweg, Erik R P

    2010-03-01

    A decade ago, Dr. L.E. Kay and co-workers described an ingenious HNCO-based triple-resonance experiment from which several protein backbone RDCs can be measured simultaneously (Yang et al. (1999) [1]). They implemented a J-scaling technique in the (15)N dimension of the 3D experiment to obtain the NH RDCs. We have used this idea to carry out J-scaling in a 2D (15)N-(1)H-TROSY experiment and have found it to be an excellent method to obtain NH RDCs for larger proteins upto 70 kDa, far superior to commonly used HSQC in-phase/anti-phase and HSQC/TROSY comparisons. Here, this method, dubbed "RDC-TROSY" is discussed in detail and the limits of its utility are assessed by simulations. Prominent in the latter analysis is the evaluation of the effect of amide proton flips on the "RDC-TROSY" linewidths. The details of the technical and computational implementations of these methods for the determination of domain orientations in 45-60 kDa Hsp70 chaperone protein constructs are described. PMID:20018538

  18. Simultaneous CT-13C and VT-15N chemical shift labelling: application to 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH.

    PubMed

    Uhrín, D; Bramham, J; Winder, S J; Barlow, P N

    2000-11-01

    Based on the HSQC scheme, we have designed a 2D heterocorrelated experiment which combines constant time (CT) 13C and variable time (VT) 15N chemical shift labelling. Although applicable to all carbons, this mode is particularly suitable for simultaneous recording of methyl-carbon and nitrogen chemical shifts at high digital resolution. The methyl carbon magnetisation is in the transverse plane during the whole CT period (1/J(CC) = 28.6 ms). The magnetisation originating from NH protons is initially stored in the 2HzNz state, then prior to the VT chemical shift labelling period is converted into 2HzNy coherence. The VT -15N mode eliminates the effect of 1J(N,CO) and 1,2J(N,CA) coupling constants without the need for band-selective carbon pulses. An optional editing procedure is incorporated which eliminates signals from CH2 groups, thus removing any potential overlap with the CH3 signals. The CT-13CH3,VT-15N HSQC building block is used to construct two 3D experiments: 3D NOESY-CH3NH and 3D 13C,15N HSQC-NOESY-CH3NH. Combined use of these experiments yields proton and heteronuclear chemical shifts for moieties experiencing NOEs with CH3 and NH protons. These NOE interactions are resolved as a consequence of the high digital resolution in the carbon and nitrogen chemical shifts of CH3 and NH groups, respectively. The techniques are illustrated using a double labelled sample of the CH domain from calponin.

  19. Decomposition and nitrogen dynamics of 15N-labeled leaf, root, and twig litter in temperate coniferous forests

    USGS Publications Warehouse

    van Huysen, Tiff L.; Harmon, Mark E.; Perakis, Steven S.; Chen, Hua

    2013-01-01

    Litter nutrient dynamics contribute significantly to biogeochemical cycling in forest ecosystems. We examined how site environment and initial substrate quality influence decomposition and nitrogen (N) dynamics of multiple litter types. A 2.5-year decomposition study was installed in the Oregon Coast Range and West Cascades using 15N-labeled litter from Acer macrophyllum, Picea sitchensis, and Pseudotsuga menziesii. Mass loss for leaf litter was similar between the two sites, while root and twig litter exhibited greater mass loss in the Coast Range. Mass loss was greatest from leaves and roots, and species differences in mass loss were more prominent in the Coast Range. All litter types and species mineralized N early in the decomposition process; only A. macrophyllum leaves exhibited a net N immobilization phase. There were no site differences with respect to litter N dynamics despite differences in site N availability, and litter N mineralization patterns were species-specific. For multiple litter × species combinations, the difference between gross and net N mineralization was significant, and gross mineralization was 7–20 % greater than net mineralization. The mineralization results suggest that initial litter chemistry may be an important driver of litter N dynamics. Our study demonstrates that greater amounts of N are cycling through these systems than may be quantified by only measuring net mineralization and challenges current leaf-based biogeochemical theory regarding patterns of N immobilization and mineralization.

  20. Insights into nitrogen allocation and recycling from nitrogen elemental analysis and 15N isotope labelling in 14 genotypes of willow.

    PubMed

    Brereton, Nicholas J B; Pitre, Frederic E; Shield, Ian; Hanley, Steven J; Ray, Michael J; Murphy, Richard J; Karp, Angela

    2014-11-01

    Minimizing nitrogen (N) fertilization inputs during cultivation is essential for sustainable production of bioenergy and biofuels. The biomass crop willow (Salix spp.) is considered to have low N fertilizer requirements due to efficient recycling of nutrients during the perennial cycle. To investigate how successfully different willow genotypes assimilate and allocate N during growth, and remobilize and consequently recycle N before the onset of winter dormancy, N allocation and N remobilization (to and between different organs) were examined in 14 genotypes of a genetic family using elemental analysis and (15)N as a label. Cuttings were established in pots in April and sampled in June, August and at onset of senescence in October. Biomass yield of the trees correlated well with yields recorded in the field. Genotype-specific variation was observed for all traits measured and general trends spanning these sampling points were identified when trees were grouped by biomass yield. Nitrogen reserves in the cutting fuelled the entirety of the canopy establishment, yet earlier cessation of this dependency was linked to higher biomass yields. The stem was found to be the major N reserve by autumn, which constitutes a major source of N loss at harvest, typically every 2-3 years. These data contribute to understanding N remobilization in short rotation coppice willow and to the identification of traits that could potentially be selected for in breeding programmes to further improve the sustainability of biomass production.

  1. Heavy water and 15N labeling with NanoSIMS analysis reveals growth-rate dependent metabolic heterogeneity in chemostats

    PubMed Central

    McGlynn, Shawn E.; Green-Saxena, Abigail

    2015-01-01

    To measure single cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labeling of microbial populations with heavy water (a passive tracer) and 15N ammonium in combination with multi-isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (∼0.1 atom % 2H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (∼6 hours, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium vs. amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments. PMID:25655651

  2. A solution NMR study of the selectively 13C, 15N-labeled peptaibol chrysospermin C in methanol.

    PubMed

    Anders, R; Wenschuh, H; Soskic, V; Fischer-Frühholz, S; Ohlenschläger, O; Dornberger, K; Brown, L R

    1998-07-01

    The conformation of the 19-residue peptaibol chrysospermin C in methanol has been investigated by NMR spectroscopy using selective 15N and 13C labeling of the alpha-aminoisobutyric acid (Aib) residues. Complete 1H and 13C sequential assignments, including stereospecific assignments for the heavily overlapped resonances from the two Cbeta methyl groups of the eight Aib residues, are reported for a peptaibol for the first time. An Aib residue followed by a Pro is an exception to previous suggestions regarding stereospecific assignment of the two Cbeta methyl groups of Aib residues. Local nuclear Overhauser effects and 3J(HNC') and 3J(HNCbeta) scalar couplings indicate that the phi angles of the Aib residues are restricted sterically to local conformations consistent with right-handed helices. Despite these constraints on the eight Aib residues, the NMR data for chrysospermin C in methanol are generally most consistent with an ensemble of transient conformations, including backbone conformations inconsistent with helical structures. Initial NMR measurements for chrysospermin C bound to micelles suggest structural and dynamic differences relative to alamethicin bound to micelles which may be related to differences in gating voltages for formation of ion channels.

  3. Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis

    PubMed Central

    Berditsch, Marina; Afonin, Sergii; Steineker, Anna; Orel, Nataliia; Jakovkin, Igor; Weber, Christian

    2015-01-01

    Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly 13C/15N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive 13C/15N-labeled amino acids. The most cost-effective production of 13C/15N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% 13C-glycerol and 0.5% 15N-ammonium sulfate, supplemented with only 0.025% of 13C/15N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state. PMID:25795666

  4. Fermentation and Cost-Effective 13C/15N Labeling of the Nonribosomal Peptide Gramicidin S for Nuclear Magnetic Resonance Structure Analysis.

    PubMed

    Berditsch, Marina; Afonin, Sergii; Steineker, Anna; Orel, Nataliia; Jakovkin, Igor; Weber, Christian; Ulrich, Anne S

    2015-06-01

    Gramicidin S (GS) is a nonribosomally synthesized decapeptide from Aneurinibacillus migulanus. Its pronounced antibiotic activity is attributed to amphiphilic structure and enables GS interaction with bacterial membranes. Despite its medical use for over 70 years, the peptide-lipid interactions of GS and its molecular mechanism of action are still not fully understood. Therefore, a comprehensive structural analysis of isotope-labeled GS needs to be performed in its biologically relevant membrane-bound state, using advanced solid-state nuclear magnetic resonance (NMR) spectroscopy. Here, we describe an efficient method for producing the uniformly (13)C/(15)N-labeled peptide in a minimal medium supplemented by selected amino acids. As GS is an intracellular product of A. migulanus, we characterized the producer strain DSM 5759 (rough-convex phenotype) and examined its biosynthetic activity in terms of absolute and biomass-dependent peptide accumulation. We found that the addition of either arginine or ornithine increases the yield only at very high supplementing concentrations (1% and 0.4%, respectively) of these expensive (13)C/(15)N-labeled amino acids. The most cost-effective production of (13)C/(15)N-GS, giving up to 90 mg per gram of dry cell weight, was achieved in a minimal medium containing 1% (13)C-glycerol and 0.5% (15)N-ammonium sulfate, supplemented with only 0.025% of (13)C/(15)N-phenylalanine. The 100% efficiency of labeling is corroborated by mass spectrometry and preliminary solid-state NMR structure analysis of the labeled peptide in the membrane-bound state.

  5. Principles of protein labeling techniques.

    PubMed

    Obermaier, Christian; Griebel, Anja; Westermeier, Reiner

    2015-01-01

    Protein labeling methods prior to separation and analysis have become indispensable approaches for proteomic profiling. Basically, three different types of tags are employed: stable isotopes, mass tags, and fluorophores. While proteins labeled with stable isotopes and mass tags are measured and differentiated by mass spectrometry, fluorescent labels are detected with fluorescence imagers. The major purposes for protein labeling are monitoring of biological processes, reliable quantification of compounds and specific detection of protein modifications and isoforms in multiplexed samples, enhancement of detection sensitivity, and simplification of detection workflows. Proteins can be labeled during cell growth by incorporation of amino acids containing different isotopes, or in biological fluids, cells or tissue samples by attaching specific groups to the ε-amino group of lysine, the N-terminus, or the cysteine residues. The principles and the modifications of the different labeling approaches on the protein level are described; benefits and shortcomings of the methods are discussed.

  6. Differential 14N/15N-Labeling of Peptides Using N-Terminal Charge Derivatization with a High-Proton Affinity for Straightforward de novo Peptide Sequencing

    PubMed Central

    Nihashi, Yoichiro; Miyashita, Masahiro; Awane, Hiroyuki; Miyagawa, Hisashi

    2013-01-01

    While de novo peptide sequencing is essential in many situations, it remains a difficult task. This is because peptide fragmentation results in complicated and often incomplete product ion spectra. In a previous study, we demonstrated that N-terminal charge derivatization with 4-amidinobenzoic acid (Aba) resulted in improved peptide fragmentation under low-energy CID conditions. However, even with this derivatization, some ambiguity exists, due to difficulties in discriminating between N- and C-terminal fragments. In this study, to specifically identify b-ions from complex product ion spectra, the differential 14N/15N-labeling of peptides was performed using Aba derivatization. 15N-Labeled Aba was synthesized in the form of a succinimide ester. Peptides were derivatized individually with 14N-Aba or 15N-Aba and analyzed by ESI-MS/MS using a linear ion trap-Orbitrap hybrid FTMS system. The N-terminal fragments (i.e., b-ions) were then identified based on m/z differences arising from isotope labeling. By comparing the spectra between 14N- and 15N-Aba derivatized peptides, b-ions could be successfully identified based on the m/z shifts, which provided reliable sequencing results for all of the peptides examined in this study. The method developed in this study allows the easy and reliable de novo sequencing of peptides, which is useful in peptidomics and proteomics studies. PMID:24860714

  7. Importance of bacterivory and preferential selection toward diatoms in larvae of Crepidula fornicata (L.) assessed by a dual stable isotope (13C, 15N) labeling approach

    NASA Astrophysics Data System (ADS)

    Leroy, Fanny; Riera, Pascal; Jeanthon, Christian; Edmond, Frédérique; Leroux, Cédric; Comtet, Thierry

    2012-05-01

    In Europe, the gastropod Crepidula fornicata is an invasive species characterized by a long reproductive period (from February to November). Thus, its larvae are exposed to variations in available food sources (in terms of quantity and quality). We aimed to investigate if bacteria could contribute to larval food both in presence or absence of phytoplankton, and to compare these results to seasonal variations of bacteria and phytoplankton abundances at a coastal site in the English Channel. First, ingestion of fluorescent beads of 0.5 to 2 μm diameter, showed that larvae were able to ingest particles of typical bacterial size. Then we used a dual stable isotope labeling approach which consisted in labeling a bacterial pelagic community with 15N and a diatom (Chaetoceros gracilis) culture with 13C, and supplying larvae with 15N-labeled bacteria, 13C-labeled diatoms, and both labeled sources. This technique has, to our knowledge, never been applied to invertebrate larvae. After 24 h of experiment, larvae were significantly enriched in all treatments: + 21.5‰ (∆δ13C) when supplied with diatoms, + 1364‰ (∆δ15N) when supplied with bacteria, and + 24‰ (∆δ13C) and + 135‰ (∆δ15N) when supplied with the two mixed sources. These results indicated that bacteria can contribute to the larval nutrition in C. fornicata, even in the presence of phytoplankton. Our results however suggested that larvae of C. fornicata preferentially used diatoms and showed that the supply of free bacteria did not alter the uptake of diatoms. Considering the seasonal variations of bacteria and phytoplankton abundances at the study site, these results suggested that bacteria may constitute a complementary resource for the larvae of C. fornicata when phytoplankton is abundant and may become a substitute resource when phytoplankton is less available. This approach offers promising perspectives to trace food sources and assess nitrogen and carbon fluxes between planktotrophic larvae

  8. Slow motions in microcrystalline proteins as observed by MAS-dependent 15N rotating-frame NMR relaxation

    NASA Astrophysics Data System (ADS)

    Krushelnitsky, Alexey; Zinkevich, Tatiana; Reif, Bernd; Saalwächter, Kay

    2014-11-01

    15N NMR relaxation rate R1ρ measurements reveal that a substantial fraction of residues in the microcrystalline chicken alpha-spectrin SH3 domain protein undergoes dynamics in the μs-ms timescale range. On the basis of a comparison of 2D site-resolved with 1D integrated 15N spectral intensities, we demonstrate that the significant fraction of broad signals in the 2D spectrum exhibits the most pronounced slow mobility. We show that 15N R1ρ's in proton-diluted protein samples are practically free from the coherent spin-spin contribution even at low MAS rates, and thus can be analysed quantitatively. Moderate MAS rates (10-30 kHz) can be more advantageous in comparison with the rates >50-60 kHz when slow dynamics are to be identified and quantified by means of R1ρ experiments.

  9. Direct measurements of protein backbone 15N spin relaxation rates from peak line-width using a fully-relaxed Accordion 3D HNCO experiment.

    PubMed

    Chen, Kang; Tjandra, Nico

    2009-03-01

    Protein backbone (15)N spin relaxation rates measured by solution NMR provide useful dynamic information with a site-specific resolution. The conventional method is to record a series of 2D (1)H-(15)N HSQC spectra with varied relaxation delays, and derive relaxation rate from the following curve fitting on the resonance intensities. Proteins with poorly resolved spectra often require several 3D HNCO spectra to be collected on a (15)N/(13)C double labeled protein sample. In order to reduce the relaxation dimension Carr et al. (P.A. Carr, D.A. Fearing, A.G. Palmer, 3D accordion spectroscopy for measuring N-15 and (CO)-Carbon-13 relaxation rates in poorly resolved NMR spectra, J. Magn. Reson. 132 (1998) 25-33) employed an Accordion type HNCO pulse sequence to obtain (15)N or (13)C T(1) relaxation rates by numerical fitting of the relaxation interfered free induction decay (FID) data. To avoid intensive analysis of the time domain data, we propose a modified protocol to measure (15)N T(1) and T(2) relaxation rates from easily obtained line-widths in an Accordion HNCO spectrum. Both T(1) and T(2) relaxation could be simultaneously convoluted into the constant-time evolution periods of (13)C' and (15)N, respectively. The relaxation delay was allowed to reach at least 3 x T(1) or 3 x T(2) so that the signal was substantially decayed by the end of the FID, and the resulting peak full-width at half height (FWHH) could be directly used to calculate relaxation rate. When applied to the 76-residue Ubiquitin and the 226-residue glutamine-binding protein (GlnBP), this method yielded T(1) and T(2) values deviating on average by 4-6% and 5-7%, respectively, from the measurements based on the conventional 2D method. In comparison, the conventional methods possessed intrinsic error ranges of 2-4% for T(1) and 3-6% for T(2). In addition to comparable accuracy, the fully-relaxed Accordion HNCO method presented here allowed measurements of relaxation rates for resonances unresolved in

  10. H/D exchange of a 15N labelled Tau fragment as measured by a simple Relax-EXSY experiment

    NASA Astrophysics Data System (ADS)

    Lopez, Juan; Ahuja, Puneet; Landrieu, Isabelle; Cantrelle, François-Xavier; Huvent, Isabelle; Lippens, Guy

    2014-12-01

    We present an equilibrium H/D exchange experiment to measure the exchange rates of labile amide protons in intrinsically unfolded proteins. By measuring the contribution of the H/D exchange to the apparent T1 relaxation rates in solvents of different D2O content, we can easily derive the rates of exchange for rapidly exchanging amide protons. The method does not require double isotope labelling, is sensitive, and requires limited fitting of the data. We demonstrate it on a functional fragment of Tau, and provide evidence for the hydrogen bond formation of the phosphate moiety of Ser214 with its own amide proton in the same fragment phosphorylated by the PKA kinase.

  11. Artifact suppression in electron paramagnetic resonance imaging of 14N- and 15N-labeled nitroxyl radicals with asymmetric absorption spectra

    NASA Astrophysics Data System (ADS)

    Takahashi, Wataru; Miyake, Yusuke; Hirata, Hiroshi

    2014-10-01

    This article describes an improved method for suppressing image artifacts in the visualization of 14N- and 15N-labeled nitroxyl radicals in a single image scan using electron paramagnetic resonance (EPR). The purpose of this work was to solve the problem of asymmetric EPR absorption spectra in spectral processing. A hybrid function of Gaussian and Lorentzian lineshapes was used to perform spectral line-fitting to successfully separate the two kinds of nitroxyl radicals. This approach can process the asymmetric EPR absorption spectra of the nitroxyl radicals being measured, and can suppress image artifacts due to spectral asymmetry. With this improved visualization method and a 750-MHz continuous-wave EPR imager, a temporal change in the distributions of a two-phase paraffin oil and water/glycerin solution system was visualized using lipophilic and hydrophilic nitroxyl radicals, i.e., 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy (16-DOXYL stearic acid) and 4-hydroxyl-2,2,6,6-tetramethylpiperidine-d17-1-15N-1-oxyl (TEMPOL-d17-15N). The results of the two-phase separation experiment verified that reasonable artifact suppression could be achieved by the present method that deals with asymmetric absorption spectra in the EPR imaging of 14N- and 15N-labeled nitroxyl radicals.

  12. Sensitivity-Enhanced MQ-HCN-CCH-TOCSY and MQ-HCN-CCH-COSY Pulse Schemes for 13C/ 15N Labeled RNA Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Hu, Weidong; Jiang, Licong; Gosser, Yuying Q.

    2000-07-01

    Sensitivity enhanced multiple-quantum 3D HCN-CCH-TOCSY and HCN-CCH-COSY experiments are presented for the ribose resonance assignment of 13C/15N-labeled RNA sample. The experiments make use of the chemical shift dispersion of N1/N9 of pyrimidine/purine to distinguish the ribose spin systems. They provide a complementary approach for the assignment of ribose resonance to the currently used HCCH-COSY and HCCH-TOCSY type experiments in which either 13C or 1H is utilized to separate the different ribose spin systems. The pulse schemes have been demonstrated on a 23-mer 13C/15N-labeled RNA aptamer complexed with neomycin and tested on a 32-mer RNA complexed with a 23-residue peptide.

  13. Individual protein balance strongly influences δ15N and δ13C values in Nile tilapia, Oreochromis niloticus

    NASA Astrophysics Data System (ADS)

    Gaye-Siessegger, Julia; Focken, Ulfert; Abel, Hansjörg; Becker, Klaus

    Although stable isotope ratios in animals have often been used as indicators of the trophic level and for the back-calculation of diets, few experiments have been done under standardized laboratory conditions to investigate factors influencing δ15N and δ13C values. An experiment using Nile tilapia [Oreochromis niloticus (L.)] was therefore carried out to test the effect of different dietary protein contents (35.4, 42.3, and 50.9%) on δ15N and δ13C values of the whole tilapia. The fish were fed the isoenergetic and isolipidic semi-synthetic diets at a relatively low level. δ15N and δ13C values of the lipid-free body did not differ between the fish fed the diets with different protein contents, but the trophic shift for N and C isotopes decreased with increasing protein accretion in the individual fish, for N from 6.5‰ to 4‰ and for C in the lipid-free body from 4‰ to 2.5‰. This is the first study showing the strong influence of the individual protein balance to the degree to which the isotopic signature of dietary protein was modified in tissue protein of fish. The extrapolation of the trophic level or the reconstruction of the diet of an animal from stable isotope ratios without knowledge of the individual physiological condition and the feeding rate may lead to erroneous results.

  14. Proton-decoupled CPMG: a better experiment for measuring (15)N R2 relaxation in disordered proteins.

    PubMed

    Yuwen, Tairan; Skrynnikov, Nikolai R

    2014-04-01

    (15)N R2 relaxation is one of the most informative experiments for characterization of intrinsically disordered proteins (IDPs). Small changes in nitrogen R2 rates are often used to determine how IDPs respond to various biologically relevant perturbations such as point mutations, posttranslational modifications and weak ligand interactions. However collecting high-quality (15)N relaxation data can be difficult. Of necessity, the samples of IDPs are often prepared with low protein concentration and the measurement time can be limited because of rapid sample degradation. Furthermore, due to hardware limitations standard experiments such as (15)N spin-lock and CPMG can sample the relaxation decay only to ca. 150ms. This is much shorter than (15)N T2 times in disordered proteins at or near physiological temperature. As a result, the sampling of relaxation decay profiles in these experiments is suboptimal, which further lowers the precision of the measurements. Here we report a new implementation of the proton-decoupled (PD) CPMG experiment which allows one to sample (15)N R2 relaxation decay up to ca. 0.5-1s. The new experiment has been validated through comparison with the well-established spin-lock measurement. Using dilute samples of denatured ubiquitin, we have demonstrated that PD-CPMG produces up to 3-fold improvement in the precision of the data. It is expected that for intrinsically disordered proteins the gains may be even more substantial. We have also shown that this sequence has a number of favorable properties: (i) the spectra are recorded with narrow linewidth in nitrogen dimension; (ii) (15)N offset correction is small and easy to calculate; (iii) the experiment is immune to various spurious effects arising from solvent exchange; (iv) the results are stable with respect to pulse miscalibration and rf field inhomogeneity; (v) with minimal change, the pulse sequence can also be used to measure R2 relaxation of (15)N(ε) spins in arginine side chains. We

  15. Effective isotope labeling of proteins in a mammalian expression system.

    PubMed

    Sastry, Mallika; Bewley, Carole A; Kwong, Peter D

    2015-01-01

    Isotope labeling of biologically interesting proteins is a prerequisite for structural and dynamics studies by NMR spectroscopy. Many of these proteins require mammalian cofactors, chaperons, or posttranslational modifications such as myristoylation, glypiation, disulfide bond formation, or N- or O-linked glycosylation; and mammalian cells have the necessary machinery to produce them in their functional forms. Here, we describe recent advances in mammalian expression, including an efficient adenoviral vector-based system, for the production of isotopically labeled proteins. This system enables expression of mammalian proteins and their complexes, including proteins that require posttranslational modifications. We describe a roadmap to produce isotopically labeled (15)N and (13)C posttranslationally modified proteins, such as the outer domain of HIV-1 gp120, which has four disulfide bonds and 15 potential sites of N-linked glycosylation. These methods should allow NMR spectroscopic analysis of the structure and function of posttranslationally modified and secreted, cytoplasmic, or membrane-bound proteins.

  16. Human baby hair amino acid natural abundance 15N-isotope values are not related to the 15N-isotope values of amino acids in mother's breast milk protein.

    PubMed

    Romek, Katarzyna M; Julien, Maxime; Frasquet-Darrieux, Marine; Tea, Illa; Antheaume, Ingrid; Hankard, Régis; Robins, Richard J

    2013-12-01

    Since exclusively breast-suckled infants obtain their nutrient only from their mother's milk, it might be anticipated that a correlation will exist between the (15)N/(14)N isotope ratios of amino acids of protein of young infants and those supplied by their mother. The work presented here aimed to determine whether amino nitrogen transfer from human milk to infant hair protein synthesized within the first month of life conserves the maternal isotopic signature or whether post-ingestion fractionation dominates the nitrogen isotope spectrum. The study was conducted at 1 month post-birth on 100 mother-infant pairs. Isotope ratios (15)N/(14)N and (13)C/(12)C were measured using isotope ratio measurement by Mass Spectrometry (irm-MS) for whole maternal milk, and infant hair and (15)N/(14)N ratios were also measured by GC-irm-MS for the N-pivaloyl-O-isopropyl esters of amino acids obtained from the hydrolysis of milk and hair proteins. The δ(15)N and δ(13)C (‰) were found to be significantly higher in infant hair than in breast milk (δ(15)N, P < 0.001; δ(13)C, P < 0.001). Furthermore, the δ(15)N (‰) of individual amino acids in infant hair was also significantly higher than that in maternal milk (P < 0.001). By calculation, the observed shift in isotope ratio was shown not to be accounted for by the amino acid composition of hair and milk proteins, indicating that it is not simply due to differences in the composition in the proteins present. Rather, it would appear that each pool-mother and infant-turns over independently, and that fractionation in infant N-metabolism even in the first month of life dominates over the nutrient N-content.

  17. Interactions of /sup 14/N:/sup 15/N stearic acid spin-label pairs: effects of host lipid alkyl chain length and unsaturation

    SciTech Connect

    Feix, J.B.; Yin, J.J.; Hyde, J.S.

    1987-06-30

    Electron-electron double resonance (ELDOR) and saturation recovery electron paramagnetic resonance (EPR) spectroscopy have been employed to examine the interactions of /sup 14/N:/sup 15/N stearic acid spin-label pairs in fluid-phase model membrane bilayers composed of a variety of phospholipids. The (/sup 14/N)-16-doxylstearate:(/sup 15/N)-16-doxylstearate (16:16) pair was utilized to measure lateral diffusion of the spin-labels, while the (/sup 14/N)-16-doxylstearate:(/sup 15/N)-5-doxylstearate (16:5) pair provided information on vertical fluctuations of the 16-doxylstearate nitroxide moiety toward the membrane surface. Three saturated host lipids of varying alkyl chain length (dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC)), an ..cap alpha..-saturated, ..beta..-unsaturated lipid (1-palmitoyl-2-oleoylphosphatidylcholine (POPC)), and phosphatidylcholine from a natural source (egg yolk phosphatidylcholine (egg PC)) were utilized as host lipids. Lateral diffusion of the stearic acid spin-labels was only slightly affected by alkyl chain length at a given reduced temperature (T/sub r/) in the saturated host lipids but was significantly decreased in POPC at the same T/sub r/. Lateral diffusion in DMPC, POPC, and egg PC was quite similar at 37/sup 0/C. A strong correlation was noted between lateral diffusion constants and rotational mobility of (/sup 14/N)-16-doxylstearate. Vertical fluctuations were likewise only slightly influenced by alklyl chain length but were strongly diminished in POPC and egg PC relative to the saturated systems. This diminution of the 16:5 interaction was observed even under conditions where no differences were discernible by conventional EPR.

  18. Combining combing and secondary ion mass spectrometry to study DNA on chips using (13)C and (15)N labeling.

    PubMed

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA - the combing, imaging by SIMS or CIS method - has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to (13)C-labeling via the detection and quantification of the (13)C (14)N (-) recombinant ion and the use of the (13)C: (12)C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  19. Combining combing and secondary ion mass spectrometry to study DNA on chips using 13C and 15N labeling

    PubMed Central

    Cabin-Flaman, Armelle; Monnier, Anne-Francoise; Coffinier, Yannick; Audinot, Jean-Nicolas; Gibouin, David; Wirtz, Tom; Boukherroub, Rabah; Migeon, Henri-Noël; Bensimon, Aaron; Jannière, Laurent; Ripoll, Camille; Norris, Victor

    2016-01-01

    Dynamic secondary ion mass spectrometry ( D-SIMS) imaging of combed DNA – the combing, imaging by SIMS or CIS method – has been developed previously using a standard NanoSIMS 50 to reveal, on the 50 nm scale, individual DNA fibers labeled with different, non-radioactive isotopes in vivo and to quantify these isotopes. This makes CIS especially suitable for determining the times, places and rates of DNA synthesis as well as the detection of the fine-scale re-arrangements of DNA and of molecules associated with combed DNA fibers. Here, we show how CIS may be extended to 13C-labeling via the detection and quantification of the 13C 14N - recombinant ion and the use of the 13C: 12C ratio, we discuss how CIS might permit three successive labels, and we suggest ideas that might be explored using CIS. PMID:27429742

  20. Selecting matched root architecture in tree pairs to be used for assessing N 2 fixation based on soil- 15N-labelling

    NASA Astrophysics Data System (ADS)

    Nasr, Hafedh; Ghorbel, Mohamed Habib; Wallander, Håkan; Dommergues, Yvon René

    2005-03-01

    It is commonly assumed that soil- 15N-labelling provides reliable estimates of N 2 fixation in trees by matching N 2-fixing and non-N 2-fixing tree pairs. As root system is a key parameter in determining suitability of the tree pairs, we compared root architecture of Acacia cyanophylla Lindl. and Casuarina glauca Sieber ex. Spreng. (two N 2-fixing trees) with Eucalyptus camaldulensis Dehn. and Ceratonia siliqua L. (two non-N 2-fixing trees) at 4-year-old in Mediterranean-semiarid zone. The rhizobium strain used appeared more motile than Frankia strain. A. cyanophylla and E. camaldulensis had extensive rooting area and volume of fine roots, and both species tended to develop marked horizontal rooting, compared to C. glauca and C. siliqua. Characteristics of fine- and horizontal-root components can be used in selecting matched root systems of N 2-fixing and reference-paired trees. Root architecture of C. glauca was more similar to C. siliqua, than to E. camaldulensis, and that of A. cyanophylla was more similar to E. camaldulensis than to C. siliqua. Accordingly, E. camaldulensis is an appropriate reference to estimate actual N 2 fixation by A. cyanophylla, and C. siliqua is an appropriate reference for C. glauca, when using soil- 15N-labelling method in the prevailing site environment.

  1. In vivo, large-scale preparation of uniformly (15)N- and site-specifically (13)C-labeled homogeneous, recombinant RNA for NMR studies.

    PubMed

    Le, My T; Brown, Rachel E; Simon, Anne E; Dayie, T Kwaku

    2015-01-01

    Knowledge of how ribonucleic acid (RNA) structures fold to form intricate, three-dimensional structures has provided fundamental insights into understanding the biological functions of RNA. Nuclear magnetic resonance (NMR) spectroscopy is a particularly useful high-resolution technique to investigate the dynamic structure of RNA. Effective study of RNA by NMR requires enrichment with isotopes of (13)C or (15)N or both. Here, we present a method to produce milligram quantities of uniformly (15)N- and site-specifically (13)C-labeled RNAs using wild-type K12 and mutant tktA Escherichia coli in combination with a tRNA-scaffold approach. The method includes a double selection protocol to obtain an E. coli clone with consistently high expression of the recombinant tRNA-scaffold. We also present protocols for the purification of the tRNA-scaffold from a total cellular RNA extract and the excision of the RNA of interest from the tRNA-scaffold using DNAzymes. Finally, we showcase NMR applications to demonstrate the benefit of using in vivo site-specifically (13)C-labeled RNA. PMID:26577743

  2. 13C- and 15N-Labeling Strategies Combined with Mass Spectrometry Comprehensively Quantify Phospholipid Dynamics in C. elegans

    PubMed Central

    Drechsler, Robin; Gafken, Philip R.; Olsen, Carissa Perez

    2015-01-01

    Membranes define cellular and organelle boundaries, a function that is critical to all living systems. Like other biomolecules, membrane lipids are dynamically maintained, but current methods are extremely limited for monitoring lipid dynamics in living animals. We developed novel strategies in C. elegans combining 13C and 15N stable isotopes with mass spectrometry to directly quantify the replenishment rates of the individual fatty acids and intact phospholipids of the membrane. Using multiple measurements of phospholipid dynamics, we found that the phospholipid pools are replaced rapidly and at rates nearly double the turnover measured for neutral lipid populations. In fact, our analysis shows that the majority of membrane lipids are replaced each day. Furthermore, we found that stearoyl-CoA desaturases (SCDs), critical enzymes in polyunsaturated fatty acid production, play an unexpected role in influencing the overall rates of membrane maintenance as SCD depletion affected the turnover of nearly all membrane lipids. Additionally, the compromised membrane maintenance as defined by LC-MS/MS with SCD RNAi resulted in active phospholipid remodeling that we predict is critical to alleviate the impact of reduced membrane maintenance in these animals. Not only have these combined methodologies identified new facets of the impact of SCDs on the membrane, but they also have great potential to reveal many undiscovered regulators of phospholipid metabolism. PMID:26528916

  3. Design and operation of a continuous 13C and 15N labeling chamber for uniform or differential, metabolic and structural, plant tissue isotope labeling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tracing heavy stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O o...

  4. Unraveling the complexity of protein backbone dynamics with combined (13)C and (15)N solid-state NMR relaxation measurements.

    PubMed

    Lamley, Jonathan M; Lougher, Matthew J; Sass, Hans Juergen; Rogowski, Marco; Grzesiek, Stephan; Lewandowski, Józef R

    2015-09-14

    Typically, protein dynamics involve a complex hierarchy of motions occurring on different time scales between conformations separated by a range of different energy barriers. NMR relaxation can in principle provide a site-specific picture of both the time scales and amplitudes of these motions, but independent relaxation rates sensitive to fluctuations in different time scale ranges are required to obtain a faithful representation of the underlying dynamic complexity. This is especially pertinent for relaxation measurements in the solid state, which report on dynamics in a broader window of time scales by more than 3 orders of magnitudes compared to solution NMR relaxation. To aid in unraveling the intricacies of biomolecular dynamics we introduce (13)C spin-lattice relaxation in the rotating frame (R1ρ) as a probe of backbone nanosecond-microsecond motions in proteins in the solid state. We present measurements of (13)C'R1ρ rates in fully protonated crystalline protein GB1 at 600 and 850 MHz (1)H Larmor frequencies and compare them to (13)C'R1, (15)N R1 and R1ρ measured under the same conditions. The addition of carbon relaxation data to the model free analysis of nitrogen relaxation data leads to greatly improved characterization of time scales of protein backbone motions, minimizing the occurrence of fitting artifacts that may be present when (15)N data is used alone. We also discuss how internal motions characterized by different time scales contribute to (15)N and (13)C relaxation rates in the solid state and solution state, leading to fundamental differences between them, as well as phenomena such as underestimation of picosecond-range motions in the solid state and nanosecond-range motions in solution.

  5. Multi-Isotope Secondary Ion Mass Spectrometry Combining Heavy Water 2H with 15N Labeling As Complementary Tracers for Metabolic Heterogeneity at the Single-Cell Level

    NASA Astrophysics Data System (ADS)

    Kopf, S.; McGlynn, S.; Cowley, E.; Green, A.; Newman, D. K.; Orphan, V. J.

    2014-12-01

    Metabolic rates of microbial communities constitute a key physiological parameter for understanding the in situ growth constraints for life in any environment. Isotope labeling techniques provide a powerful approach for measuring such biological activity, due to the use of isotopically enriched substrate tracers whose incorporation into biological materials can be detected with high sensitivity by isotope-ratio mass spectrometry. Nano-meter scale secondary ion mass spectrometry (NanoSIMS) combined with stable isotope labeling provides a unique tool for studying the spatiometabolic activity of microbial populations at the single cell level in order to assess both community structure and population diversity. However, assessing the distribution and range of microbial activity in complex environmental systems with slow-growing organisms, diverse carbon and nitrogen sources, or heterotrophic subpopulations poses a tremendous technical challenge because the introduction of isotopically labeled substrates frequently changes the nutrient availability and can inflate or bias measures of activity. Here, we present the use of hydrogen isotope labeling with deuterated water as an important new addition to the isotopic toolkit and apply it for the determination of single cell microbial activities by NanoSIMS imaging. This tool provides a labeling technique that minimally alters any aquatic chemical environment, can be administered with strong labels even in minimal addition (natural background is very low), is an equally universal substrate for all forms of life even in complex, carbon and nitrogen saturated systems, and can be combined with other isotopic tracers. The combination of heavy water labeling with the most commonly used NanoSIMS tracer, 15N, is technically challenging but opens up a powerful new set of multi-tracer experiments for the study of microbial activity in complex communities. We present the first truly simultaneous single cell triple isotope system

  6. 1H- and 15N-NMR assignment and solution structure of the chemotactic Escherichia coli Che Y protein.

    PubMed

    Bruix, M; Pascual, J; Santoro, J; Prieto, J; Serrano, L; Rico, M

    1993-08-01

    Che Y is a 129-residue parallel alpha/beta protein involved in bacterial chemotaxis. We have used this protein as a model to study the folding reaction of parallel alpha/beta proteins. As a first step we carried out the complete assignment of the 1H and 15N spectra from Escherichia coli Che Y protein on the basis of two-dimensional 1H homonuclear and 1H-15N heteronuclear experiments by using sequence-specific methods. Our assignments differ from the preliminary assignments made by Kar et al. [Kar, L., Matsumura, P. & Johnson, M.E. (1992) Biochem. J. 287, 521-531] of aromatic residues obtained by comparison of NOEs with short proton-proton distances in the crystal structure of Che Y. The analysis of the extension of the secondary elements, as well as a preliminary calculation of the three-dimensional structure, indicate that the solution structure is closely coincident with the single crystal structure determined by X-ray diffraction.

  7. NMR studies on /sup 15/N-labeled creatine (CR), creatinine (CRN), phosphocreatine (PCR), and phosphocreatinine (PCRN), and on barriers to rotation in creatine kinase-bound creatine in the enzymatic reaction

    SciTech Connect

    Kenyon, G.L.; Reddick, R.E.

    1986-05-01

    Recently, the authors have synthesized /sup 15/N-2-Cr, /sup 15/N-3-Crn, /sup 15/N-2-Crn, /sup 15/N-3-PCrn, /sup 15/N-3-PCr, and /sup 15/N-2-PCr. /sup 1/H, /sup 15/N, /sup 31/P NMR data show that Crn protonates exclusively at the non-methylated ring nitrogen, confirm that PCrn is phosphorylated at the exocyclic nitrogen, and demonstrate that the /sup 31/P-/sup 15/N one-bond coupling constant in /sup 15/N-3-PCr is 18 Hz, not 3 Hz as previously reported by Brindle, K.M., Porteous, R. and Radda, G.K.. The authors have found that creatine kinase is capable of catalyzing the /sup 14/N//sup 15/N positional isotope exchange of 3-/sup 15/N-PCr in the presence of MgADP, but not in its absence. Further, the exchange does not take place when labeled PCr is resynthesized exclusively from the ternary complex E X Cr X MgATP as opposed to either E X Cr or free Cr. This suggests that the enzyme both imparts an additional rotational barrier to creatine in the complex and catalyzes the transfer of phosphoryl group with essentially complete regiospecificity.

  8. Balancing the (carbon) budget: Using linear inverse models to estimate carbon flows and mass-balance 13C:15N labelling experiments in low oxygen sediments.

    NASA Astrophysics Data System (ADS)

    Hunter, William Ross; Van Oevelen, Dick; Witte, Ursula

    2013-04-01

    Over 1 million km2 of seafloor experience permanent low-oxygen conditions within oxygen minimum zones (OMZs). OMZs are predicted to grow as a consequence of climate change, potentially affecting oceanic biogeochemical cycles. The Arabian Sea OMZ impinges upon the western Indian continental margin at bathyal depths (150 - 1500m) producing a strong depth dependent oxygen gradient at the sea floor. The influence of the OMZ upon the short term processing of organic matter by sediment ecosystems was investigated using in situ stable isotope pulse chase experiments. These deployed doses of 13C:15N labeled organic matter onto the sediment surface at four stations from across the OMZ (water depth 540 - 1100 m; [O2] = 0.35 - 15 μM). In order to prevent experimentally anoxia, the mesocosms were not sealed. 13C and 15N labels were traced into sediment, bacteria, fauna and 13C into sediment porewater DIC and DOC. However, the DIC and DOC flux to the water column could not be measured, limiting our capacity to obtain mass-balance for C in each experimental mesocosm. Linear Inverse Modeling (LIM) provides a method to obtain a mass-balanced model of carbon flow that integrates stable-isotope tracer data with community biomass and biogeochemical flux data from a range of sources. Here we present an adaptation of the LIM methodology used to investigate how ecosystem structure influenced carbon flow across the Indian margin OMZ. We demonstrate how oxygen conditions affect food-web complexity, affecting the linkages between the bacteria, foraminifera and metazoan fauna, and their contributions to benthic respiration. The food-web models demonstrate how changes in ecosystem complexity are associated with oxygen availability across the OMZ and allow us to obtain a complete carbon budget for the stationa where stable-isotope labelling experiments were conducted.

  9. Estimation of internal and external nitrogen for corals with a long-term 15N-labelling experiment and subsequent model calculations

    NASA Astrophysics Data System (ADS)

    Tanaka, Yasuaki; Grottoli, Andréa; Matsui, Yohei; Suzuki, Atsushi; Sakai, Kazuhiko

    2014-05-01

    Coral reef ecosystems maintain high primary productivity though the seawater is extremely oligotrophic. One of the hypotheses to explain this paradox is the recycling of nutrients in animal-algal symbiotic organisms such as corals. It is relatively easy to measure nutrient uptake rates by corals from seawater, but the proportion of internally circulating nutrients between the coral host and the endosymbiotic algae (zooxanthellae) is more challenging. Here, we performed a long-term and continuous 15N-labelling experiment to quantify the proportionate contribution of seawater (external N source) and the animal host (internal N source) to the total N influx in the endosymbiotic algae. Branches from the scleractinian corals Porites cylindrica and Montipora digitata from Okinawa, Japan, were cultured for 2 months in indoor, flow-through, filtered seawater tanks with the continuous supply of 15N-labelled nitrate. At the initial and after 2, 4, and 9 weeks of the study, coral branches were collected and the algal and animal fractions were separated for isotopic analyses. In both corals, the N isotope ratio of symbiotic algae exponentially increased and the values were much higher than those of the host tissue, suggesting that the algae had a faster turnover N time than the animal host. Algal and host N biomass normalized to the coral surface area slowly decreased in both coral species over the study period. To calculate the contribution of internal and external N, a simple mixing model of algal N metabolism was designed. Using differential equations of 15N balance and N biomass balance, F1 and F2 (external and internal N fluxes to symbiotic algae, respectively) were expressed as the functions of time. The model calculations showed that F2 was much higher than F1 in P. cylindrica and the percentage of internal N to the total influx N (PIN) was >70%. On the other hand, the contribution of F1 and F2 was comparable in M. digitata and the PIN was 40-70%. These results

  10. Post-translational heterocyclic backbone modifications in the 43-peptide antibiotic microcin B17. Structure elucidation and NMR study of a 13C,15N-labelled gyrase inhibitor.

    PubMed

    Bayer, A; Freund, S; Jung, G

    1995-12-01

    Microcin B17 (McB17), the first known gyrase inhibitor of peptidic nature, is produced by ribosomal synthesis and post-translational modification of the 69-residue precursor protein by an Escherichia coli strain. To elucidate the chemical structure of the mature 43-residue peptide antibiotic, fermentation and purification protocols were established and optimized which allowed the isolation and purification of substantial amounts of highly pure McB17 (non-labelled, 15N-labelled and 13C/15N-labelled peptide. By ultraviolet-absorption spectroscopy. HPLC-electrospray mass spectrometry and GC-mass spectrometry, amino acid analysis, protein sequencing, and, in particular, multidimensional NMR, we could demonstrate and unequivocally prove that the enzymic modification of the precursor backbone at Gly-Cys and Gly-Ser segments leads to the formation of 2-aminomethylthiazole-4-carboxylic acid and 2-aminomethyloxazole-4-carboxylic acid, respectively. In addition, two bicyclic modifications 2-(2-aminomethyloxazolyl)thiazole-4-carboxylic acid and 2-(2-aminomethylthiazolyl)oxazole-4-carboxylic acid were found that consist of directly linked thiazole and oxazole rings derived from one Gly-Ser-Cys and one Gly-Cys-Ser segment. Analogous to the thiazole and oxazole rings found in antitumor peptides of microbial and marine origin, these heteroaromatic ring systems of McB17 presumably play an important role in its gyrase-inhibiting activity, e.g. interacting with the DNA to trap the covalent protein-DNA intermediate of the breakage-reunion reaction of the gyrase.

  11. α-Ketoacids as precursors for phenylalanine and tyrosine labelling in cell-based protein overexpression.

    PubMed

    Lichtenecker, Roman J; Weinhäupl, Katharina; Schmid, Walther; Konrat, Robert

    2013-12-01

    (13)C-α-ketoacid metabolic precursors of phenylalanine and tyrosine effectively enter the metabolism of a protein overexpressing E. coli strain to label Phe- and Tyr-residues devoid of any cross-labelling. The methodology gives access to highly selective labelling patterns as valuable tools in protein NMR spectroscopy without the need of (15)N-chiral amino acid synthesis using organic chemistry.

  12. Modeling (15)N NMR chemical shift changes in protein backbone with pressure.

    PubMed

    La Penna, Giovanni; Mori, Yoshiharu; Kitahara, Ryo; Akasaka, Kazuyuki; Okamoto, Yuko

    2016-08-28

    Nitrogen chemical shift is a useful parameter for determining the backbone three-dimensional structure of proteins. Empirical models for fast calculation of N chemical shift are improving their reliability, but there are subtle effects that cannot be easily interpreted. Among these, the effects of slight changes in hydrogen bonds, both intramolecular and with water molecules in the solvent, are particularly difficult to predict. On the other hand, these hydrogen bonds are sensitive to changes in protein environment. In this work, the change of N chemical shift with pressure for backbone segments in the protein ubiquitin is correlated with the change in the population of hydrogen bonds involving the backbone amide group. The different extent of interaction of protein backbone with the water molecules in the solvent is put in evidence. PMID:27586953

  13. Modeling 15N NMR chemical shift changes in protein backbone with pressure

    NASA Astrophysics Data System (ADS)

    La Penna, Giovanni; Mori, Yoshiharu; Kitahara, Ryo; Akasaka, Kazuyuki; Okamoto, Yuko

    2016-08-01

    Nitrogen chemical shift is a useful parameter for determining the backbone three-dimensional structure of proteins. Empirical models for fast calculation of N chemical shift are improving their reliability, but there are subtle effects that cannot be easily interpreted. Among these, the effects of slight changes in hydrogen bonds, both intramolecular and with water molecules in the solvent, are particularly difficult to predict. On the other hand, these hydrogen bonds are sensitive to changes in protein environment. In this work, the change of N chemical shift with pressure for backbone segments in the protein ubiquitin is correlated with the change in the population of hydrogen bonds involving the backbone amide group. The different extent of interaction of protein backbone with the water molecules in the solvent is put in evidence.

  14. Quantitative Shotgun Proteomics Using a Uniform 15N-Labeled Standard to Monitor Proteome Dynamics in Time Course Experiments Reveals New Insights into the Heat Stress Response of Chlamydomonas reinhardtii*

    PubMed Central

    Mühlhaus, Timo; Weiss, Julia; Hemme, Dorothea; Sommer, Frederik; Schroda, Michael

    2011-01-01

    Crop-plant-yield safety is jeopardized by temperature stress caused by the global climate change. To take countermeasures by breeding and/or transgenic approaches it is essential to understand the mechanisms underlying plant acclimation to heat stress. To this end proteomics approaches are most promising, as acclimation is largely mediated by proteins. Accordingly, several proteomics studies, mainly based on two-dimensional gel-tandem MS approaches, were conducted in the past. However, results often were inconsistent, presumably attributable to artifacts inherent to the display of complex proteomes via two-dimensional-gels. We describe here a new approach to monitor proteome dynamics in time course experiments. This approach involves full 15N metabolic labeling and mass spectrometry based quantitative shotgun proteomics using a uniform 15N standard over all time points. It comprises a software framework, IOMIQS, that features batch job mediated automated peptide identification by four parallelized search engines, peptide quantification and data assembly for the processing of large numbers of samples. We have applied this approach to monitor proteome dynamics in a heat stress time course using the unicellular green alga Chlamydomonas reinhardtii as model system. We were able to identify 3433 Chlamydomonas proteins, of which 1116 were quantified in at least three of five time points of the time course. Statistical analyses revealed that levels of 38 proteins significantly increased, whereas levels of 206 proteins significantly decreased during heat stress. The increasing proteins comprise 25 (co-)chaperones and 13 proteins involved in chromatin remodeling, signal transduction, apoptosis, photosynthetic light reactions, and yet unknown functions. Proteins decreasing during heat stress were significantly enriched in functional categories that mediate carbon flux from CO2 and external acetate into protein biosynthesis, which also correlated with a rapid, but fully

  15. Synthesis of 14N and 15N-labeled trityl-nitroxide biradicals with strong spin-spin interaction and improved sensitivity to redox status and oxygen

    PubMed Central

    Liu, Yangping; Villamena, Frederick A.; Song, Yuguang; Sun, Jian; Rockenbauer, Antal

    2014-01-01

    Simultaneous evaluation redox status and oxygenation in biological systems is of great importance for the understanding of biological functions. Electron paramagnetic resonance spectroscopy coupled with the use of the nitroxide radicals have been an indispensable technique for this application but are still limited by its low oxygen sensitivity, and low EPR resolution in part due to the moderately broad EPR triplet and spin quenching through bioreduction. In this study, we showed that these drawbacks can be overcome through the use of trityl-nitroxide biradicals allowing for the simultaneous measurement of redox status and oxygenation. A new trityl-nitroxide biradical TNN14 composed of a pyrrolidinyl-nitroxide and a trityl, and its isotopically labeled 15N analogue TNN15 were synthesized and characterized. Both biradicals exhibited much stronger spin-spin interaction with J > 400 G than the previous synthesized trityl-nitroxide biradicals TN1 (~160 G) and TN2 (~52 G) with longer linker chain length. The enhanced stability of TNN14 was evaluated using ascorbate as reductant and the effect of different types of cyclodextrins on its stability in the presence of ascorbate was also investigated. Both biradicals are sensitive to redox status, and their corresponding trityl-hydroxylamines resulting from the reduction of the biradicals by ascorbate share the same oxygen sensitivity. Of note is that the 15N-labeled TNN15-H with an EPR doublet exhibits improved EPR signal amplitude as compared to TNN14-H with an EPR triplet. In addition, cyclic voltammetric studies verify the characteristic electrochemical behaviors of the trityl-nitroxide biradicals. PMID:21028905

  16. Characterization of the overall rotational diffusion of a protein from 15N relaxation measurements and hydrodynamic calculations.

    PubMed

    Blake-Hall, Jennifer; Walker, Oliver; Fushman, David

    2004-01-01

    In this chapter, we discuss experimental and theoretical methods for characterizing the overall rotational diffusion of molecules in solution. The methods are illustrated for the B3 domain of protein G, a small protein with rotational anisotropy of Dpar/Dperp = 1.4. The rotational diffusion tensor of the protein is determined directly from 15N relaxation measurements. The experimental data are treated assuming various possible models for the overall tumbling: isotropic, axially symmetric, and fully anisotropic, and the results of these analyses are compared to determine an adequate diffusion model for the protein. These experimentally derived characteristics of the protein are compared with the results of theoretical calculations of the diffusion tensor using various hydrodynamic models, to find optimal models and parameter sets for theoretical predictions. We also derive model-free characteristics of internal backbone motions in the protein, to show that different models for the overall motion can result in significantly different pictures of motion. This emphasizes the necessity of accurately characterizing the overall tumbling of a molecule to determine its local dynamics.

  17. Through-space (19) F-(15) N couplings for the assignment of stereochemistry in flubenzimine.

    PubMed

    Ghiviriga, Ion; Rubinski, Miles A; Dolbier, William R

    2016-07-01

    Through-space (19) F-(15) N couplings revealed the configuration of flubenzimine, with the CF3 group on N4 pointing towards the lone pair of N5. The (19) F-(15) N coupling constants were measured at natural abundance using a spin-state selective indirect-detection pulse sequence. As (15) N-labelled proteins are routinely synthesized for NMR studies, through-space (19) F-(15) N couplings have the potential to probe the stereochemistry of these proteins by (19) F labelling of some amino acids or can reveal the site of docking of fluorine-containing drugs. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Label and Label-Free Detection Techniques for Protein Microarrays

    PubMed Central

    Syahir, Amir; Usui, Kenji; Tomizaki, Kin-ya; Kajikawa, Kotaro; Mihara, Hisakazu

    2015-01-01

    Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano-biological events.

  19. Use of a novel nitrification inhibitor to reduce nitrous oxide emission from (15)N-labelled dairy slurry injected into soil.

    PubMed

    Dittert, K; Bol, R; King, R; Chadwick, D; Hatch, D

    2001-01-01

    Recent recommendations for environmentally sound use of liquid animal manure often include injection of slurry into soil. Two of the most important undesired side effects, ammonia (NH(3)) volatilisation and odour emissions, are usually significantly reduced by slurry injection. On the other hand, because of the higher amount of nitrogen (N) remaining in soil, the risk of nitrate (NO(3)(-)) leaching and nitrous oxide (N(2)O) emissions is increased. Thus, the reduction of local effects caused by NH(3) deposition, e.g. N enrichment and soil acidification, may be at the cost of large-scale effects such as ozone depletion and global warming as a result of emitted N(2)O. In this context, nitrification inhibitors can contribute significantly to a reduction in NO(3)(-) leaching and N(2)O production. A field experiment was carried out at IGER, North Wyke, which aimed to evaluate the effect of the new nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP/ENTEC). For this experiment, (15)N enriched dairy slurry was used and the isotopic label in soil N as well as in N(2)O were studied. After slurry injection into the grassland soil in August 2000, the major emissions of N(2)O occurred during the first ten days. As expected, high N(2)O emission rates and (15)N content of the emissions were concentrated on the slurry injection slots, showing a steep decrease towards the untreated centre-point between slurry injection slots. The nitrification inhibitor DMPP proved to be very efficient in reducing N(2)O emissions. At a rate of 2 kg DMPP ha(-1), the total amount of N(2)O emitted was reduced by 32%, when compared with slurry injection without DMPP. The isotopic label of the emitted N(2)O showed that during the 22-day experimental period, emissions from the slurry N pool were strongly reduced by DMPP from 0.93 kg N(2)O-N ha(-1) (-DMPP) to 0.50 kg N(2)O-N ha(-1) (+DMPP), while only a minor effect on emissions from the soil N pool was observed (0.69 to 0.60 kg N(2)O-N ha(-1

  20. Dynamics of exogenous nitrogen partitioning and nitrogen remobilization from vegetative organs in pea revealed by 15N in vivo labeling throughout seed filling.

    PubMed

    Schiltz, Séverine; Munier-Jolain, Nathalie; Jeudy, Christian; Burstin, Judith; Salon, Christophe

    2005-04-01

    The fluxes of (1) exogenous nitrogen (N) assimilation and (2) remobilization of endogenous N from vegetative plant compartments were measured by 15N labeling during the seed-filling period in pea (Pisum sativum L. cv Cameor), to better understand the mechanism of N remobilization. While the majority (86%) of exogenous N was allocated to the vegetative organs before the beginning of seed filling, this fraction decreased to 45% at the onset of seed filling, the remainder being directed to seeds. Nitrogen remobilization from vegetative parts contributed to 71% of the total N in mature seeds borne on the first two nodes (first stratum). The contribution of remobilized N to total seed N varied, with the highest proportion at the beginning of filling; it was independent of the developmental stage of each stratum of seeds, suggesting that remobilized N forms a unique pool, managed at the whole-plant level and supplied to all filling seeds whatever their position on the plant. Once seed filling starts, N is remobilized from all vegetative organs: 30% of the total N accumulated in seeds was remobilized from leaves, 20% from pod walls, 11% from roots, and 10% from stems. The rate of N remobilization was maximal when seeds of all the different strata were filling, consistent with regulation according to the N demand of seeds. At later stages of seed filling, the rate of remobilization decreases and may become controlled by the amount of residual N in vegetative tissues.

  1. NMR studies of active-site properties of human carbonic anhydrase II by using (15) N-labeled 4-methylimidazole as a local probe and histidine hydrogen-bond correlations.

    PubMed

    Shenderovich, Ilya G; Lesnichin, Stepan B; Tu, Chingkuang; Silverman, David N; Tolstoy, Peter M; Denisov, Gleb S; Limbach, Hans-Heinrich

    2015-02-01

    By using a combination of liquid and solid-state NMR spectroscopy, (15) N-labeled 4-methylimidazole (4-MI) as a local probe of the environment has been studied: 1) in the polar, wet Freon CDF3 /CDF2 Cl down to 130 K, 2) in water at pH 12, and 3) in solid samples of the mutant H64A of human carbonic anhydrase II (HCA II). In the latter, the active-site His64 residue is replaced by alanine; the catalytic activity is, however, rescued by the presence of 4-MI. For the Freon solution, it is demonstrated that addition of water molecules not only catalyzes proton tautomerism but also lifts its quasidegeneracy. The possible hydrogen-bond clusters formed and the mechanism of the tautomerism are discussed. Information about the imidazole hydrogen-bond geometries is obtained by establishing a correlation between published (1) H and (15) N chemical shifts of the imidazole rings of histidines in proteins. This correlation is useful to distinguish histidines embedded in the interior of proteins and those at the surface, embedded in water. Moreover, evidence is obtained that the hydrogen-bond geometries of His64 in the active site of HCA II and of 4-MI in H64A HCA II are similar. Finally, the degeneracy of the rapid tautomerism of the neutral imidazole ring His64 reported by Shimahara et al. (J. Biol. Chem.- 2007, 282, 9646) can be explained with a wet, polar, nonaqueous active-site conformation in the inward conformation, similar to the properties of 4-MI in the Freon solution. The biological implications for the enzyme mechanism are discussed. PMID:25521423

  2. NMR studies of active-site properties of human carbonic anhydrase II by using (15) N-labeled 4-methylimidazole as a local probe and histidine hydrogen-bond correlations.

    PubMed

    Shenderovich, Ilya G; Lesnichin, Stepan B; Tu, Chingkuang; Silverman, David N; Tolstoy, Peter M; Denisov, Gleb S; Limbach, Hans-Heinrich

    2015-02-01

    By using a combination of liquid and solid-state NMR spectroscopy, (15) N-labeled 4-methylimidazole (4-MI) as a local probe of the environment has been studied: 1) in the polar, wet Freon CDF3 /CDF2 Cl down to 130 K, 2) in water at pH 12, and 3) in solid samples of the mutant H64A of human carbonic anhydrase II (HCA II). In the latter, the active-site His64 residue is replaced by alanine; the catalytic activity is, however, rescued by the presence of 4-MI. For the Freon solution, it is demonstrated that addition of water molecules not only catalyzes proton tautomerism but also lifts its quasidegeneracy. The possible hydrogen-bond clusters formed and the mechanism of the tautomerism are discussed. Information about the imidazole hydrogen-bond geometries is obtained by establishing a correlation between published (1) H and (15) N chemical shifts of the imidazole rings of histidines in proteins. This correlation is useful to distinguish histidines embedded in the interior of proteins and those at the surface, embedded in water. Moreover, evidence is obtained that the hydrogen-bond geometries of His64 in the active site of HCA II and of 4-MI in H64A HCA II are similar. Finally, the degeneracy of the rapid tautomerism of the neutral imidazole ring His64 reported by Shimahara et al. (J. Biol. Chem.- 2007, 282, 9646) can be explained with a wet, polar, nonaqueous active-site conformation in the inward conformation, similar to the properties of 4-MI in the Freon solution. The biological implications for the enzyme mechanism are discussed.

  3. Plasma free amino acid kinetics in rainbow trout (Oncorhynchus mykiss) using a bolus injection of 15N-labeled amino acids.

    PubMed

    Robinson, Jacob William; Yanke, Dan; Mirza, Jeff; Ballantyne, James Stuart

    2011-02-01

    To gain insight into the metabolic design of the amino acid carrier systems in fish, we injected a bolus of (15)N amino acids into the dorsal aorta in mature rainbow trout (Oncorhynchus mykiss). The plasma kinetic parameters including concentration, pool size, rate of disappearance (R(d)), half-life and turnover rate were determined for 15 amino acids. When corrected for metabolic rate, the R(d) values obtained for trout for most amino acids were largely comparable to human values, with the exception of glutamine (which was lower) and threonine (which was higher). R(d) values ranged from 0.9 μmol 100 g(-1) h(-1) (lysine) to 22.1 μmol 100 g(-1) h(-1) (threonine) with most values falling between 2 and 6 μmol 100 g(-1) h(-1). There was a significant correlation between R(d) and the molar proportion of amino acids in rainbow trout whole body protein hydrolysate. Other kinetic parameters did not correlate significantly with whole body amino acid composition. This indicates that an important design feature of the plasma-free amino acids system involves proportional delivery of amino acids to tissues for protein synthesis.

  4. Selective observation of biologically important 15N-labeled metabolites in isolated rat brain and liver by 1H-detected multiple-quantum-coherence spectroscopy

    NASA Astrophysics Data System (ADS)

    Kanamori, Keiko; Ross, Brian D.; Parivar, Farhad

    Four cerebral metabolites of importance in neurotransmission, serotonin, L-tryptophan, L-glutamine, and N-acetyl- L-aspartate, and two hepatic urea-cycle intermediates, citrulline and urea, were found to be observable by 1H- 15N heteronuclear multiple-quantum-coherence (HMQC) spectroscopy in aqueous solution at physiological pH and temperature, through the protons spin-coupled to their indole, amide, or ureido nitrogen. Their 1H chemical shifts were well dispersed over a 5-10 ppm region while the 1J 15N- 1H values were 87-99 Hz. For [γ- 15N]glutamine, a 50- to 100-fold increase in sensitivity over direct 15N detection was achieved, in contrast to a 2-fold increase by the polarization-transfer method. In the isolated brain of portacaval-shunted rats, the amide protons of biologically 15N-enriched [γ- 15N]glutamine were observed in 2 min of acquisition, with suppression of proton signals from all other cerebral metabolites. In isolated liver of 15N-enriched control rats, [ 15NIurea protons were observed in 16 min. The HMQC method is likely to be effective for the in vivo study of cerebral and hepatic nitrogen metabolism.

  5. sup 15 N and sup 13 C NMR studies of ligands bound to the 280,000-dalton protein porphobilinogen synthase elucidate the structures of enzyme-bound product and a Schiff base intermediate

    SciTech Connect

    Jaffe, E.K.; Rajagopalan, J.S. ); Markham, G.D. )

    1990-09-11

    Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). Despite the 280,000-dalton size of PBGS, much can be learned about the reaction mechanism through {sup 13}C and {sup 15}N NMR. The authors knowledge, these studies represent the largest protein complex for which individual nuclei have been characterized by {sup 13}C or {sup 15}N NMR. Here they extend their {sup 13}C NMR studies to PBGS complexes with (3,3-{sup 2}H{sub 2},3-{sup 13}C)ALA and report {sup 15}N NMR studies of ({sup 15}N)ALA bound to PBGS. As in their previous {sup 13}C NMR studies, observation of enzyme-bound {sup 15}N-labeled species was facilitated by deuteration at nitrogens that are attached to slowly exchanging hydrogens. For holo-PBGS at neutral pH, the NMR spectra reflect the structure of the enzyme-bound product porphobilinogen (PBG), whose chemical shifts are uniformly consistent with deprotonation of the amino group whose solution pK{sub a} is 11. Despite this local environment, the protons of the amino group are in rapid exchange with solvent. For methyl methanethiosulfonate (MMTS) modified PBGS, the NMR spectra reflect the chemistry of an enzyme-bound Schiff base intermediate that is formed between C{sub 4} of ALA and an active-site lysine. The {sup 13}C chemical shift of (3,3-{sup 2}H{sub 2},3-{sup 13}C)ALA confirms that the Schiff base is an imine of E stereochemistry. By comparison to model imines formed between ({sup 15}N)ALA and hydrazine or hydroxylamine, the {sup 15}N chemical shift of the enzyme-bound Schiff base suggests that the free amino group is an environment resembling partial deprotonation. Deprotonation of the amino group would facilitate formation of a Schiff base between the amino group of the enzyme-bound Schiff base and C{sub 4} of the second ALA substrate. This is the first evidence supporting carbon-nitrogen bond formation as the initial site of interaction between the two substrate molecules.

  6. Algal autolysate medium to label proteins for NMR in mammalian cells.

    PubMed

    Fuccio, Carmelo; Luchinat, Enrico; Barbieri, Letizia; Neri, Sara; Fragai, Marco

    2016-04-01

    In-cell NMR provides structural and functional information on proteins directly inside living cells. At present, the high costs of the labeled media for mammalian cells represent a limiting factor for the development of this methodology. Here we report a protocol to prepare a homemade growth medium from Spirulina platensis autolysate, suitable to express uniformly labeled proteins inside mammalian cells at a reduced cost-per-sample. The human proteins SOD1 and Mia40 were overexpressed in human cells grown in (15)N-enriched S. platensis algal-derived medium, and high quality in-cell NMR spectra were obtained. PMID:27106902

  7. Effect of 15 N Labeled Riparian Fertilization And Salmon Carcass Analog Addition On Food Web Dynamics And Productivity In Four Idaho Streams

    NASA Astrophysics Data System (ADS)

    Rugenski, A. T.; Kohler, A.; Minshall, G. W.; Danehy, R. J.; Taki, D.

    2005-05-01

    Nutrient budgets of stream/riparian ecosystems in the Intermountain West have been depleted through declining salmon populations and certain anthropogenic disturbances (e.g. forestry practices). We measured stream food web responses to a riparian fertilization and an in-stream carcass analog addition in 4 Idaho streams with a 15 N tracer. Aerial application of fertilizer pellets to light (224 kg/ha) and heavy (448 kg/ha) treatment sections of 2 streams and carcass analog additions to 2 others were completed in autumn. Periphyton response was measured through chlorophyll a, nutrient diffusing substrata, and stable isotope analyses. Macroinvertebrates were analyzed for abundance, biomass, community structure, and stable isotope composition. Also, willow (Salix) breakdown rates were determined. Pre-treatment chlorophyll a values showed no significant difference between treatment and reference reaches. Post-treatment results showed significantly higher chlorophyll a and δ 15 N values in treatment reaches compared to reference reaches. Macroinvertebrate abundance, richness, biomass, and δ 15 N values also increased in treated reaches. No significant differences were detected in leaf breakdown rates between reaches. Riparian fertilization effects were longer lasting than the in-stream treatment. These results suggest that nutrient addition to streams and riparian areas can be used as a management tool to increase stream productivity where nutrients are limiting.

  8. Labeling strategies for 13C-detected aligned-sample solid-state NMR of proteins

    NASA Astrophysics Data System (ADS)

    Filipp, Fabian V.; Sinha, Neeraj; Jairam, Lena; Bradley, Joel; Opella, Stanley J.

    2009-12-01

    13C-detected solid-state NMR experiments have substantially higher sensitivity than the corresponding 15N-detected experiments on stationary, aligned samples of isotopically labeled proteins. Several methods for tailoring the isotopic labeling are described that result in spatially isolated 13C sites so that dipole-dipole couplings among the 13C are minimized, thus eliminating the need for homonuclear 13C- 13C decoupling in either indirect or direct dimensions of one- or multi-dimensional NMR experiments that employ 13C detection. The optimal percentage for random fractional 13C labeling is between 25% and 35%. Specifically labeled glycerol and glucose can be used at the carbon sources to tailor the isotopic labeling, and the choice depends on the resonances of interest for a particular study. For investigations of the protein backbone, growth of the bacteria on [2- 13C]-glucose-containing media was found to be most effective.

  9. Quantification of soy protein using the isotope method (δ(13)C and δ(15)N) for commercial brands of beef hamburger.

    PubMed

    Ducatti, Rhani; de Almeida Nogueira Pinto, José Paes; Sartori, Maria Márcia Pereira; Ducatti, Carlos

    2016-12-01

    Hamburgers (beef patties) may be adulterated through the overuse of protein extenders. Among vegetables, soy protein is the best substitute for animal protein. These ingredients help to reduce the cost of producing a final product, and they maximize profits for fraudulent industries. Moreover, the ingestion of soy or other non-meat proteins by allergic individuals may present a health risk. In addition, monitoring by supervisory bodies is hampered by a lack of appropriate analytical methodologies. Within this context, the aim of this study was to determine and quantify the levels of added soy protein by determination of (15)N and (13)C stable isotopes. A total of 100 beef hamburger samples from 10 commercial brands were analyzed. Only three samples of the G brand were within the standards set the Brazilian legislation. The remaining 97 samples from 10 commercial brands contained >4% soy protein; therefore, they are adulterated and not in compliance with the current legislation. PMID:27501234

  10. Computer assignment of the backbone resonances of labelled proteins using two-dimensional correlation experiments.

    PubMed

    Morelle, N; Brutscher, B; Simorre, J P; Marion, D

    1995-02-01

    We present ALPS (Assignment for Labelled Protein Spectra), a flexible computer program for the automatic assignment of backbone NMR resonances of (15)N/(13)C-labelled proteins. The program constructs pseudoresidues from peak-picking lists of a set of two-dimensional triple resonance experiments and uses either a systematic search or a simulated annealing-based optimization to perform the assignment. This method has been successfully tested on two-dimensional triple resonance spectra of Rhodobacter capsulatus ferrocytochrome c (2) (116 amino acids).

  11. Investigation of the backbone dynamics of the IgG-binding domain of streptococcal protein G by heteronuclear two-dimensional 1H-15N nuclear magnetic resonance spectroscopy.

    PubMed

    Barchi, J J; Grasberger, B; Gronenborn, A M; Clore, G M

    1994-01-01

    The backbone dynamics of the immunoglobulin-binding domain (B1) of streptococcal protein G, uniformly labeled with 15N, have been investigated by two-dimensional inverse detected heteronuclear 1H-15N NMR spectroscopy at 500 and 600 MHz. 15N T1, T2, and nuclear Overhauser enhancement data were obtained for all 55 backbone NH vectors of the B1 domain at both field strengths. The overall correlation time obtained from an analysis of the T1/T2 ratios was 3.3 ns at 26 degrees C. Overall, the B1 domain is a relatively rigid protein, consistent with the fact that over 95% of the residues participate in secondary structure, comprising a four-stranded sheet arranged in a -1, +3x, -1 topology, on top of which lies a single helix. Residues in the turns and loops connecting the elements of secondary structure tend to exhibit a higher degree of mobility on the picosecond time scale, as manifested by lower values of the overall order parameter. A number of residues at the ends of the secondary structure elements display two distinct internal motions that are faster than the overall rotational correlation time: one is fast (< 20 ps) and lies in the extreme narrowing limit, whereas the other is one to two orders of magnitude slower (1-3 ns) and lies outside the extreme narrowing limit. The slower motion can be explained by large-amplitude (20-40 degrees) jumps in the N-H vectors between states with well-defined orientations that are stabilized by hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Measurement of 13C and 15N isotope labeling by gas chromatography/combustion/isotope ratio mass spectrometry to study amino acid fluxes in a plant-microbe symbiotic association.

    PubMed

    Molero, Gemma; Aranjuelo, Iker; Teixidor, Pilar; Araus, José Luis; Nogués, Salvador

    2011-03-15

    We have developed a method based on a double labeling with stable isotopes and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses to study amino acid exchange in a symbiotic plant-microbe association. Isotopic precision was studied for 21 standards including 15 amino acid derivatives, three N-protected amino acid methyl esters, three amines and one international standard. High correlations were observed between the δ(13)C and δ(15)N values obtained by GC/C/IRMS and those obtained by an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (R(2) = 0.9868 and 0.9992, respectively). The mean precision measured was 0.04‰ for δ(13)C and 0.28‰ for δ(15)N (n = 15). This method was applied in vivo to the symbiotic relationship between alfalfa (Medicago sativa L.) and N(2)-fixing bacteria. Plants were simultaneously labeled over 10 days with (13)C-depleted CO(2) ((12)CO(2)), which was assimilated through photosynthesis by leaves, and (15)N(2) fixed via nodules. Subsequently, the C and N isotope compositions (i.e. δ(13)C and δ(15)N) of free amino acids were analyzed in leaves and nodules by GC/C/IRMS. The method revealed the pattern of C and N exchange between leaves and nodules, highlighting that γ-aminobutanoic acid and glycine may represent an important form of C transport from leaves to the nodules. The results confirmed the validity, reliability and accuracy of the method for assessing C and N fluxes between plants and symbiotic bacteria and support the use of this technique in a broad range of metabolic and fluxomic studies.

  13. Trace fluorescent labeling for protein crystallization

    PubMed Central

    Pusey, Marc; Barcena, Jorge; Morris, Michelle; Singhal, Anuj; Yuan, Qunying; Ng, Joseph

    2015-01-01

    Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent. PMID:26144224

  14. The fates of 15N-labeled fertilizer in a wheat–soil system as influenced by fertilization practice in a loamy soil

    NASA Astrophysics Data System (ADS)

    Chen, Zhaoming; Wang, Huoyan; Liu, Xiaowei; Lu, Dianjun; Zhou, Jianmin

    2016-10-01

    Appropriate fertilization practice is crucial to achieve maximum wheat grain yield with minimum nitrogen (N) loss. A field 15N micro-plot experiment was conducted to determine the effects of application methods [split application (SA) and band application (BA)] and N rates (60, 150 and 240 kg ha‑1) on the wheat grain yield, urea-15N fate and N efficiency in Jiangyan County, China. At high N rates, wheat grain yield was significantly higher for SA than BA treatment, but there was no difference at the lower N rates. Plant N derived from fertilizer was higher in SA than in BA treatment. The high N fertilizer application increased total N uptake by wheat derived from fertilizer, but wheat plant N derived from soil was not affected by the N rate. Fertilizer-N recovery in SA treatment was higher than in BA treatment. Residual N recovery in the 0–80 cm soil layer was 31–51%, which decreased with increasing N rate. The highest N loss was found for BA treatment at the N application of 240 kg ha‑1. The one-time BA of N fertilizer, especially for higher N rates, led to reduced wheat grain yield and N efficiency, and increased the N loss.

  15. The fates of 15N-labeled fertilizer in a wheat–soil system as influenced by fertilization practice in a loamy soil

    PubMed Central

    Chen, Zhaoming; Wang, Huoyan; Liu, Xiaowei; Lu, Dianjun; Zhou, Jianmin

    2016-01-01

    Appropriate fertilization practice is crucial to achieve maximum wheat grain yield with minimum nitrogen (N) loss. A field 15N micro-plot experiment was conducted to determine the effects of application methods [split application (SA) and band application (BA)] and N rates (60, 150 and 240 kg ha−1) on the wheat grain yield, urea-15N fate and N efficiency in Jiangyan County, China. At high N rates, wheat grain yield was significantly higher for SA than BA treatment, but there was no difference at the lower N rates. Plant N derived from fertilizer was higher in SA than in BA treatment. The high N fertilizer application increased total N uptake by wheat derived from fertilizer, but wheat plant N derived from soil was not affected by the N rate. Fertilizer-N recovery in SA treatment was higher than in BA treatment. Residual N recovery in the 0–80 cm soil layer was 31–51%, which decreased with increasing N rate. The highest N loss was found for BA treatment at the N application of 240 kg ha−1. The one-time BA of N fertilizer, especially for higher N rates, led to reduced wheat grain yield and N efficiency, and increased the N loss. PMID:27713476

  16. REDOR NMR of stable-isotope-labeled protein binding sites

    SciTech Connect

    Schaefer, J.

    1994-12-01

    Rotational-echo, double resonance (REDOR) NMR, a new analytical spectroscopic technique for solids spinning at the magic angle, has been developed over the last 5 years. REDOR provides a direct measure of heteronuclear dipolar coupling between isolated pairs of labeled nuclei. In a solid with a {sup 13}C-{sup 15}N labeled pair, for example, the {sup 13}C rotational echoes that form each rotor period following a{sup 1}H-{sup 13}C cross-polarization transfer can be prevented from reaching full intensity by insertion of a {sup 15}N {pi} pulse each half rotor period. The REDOR difference (the difference between a {sup 13}C NMR spectrum obtained under these conditions and one obtained with no {sup 15}N {pi} pulses) has a strong dependence on the {sup 13}C-{sup 15}N dipolar coupling, and hence, the {sup 13}C-{sup 15}N internuclear distance. REDOR is described as double-resonance even though three radio frequencies (typically {sup 1}H, {sup 13}C, and {sup 15}N) are used because the protons are removed from the important evolution part of the experiment by resonant decoupling. The dephasing of magnetization in REDOR arises from a local dipolar {sup 13}C-{sup 15}N field gradient and involves no polarization transfer. REDOR has no dependence on {sup 13}C or {sup 15}N chemical-shift tensors and does not require resolution of a {sup 13}C-{sup 15}N coupling in the chemical-shift dimension.

  17. 1H, 13C, and 15N resonance assignments for Escherichia coli ytfP, a member of the broadly conserved UPF0131 protein domain family

    SciTech Connect

    Aramini, James M.; Swapna, G.V.T.; Huang, Yuanpeng; Rajan, Paranji K.; Xiao, Rong; Shastry, Ritu; Acton, Thomas; Cort, John R.; Kennedy, Michael A.; Montelione, Gaetano

    2005-11-01

    Protein ytfP from Escherichia coli (Swiss-Prot ID: YTFP-ECOLI; NESG target ID: ER111; Wunderlich et al., 2004) is a 113-residue member of the UPF0131 protein family (Pfam ID: PF03674) of unknown function. This domain family is found in organisms from all three kingdoms, archaea, eubacteria and eukaryotes. Using triple resonance NMR techniques, we have determined 97% of backbone and 91% of side chain 1H, 13C, and 15N resonance assignments. The chemical shift and 3J(HN?Ha) scalar coupling data reveal a mixed a/b topology,????????. BMRB deposit with Accession No. 6448. Reference: Wunderlich et al. (2004) Proteins, 56, 181?187.

  18. 1H, 13C, and 15N resonance assignments for the protein coded by gene locus BB0938 of Bordetella bronchiseptica

    SciTech Connect

    Rossi, Paolo; Ramelot, Theresa A.; Xiao, Rong; Ho, Chi K.; Ma, LiChung; Acton, Thomas; Kennedy, Michael A.; Montelione, Gaetano

    2005-11-01

    The product of gene locus BB0938 from Bordetella bronchiseptica (Swiss-Prot ID: Q7WNU7-BORBR; NESG target ID: BoR11; Wunderlich et al., 2004; Pfam ID: PF03476) is a 128-residue protein of unknown function. This broadly conserved protein family is found in eubacteria and eukaryotes. Using triple resonance NMR techniques, we have determined 98% of backbone and 94% of side chain 1H, 13C, and 15N resonance assignments. The chemical shift and 3J(HN?Ha) scalar coupling data reveal a b topology with a seven-residue helical insert, ??????????. BMRB deposit with accession number 6693. Reference: Wunderlich et al. (2004) Proteins, 56, 181?187.

  19. RFP tags for labeling secretory pathway proteins

    SciTech Connect

    Han, Liyang; Zhao, Yanhua; Xu, Pingyong; Huan, Shuangyan

    2014-05-09

    Highlights: • Membrane protein Orai1 can be used to report the fusion properties of RFPs. • Artificial puncta are affected by dissociation constant as well as pKa of RFPs. • Among tested RFPs mOrange2 is the best choice for secretory protein labeling. - Abstract: Red fluorescent proteins (RFPs) are useful tools for live cell and multi-color imaging in biological studies. However, when labeling proteins in secretory pathway, many RFPs are prone to form artificial puncta, which may severely impede their further uses. Here we report a fast and easy method to evaluate RFPs fusion properties by attaching RFPs to an environment sensitive membrane protein Orai1. In addition, we revealed that intracellular artificial puncta are actually colocalized with lysosome, thus besides monomeric properties, pKa value of RFPs is also a key factor for forming intracellular artificial puncta. In summary, our current study provides a useful guide for choosing appropriate RFP for labeling secretory membrane proteins. Among RFPs tested, mOrange2 is highly recommended based on excellent monomeric property, appropriate pKa and high brightness.

  20. Grass species influence on plant N uptake - Determination of atmospheric N deposition to a semi-natural peat bog site using a 15N labelling approach

    NASA Astrophysics Data System (ADS)

    Hurkuck, Miriam; Brümmer, Christian; Spott, Oliver; Flessa, Heinz; Kutsch, Werner L.

    2014-05-01

    Large areas of natural peat bogs in Northwestern Germany have been converted to arable land and were subjected to draining and peat cutting in the past. The few protected peatland areas remaining are affected by high nitrogen (N) deposition. Our study site - a moderately drained raised bog - is surrounded by highly fertilized agricultural land and livestock production. In this study, we used a 15N pool dilution technique called 'Integrated Total Nitrogen Input' (ITNI) to quantify annual deposition of atmospheric N into biomonitoring pots over a two-year period. Since it considers direct N uptake by plants, it was expected to result in higher N input than conventional methods for determination of N deposition (e.g. micrometeorological approaches, bulk N samplers). Using Lolium multiflorum and Eriophorum vaginatum as monitor plants and low, medium and high levels of fertilization, we aimed to simulate increasing N deposition to planted pots and to allocate airborne N after its uptake by the soil-plant system in aboveground biomass, roots and soil. Increasing N fertilization was positively correlated with biomass production of Eriophorum vaginatum, whereas atmospheric plant N uptake decreased and highest airborne N input of 899.8 ± 67.4 µg N d-1 pot-1 was found for low N fertilization. In contrast, Lolium multiflorum showed a clear dependency of N supply on plant N uptake and was highest (688.7 ± 41.4 µg N d-1 pot-1) for highly fertilized vegetation pots. Our results suggest that grass species respond differently to increasing N input. While crop grasses such as Lolium multiflorum take up N according to N availability, species adopted to nutrient-limited conditions like Eriophorum vaginatum show N saturation effects with increasing N supply. Total airborne N input ranged from about 24 to 66 kg N ha-1 yr-1 dependent on the used indicator plant and the amount of added fertilizer. Parallel determination of atmospheric N deposition using a micrometeorological approach

  1. Simultaneous Site-Specific Dual Protein Labeling Using Protein Prenyltransferases.

    PubMed

    Zhang, Yi; Blanden, Melanie J; Sudheer, Ch; Gangopadhyay, Soumyashree A; Rashidian, Mohammad; Hougland, James L; Distefano, Mark D

    2015-12-16

    Site-specific protein labeling is an important technique in protein chemistry and is used for diverse applications ranging from creating protein conjugates to protein immobilization. Enzymatic reactions, including protein prenylation, have been widely exploited as methods to accomplish site-specific labeling. Enzymatic prenylation is catalyzed by prenyltransferases, including protein farnesyltransferase (PFTase) and geranylgeranyltransferase type I (GGTase-I), both of which recognize C-terminal CaaX motifs with different specificities and transfer prenyl groups from isoprenoid diphosphates to their respective target proteins. A number of isoprenoid analogues containing bioorthogonal functional groups have been used to label proteins of interest via PFTase-catalyzed reaction. In this study, we sought to expand the scope of prenyltransferase-mediated protein labeling by exploring the utility of rat GGTase-I (rGGTase-I). First, the isoprenoid specificity of rGGTase-I was evaluated by screening eight different analogues and it was found that those with bulky moieties and longer backbone length were recognized by rGGTase-I more efficiently. Taking advantage of the different substrate specificities of rat PFTase (rPFTase) and rGGTase-I, we then developed a simultaneous dual labeling method to selectively label two different proteins by using isoprenoid analogue and CaaX substrate pairs that were specific to only one of the prenyltransferases. Using two model proteins, green fluorescent protein with a C-terminal CVLL sequence (GFP-CVLL) and red fluorescent protein with a C-terminal CVIA sequence (RFP-CVIA), we demonstrated that when incubated together with both prenyltransferases and the selected isoprenoid analogues, GFP-CVLL was specifically modified with a ketone-functionalized analogue by rGGTase-I and RFP-CVIA was selectively labeled with an alkyne-containing analogue by rPFTase. By switching the ketone-containing analogue to an azide-containing analogue, it was

  2. Trace fluorescent labeling for protein crystallization

    SciTech Connect

    Pusey, Marc Barcena, Jorge; Morris, Michelle; Singhal, Anuj; Yuan, Qunying; Ng, Joseph

    2015-06-27

    The presence of a covalently bound fluorescent probe at a concentration of <0.5% does not affect the outcome of macromolecule crystallization screening experiments. Additionally, the fluorescence can be used to determine new, not immediately apparent, lead crystallization conditions. Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent.

  3. Chemoenzymatic Labeling of Proteins: Techniques and Approaches

    PubMed Central

    Rashidian, Mohammad; Dozier, Jonathan K.; Distefano, Mark D.

    2013-01-01

    Site-specific modification of proteins is a major challenge in modern chemical biology due to the large number of reactive functional groups typically present in polypeptides. Because of its importance in biology and medicine, the development of methods for site-specific modification of proteins is an area of intense research. Selective protein modification procedures have been useful for oriented protein immobilization, for studies of naturally-occurring post-translational modifications, for creating antibody-drug conjugates, for the introduction of fluorophores and other small molecules on to proteins, for examining protein structure, folding, dynamics and protein-protein interactions and for the preparation of protein-polymer conjugates. One of the most important approaches for protein labeling is to incorporate bioorthogonal functionalities into proteins at specific sites via enzymatic reactions. The incorporated tags then enable reactions that are chemoselective, whose functional groups are not only inert in biological media, but also do not occur natively in proteins or other macromolecules. This review article summarizes the enzymatic strategies, which enable site-specific functionalization of proteins with a variety of different functional groups. The enzymes covered in this review include formylglycine generating enzyme, sialyltransferases, phosphopantetheinyltransferases, O-GlcNAc post-translational modification, sortagging, transglutaminase, farnesyltransferase, biotin ligase, lipoic acid ligase and N-myristoyl transferase. PMID:23837885

  4. Isotopic labeling of mammalian G protein-coupled receptors (GPCRs) heterologously expressed in Caenorhabditis elegans*

    PubMed Central

    Salom, David; Cao, Pengxiu; Yuan, Yiyuan; Miyagi, Masaru; Feng, Zhaoyang; Palczewski, Krzysztof

    2015-01-01

    High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack posttranslational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with 15N,13C by providing them with isotopically labeled bacteria. 2H labeling also was achieved by growing C. elegans in presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the ‘test’ GPCR to demonstrate the viability of this approach. Although the worms’ cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization. PMID:25461480

  5. 15N-15N Proton Assisted Recoupling in Magic Angle Spinning NMR

    PubMed Central

    Lewandowski, Józef R.; De Paëpe, Gaël; Eddy, Matthew T.; Griffin, Robert G.

    2009-01-01

    We describe a new magic angle spinning (MAS) NMR experiment for obtaining 15N-15N correlation spectra. The approach yields direct information about the secondary and tertiary structure of proteins, including identification of α-helical stretches and inter-strand connectivity in antiparallel β-sheets, which are of major interest for structural studies of membrane proteins and amyloid fibrils. The method, 15N-15N proton assisted recoupling (PAR), relies on a second order mechanism, third spin assisted recoupling (TSAR), used previously in the context of 15N-13C and 13C-13C polarization transfer schemes. In comparison to 15N-15N proton driven spin diffusion experiments, the PAR technique accelerates polarization transfer between 15N’s by a factor of ~102−103, and is furthermore applicable over the entire range of currently available MAS frequencies (10–70 kHz). PMID:19334788

  6. Selectively Labeling the Heterologous Protein in Escherichia coli for NMR Studies: A Strategy to Speed Up NMR Spectroscopy

    NASA Astrophysics Data System (ADS)

    Almeida, F. C. L.; Amorim, G. C.; Moreau, V. H.; Sousa, V. O.; Creazola, A. T.; Américo, T. A.; Pais, A. P. N.; Leite, A.; Netto, L. E. S.; Giordano, R. J.; Valente, A. P.

    2001-01-01

    Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The 1H/15N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective 15N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good 1H/15N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The 15N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the 1H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.

  7. (1)H, (15)N and (13)C resonance assignments of the conserved region in the middle domain of S. pombe Sin1 protein.

    PubMed

    Kataoka, Saori; Furuita, Kyoko; Hattori, Yoshikazu; Kobayashi, Naohiro; Ikegami, Takahisa; Shiozaki, Kazuhiro; Fujiwara, Toshimichi; Kojima, Chojiro

    2015-04-01

    SAPK-interacting protein 1 (Sin1) is an important component of the target of rapamycin (TOR) complex 2 (TORC2). TOR is a serine/threonine-specific protein kinase and forms functionally distinct protein complexes referred to as TORC1 and TORC2. TORC2, conserved from yeast to humans, phosphorylates AGC-family protein kinases and has many cellular functions including the regulation of actin cytoskeleton. The Sin1 subunit of TORC2 is required for the binding of TORC2 to substrates, and the conserved region in the middle (CRIM) domain of Sin1 is important in the substrate recognition of TORC2. Here, we report on the (1)H, (13)C and (15)N resonance assignments of fission yeast Schizosaccharomyces pombe Sin1 (amino acids 247-400) (Sin1CRIM), which possesses the CRIM domain. These data contribute toward the structure determination of Sin1CRIM and an understanding of the interactions of Sin1CRIM with substrates of TORC2.

  8. Time-shared experiments for efficient assignment of triple-selectively labeled proteins.

    PubMed

    Löhr, Frank; Laguerre, Aisha; Bock, Christoph; Reckel, Sina; Connolly, Peter J; Abdul-Manan, Norzehan; Tumulka, Franz; Abele, Rupert; Moore, Jonathan M; Dötsch, Volker

    2014-11-01

    Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially. Sign encoding by the phases of (13)C 90° pulses allows to either select or discriminate against (13)C' or (13)C(α) spins coupled to (15)N. As a result, (1)H-(15)N correlation maps of the various isotopomeric species present in triple-selectively labeled proteins are deconvoluted which in turn reduces problems due to spectral overlap. The new methods are demonstrated with four different membrane proteins with rotational correlation times ranging from 18 to 52 ns.

  9. Time-shared experiments for efficient assignment of triple-selectively labeled proteins

    PubMed Central

    Löhr, Frank; Laguerre, Aisha; Bock, Christoph; Reckel, Sina; Connolly, Peter J.; Abdul-Manan, Norzehan; Tumulka, Franz; Abele, Rupert; Moore, Jonathan M.; Dötsch, Volker

    2014-01-01

    Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially. Sign encoding by the phases of 13C 90° pulses allows to either select or discriminate against 13C’ or 13Cα spins coupled to 15N. As a result, 1H-15N correlation maps of the various isotopomeric species present in triple-selectively labeled proteins are deconvoluted which in turn reduces problems due to spectral overlap. The new methods are demonstrated with four different membrane proteins with rotational correlation times ranging from 18 to 52 ns. PMID:25442777

  10. Method for the Determination of ¹⁵N Incorporation Percentage in Labeled Peptides and Proteins.

    PubMed

    Kilpatrick, Eric L

    2016-01-01

    Use of labeled (15)N proteins and peptides as internal standards in isotope-dilution mass spectrometry for the quantification of proteins has been increasing and is now accepted as a gold standard for this analysis. As a necessary reagent in this process, stable heavy isotope-labeled internal standards must be rigorously characterized in a number of ways including identity, concentration, purity, and structure. Additionally, the degree of the incorporation of the heavy isotope is a critical feature to consider. For proteins that are (15)N labeled, the percentage of incorporation is a valid measurement used to assess the fitness-to-purpose of the material. This measurement should be objective, repeatable, and based on empirical analysis. One means of assigning this value is to compare a mass spectrum of the isotopic profile of a peptide against a series of theoretical profiles containing different enrichment rates. This comparison can be made using the Pearson product-moment correlation coefficient (r) to find the best match between the empirical and theoretical profiles. Theoretical profiles can be generated using probability multinomial analysis but are computationally intensive and require the use of computers for practical use. The method described in this chapter describes the development and use of a computer program to calculate the percentage of (15)N enrichment of a labeled internal standard. Additionally, methods will be described for the empirical determination of an isotopic profile using a variety of mass spectrometry techniques.

  11. Production of selenomethionine-labeled proteins in two-liter plastic bottles for structure determination.

    SciTech Connect

    Stols, L.; Sanville Millard, C.; Dementieva, I.; Donnelly, M. I.

    2004-03-01

    A simplified approach developed recently for the production of heterologous proteins in Escherichia coli uses 2-liter polyethylene terephthalate beverage bottles as disposable culture vessels [Sanville Millard, C. et al. 2003. Protein Expr. Purif. 29, 311-320]. The method greatly reduces the time and effort needed to produce native proteins for structural or functional studies. We now demonstrate that the approach is also well suited for production of proteins in defined media with incorporation of selenomethionine to facilitate structure determination by multiwavelength anomalous diffraction. Induction of a random set of Bacillus stearothermophilus target genes under the new protocols generated soluble selenomethionyl proteins in good yield. Several selenomethionyl proteins were purified in good yields and three were subjected to amino acid analysis. Incorporation of selenomethionine was determined to be greater than 95% in one protein and greater than 98% in the other two. In the preceding paper [Zhao et al., this issue, pp. 87-93], the approach is further extended to production of [U-15N]- or [U-13C, U-15N]-labeled proteins. The approach thus appears suitable for high-throughput production of proteins for structure determination by X-ray crystallography or nuclear magnetic resonance spectroscopy.

  12. Combined ligand-observe 19F and protein-observe 15N,1H-HSQC NMR suggests phenylalanine as the key Δ-somatostatin residue recognized by human protein disulfide isomerase

    PubMed Central

    Richards, Kirsty L.; Rowe, Michelle L.; Hudson, Paul B.; Williamson, Richard A.; Howard, Mark J.

    2016-01-01

    Human protein disulphide isomerase (hPDI) is an endoplasmic reticulum (ER) based isomerase and folding chaperone. Molecular detail of ligand recognition and specificity of hPDI are poorly understood despite the importance of the hPDI for folding secreted proteins and its implication in diseases including cancer and lateral sclerosis. We report a detailed study of specificity, interaction and dissociation constants (Kd) of the peptide-ligand Δ-somatostatin (AGSKNFFWKTFTSS) binding to hPDI using 19F ligand-observe and 15N,1H-HSQC protein-observe NMR methods. Phe residues in Δ-somatostatin are hypothesised as important for recognition by hPDI therefore, step-wise peptide Phe-to-Ala changes were progressively introduced and shown to raise the Kd from 103 + 47 μM until the point where binding was abolished when all Phe residues were modified to Ala. The largest step-changes in Kd involved the F11A peptide modification which implies the C-terminus of Δ-somatostatin is a prime recognition region. Furthermore, this study also validated the combined use of 19F ligand-observe and complimentary 15N,1H-HSQC titrations to monitor interactions from the protein’s perspective. 19F ligand-observe NMR was ratified as mirroring 15N protein-observe but highlighted the advantage that 19F offers improved Kd precision due to higher spectrum resolution and greater chemical environment sensitivity. PMID:26786784

  13. Efficient isotopic tryptophan labeling of membrane proteins by an indole controlled process conduct.

    PubMed

    Berger, Christian; Berndt, Sandra; Pichert, Annelie; Theisgen, Stephan; Huster, Daniel

    2013-06-01

    A protocol for the efficient isotopic labeling of large G protein-coupled receptors with tryptophan in Escherichia coli as expression host was developed that sufficiently suppressed the naturally occurring L-tryptophan indole lyase, which cleaves tryptophan into indole, pyruvate, and ammonia resulting in scrambling of the isotopic label in the protein. Indole produced by the tryptophanase is naturally used as messenger for cell-cell communication. Detailed analysis of different process conducts led to the optimal expression strategy, which mimicked cell-cell communication by the addition of indole during expression. Discrete concentrations of indole and (15) N2 -L-tryptophan at dedicated time points in the fermentation drastically increased the isotopic labeling efficiency. Isotope scrambling was only observed in glutamine, asparagine, and arginine side chains but not in the backbone. This strategy allows producing specifically tryptophan labeled membrane proteins at high concentrations avoiding the disadvantages of the often low yields of auxotrophic E. coli strains. In the fermentation process carried out according to this protocol, we produced ∼15 mg of tryptophan labeled neuropeptide Y receptor type 2 per liter medium.

  14. SIMS ion microscopy imaging of boronophenylalanine (BPA) and 13C15N-labeled phenylalanine in human glioblastoma cells: Relevance of subcellular scale observations to BPA-mediated boron neutron capture therapy of cancer

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash; Lorey, Daniel R., II

    2007-02-01

    p-Boronophenylalanine (BPA) is a clinically approved boron neutron capture therapy (BNCT) agent currently being used in clinical trials of glioblastoma multiforme, melanoma and liver metastases. Secondary ion mass spectrometry (SIMS) observations from the Cornell SIMS Laboratory provided support for using a 6 h infusion of BPA, instead of a 2 h infusion, for achieving higher levels of boron in brain tumor cells. These observations were clinically implemented in Phase II experimental trials of glioblastoma multiforme in Sweden. However, the mechanisms for higher BPA accumulation with longer infusions have remained unknown. In this work, by using 13C15N-labeled phenylalanine and T98G human glioblastoma cells, comparisons between the 10B-delivery of BPA and the accumulation of labeled phenylalanine after 2 and 6 h treatments were made with a Cameca IMS-3f SIMS ion microscope at 500 nm spatial resolution in fast frozen, freeze-fractured, freeze-dried cells. Due to the presence of the Na-K-ATPase in the plasma membrane of most mammalian cells, the cells maintain an approximately 10/1 ratio of K/Na in the intracellular milieu. Therefore, the quantitative imaging of these highly diffusible species in the identical cell in which the boron or labeled amino acid was imaged provides a rule-of-thumb criterion for validation of SIMS observations and the reliability of the cryogenic sampling. The labeled phenylalanine was detected at mass 28, as the 28(13C15N)- molecular ion. Correlative analysis with optical and confocal laser scanning microscopy revealed that fractured freeze-dried glioblastoma cells contained well-preserved ultrastructural details with three discernible subcellular regions: a nucleus or multiple nuclei, a mitochondria-rich perinuclear cytoplasmic region and the remaining cytoplasm. SIMS analysis revealed that the overall cellular signals of both 10B from BPA and 28CN- from labeled phenylalanine increased approximately 1.6-fold between the 2 and 6 h exposures

  15. Improved protein labeling by stannous tartrate reduction of pertechnetate

    SciTech Connect

    Pettit, W.A.; DeLand, F.H.; Bennett, S.J.; Goldenberg, D.M.

    1980-01-01

    A procedure has been developed whereby small amounts of protein - specifically human serum albumin and immunoglobulin G - can be labeled with Tc-99m. Artifactual problems associated with electrolytic and stannous chloride labeling procedures are virtually eliminated. The procedure is satisfactory for labeling human serum albumin, normal goat immunoglobulin G, and goat anti-carcinoembryonic antigen immunoglobulin G.

  16. Strategies for labeling proteins with PARACEST agents

    PubMed Central

    Vasalatiy, Olga; Zhao, Piyu; Woods, Mark; Marconescu, Andrei; Castillo-Muzquiz, Aminta; Thorpe, Philip; Kiefer, Garry E.; Sherry, A. Dean

    2011-01-01

    Reactive surface lysine groups on the chimeric monoclonal antibody (3G4) and on human serum albumin (HSA) were labeled with two different PARACEST chelates. Between 7.4 – 10.1 chelates were added per 3G4 molecule and between 5.6 – 5.9 chelates per molecule of HSA, depending upon which conjugation chemistry was used. The immunoreactivity of 3G4 as measured by ELISA assays was highly dependent upon the number of attached chelates: 88% immunoreactivity with 7.4 chelates per antibody versus only 17% immunoreactivity with 10.1 chelates per antibody. Upon conjugation to 3G4, the bound water lifetime of Eu-1 increased only marginally, up from 53 μs for the non-conjugated chelate to 65–77 μs for conjugated chelates. Conjugation of a chelate Eu-2 to HSA via a single side-chain group also resulted in little or no change in bound water lifetime (73–75 μs for both the conjugated and non-conjugated forms). These data indicate that exchange of water molecules protons between the inner-sphere site on covalently attached PARACEST agent and bulk water is largely unaffected by the mode of attachment of the agent to the protein and likely its chemical surroundings on the surface of the protein. PMID:20621494

  17. Photoactivatable protein labeling by singlet oxygen mediated reactions.

    PubMed

    To, Tsz-Leung; Medzihradszky, Katalin F; Burlingame, Alma L; DeGrado, William F; Jo, Hyunil; Shu, Xiaokun

    2016-07-15

    Protein-protein interactions regulate many biological processes. Identification of interacting proteins is thus an important step toward molecular understanding of cell signaling. The aim of this study was to investigate the use of photo-generated singlet oxygen and a small molecule for proximity labeling of interacting proteins in cellular environment. The protein of interest (POI) was fused with a small singlet oxygen photosensitizer (miniSOG), which generates singlet oxygen ((1)O2) upon irradiation. The locally generated singlet oxygen then activated a biotin-conjugated thiol molecule to form a covalent bond with the proteins nearby. The labeled proteins can then be separated and subsequently identified by mass spectrometry. To demonstrate the applicability of this labeling technology, we fused the miniSOG to Skp2, an F-box protein of the SCF ubiquitin ligase, and expressed the fusion protein in mammalian cells and identified that the surface cysteine of its interacting partner Skp1 was labeled by the biotin-thiol molecule. This photoactivatable protein labeling method may find important applications including identification of weak and transient protein-protein interactions in the native cellular context, as well as spatial and temporal control of protein labeling.

  18. Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling1[W][OPEN

    PubMed Central

    Nelson, Clark J.; Alexova, Ralitza; Jacoby, Richard P.; Millar, A. Harvey

    2014-01-01

    Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used 15N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of 15N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of 14N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until 15N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that 15N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis. PMID:25082890

  19. Engineering Protein Farnesyltransferase for Enzymatic Protein Labeling Applications

    PubMed Central

    2015-01-01

    Creating covalent protein conjugates is an active area of research due to the wide range of uses for protein conjugates spanning everything from biological studies to protein therapeutics. Protein Farnesyltransferase (PFTase) has been used for the creation of site-specific protein conjugates, and a number of PFTase substrates have been developed to facilitate that work. PFTase is an effective catalyst for protein modification because it transfers Farnesyl diphosphate (FPP) analogues to protein substrates on a cysteine four residues from the C-terminus. While much work has been done to synthesize various FPP analogues, there are few reports investigating how mutations in PFTase alter the kinetics with these unnatural analogues. Herein we examined how different mutations within the PFTase active site alter the kinetics of the PFTase reaction with a series of large FPP analogues. We found that mutating either a single tryptophan or tyrosine residue to alanine results in greatly improved catalytic parameters, particularly in kcat. Mutation of tryptophan 102β to alanine caused a 4-fold increase in kcat and a 10-fold decrease in KM for a benzaldehyde-containing FPP analogue resulting in an overall 40-fold increase in catalytic efficiency. Similarly, mutation of tyrosine 205β to alanine caused a 25-fold increase in kcat and a 10-fold decrease in KM for a coumarin-containing analogue leading to a 300-fold increase in catalytic efficiency. Smaller but significant changes in catalytic parameters were also obtained for cyclo-octene- and NBD-containing FPP analogues. The latter compound was used to create a fluorescently labeled form of Ciliary Neurotrophic Factor (CNTF), a protein of therapeutic importance. Additionally, computational modeling was performed to study how the large non-natural isoprenoid analogues can fit into the active sites enlarged via mutagenesis. Overall, these results demonstrate that PFTase can be improved via mutagenesis in ways that will be useful

  20. Identification of ejaculated proteins in the house mouse (Mus domesticus) via isotopic labeling

    PubMed Central

    2011-01-01

    Background Seminal fluid plays an important role in successful fertilization, but knowledge of the full suite of proteins transferred from males to females during copulation is incomplete. The list of ejaculated proteins remains particularly scant in one of the best-studied mammalian systems, the house mouse (Mus domesticus), where artificial ejaculation techniques have proven inadequate. Here we investigate an alternative method for identifying ejaculated proteins, by isotopically labeling females with 15N and then mating them to unlabeled, vasectomized males. Proteins were then isolated from mated females and identified using mass spectrometry. In addition to gaining insights into possible functions and fates of ejaculated proteins, our study serves as proof of concept that isotopic labeling is a powerful means to study reproductive proteins. Results We identified 69 male-derived proteins from the female reproductive tract following copulation. More than a third of all spectra detected mapped to just seven genes known to be structurally important in the formation of the copulatory plug, a hard coagulum that forms shortly after mating. Seminal fluid is significantly enriched for proteins that function in protection from oxidative stress and endopeptidase inhibition. Females, on the other hand, produce endopeptidases in response to mating. The 69 ejaculated proteins evolve significantly more rapidly than other proteins that we previously identified directly from dissection of the male reproductive tract. Conclusion Our study attempts to comprehensively identify the proteins transferred from males to females during mating, expanding the application of isotopic labeling to mammalian reproductive genomics. This technique opens the way to the targeted monitoring of the fate of ejaculated proteins as they incubate in the female reproductive tract. PMID:21663664

  1. Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility.

    PubMed

    Kobayashi, Hiroshi; Swapna, G V T; Wu, Kuen-Phon; Afinogenova, Yuliya; Conover, Kenith; Mao, Binchen; Montelione, Gaetano T; Inouye, Masayori

    2012-04-01

    A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS(2)) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS(2)-tag is replaced with non-isotope labeled PrS(2)-tag, silencing the NMR signals from PrS(2)-tag in isotope-filtered (1)H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS(2)-tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS(2) (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS(2)-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS(2)-tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone (1)H, (15)N and (13)C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear (1)H-(15)N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct.

  2. Backbone and sidechain 1H, 15N and 13C assignments of the KSR1 CA1 domain

    PubMed Central

    Koveal, Dorothy; Pinheiro, Anderson S.; Peti, Wolfgang; Page, Rebecca

    2014-01-01

    The backbone and side chain resonance assignments of the murine KSR1 CA1 domain have been determined based on triple-resonance experiments using uniformly [13C, 15N]-labeled protein. This assignment is the first step towards the determination of the three-dimensional structure of the unique KSR1 CA1 domain. PMID:20737253

  3. Fluorescein-labeled glutathione to study protein S-glutathionylation.

    PubMed

    Landino, Lisa M; Brown, Carolyn M; Edson, Carolyn A; Gilbert, Laura J; Grega-Larson, Nathan; Wirth, Anna Jean; Lane, Kelly C

    2010-07-01

    Numerous studies of S-glutathionylation of cysteine thiols indicate that this protein modification plays a key role in redox regulation of proteins. To facilitate the study of protein S-glutathionylation, we developed a synthesis and purification to produce milligram quantities of fluorescein-labeled glutathione. The amino terminus of the glutathione tripeptide reacted with fluorescein isothiocyanate readily in ammonium bicarbonate. Purification by solid phase extraction on C8 and C18 columns separated excess reactants from desired products. Both oxidized and reduced fluorescein-labeled glutathione reacted with a variety of thiol-containing proteins to yield fluorescent proteins. PMID:20156418

  4. Fluorescein-labeled glutathione to study protein S-glutathionylation.

    PubMed

    Landino, Lisa M; Brown, Carolyn M; Edson, Carolyn A; Gilbert, Laura J; Grega-Larson, Nathan; Wirth, Anna Jean; Lane, Kelly C

    2010-07-01

    Numerous studies of S-glutathionylation of cysteine thiols indicate that this protein modification plays a key role in redox regulation of proteins. To facilitate the study of protein S-glutathionylation, we developed a synthesis and purification to produce milligram quantities of fluorescein-labeled glutathione. The amino terminus of the glutathione tripeptide reacted with fluorescein isothiocyanate readily in ammonium bicarbonate. Purification by solid phase extraction on C8 and C18 columns separated excess reactants from desired products. Both oxidized and reduced fluorescein-labeled glutathione reacted with a variety of thiol-containing proteins to yield fluorescent proteins.

  5. Robust method for investigating nitrogen metabolism of 15N labeled amino acids using AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry: application to a parasitic plant-plant interaction.

    PubMed

    Gaudin, Zachary; Cerveau, Delphine; Marnet, Nathalie; Bouchereau, Alain; Delavault, Philippe; Simier, Philippe; Pouvreau, Jean-Bernard

    2014-01-21

    An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of (15)N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa. PMID:24359440

  6. 13C-13C and 15N-13C correlation spectroscopy of membrane-associated and uniformly labeled human immunodeficiency virus and influenza fusion peptides: Amino acid-type assignments and evidence for multiple conformations

    NASA Astrophysics Data System (ADS)

    Bodner, Michele L.; Gabrys, Charles M.; Struppe, Jochem O.; Weliky, David P.

    2008-02-01

    Many viruses which cause disease including human immunodeficiency virus (HIV) and influenza are "enveloped" by a membrane and infection of a host cell begins with joining or "fusion" of the viral and target cell membranes. Fusion is catalyzed by viral proteins in the viral membrane. For HIV and for the influenza virus, these fusion proteins contain an ˜20-residue apolar "fusion peptide" that binds to target cell membranes and plays a critical role in fusion. For this study, the HIV fusion peptide (HFP) and influenza virus fusion peptide (IFP) were chemically synthesized with uniform C13, N15 labeling over large contiguous regions of amino acids. Two-dimensional C13-C13 and N15-C13 spectra were obtained for the membrane-bound fusion peptides and an amino acid-type C13 assignment was obtained for the labeled residues in HFP and IFP. The membrane used for the HFP sample had a lipid headgroup and cholesterol composition comparable to that of host cells of the virus, and the C13 chemical shifts were more consistent with β strand conformation than with helical conformation. The membrane used for the IFP sample did not contain cholesterol, and the chemical shifts of the dominant peaks were more consistent with helical conformation than with β strand conformation. There were additional peaks in the IFP spectrum whose shifts were not consistent with helical conformation. An unambiguous C13 and N15 assignment was obtained in an HFP sample with more selective labeling, and two shifts were identified for the Leu-9 CO, Gly-10 N, and Gly-10 Cα nuclei. These sets of two shifts may indicate two β strand registries such as parallel and antiparallel. Although most spectra were obtained on a 9.4T instrument, one C13-C13 correlation spectrum was obtained on a 16.4T instrument and was better resolved than the comparable 9.4T spectrum. More selective labeling and higher field may, therefore, be approaches to obtaining unambiguous assignments for membrane-associated fusion peptides.

  7. Computer Modeling of Nitroxide Spin Labels on Proteins

    PubMed Central

    Hatmal, Ma’mon M.; Li, Yiyu; Hegde, Balachandra G.; Hegde, Prabhavati B.; Jao, Christine C.; Langen, Ralf; Haworth, Ian S.

    2012-01-01

    Electron paramagnetic resonance (EPR) using site-directed spin-labeling (SDSL) can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of inter-label distances using the double electron-electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of inter-label distances. We describe an algorithm, PRONOX, for rapid computation of inter-label distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12, and with published data for FCHo2 (F-BAR), endophilin, and α-synuclein; a total of 44 inter-label distances. The program reproduced these distances accurately (r2=0.94, slope=0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggests that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure. PMID:21792846

  8. Compound-specific 15N stable isotope probing of N assimilation by the soil microbial biomass: a new methodological paradigm in soil N cycling

    NASA Astrophysics Data System (ADS)

    Charteris, A. F.; Knowles, T. D. J.; Michaelides, K.; Evershed, R. P.

    2015-10-01

    A compound-specific nitrogen-15 stable isotope probing (15N-SIP) technique is described which allows investigation of the fate of inorganic- or organic-N amendments to soils. The technique uses gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to determine the δ15N values of individual amino acids (AAs; determined as N-acetyl, O-isopropyl derivatives) as proxies of biomass protein production. The δ15N values are used together with AA concentrations to quantify N assimilation of 15N-labelled substrates by the soil microbial biomass. The utility of the approach is demonstrated through incubation experiments using inorganic 15N-labelled substrates ammonium (15NH4+) and nitrate (15NO3-) and an organic 15N-labelled substrate, glutamic acid (15N-Glu). Assimilation of all the applied substrates was undetectable based on bulk soil properties, i.e. % total N (% TN), bulk soil N isotope composition and AA concentrations, all of which remained relatively constant throughout the incubation experiments. In contrast, compound-specific AA δ15N values were highly sensitive to N assimilation, providing qualitative and quantitative insights into the cycling and fate of the applied 15N-labelled substrates. The utility of this 15N-AA-SIP technique is considered in relation to other currently available methods for investigating the microbially-mediated assimilation of nitrogenous substrates into the soil organic N pool. This approach will be generally applicable to the study of N cycling in any soil, or indeed, in any complex ecosystem.

  9. Site-specific protein labeling with SNAP-tags.

    PubMed

    Cole, Nelson B

    2013-09-24

    Site-specific labeling of cellular proteins with chemical probes is a powerful tool for studying protein function in living cells. A number of small peptide and protein tags have been developed that can be labeled with synthetic probes with high efficiencies and specificities and provide flexibility not available with fluorescent proteins. The SNAP-tag is a modified form of the DNA repair enzyme human O(6)-alkylguanine-DNA-alkyltransferase, and undergoes a self-labeling reaction to form a covalent bond with O(6)-benzylguanine (BG) derivatives. BG can be modified with a wide variety of fluorophores and other reporter compounds, generally without affecting the reaction with the SNAP-tag. In this unit, basic strategies for labeling SNAP-tag fusion proteins, both for live cell imaging and for in vitro analysis, are described. This includes a description of a releasable SNAP-tag probe that allows the user to chemically cleave the fluorophore from the labeled SNAP-tag fusion. In vitro labeling of purified SNAP-tag fusions is briefly described.

  10. Interfacial polymerization for colorimetric labeling of protein expression in cells.

    PubMed

    Lilly, Jacob L; Sheldon, Phillip R; Hoversten, Liv J; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium.

  11. Sequence-specific {sup 1}H, {sup 13}C, and {sup 15}N resonance assignments for intestinal fatty-acid-binding protein complexed with palmitate (15.4 kDA)

    SciTech Connect

    Hodsdon, M.E.; Toner, J.J.; Cistola, D.P.

    1994-12-01

    Intestinal fatty-acid-binding protein (I-FABP) belongs to a family of soluble, cytoplasmic proteins that are thought to function in the intracellular transport and trafficking of polar lipids. Individual members of this protein family have distinct specificities and affinities for fatty acids, cholesterol, bile salts, and retinoids. We are comparing several retinol- and fatty-acid-binding proteins from intestine in order to define the factors that control molecular recognition in this family of proteins. We have established sequential resonance assignments for uniformly {sup 13}C/{sup 15}N-enriched I-FABP complexed with perdeuterated palmitate at pH7.2 and 37{degrees}C. The assignment strategy was similar to that introduced for calmodulin. We employed seven three-dimensional NMR experiments to establish scalar couplings between backbone and sidechain atoms. Backbone atoms were correlated using triple-resonance HNCO, HNCA, TOCSY-HMQC, HCACO, and HCA(CO)N experiments. Sidechain atoms were correlated using CC-TOCSY, HCCH-TOCSY, and TOCSY-HMQC. The correlations of peaks between three-dimensional spectra were established in a computer-assisted manner using NMR COMPASS (Molecular Simulations, Inc.) Using this approach, {sup 1}H, {sup 13}C, and {sup 15}N resonance assignments have been established for 120 of the 131 residues of I-FABP. For 18 residues, amide {sup 1}H and {sup 15}N resonances were unobservable, apparently because of the rapid exchange of amide protons with bulk water at pH 7.2. The missing amide protons correspond to distinct amino acid patterns in the protein sequence, which will be discussed. During the assignment process, several sources of ambiguity in spin correlations were observed. To overcome this ambiguity, the additional inter-residue correlations often observed in the HNCA experiment were used as cross-checks for the sequential backbone assignments.

  12. NMR study of non-structural proteins--part I: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Mayaro virus (MAYV).

    PubMed

    Melekis, Efstathios; Tsika, Aikaterini C; Lichière, Julie; Chasapis, Christos T; Margiolaki, Irene; Papageorgiou, Nicolas; Coutard, Bruno; Bentrop, Detlef; Spyroulias, Georgios A

    2015-04-01

    Macro domains are ADP-ribose-binding modules present in all eukaryotic organisms, bacteria and archaea. They are also found in non-structural proteins of several positive strand RNA viruses such as alphaviruses. Here, we report the high yield expression and preliminary structural analysis through solution NMR spectroscopy of the macro domain from New World Mayaro Alphavirus. The recombinant protein was well-folded and in a monomeric state. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure determined by TALOS+.

  13. Technological advances in site-directed spin labeling of proteins.

    PubMed

    Hubbell, Wayne L; López, Carlos J; Altenbach, Christian; Yang, Zhongyu

    2013-10-01

    Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion.

  14. Labeling proteins by affinity-guided DMAP chemistry.

    PubMed

    Tamura, Tomonori; Hamachi, Itaru

    2015-01-01

    Catalysts have long played an essential role in organic synthesis and thus hold potential as tools for chemical protein modification. However, there are only a few examples of catalyst-mediated protein labeling under biological conditions because of the difficulty of designing molecular catalysts that work in aqueous environments with high target selectivity and reaction efficiency. To overcome this situation, we have previously developed a new catalyst-based method, termed affinity-guided DMAP (4-dimethylaminopyridine) (AGD) chemistry, for site-specific protein labeling in a target-selective manner using an acyl transfer reaction. More recently, we discovered that the labeling rate and efficiency can be greatly enhanced by using "multivalent" DMAP groups. Here, we describe the principle of the multivalent AGD chemistry and the protocol for chemical labeling of FK506-binding protein 12 (FKBP12) in test tubes. In this method, the FKBP12 labeling is completed within 30 min and occurs site specifically at the vicinity of the ligand-binding pocket of the protein.

  15. Dark dyes-bright complexes: fluorogenic protein labeling.

    PubMed

    Bruchez, Marcel P

    2015-08-01

    Complexes formed between organic dyes and genetically encoded proteins combine the advantages of stable and tunable fluorescent molecules and targetable, biologically integrated labels. To overcome the challenges imposed by labeling with bright fluorescent dyes, a number of approaches now exploit chemical or environmental changes to control the properties of a bound dye, converting dyes from a weakly fluorescent state to a bright, easily detectable complex. Optimized, such approaches avoid the need for removal of unbound dyes, facilitate rapid and simple assays in cultured cells and enable hybrid labeling to function more robustly in living model organisms.

  16. Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta.

    PubMed

    Marion, D; Driscoll, P C; Kay, L E; Wingfield, P T; Bax, A; Gronenborn, A M; Clore, G M

    1989-07-25

    The application of three-dimensional (3D) heteronuclear NMR spectroscopy to the sequential assignment of the 1H NMR spectra of larger proteins is presented, using uniformly labeled (approximately 95%) [15N]interleukin 1 beta, a protein of 153 residues and molecular mass of 17.4 kDa, as an example. The two-dimensional (2D) 600-MHz spectra of interleukin 1 beta are too complex for complete analysis, owing to extensive cross-peak overlap and chemical shift degeneracy. We show that the combined use of 3D 1H-15N Hartmann-Hahn-multiple quantum coherence (HOHAHA-HMQC) and nuclear Overhauser-multiple quantum coherence (NOESY-HMQC) spectroscopy, designed to provide the necessary through-bond and through-space correlations for sequential assignment, provides a practical general-purpose method for resolving ambiguities which severely limit the analysis of conventional 2D NMR spectra. The absence of overlapping cross-peaks in these 3D spectra allows the unambiguous identification of C alpha H(i)-NH(i+1) and NH(i)-NH(i+1) through-space nuclear Overhauser connectivities necessary for connecting a particular C alpha H(i)-NH(i) through-bond correlation with its associated through-space sequential cross-peak The problem of amide NH chemical shift degeneracy in the 1H NMR spectrum is therefore effectively removed, and the assignment procedure simply involves inspecting a series of 2D 1H-1H slices edited by the chemical shift of the directly bonded 15N atom. Connections between residues can be identified almost without any knowledge of the spin system types involved, though this type of information is clearly required for the eventual placement of the connected residues within the primary sequence.

  17. An Overview of Label-free Electrochemical Protein Sensors

    PubMed Central

    Vestergaard, Mun'delanji; Kerman, Kagan; Tamiya, Eiichi

    2007-01-01

    Electrochemical-based protein sensors offer sensitivity, selectivity and reliability at a low cost, making them very attractive tools for protein detection. Although the sensors use a broad range of different chemistries, they all depend on the solid electrode surface, interactions with the target protein and the molecular recognition layer. Traditionally, redox enzymes have provided the molecular recognition elements from which target proteins have interacted with. This necessitates that the redox-active enzymes couple with electrode surfaces and usually requires the participation of added diffusional components, or assembly of the enzymes in functional chemical matrices. These complications, among many others, have seen a trend towards non-enzymatic-based electrochemical protein sensors. Several electrochemical detection approaches have been exploited. Basically, these have fallen into two categories: labeled and label-free detection systems. The former rely on a redox-active signal from a reporter molecule or a label, which changes upon the interaction of the target protein. In this review, we discuss the label-free electrochemical detection of proteins, paying particular emphasis to those that exploit intrinsic redox-active amino acids.

  18. Characterization of mammalian glucose transport proteins using photoaffinity labeling techniques

    SciTech Connect

    Wadzinski, B.E.

    1989-01-01

    A carrier-free radioiodinated phenylazide derivative of forskolin, 3-iodo-4-azidophenethylamido-7-O-succinyl-deacetyl-forskolin (({sup 125}I)IAPS-forskolin), has been shown to be a highly selective photoaffinity probe for the human erythrocyte glucose transported and the glucose transport proteins found in several mammalian tissues and cultured cells where the glucose transport protein is present at a low concentration. The photoincorporation of ({sup 125}I)IAPS-forskolin into these glucose transporters was blocked by D- (but not L-) glucose, cytochalasin B, and forskolin. In addition to labeling the mammalian glucose transport proteins, ({sup 125}I)IAPS-forskolin also labeled the L-arabinose transporter from E. coli. In muscle and adipose tissues, glucose transport is markedly increased in response to insulin. ({sup 125}I)IAPS-forskolin was shown to selectivity tag the glucose transporter in membranes derived from these cells. In addition, the covalent derivatization of the transport protein in subcellular fractions of the adipocyte has provided a means to study the hormonal regulation of glucose transport. ({sup 125}I)IAPS-forskolin has also been used to label the purified human erythrocyte glucose transporter. The site of insertion has therefore been localized by analysis of the radiolabeled peptides which were produced following chemical and proteolytic digestion of the labeled transport protein.

  19. Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads.

    PubMed

    Nath, Nidhi; Godat, Becky; Urh, Marjeta

    2016-01-01

    Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput. PMID:27685323

  20. Affinity labeling of GTP-binding proteins in cellular extracts.

    PubMed

    Löw, A; Faulhammer, H G; Sprinzl, M

    1992-05-25

    GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [alpha-32P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross-linking of [alpha-32P]GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. thermophilus and initiation factor 2 (IF2) from T. thermophilus. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins. PMID:1592117

  1. The measurement of protein turnover by density labelling

    PubMed Central

    Boudet, Alain; Humphrey, Thomas J.; Davies, David D.

    1975-01-01

    A method for measuring the rate of protein degradation in plant tissue is described. The method uses density labelling to avoid difficulties associated with compartmentation and recycling of amino acids. Although the technique cannot be readily adapted to measure the rate of degradation of single proteins, it avoids difficulties of interpretation due to enzyme activation or inactivation. Values for the half-life of Lemna minor protein obtained by this method are compared with values obtained by a number of other methods. To obtain satisfactory results it was necessary to improve the method of isopycnic centrifugation in CsCl gradients. A considerable improvement was achieved by using KBr gradients, and the advantages of using KBr rather than CsCl for the separation of density-labelled protein are discussed. PMID:1220694

  2. Aqueous Oxidative Heck Reaction as a Protein-Labeling Strategy

    PubMed Central

    Ourailidou, Maria Eleni; van der Meer, Jan-Ytzen; Baas, Bert-Jan; Jeronimus-Stratingh, Margot; Gottumukkala, Aditya L; Poelarends, Gerrit J; Minnaard, Adriaan J; Dekker, Frank J

    2014-01-01

    An increasing number of chemical reactions are being employed for bio-orthogonal ligation of detection labels to protein-bound functional groups. Several of these strategies, however, are limited in their application to pure proteins and are ineffective in complex biological samples such as cell lysates. Here we present the palladium-catalyzed oxidative Heck reaction as a new and robust bio-orthogonal strategy for linking functionalized arylboronic acids to protein-bound alkenes in high yields and with excellent chemoselectivity even in the presence of complex protein mixtures from living cells. Advantageously, this reaction proceeds under aerobic conditions, whereas most other metal-catalyzed reactions require inert atmosphere. PMID:24376051

  3. Protein specific fluorescent microspheres for labelling a protein

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor)

    1982-01-01

    Highly fluorescent, stable and biocompatible microspheres are obtained by copolymerizing an acrylic monomer containing a covalent bonding group such as hydroxyl, amine or carboxyl, for example, hydroxyethylmethacrylate, with an addition polymerizable fluorescent comonomer such as dansyl allyl amine. A lectin or antibody is bound to the covalent site to provide cell specificity. When the microspheres are added to a cell suspension the marked microspheres will specifically label a cell membrane by binding to a specific receptor site thereon. The labeled membrane can then be detected by fluorescence of the fluorescent monomer.

  4. sup 14,15 N, sup 13 C, sup 57 Fe, and sup 1,2 H Q-band ENDOR study of Fe-S proteins with clusters that have endogenous sulfur ligands

    SciTech Connect

    Houseman, A.L.P.; Chaoliang Fan; Werst, M.M.; Hoffman, B.M. ); Byungha Oh; Markley, J.L. ); Kennedy, M.C.; Beinert, H. )

    1992-02-25

    The benefits of performing ENDOR experiments at higher microwave frequency are demonstrated in a Q-band (35 GHz) ENDOR investigation of a number of proteins with (nFe-mS) clusters, n = 2,3,4. Each protein displays several resonances in the frequency range of 0-20 MHz. In all instances, features are seen near {nu} {approx} 13 and 8 MHz that can be assigned, respectively, to distant ENDOR from {sup 13}C in natural-abundance (1.1%) and from {sup 14}N. In addition, a number of proteins show local {sup 13}C ENDOR signals with resolved hyperfine interactions; these are assigned to the {beta} carbons of cysteines bound to the cluster. Quadrupole coupling constants are derived for both local and distant {sup 14}N signals. The interpretation of the data is supported by studies on {sup 15}N- and {sup 13}C-enriched ferredoxin (Fd) from Anabaena 7120, where the {sup 15}N signals can be clearly correlated with the corresponding {sup 14}N signals and where the {sup 13}C signals are strongly enhanced. Thus, the observation of {sup 14}N {Delta}m{sub I} = {plus minus} 2 signals at Q-band provides a new technique for examining weak interactions with a cluster. Six proteins show an additional pattern near {nu} {approx} 18 MHz that arises from {sup 57}Fe in natural abundance (2.2%) with A({sup 57}Fe) {approx} 36 MHz, which opens the possibility of studying proteins for which enrichment is impractical. Q-band ENDOR studies also have been carried out on four {sup 2}H-exchanged Fe-S proteins, and ENDOR detects exchangeable protons in each. The importance of these findings for the interpretation of X- and Q-band ENDOR at low radiofrequencies is discussed.

  5. Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.

    PubMed

    Fan, Ying; Shi, Lichi; Ladizhansky, Vladimir; Brown, Leonid S

    2011-02-01

    Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is yeast expression systems, particularly Pichia pastoris. We report on successful implementation and optimization of isotope labeling protocols, previously used for soluble secreted proteins, to produce homogeneous samples of a eukaryotic seven-transmembrane helical protein, rhodopsin from Leptosphaeria maculans. Even in shake-flask cultures, yields exceeded 5 mg of purified uniformly (13)C,(15)N-labeled protein per liter of culture. The protein was stable (at least several weeks at 5°C) and functionally active upon reconstitution into lipid membranes at high protein-to-lipid ratio required for solid-state NMR. The samples gave high-resolution (13)C and (15)N solid-state magic angle spinning NMR spectra, amenable to a detailed structural analysis. We believe that similar protocols can be adopted for challenging mammalian targets, which often resist characterization by other structural methods.

  6. TRAMPLE: the transmembrane protein labelling environment.

    PubMed

    Fariselli, Piero; Finelli, Michele; Rossi, Ivan; Amico, Mauro; Zauli, Andrea; Martelli, Pier Luigi; Casadio, Rita

    2005-07-01

    TRAMPLE (http://gpcr.biocomp.unibo.it/biodec/) is a web application server dedicated to the detection and the annotation of transmembrane protein sequences. TRAMPLE includes different state-of-the-art algorithms for the prediction of signal peptides, transmembrane segments (both beta-strands and alpha-helices), secondary structure and fast fold recognition. TRAMPLE also includes a complete content management system to manage the results of the predictions. Each user of the server has his/her own workplace, where the data can be stored, organized, accessed and annotated with documents through a simple web-based interface. In this manner, TRAMPLE significantly improves usability with respect to other more traditional web servers. PMID:15980454

  7. Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

    PubMed Central

    Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

    2013-01-01

    Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

  8. PSEA-Quant: a protein set enrichment analysis on label-free and label-based protein quantification data.

    PubMed

    Lavallée-Adam, Mathieu; Rauniyar, Navin; McClatchy, Daniel B; Yates, John R

    2014-12-01

    The majority of large-scale proteomics quantification methods yield long lists of quantified proteins that are often difficult to interpret and poorly reproduced. Computational approaches are required to analyze such intricate quantitative proteomics data sets. We propose a statistical approach to computationally identify protein sets (e.g., Gene Ontology (GO) terms) that are significantly enriched with abundant proteins with reproducible quantification measurements across a set of replicates. To this end, we developed PSEA-Quant, a protein set enrichment analysis algorithm for label-free and label-based protein quantification data sets. It offers an alternative approach to classic GO analyses, models protein annotation biases, and allows the analysis of samples originating from a single condition, unlike analogous approaches such as GSEA and PSEA. We demonstrate that PSEA-Quant produces results complementary to GO analyses. We also show that PSEA-Quant provides valuable information about the biological processes involved in cystic fibrosis using label-free protein quantification of a cell line expressing a CFTR mutant. Finally, PSEA-Quant highlights the differences in the mechanisms taking place in the human, rat, and mouse brain frontal cortices based on tandem mass tag quantification. Our approach, which is available online, will thus improve the analysis of proteomics quantification data sets by providing meaningful biological insights. PMID:25177766

  9. Hydrazide Reactive Peptide Tags for Site-Specific Protein Labeling

    PubMed Central

    Eldridge, Glenn M.; Weiss, Gregory A.

    2011-01-01

    New site-specific protein labeling (SSPL) reactions for targeting specific, short peptides could be useful for the real time detection of proteins inside of living cells. One SSPL approach matches bioorthogonal reagents with complementary peptides. Here, hydrazide reactive peptides were selected from phage-displayed libraries using reaction-based selections. Selection conditions included washes of varying pH and treatment with NaCNBH3 in order to specifically select reactive carbonyl containing peptides. Selected peptides were fused to T4 lysozyme or synthesized on filter paper for colorimetric assays of the peptide-hydrazide interaction. A peptide-lysozyme protein fusion demonstrated specific, covalent labeling by the Hydrazide Reactive (HyRe) peptides in crude bacterial cell lysates, sufficient for the specific detection of an over-expressed protein fusion. Chemical synthesis of a short HyRe tag variant and subsequent reaction with two structurally distinct hydrazide probes produced covalent adducts observable by MALDI-TOF MS and MS/MS. Rather than isolating reactive carbonyl-containing peptides, we observed reaction with the N–terminal His of HyRe tag 114, amino acid sequence HKSNHSSKNRE, which attacks the hydrazide carbonyl at neutral pH. However, at the pH used during selection wash steps (<6.0), an alternative imine-containing product is formed that can be reduced with sodium cyanoborohydride. MSMS further reveals that this low pH product forms an adduct on Ser6. Further optimization of the novel bimolecular reaction described here could provide a useful tool for in vivo protein labeling and bioconjugate synthesis. The reported selection and screening methods could be widely applicable to the identification of peptides capable of other site-specific protein labeling reactions with bioorthogonal reagents. PMID:21905743

  10. Label-Free Identification and Quantification of SUMO Target Proteins.

    PubMed

    Hendriks, Ivo A; Vertegaal, Alfred C O

    2016-01-01

    Mass spectrometry-based approaches are utilized with increasing frequency to facilitate identification of novel SUMO target proteins and to elucidate the dynamics of SUMOylation in response to cellular stresses. Here, we describe a robust method for the identification of SUMO target proteins, and the relative quantification of SUMOylation dynamics, using a label-free approach. The method relies on a decahistidine (His10)-tagged SUMO, which is expressed at a low level in a mammalian cell line or model organism. The His10-tag allows for a single-step, high-yield, and high-purity enrichment of SUMOylated proteins, which are then digested and analyzed by high-resolution mass spectrometry. Matching between runs and label-free quantification integrated in the freely available MaxQuant software allow for a high rate and accuracy of quantification, providing a strong alternative to laborious sample or cell labeling techniques. The method described here allows for identification of >1000 SUMO target proteins, and characterization of their SUMOylation dynamics, without requiring sample fractionation. The purification procedure, starting from total lysate, can be performed in ~4 days. PMID:27631806

  11. Toward hyperpolarized molecular imaging of HIV: synthesis and longitudinal relaxation properties of 15N-Azidothymidine

    PubMed Central

    Shchepin, Roman V.; Chekmenev, Eduard Y.

    2015-01-01

    Previously unreported 15N labeled Azidothymidine (AZT) was prepared as an equimolar mixture of two isotopomers: 1-15N-AZT and 3-15N-AZT. Polarization decay of 15N NMR signal was studied in high (9.4 T) and low (~50 mT) magnetic fields. 15N T1 values were 45 ± 5 s (1-15N-AZT) and 37 ± 2 s (3-15N-AZT) at 9.4 T, and 140 ± 16 s (3-15N-AZT) at 50 mT. 15N-AZT can be potentially 15N hyperpolarized by several methods. These sufficiently long 15N-AZT T1 values potentially enable hyperpolarized in vivo imaging of 15N-AZT, because of the known favorable efficient (i.e., of the time scale shorter than the longest reported here 15N T1) kinetics of uptake of injected AZT. Therefore, 3-15N-AZT can be potentially used for HIV molecular imaging using hyperpolarized magnetic resonance imaging. PMID:25156931

  12. Sparse labeling of proteins: Structural characterization from long range constraints

    NASA Astrophysics Data System (ADS)

    Prestegard, James H.; Agard, David A.; Moremen, Kelley W.; Lavery, Laura A.; Morris, Laura C.; Pederson, Kari

    2014-04-01

    Structural characterization of biologically important proteins faces many challenges associated with degradation of resolution as molecular size increases and loss of resolution improving tools such as perdeuteration when non-bacterial hosts must be used for expression. In these cases, sparse isotopic labeling (single or small subsets of amino acids) combined with long range paramagnetic constraints and improved computational modeling offer an alternative. This perspective provides a brief overview of this approach and two discussions of potential applications; one involving a very large system (an Hsp90 homolog) in which perdeuteration is possible and methyl-TROSY sequences can potentially be used to improve resolution, and one involving ligand placement in a glycosylated protein where resolution is achieved by single amino acid labeling (the sialyltransferase, ST6Gal1). This is not intended as a comprehensive review, but as a discussion of future prospects that promise impact on important questions in the structural biology area.

  13. Elucidation of protein-protein interactions using chemical cross-linking or label transfer techniques.

    PubMed

    Fancy, D A

    2000-02-01

    Understanding the architectures of multiprotein complexes is a central problem in biology. Of the many chemical methods available, label transfer and cross-linking are becoming more popular. Recently, label transfer has been applied to very large protein complexes with great success, and new oxidative methods for protein cross-linking have been developed that are fast and highly efficient. Advances in these techniques should increase the understanding of biological structures and mechanisms.

  14. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    PubMed

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy.

  15. A general approach to visualize protein binding and DNA conformation without protein labelling.

    PubMed

    Song, Dan; Graham, Thomas G W; Loparo, Joseph J

    2016-03-08

    Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein-DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein-DNA interactions.

  16. NMR study of non-structural proteins--part II: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Venezuelan equine encephalitis virus (VEEV).

    PubMed

    Makrynitsa, Garyfallia I; Ntonti, Dioni; Marousis, Konstantinos D; Tsika, Aikaterini C; Lichière, Julie; Papageorgiou, Nicolas; Coutard, Bruno; Bentrop, Detlef; Spyroulias, Georgios A

    2015-10-01

    Macro domains consist of 130-190 amino acid residues and appear to be highly conserved in all kingdoms of life. Intense research on this field has shown that macro domains bind ADP-ribose and other similar molecules, but their exact function still remains intangible. Macro domains are highly conserved in the Alphavirus genus and the Venezuelan equine encephalitis virus (VEEV) is a member of this genus that causes fatal encephalitis to equines and humans. In this study we report the high yield recombinant expression and preliminary solution NMR study of the macro domain of VEEV. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure predicted by TALOS+. The protein shows a unique mixed α/β-fold.

  17. Production, Purification, and Characterization of ¹⁵N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements.

    PubMed

    Reddy, Prasad T; Jaruga, Pawel; Nelson, Bryant C; Lowenthal, Mark S; Jemth, Ann-Sofie; Loseva, Olga; Coskun, Erdem; Helleday, Thomas; Dizdaroglu, Miral

    2016-01-01

    Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA-protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length (15)N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of (15)N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented.

  18. Selective {sup 2}H and {sup 13}C labeling in NMR analysis of solution protein structure and dynamics

    SciTech Connect

    LeMaster, D.M.

    1994-12-01

    Preparation of samples bearing combined isotope enrichment patterns has played a central role in the recent advances in NMR analysis of proteins in solution. In particular, uniform {sup 13}C, {sup 15}N enrichment has made it possible to apply heteronuclear multidimensional correlation experiments for the mainchain assignments of proteins larger than 30 KDa. In contrast, selective labeling approaches can offer advantages in terms of the directedness of the information provided, such as chirality and residue type assignments, as well as through enhancements in resolution and sensitivity that result from editing the spectral complexity, the relaxation pathways and the scalar coupling networks. In addition, the combination of selective {sup 13}C and {sup 2}H enrichment can greatly facilitate the determination of heteronuclear relaxation behavior.

  19. A Monte Carlo/Simulated Annealing Algorithm for Sequential Resonance Assignment in Solid State NMR of Uniformly Labeled Proteins with Magic-Angle Spinning

    PubMed Central

    Tycko, Robert; Hu, Kan-Nian

    2010-01-01

    We describe a computational approach to sequential resonance assignment in solid state NMR studies of uniformly 15N,13C-labeled proteins with magic-angle spinning. As input, the algorithm uses only the protein sequence and lists of 15N/13Cα crosspeaks from 2D NCACX and NCOCX spectra that include possible residue-type assignments of each crosspeak. Assignment of crosspeaks to specific residues is carried out by a Monte Carlo/simulated annealing algorithm, implemented in the program MC_ASSIGN1. The algorithm tolerates substantial ambiguity in residue-type assignments and coexistence of visible and invisible segments in the protein sequence. We use MC_ASSIGN1 and our own 2D spectra to replicate and extend the sequential assignments for uniformly labeled HET-s(218-289) fibrils previously determined manually by Siemer et al. (J. Biomolec. NMR, vol. 34, pp. 75-87, 2006) from a more extensive set of 2D and 3D spectra. Accurate assignments by MC_ASSIGN1 do not require data that are of exceptionally high quality. Use of MC_ASSIGN1 (and its extensions to other types of 2D and 3D data) is likely to alleviate many of the difficulties and uncertainties associated with manual resonance assignments in solid state NMR studies of uniformly labeled proteins, where spectral resolution and signal-to-noise are often sub-optimal. PMID:20547467

  20. Single-molecule mechanics of protein-labelled DNA handles.

    PubMed

    Jadhav, Vivek S; Brüggemann, Dorothea; Wruck, Florian; Hegner, Martin

    2016-01-01

    DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA-protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot-streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein-DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time

  1. A general approach to visualize protein binding and DNA conformation without protein labelling

    PubMed Central

    Song, Dan; Graham, Thomas G. W.; Loparo, Joseph J.

    2016-01-01

    Single-molecule manipulation methods, such as magnetic tweezers and flow stretching, generally use the measurement of changes in DNA extension as a proxy for examining interactions between a DNA-binding protein and its substrate. These approaches are unable to directly measure protein–DNA association without fluorescently labelling the protein, which can be challenging. Here we address this limitation by developing a new approach that visualizes unlabelled protein binding on DNA with changes in DNA conformation in a relatively high-throughput manner. Protein binding to DNA molecules sparsely labelled with Cy3 results in an increase in fluorescence intensity due to protein-induced fluorescence enhancement (PIFE), whereas DNA length is monitored under flow of buffer through a microfluidic flow cell. Given that our assay uses unlabelled protein, it is not limited to the low protein concentrations normally required for single-molecule fluorescence imaging and should be broadly applicable to studying protein–DNA interactions. PMID:26952553

  2. Far-red fluorescent tag for protein labelling.

    PubMed

    Fradkov, Arkady F; Verkhusha, Vladislav V; Staroverov, Dmitry B; Bulina, Maria E; Yanushevich, Yurii G; Martynov, Vladimir I; Lukyanov, Sergey; Lukyanov, Konstantin A

    2002-11-15

    Practical applications of green fluorescent protein ('GFP')-like fluorescent proteins (FPs) from species of the class Anthozoa (sea anemones, corals and sea pens) are strongly restricted owing to their oligomeric nature. Here we suggest a strategy to overcome this problem by the use of two covalently linked identical red FPs as non-oligomerizing fusion tags. We have applied this approach to the dimeric far-red fluorescent protein HcRed1 and have demonstrated superiority of the tandem tag in the in vivo labelling of fine cytoskeletal structures and tiny nucleoli. In addition, a possibility of effective fluorescence resonance energy transfer ('FRET') between enhanced yellow FP mutant ('EYFP') and tandem HcRed1 was demonstrated in a protease assay.

  3. Aptamer-mediated indirect quantum dot labeling and fluorescent imaging of target proteins in living cells

    NASA Astrophysics Data System (ADS)

    Liu, Jianbo; Zhang, Pengfei; Yang, Xiaohai; Wang, Kemin; Guo, Qiuping; Huang, Jin; Li, Wei

    2014-12-01

    Protein labeling for dynamic living cell imaging plays a significant role in basic biological research, as well as in clinical diagnostics and therapeutics. We have developed a novel strategy in which the dynamic visualization of proteins within living cells is achieved by using aptamers as mediators for indirect protein labeling of quantum dots (QDs). With this strategy, the target protein angiogenin was successfully labeled with fluorescent QDs in a minor intactness model, which was mediated by the aptamer AL6-B. Subsequent living cell imaging analyses indicated that the QDs nanoprobes were selectively bound to human umbilical vein endothelial cells, gradually internalized into the cytoplasm, and mostly localized in the lysosome organelle, indicating that the labeled protein retained high activity. Compared with traditional direct protein labeling methods, the proposed aptamer-mediated strategy is simple, inexpensive, and provides a highly selective, stable, and intact labeling platform that has shown great promise for future biomedical labeling and intracellular protein dynamic analyses.

  4. Single-molecule mechanics of protein-labelled DNA handles

    PubMed Central

    Wruck, Florian

    2016-01-01

    Summary DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition

  5. 1H and 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease alpha-Sarcin.

    PubMed Central

    Campos-Olivas, R.; Bruix, M.; Santoro, J.; Martínez del Pozo, A.; Lacadena, J.; Gavilanes, J. G.; Rico, M.

    1996-01-01

    The ribosome-inactivating protein alpha-Sarcin (alpha S) is a 150-residue fungal ribonuclease that, after entering sensitive cells, selectively cleaves a single phosphodiester bond in an universally conserved sequence of the major rRNA to inactivate the ribosome and thus exert its cytotoxic action. As a first step toward establishing the structure-dynamics-function relationships in this system, we have carried out the assignment of the 1H and 15N NMR spectrum of alpha S on the basis of homonuclear (1H-1H) and heteronuclear (1H-15N) two-dimensional correlation spectra of a uniformly 15N-labeled sample, and two selectively 15N-labeled (Tyr and Phe) samples, as well as a single three-dimensional experiment. The secondary structure of alpha S, as derived from the characteristic patterns of dipolar connectivities between backbone protons, conformational chemical shifts, and the protection of backbone amide protons against exchange, consists of a long N-terminal beta-hairpin, a short alpha-helical segment, and a C-terminal beta-sheet of five short strands arranged in a + 1, + 1, + 1, + 1 topology, connected by long loops in which the 13 Pro residues are located. PMID:8732769

  6. Effect of Fluorescently Labeling Protein Probes on Kinetics of Protein-Ligand Reactions

    PubMed Central

    Sun, Y.S.; Landry, J.P.; Fei, Y.Y.; Luo, J.T.; Wang, X.B.; Lam, K.S.

    2009-01-01

    We studied the effect of fluorescently labeling proteins on protein-ligand reactions. Un-labeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an oblique-incidence reflectivity difference scanning microscope (a label-free detector), we measured kon and koff for streptavidin-peptide reactions and antibody-antigen reaction. We found that (1) equilibrium dissociation constants, defined as KD = koff/kon, for streptavidin-peptide reactions increases by a factor of 3 ~ 4 when the solution-phase streptavidin is labeled with Cy3 dye; and (2) KD for reactions of solution-phase goat anti-rabbit antibodies with rabbit IgG targets also change significantly when the goat antibodies are labeled with Cy3 dye. PMID:18991423

  7. Optimal isotope labelling for NMR protein structure determinations.

    PubMed

    Kainosho, Masatsune; Torizawa, Takuya; Iwashita, Yuki; Terauchi, Tsutomu; Mei Ono, Akira; Güntert, Peter

    2006-03-01

    Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Here we present stereo-array isotope labelling (SAIL), a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination.

  8. Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

    PubMed Central

    Volkmann, Gerrit; Liu, Xiang-Qin

    2009-01-01

    Background Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. Methodology A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. Conclusions We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups. PMID:20027230

  9. Protein Retention Assessment of Four Levels of Poultry By-Product Substitution of Fishmeal in Rainbow Trout (Oncorhynchus mykiss) Diets Using Stable Isotopes of Nitrogen (δ15N) as Natural Tracers

    PubMed Central

    Badillo, Daniel; Herzka, Sharon Z.; Viana, Maria Teresa

    2014-01-01

    This is second part from an experiment where the nitrogen retention of poultry by-product meal (PBM) compared to fishmeal (FM) was evaluated using traditional indices. Here a quantitative method using stable isotope ratios of nitrogen (δ15N values) as natural tracers of nitrogen incorporation into fish biomass is assessed. Juvenile rainbow trout (Oncorhynchus mykiss) were fed for 80 days on isotopically distinct diets in which 0, 33, 66 and 100% of FM as main protein source was replaced by PBM. The diets were isonitrogenous, isolipidic and similar in gross energy content. Fish in all treatments reached isotopic equilibrium by the end of the experiment. Two-source isotope mixing models that incorporated the isotopic composition of FM and PBM as well as that of formulated feeds, empirically derived trophic discrimination factors and the isotopic composition of fish that had reached isotopic equilibrium to the diets were used to obtain a quantitative estimate of the retention of each source of nitrogen. Fish fed the diets with 33 and 66% replacement of FM by PBM retained poultry by-product meal roughly in proportion to its level of inclusion in the diets, whereas no differences were detected in the protein efficiency ratio. Coupled with the similar biomass gain of fishes fed the different diets, our results support the inclusion of PBM as replacement for fishmeal in aquaculture feeds. A re-feeding experiment in which all fish were fed a diet of 100% FM for 28 days indicated isotopic turnover occurred very fast, providing further support for the potential of isotopic ratios as tracers of the retention of specific protein sources into fish tissues. Stable isotope analysis is a useful tool for studies that seek to obtain quantitative estimates of the retention of different protein sources. PMID:25226392

  10. Analysis of proteome dynamics in mice by isotopic labeling.

    PubMed

    Price, John C; Ghaemmaghami, Sina

    2014-01-01

    Recent advances in mass spectrometry and in vivo isotopic labeling have enabled proteome-wide analyses of protein turnover in complex organisms. Here, we describe a protocol for analyzing protein turnover rates in mouse tissues by comprehensive (15)N labeling. The procedure involves the complete isotopic labeling of blue green algae (Spirulina platensis) with (15)N and utilizing it as a source of dietary nitrogen for mice. We outline a detailed protocol for in-house production of (15)N-labeled algae, labeling of mice, and analysis of isotope incorporation kinetics by mass spectrometry. The methodology can be adapted to analyze proteome dynamics in most murine tissues and may be particularly useful in the analysis of proteostatic disruptions in mouse models of disease. PMID:24791984

  11. Fate and metabolism of [15N]2,4,6-trinitrotoluene in soil.

    PubMed

    Weiss, Martin; Geyer, Roland; Russow, Rolf; Richnow, Hans H; Kästner, Matthias

    2004-08-01

    The fates of the labels from [14C] and [15N] trinitrotoluene were analyzed in bioreactors under aerobic conditions in soil treated by a fungal bioremediation process with Stropharia rugosoannulata and in control soil. Up to 17.5% of the 15N label had a different fate than the 14C label. Three N-mineralization processes were identified in detailed experiments with [15N]TNT. About 2% of the 15N label was found as NO3- and NH4+, showing simultaneous processes of direct TNT denitration (I) and reduction with cleavage of the amino groups (II). The enrichment of NO2-/NO3- (up to 7.5 atom% 15N abundance) indicates the formation of Meisenheimer complexes with a denitration of [15N]TNT. A 1.4% of the label was found distributed between N2O and N2. However, the 15N enrichment of the N2O (up to 38 atom%) demonstrated that both N atoms were generated from the labeled TNT and clearly indicates a novel formation process (III). We propose, as an explanation, the generation of N2O by cleavage from condensed azoxy metabolites. In addition, 1.7% of the 15N label was detected as biogenic amino acids in the wheat straw containing the fungus. Overall, 60 to 85% of the applied [15N]TNT was degraded and 52 to 64% was found as nonextractable residues in the soil matrix. Three percent was detected as 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene.

  12. Effect of fluorescently labeling protein probes on kinetics of protein-ligand reactions.

    PubMed

    Sun, Y S; Landry, J P; Fei, Y Y; Zhu, X D; Luo, J T; Wang, X B; Lam, K S

    2008-12-01

    We studied the effect of fluorescently labeling proteins on protein-ligand reactions. Unlabeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an oblique-incidence reflectivity difference scanning microscope (a label-free detector), we measured k(on) and k(off) for streptavidin-peptide reactions and antibody-antigen reaction. We found that (1) equilibrium dissociation constants, defined as K(D) = k(off)/k(on), for streptavidin-peptide reactions increases by a factor of 3-4 when the solution-phase streptavidin is labeled with Cy3 dye and (2) K(D) for reactions of solution-phase goat anti-rabbit antibodies with rabbit IgG targets also change significantly when the goat antibodies are labeled with Cy3 dye. PMID:18991423

  13. Nanoscale Label-free Bioprobes to Detect Intracellular Proteins in Single Living Cells

    PubMed Central

    Hong, Wooyoung; Liang, Feng; Schaak, Diane; Loncar, Marko; Quan, Qimin

    2014-01-01

    Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP). PMID:25154394

  14. Nanoscale Label-free Bioprobes to Detect Intracellular Proteins in Single Living Cells

    NASA Astrophysics Data System (ADS)

    Hong, Wooyoung; Liang, Feng; Schaak, Diane; Loncar, Marko; Quan, Qimin

    2014-08-01

    Fluorescent labeling techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for use in non-transfectable cells, and labels can interfere with protein functions. While label-free biosensors have been realized by nanofabrication, a method to track intracellular protein dynamics in real-time, in situ and in living cells has not been found. Here we present the first demonstration of label-free detection of intracellular p53 protein dynamics through a nanoscale surface plasmon-polariton fiber-tip-probe (FTP).

  15. 3D HCCH 3-TOCSY for Resonance Assignment of Methyl-Containing Side Chains in 13C-Labeled Proteins

    NASA Astrophysics Data System (ADS)

    Uhrín, Dušan; Uhrínová, Stanislava; Leadbeater, Claire; Nairn, Jacqueline; Price, Nicholas C.; Barlow, Paul N.

    2000-02-01

    Two 3D experiments, (H)CCH3-TOCSY and H(C)CH3-TOCSY, are proposed for resonance assignment of methyl-containing amino acid side chains. After the initial proton-carbon INEPT step, during which either carbon or proton chemical shift labeling is achieved (t1), the magnetization is spread along the amino acid side chains by a carbon spin lock. The chemical shifts of methyl carbons are labeled (t2) during the following constant time interval. Finally the magnetization is transferred, in a reversed INEPT step, to methyl protons for detection (t3). The proposed experiments are characterized by high digital resolution in the methyl carbon dimension (t2max = 28.6 ms), optimum sensitivity due to the use of proton decoupling during the long constant time interval, and an optional removal of CH2, or CH2 and CH, resonances from the F2F3 planes. The building blocks used in these experiments can be implemented in a range of heteronuclear experiments focusing on methyl resonances in proteins. The techniques are illustrated using a 15N, 13C-labeled E93D mutant of Schizosacharomyces pombe phosphoglycerate mutase (23.7 kDa).

  16. Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

    PubMed Central

    Ke, Na; Landgraf, Dirk; Paulsson, Johan

    2016-01-01

    ABSTRACT The use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryote Escherichia coli. We show that functional protein fusions of the HaloTag can be detected both in vivo and in vitro when expressed within the cytoplasmic or periplasmic compartments of E. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications. IMPORTANCE Visualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryote Escherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells. PMID:26787765

  17. Frequency-Selective Heteronuclear Dephasing and Selective Carbonyl Labeling to Deconvolute Crowded Spectra of Membrane Proteins By Magic Angle Spinning NMR

    PubMed Central

    Traaseth, Nathaniel J.; Veglia, Gianluigi

    2011-01-01

    We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR spectra of membrane proteins in fluid lipid membranes with broad lines and high redundancy in the primary sequence. We implemented this approach in both heteronuclear 15N-13Cα and homonuclear 13C-13C dipolar assisted rotational resonance (DARR) correlation experiments. We demonstrate its efficacy for the membrane protein phospholamban reconstituted in fluid PC/PE/PA lipid bilayers. The main advantage of this method is to discriminate overlapped 13Cα resonances by strategically labeling the preceding residue. This method is highly complementary to 13C′i-1-15Ni-13Cαi and 13Cαi-1-15Ni-1-13C′i experiments to discriminate inter-residue spin systems at a minimal cost to signal-to-noise. PMID:21482162

  18. A novel method for observing proteins in vivo using a small fluorescent label and multiphoton imaging.

    PubMed

    Botchway, Stanley W; Barba, Ignasi; Jordan, Randolf; Harmston, Rebecca; Haggie, Peter M; Williams, Simon-Peter; Fulton, Alexandra M; Parker, Anthony W; Brindle, Kevin M

    2005-09-15

    A novel method for the fluorescence detection of proteins in cells is described in the present study. Proteins are labelled by the selective biosynthetic incorporation of 5-hydroxytryptophan and the label is detected via selective two-photon excitation of the hydroxyindole and detection of its fluorescence emission at 340 nm. The method is demonstrated in this paper with images of a labelled protein in yeast cells.

  19. Advances in chemical labeling of proteins in living cells

    PubMed Central

    Yan, Qi; Bruchez, Marcel P.

    2015-01-01

    Summary The pursuit of quantitative biological information with imaging requires robust labeling approaches that can be used in multiple applications and with a variety of detectable colors and properties. In addition to conventional fluorescent proteins, chemists and biologists have come together to provide a range of approaches that combine dye chemistry with the convenience of genetic targeting. This hybrid-tagging approach combines the rational design of properties available through synthetic dye chemistry with the robust biological targeting available with genetic encoding. In this review, we discuss the current range of approaches that have been exploited for dye targeting, or targeting and activation, and some of the recent applications that are uniquely enabled by these hybrid-tagging approaches. PMID:25743694

  20. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

    PubMed

    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  1. Monitoring Protein Misfolding by Site-Specific Labeling of Proteins In Vivo

    PubMed Central

    Hsieh, Tzung-yang; Nillegoda, Nadinath B.; Tyedmers, Jens; Bukau, Bernd; Mogk, Axel; Kramer, Günter

    2014-01-01

    Incorporating fluorescent amino acids by suppression of the TAG amber codon is a useful tool for site-specific labeling of proteins and visualizing their localization in living cells. Here we use a plasmid encoded orthogonal tRNA/aminoacyl-tRNA synthetase pair to site-specifically label firefly luciferase with the environmentally sensitive fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2- aminopropanoic acid (ANAP) and explore the detectability of conformational changes in labeled luciferase in the yeast cytoplasm. We find that ANAP labeling efficiency is greatly increased in [PSI+] cells and show that analysis of the ANAP fluorescence emission by confocal imaging allows for tracking the thermal unfolding and aggregation of luciferase in vivo. Furthermore we demonstrate that flow cytometry can be used to study conformational changes in luciferase and chaperone-mediated refolding in quantitative terms and at the level of single cells. This experimental setup for the first time allows for the direct analysis of the folding state of a protein in living cells and may serve as valuable new tool for examining mechanisms of protein folding, misfolding and aggregation. PMID:24915041

  2. Development of a Split SNAP-CLIP Double Labeling System for Tracking Proteins Following Dissociation from Protein-Protein Complexes in Living Cells.

    PubMed

    Mie, Masayasu; Naoki, Tatsuhiko; Kobatake, Eiry

    2016-08-16

    The split SNAP-tag protein-fragment complementation assay (PCA) is a useful tool for imaging protein-protein interactions (PPIs) in living cells. In contrast to conventional methods employed for imaging PPIs, the split SNAP-tag PCA enables tracking of proteins following dissociation from protein-protein complexes. A limitation of this system, however, is that it only allows for labeling and tracking of one of the proteins forming the protein-protein complex. To track both proteins forming a protein-protein complex, each protein needs to be appropriately labeled. In this study, a split SNAP-CLIP double labeling system is developed and applied for tracking of each protein forming a protein-protein complex. As a proof-of concept, FM protein for PPIs and protein kinase C alpha (PKCα) for translocation are introduced to a split SNAP-CLIP double labeling system. The results show a split SNAP-CLIP double labeling system enables labeling of both proteins in a protein-protein complex and subsequent tracking of each of the proteins following dissociation from the protein-protein complexes in living cells.

  3. Isotope labeling of eukaryotic membrane proteins in yeast for solid-state NMR.

    PubMed

    Fan, Ying; Emami, Sanaz; Munro, Rachel; Ladizhansky, Vladimir; Brown, Leonid S

    2015-01-01

    Solid-state NMR (ssNMR) is a rapidly developing technique for exploring structure and dynamics of membrane proteins, but its progress is hampered by its low sensitivity. Despite the latest technological advances, routine ssNMR experiments still require several milligrams of isotopically labeled protein. While production of bacterial membrane proteins on this scale is usually feasible, obtaining such quantities of eukaryotic membrane proteins is often impossible or extremely costly. We have demonstrated that, by using isotopic labeling in yeast Pichia pastoris, one can inexpensively produce milligram quantities of doubly labeled functional samples, which yield multidimensional ssNMR spectra of high resolution suitable for detailed structural investigation. This was achieved by combining protocols of economical isotope labeling of soluble proteins previously used for solution NMR with protocols of expression of eukaryotic membrane proteins successfully employed for other methods. We review two cases of such isotope labeling, of fungal rhodopsin from Leptosphaeria maculans and human aquaporin-1. PMID:26577733

  4. Isotope labeling of eukaryotic membrane proteins in yeast for solid-state NMR.

    PubMed

    Fan, Ying; Emami, Sanaz; Munro, Rachel; Ladizhansky, Vladimir; Brown, Leonid S

    2015-01-01

    Solid-state NMR (ssNMR) is a rapidly developing technique for exploring structure and dynamics of membrane proteins, but its progress is hampered by its low sensitivity. Despite the latest technological advances, routine ssNMR experiments still require several milligrams of isotopically labeled protein. While production of bacterial membrane proteins on this scale is usually feasible, obtaining such quantities of eukaryotic membrane proteins is often impossible or extremely costly. We have demonstrated that, by using isotopic labeling in yeast Pichia pastoris, one can inexpensively produce milligram quantities of doubly labeled functional samples, which yield multidimensional ssNMR spectra of high resolution suitable for detailed structural investigation. This was achieved by combining protocols of economical isotope labeling of soluble proteins previously used for solution NMR with protocols of expression of eukaryotic membrane proteins successfully employed for other methods. We review two cases of such isotope labeling, of fungal rhodopsin from Leptosphaeria maculans and human aquaporin-1.

  5. The effect of noncollinearity of 15N-1H dipolar and 15N CSA tensors and rotational anisotropy on 15N relaxation, CSA/dipolar cross correlation, and TROSY.

    PubMed

    Fushman, D; Cowburn, D

    1999-02-01

    Current approaches to 15N relaxation in proteins assume that the 15N-1H dipolar and 15N CSA tensors are collinear. We show theoretically that, when there is significant anisotropy of molecular rotation, different orientations of the two tensors, experimentally observed in proteins, nucleic acids, and small peptides, will result in differences in site-specific correlation functions and spectral densities. The standard treatments of the rates of longitudinal and transverse relaxation of amide 15N nuclei, of the 15N CSA/15N-1H dipolar cross correlation, and of the TROSY experiment are extended to account for the effect of noncollinearity of the 15N-1H dipolar and 15N CSA (chemical shift anisotropy) tensors. This effect, proportional to the degree of anisotropy of the overall motion, (D parallel/D perpendicular - 1), is sensitive to the relative orientation of the two tensors and to the orientation of the peptide plane with respect to the diffusion coordinate frame. The effect is negligible at small degrees of anisotropy, but is predicted to become significant for D parallel/D perpendicular > or = 1.5, and at high magnetic fields. The effect of noncollinearity of 15N CSA and 15N-1H dipolar interaction is sensitive to both gross (hydrodynamic) properties and atomic-level details of protein structure. Incorporation of this effect into relaxation data analysis is likely to improve both precision and accuracy of the derived characteristics of protein dynamics, especially at high magnetic fields and for molecules with a high degree of anisotropy of the overall motion. The effect will also make TROSY efficiency dependent on local orientation in moderately anisotropic systems.

  6. Rapid removal of unincorporated label and proteins from DNA sequencing reactions.

    PubMed

    Kaczorowski, T; Sektas, M

    1996-04-01

    This article presents a simple and rapid method for removal of unincorporated label and proteins from DNA sequencing reactions by using Wizard purification resin. This method can be successfully applied for preparation of end-labeled oligonucleotides free of unincorporated label, which is important in experiments (including DNA sequencing) when the level of background should be as low as possible. Also, this method is effective in removal of proteins from DNA sequencing reactions. PMID:8734430

  7. Dye-Doped Silica Nanoparticle Labels/Protein Microarray for Detection of Protein Biomarkers

    SciTech Connect

    Wu, Hong; Huo, Qisheng; Varnum, Susan M.; Liu, Guodong; Wang, Jun; Nie, Zimin; Liu, Jun; Lin, Yuehe

    2008-10-20

    Biomarkers serve as indicators of biological and pathological processes, or physiological and pharmacological responses to a drug treatment. Interleukin-6 (IL-6), a biomarker with its important biological and pathological functions, has been studied for decades. Conventional fluorescence immunoassay has been widely used for analysis of biomakers like IL-6. However, single fluorophore labeling shows its limitations of low intensity and poor stability. We report a dye-encapsulated silica nanoparticle as a label, with the advantages of high fluorescence intensity, photostability, and biocompatibility, in conjunction with microarray technology for sensitive immunoassay of IL-6 on a microarray format. The tris (2,2’-bipyridyl)ruthenium (II)chloride hexahydrate (Rubpy) dye incorporated into silica nanoparticles using a simple one-step microemulsion synthesis step. The nanoparticles are uniform in size with a diameter of 50 nm. The microarray fluorescent immunoassay approach based on dye-doped silica nanoparticle labels has high sensitivity for practical applications with a limit of detection for IL-6 down to 0.1 ng mL-1. The calibration curve is linear over the range from 0.1 ng mL-1 to 10 ng mL-1. Furthermore, results illustrated that the assay is highly specific for IL-6 in the presence of range of cytokines or proteins. The RuDS dye-labeled nanoparticles in connection with protein microarrays show the promise for clinical diagnosis of biomarkers.

  8. Absolute quantification of protein and post-translational modification abundance with stable isotope–labeled synthetic peptides

    PubMed Central

    Kettenbach, Arminja N; Rush, John; Gerber, Scott A

    2013-01-01

    In the analysis of biological systems, it is of interest to identify the components of the system and to monitor their changes in abundance under different conditions. The AQUA (for ‘absolute quantification’) method allows sensitive and specific targeted quantification of protein and post-translational modifications in complex protein mixtures using stable isotope–labeled peptides as internal standards. Each AQUA experiment is composed of two stages: method development and application to a biological scenario. In the method development stage, peptides from the protein of interest are chosen and then synthesized with stable isotopes such as 13C, 2H or 15N. The abundance of these internal standards and their endogenous counterparts can be measured by mass spectrometry with selected reaction monitoring or selected ion monitoring methods. Once an AQUA method is established, it can be rapidly applied to a wide range of biological samples, from tissue culture cells to human plasma and tissue. After AQUA peptide synthesis, the development, optimization and application of AQUA analyses to a specific biological problem can be achieved in ~1 week. Here we demonstrate the usefulness of this method by monitoring both Polo-like kinase 1 (Plk1) protein abundance in multiple lung cancer cell lines and the extent of Plk1 activation loop phosphorylation (pThr-210) during release from S phase. PMID:21293459

  9. Heterogeneous distribution of dye-labelled biomineralizaiton proteins in calcite crystals

    NASA Astrophysics Data System (ADS)

    Liu, Chuang; Xie, Liping; Zhang, Rongqing

    2015-12-01

    Biominerals are highly ordered crystals mediated by organic matters especially proteins in organisms. However, how specific proteins are distributed inside biominerals are not well understood. In the present study, we use fluorescein isothiocyanate (FITC) to label extracted proteins from the shells of bivalve Pinctada fucata. By confocal laser scanning microscopy (CLSM), we observe a heterogeneous distribution of dye-labelled proteins inside synthetic calcite at the microscale. Proteins from the prismatic calcite layers accumulate at the edge of crystals while proteins from the nacreous aragonite layers accumulate at the center of crystals. Raman and X-ray powder diffraction show that both the proteins cannot alter the crystal phase. Scanning electron microscope demonstrates both proteins are able to affect the crystal morphology. This study may provide a direct approach for the visualization of protein distributions in crystals by small-molecule dye-labelled proteins as the additives in the crystallization process and improve our understanding of intracrystalline proteins distribution in biogenic calcites.

  10. In cellulo protein labelling with Ru-conjugate for luminescence imaging and bioorthogonal photocatalysis.

    PubMed

    Sadhu, Kalyan K; Lindberg, E; Winssinger, N

    2015-12-01

    Labelling of proteins with a luminescent ruthenium complex enables the direct visualization and photocatalytic reduction of aryl azide in live cells. The confinement of catalysis to the labeled proteins was visualized using an azide-based immolative linker releasing a precipitating dye. PMID:26426098

  11. Label-Free Protein Quantification for Plant Golgi Protein Localization and Abundance1[W

    PubMed Central

    Nikolovski, Nino; Shliaha, Pavel V.; Gatto, Laurent; Dupree, Paul; Lilley, Kathryn S.

    2014-01-01

    The proteomic composition of the Arabidopsis (Arabidopsis thaliana) Golgi apparatus is currently reasonably well documented; however, little is known about the relative abundances between different proteins within this compartment. Accurate quantitative information of Golgi resident proteins is of great importance: it facilitates a better understanding of the biochemical processes that take place within this organelle, especially those of different polysaccharide synthesis pathways. Golgi resident proteins are challenging to quantify because the abundance of this organelle is relatively low within the cell. In this study, an organelle fractionation approach targeting the Golgi apparatus was combined with a label-free quantitative mass spectrometry (data-independent acquisition method using ion mobility separation known as LC-IMS-MSE [or HDMSE]) to simultaneously localize proteins to the Golgi apparatus and assess their relative quantity. In total, 102 Golgi-localized proteins were quantified. These data show that organelle fractionation in conjunction with label-free quantitative mass spectrometry is a powerful and relatively simple tool to access protein organelle localization and their relative abundances. The findings presented open a unique view on the organization of the plant Golgi apparatus, leading toward unique hypotheses centered on the biochemical processes of this organelle. PMID:25122472

  12. Quantitative Fluorescent Labeling of Aldehyde-Tagged Proteins for Single-Molecule Imaging

    PubMed Central

    Shi, Xinghua; Jung, Yonil; Lin, Li-Jung; Liu, Cheng; Wu, Cong; Cann, Isaac K. O.; Ha, Taekjip

    2012-01-01

    A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescent labeling with high efficiency, specificity, and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ~100% efficiency while maintaining the biological function. We demonstrate that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. We further show the unique power of our method in a series of single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor. PMID:22466795

  13. Hydroponic isotope labeling of entire plants and high-performance mass spectrometry for quantitative plant proteomics.

    PubMed

    Bindschedler, Laurence V; Mills, Davinia J S; Cramer, Rainer

    2012-01-01

    Hydroponic isotope labeling of entire plants (HILEP) combines hydroponic plant cultivation and metabolic labeling with stable isotopes using (15)N-containing inorganic salts to label whole and mature plants. Employing (15)N salts as the sole nitrogen source for HILEP leads to the production of healthy-looking plants which contain (15)N proteins labeled to nearly 100%. Therefore, HILEP is suitable for quantitative plant proteomic analysis, where plants are grown in either (14)N- or (15)N-hydroponic media and pooled when the biological samples are collected for relative proteome quantitation. The pooled (14)N-/(15)N-protein extracts can be fractionated in any suitable way and digested with a protease for shotgun proteomics, using typically reverse phase liquid chromatography nanoelectrospray ionization tandem mass spectrometry (RPLC-nESI-MS/MS). Best results were obtained with a hybrid ion trap/FT-MS mass spectrometer, combining high mass accuracy and sensitivity for the MS data acquisition with speed and high-throughput MS/MS data acquisition, increasing the number of proteins identified and quantified and improving protein quantitation. Peak processing and picking from raw MS data files, protein identification, and quantitation were performed in a highly automated way using integrated MS data analysis software with minimum manual intervention, thus easing the analytical workflow. In this methodology paper, we describe how to grow Arabidopsis plants hydroponically for isotope labeling using (15)N salts and how to quantitate the resulting proteomes using a convenient workflow that does not require extensive bioinformatics skills.

  14. Characterization of protein expression levels with label-free detected reverse phase protein arrays.

    PubMed

    Guo, Xuexue; Deng, Yihong; Zhu, Chenggang; Cai, Junlong; Zhu, Xiangdong; Landry, James P; Zheng, Fengyun; Cheng, Xunjia; Fei, Yiyan

    2016-09-15

    In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies. PMID:27372609

  15. Nitrate Reduction in a Groundwater Microcosm Determined by 15N Gas Chromatography-Mass Spectrometry

    PubMed Central

    Bengtsson, Göran; Annadotter, Heléne

    1989-01-01

    Aerobic and anaerobic groundwater continuous-flow microcosms were designed to study nitrate reduction by the indigenous bacteria in intact saturated soil cores from a sandy aquifer with a concentration of 3.8 mg of NO3−-N liter−1. Traces of 15NO3− were added to filter-sterilized groundwater by using a Darcy flux of 4 cm day−1. Both assimilatory and dissimilatory reduction rates were estimated from analyses of 15N2, 15N2O, 15NH4+, and 15N-labeled protein amino acids by capillary gas chromatography-mass spectrometry. N2 and N2O were separated on a megabore fused-silica column and quantified by electron impact-selected ion monitoring. NO3− and NH4+ were analyzed as pentafluorobenzoyl amides by multiple-ion monitoring and protein amino acids as their N-heptafluorobutyryl isobutyl ester derivatives by negative ion-chemical ionization. The numbers of bacteria and their [methyl-3H]thymidine incorporation rates were simultaneously measured. Nitrate was completely reduced in the microcosms at a rate of about 250 ng g−1 day−1. Of this nitrate, 80 to 90% was converted by aerobic denitrification to N2, whereas only 35% was denitrified in the anaerobic microcosm, where more than 50% of NO3− was reduced to NH4+. Assimilatory reduction was recorded only in the aerobic microcosm, where N appeared in alanine in the cells. The nitrate reduction rates estimated for the aquifer material were low in comparison with rates in eutrophic lakes and coastal sediments but sufficiently high to remove nitrate from an uncontaminated aquifer of the kind examined in less than 1 month. PMID:16348048

  16. Probing Protein Structure by Amino Acid-Specific Covalent Labeling and Mass Spectrometry

    PubMed Central

    Mendoza, Vanessa Leah; Vachet, Richard W.

    2009-01-01

    For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. PMID:19016300

  17. Dye-Doped Silica Nanoparticle Labels/Protein Microarray for Detection of Protein Biomarkers

    PubMed Central

    Wu, Hong; Huo, Qisheng; Varnum, Susan; Wang, Jun; Liu, Guodong; Nie, Zimin; Liu, Jun; Lin, Yuehe

    2008-01-01

    We report a dye-encapsulated silica nanoparticle as a label, with the advantages of high fluorescence intensity, photostability, and biocompatibility, in conjunction with microarray technology for sensitive immunoassay of a biomarker, Interleukin-6 (IL-6), on a microarray format. The tris (2,2’-bipyridyl)ruthenium (II)chloride hexahydrate (Rubpy) dye was incorporated into silica nanoparticles using a simple one-step microemulsion synthesis. In this synthesis process, Igepal CA520 was used as the surfactant, therefore, no requirement of cosolvent during the synthesis and the particle size was reduced comparing to the commonly used Triton surfactant system. The nanoparticles are uniform in size with a diameter of 50 nm. The microarray fluorescent immunoassay approach based on dye-doped silica nanoparticle labels has high sensitivity for practical applications with a limit of detection for IL-6 down to 0.1 ng mL−1. The calibration curve is linear over the range from 0.1 ng mL−1 to 10 ng mL−1. Furthermore, results illustrated that the assay is highly specific for IL-6 in the presence of range of cytokines or proteins. The RuDS dye-labeled nanoparticles in connection with protein microarrays show the promise for clinical diagnosis of biomarkers. PMID:18936832

  18. Preparation and characterization of 15N-enriched, size-defined heparan sulfate precursor oligosaccharides

    PubMed Central

    Sigulinsky, Crystal; Babu, Ponnusamy; Victor, Xylophone V.; Kuberan, Balagurunathan

    2009-01-01

    We report the preparation of size-defined [15N]N-acetylheparosan oligosaccharides from Escherichia coli-derived 15N-enriched N-acetylheparosan. Optimized growth conditions of E. coli in minimal media containing 15NH4Cl yielded [15N]N-acetylheparosan on a preparative scale. Depolymerization of [15N]N-acetylheparosan by heparitinase I yielded resolvable, even-numbered oligosaccharides ranging from disaccharide to icosaccharide. Anion-exchange chromatography-assisted fractionation afforded size-defined [15N]N-acetylheparosan oligosaccharides identifiable by ESI-TOFMS. These isotopically labeled oligosaccharides will prove to be valuable research tools for the chemoenzymatic synthesis of heparin and heparan sulfate oligosaccharides and for the study of their structural biology. PMID:19945695

  19. 15N and13C NMR investigation of hydroxylamine-derivatized humic substances

    USGS Publications Warehouse

    Thorn, K.A.; Arterburn, J.B.; Mikita, M.A.

    1992-01-01

    Five fulvic and humic acid samples of diverse origins were derivatized with 15N-labeled hydroxylamine and analyzed by liquid-phase 15N NMR spectrometry. The 15N NMR spectra indicated that hydroxylamine reacted similarly with all samples and could discriminate among carbonyl functional groups. Oximes were the major derivatives; resonances attributable to hydroxamic acids, the reaction products of hydroxylamine with esters, and resonances attributable to the tautomeric equilibrium position between the nitrosophenol and monoxime derivatives of quinones, the first direct spectroscopic evidence for quinones, also were evident. The 15N NMR spectra also suggested the presence of nitriles, oxazoles, oxazolines, isocyanides, amides, and lactams, which may all be explained in terms of Beckmann reactions of the initial oxime derivatives. INEPT and ACOUSTIC 15N NMR spectra provided complementary information on the derivatized samples. 13C NMR spectra of derivatized samples indicated that the ketone/quinone functionality is incompletely derivatized with hydroxylamine. ?? 1991 American Chemical Society.

  20. Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy

    PubMed Central

    Greco, Todd M.; Guise, Amanda J.; Cristea, Ileana M.

    2016-01-01

    In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. PMID:26867737

  1. Backbone dynamics of barstar: a (15)N NMR relaxation study.

    PubMed

    Sahu, S C; Bhuyan, A K; Majumdar, A; Udgaonkar, J B

    2000-12-01

    Backbone dynamics of uniformly (15)N-labeled barstar have been studied at 32 degrees C, pH 6.7, by using (15)N relaxation data obtained from proton-detected 2D (1)H-(15)N NMR spectroscopy. (15)N spin-lattice relaxation rate constants (R(1)), spin-spin relaxation rate constants (R(2)), and steady-state heteronuclear (1)H-(15)N NOEs have been determined for 69 of the 86 (excluding two prolines and the N-terminal residue) backbone amide (15)N at a magnetic field strength of 14.1 Tesla. The primary relaxation data have been analyzed by using the model-free formalism of molecular dynamics, using both isotropic and axially symmetric diffusion of the molecule, to determine the overall rotational correlation time (tau(m)), the generalized order parameter (S(2)), the effective correlation time for internal motions (tau(e)), and NH exchange broadening contributions (R(ex)) for each residue. As per the axially symmetric diffusion, the ratio of diffusion rates about the unique and perpendicular axes (D( parallel)/D( perpendicular)) is 0.82 +/- 0.03. The two results have only marginal differences. The relaxation data have also been used to map reduced spectral densities for the NH vectors of these residues at three frequencies: 0, omega(H), and omega(N), where omega(H),(N) are proton and nitrogen Larmor frequencies. The value of tau(m) obtained from model-free analysis of the relaxation data is 5.2 ns. The reduced spectral density analysis, however, yields a value of 5.7 ns. The tau(m) determined here is different from that calculated previously from time-resolved fluorescence data (4.1 ns). The order parameter ranges from 0.68 to 0.98, with an average value of 0.85 +/- 0.02. A comparison of the order parameters with the X-ray B-factors for the backbone nitrogens of wild-type barstar does not show any considerable correlation. Model-free analysis of the relaxation data for seven residues required the inclusion of an exchange broadening term, the magnitude of which ranges from 2

  2. Label-free measuring and mapping of binding kinetics of membrane proteins in single living cells

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Yang, Yunze; Wang, Shaopeng; Nagaraj, Vinay J.; Liu, Qiang; Wu, Jie; Tao, Nongjian

    2012-10-01

    Membrane proteins mediate a variety of cellular responses to extracellular signals. Although membrane proteins are studied intensively for their values as disease biomarkers and therapeutic targets, in situ investigation of the binding kinetics of membrane proteins with their ligands has been a challenge. Traditional approaches isolate membrane proteins and then study them ex situ, which does not reflect accurately their native structures and functions. We present a label-free plasmonic microscopy method to map the local binding kinetics of membrane proteins in their native environment. This analytical method can perform simultaneous plasmonic and fluorescence imaging, and thus make it possible to combine the strengths of both label-based and label-free techniques in one system. Using this method, we determined the distribution of membrane proteins on the surface of single cells and the local binding kinetic constants of different membrane proteins. Furthermore, we studied the polarization of the membrane proteins on the cell surface during chemotaxis.

  3. Determination of methyl 13C-15N dipolar couplings in peptides and proteins by three-dimensional and four-dimensional magic-angle spinning solid-state NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Helmus, Jonathan J.; Nadaud, Philippe S.; Höfer, Nicole; Jaroniec, Christopher P.

    2008-02-01

    We describe three- and four-dimensional semiconstant-time transferred echo double resonance (SCT-TEDOR) magic-angle spinning solid-state nuclear magnetic resonance (NMR) experiments for the simultaneous measurement of multiple long-range N15-Cmethyl13 dipolar couplings in uniformly C13, N15-enriched peptides and proteins with high resolution and sensitivity. The methods take advantage of C13 spin topologies characteristic of the side-chain methyl groups in amino acids alanine, isoleucine, leucine, methionine, threonine, and valine to encode up to three distinct frequencies (N15-Cmethyl13 dipolar coupling, N15 chemical shift, and Cmethyl13 chemical shift) within a single SCT evolution period of initial duration ˜1/JCC1 (where JCC1≈35Hz, is the one-bond Cmethyl13-C13 J-coupling) while concurrently suppressing the modulation of NMR coherences due to C13-C13 and N15-C13 J-couplings and transverse relaxation. The SCT-TEDOR schemes offer several important advantages over previous methods of this type. First, significant (approximately twofold to threefold) gains in experimental sensitivity can be realized for weak N15-Cmethyl13 dipolar couplings (corresponding to structurally interesting, ˜3.5Å or longer, distances) and typical Cmethyl13 transverse relaxation rates. Second, the entire SCT evolution period can be used for Cmethyl13 and/or N15 frequency encoding, leading to increased spectral resolution with minimal additional coherence decay. Third, the experiments are inherently "methyl selective," which results in simplified NMR spectra and obviates the use of frequency-selective pulses or other spectral filtering techniques. Finally, the N15-C13 cross-peak buildup trajectories are purely dipolar in nature (i.e., not influenced by J-couplings or relaxation), which enables the straightforward extraction of N15-Cmethyl13 distances using an analytical model. The SCT-TEDOR experiments are demonstrated on a uniformly C13, N15-labeled peptide, N-acetyl-valine, and a 56

  4. Differentiation of histidine tautomeric states using 15N selectively filtered 13C solid-state NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Miao, Yimin; Cross, Timothy A.; Fu, Riqiang

    2014-08-01

    The histidine imidazole ring in proteins usually contains a mixture of three possible tautomeric states (two neutral - τ and π states and a charged state) at physiological pHs. Differentiating the tautomeric states is critical for understanding how the histidine residue participates in many structurally and functionally important proteins. In this work, one dimensional 15N selectively filtered 13C solid-state NMR spectroscopy is proposed to differentiate histidine tautomeric states and to identify all 13C resonances of the individual imidazole rings in a mixture of tautomeric states. When 15N selective 180° pulses are applied to the protonated or non-protonated nitrogen region, the 13C sites that are bonded to the non-protonated or protonated nitrogen sites can be identified, respectively. A sample of 13C, 15N labeled histidine powder lyophilized from a solution at pH 6.3 has been used to illustrate the usefulness of this scheme by uniquely assigning resonances of the neutral τ and charged states from the mixture.

  5. Efficient and selective isotopic labeling of hemes to facilitate the study of multiheme proteins

    SciTech Connect

    Fonseca, Bruno M.; Tien, Ming; Rivera, Mario; Shi, Liang; Louro, Ricardo O.

    2012-04-02

    Specific isotopic labeling of hemes provides a unique opportunity to characterize the structure and function of heme-proteins. Unfortunately, present day methods do not allow efficient labeling in high yields of multiheme cytochromes c, which are of great biotechnological interest. Here, a method for production of recombinant multiheme cytochromes c in Escherichia coli with isotopically labeled hemes is reported. A small tetraheme cytochrome of 12 kDa from Shewanella oneidensis MR-1 was used to demonstrate the method, achieving a production of 4 mg of pure protein per liter. This method achieves, in a single step, efficient expression and incorporation of hemes isotopically labeled in specific atom positions adequate for spectroscopic characterization of these complex heme proteins. It is, furthermore, of general application to heme proteins opening new possibilities in the characterization of this important class of proteins.

  6. Selective Methyl Labeling of Eukaryotic Membrane Proteins Using Cell-Free Expression

    PubMed Central

    2015-01-01

    Structural characterization of membrane proteins and other large proteins with NMR relies increasingly on perdeuteration combined with incorporation of specifically protonated amino acid moieties, such as methyl groups of isoleucines, valines, or leucines. The resulting proton dilution reduces dipolar broadening producing sharper resonance lines, ameliorates spectral crowding, and enables measuring of crucial distances between and to methyl groups. While incorporation of specific methyl labeling is now well established for bacterial expression using suitable precursors, corresponding methods are still lacking for cell-free expression, which is often the only choice for producing labeled eukaryotic membrane proteins in mg quantities. Here we show that we can express methyl-labeled human integral membrane proteins cost-effectively by cell-free expression based of crude hydrolyzed ILV-labeled OmpX inclusion bodies. These are obtained in Escherichia coli with very high quantity and represent an optimal intermediate to channel ILV precursors into the eukaryotic proteins. PMID:24937763

  7. Steroselective synthesis and application of L-( sup 15 N) amino acids

    SciTech Connect

    Unkefer, C.J. ); Lodwig, S.N. . Div. of Science)

    1991-01-01

    We have developed two general approaches to the stereoselective synthesis of {sup 15}N- and {sup 13}C-labeled amino acids. First, labeled serine, biosynthesized using the methylotrophic bacterium M. extorquens AM1, serves as a chiral precursor for the synthesis of other amino acids. For example, pyridoxal phosphate enzymes can be used for the conversion of L-({alpha}-{sup 15}N)serine to L-({alpha}-{sup 15}N)tyrosine, L-({alpha}-{sup 15}N)tryptophan, and L-({alpha}-{sup 15}N)cysteine. In the second approach, developed by Oppolzer and Tamura, an electrophilic amination'' reagent, 1-chloro-1-nitrosocyclohexane, was used to convert chiral enolates into L-{alpha}-amino acids. We prepared 1-chloro-1-({sup 15}N) nitrosocyclohexane and used it to aminate chiral enolates to produce L-({alpha}-{sup 15}N)amino acids. The stereoselectivity of this scheme using the Oppolzer sultam chiral auxiliary is remarkable, producing enantiomer ratios of 200 to 1. 22 refs., 4 figs.

  8. Site-specific labeling of proteins via sortase: protocols for the molecular biologist.

    PubMed

    Popp, Maximilian Wei-Lin

    2015-01-01

    Creation of site-specifically labeled protein bioconjugates is an important tool for the molecular biologist and cell biologist. Chemical labeling methods, while versatile with respect to the types of moieties that can be attached, suffer from lack of specificity, often targeting multiple positions within a protein. Here we describe protocols for the chemoenzymatic labeling of proteins at the C-terminus using the bacterial transpeptidase, sortase A. We detail a protocol for the purification of an improved pentamutant variant of the Staphylococcus aureus enzyme (SrtA 5(o)) that exhibits vastly improved kinetics relative to the wild-type enzyme. Importantly, a protocol for the construction of peptide probes compatible with sortase labeling using techniques that can be adapted to any cellular/molecular biology lab with no existing infrastructure for synthetic chemistry is described. Finally, we provide an example of how to optimize the labeling reaction using the improved SrtA 5(o) variant. PMID:25560076

  9. Label-free detection repeatability of protein microarrays by oblique-incidence reflectivity difference method

    NASA Astrophysics Data System (ADS)

    Dai, Jun; Li, Lin; Wang, JingYi; He, LiPing; Lu, HuiBin; Ruan, KangCheng; Jin, KuiJuan; Yang, GuoZhen

    2012-12-01

    We examine the repeatabilities of oblique-incidence reflectivity difference (OIRD) method for label-free detecting biological molecular interaction using protein microarrays. The experimental results show that the repeatabilities are the same in a given microarray or microarray-microarray and are consistent, indicating that OIRD is a promising label-free detection technique for biological microarrays.

  10. Labeling Cell Surface GPIs and GPI-Anchored Proteins through Metabolic Engineering with Artificial Inositol Derivatives.

    PubMed

    Lu, Lili; Gao, Jian; Guo, Zhongwu

    2015-08-10

    Glycosylphosphatidylinositol (GPI) anchoring of proteins to the cell surface is important for various biological processes, but GPI-anchored proteins are difficult to study. An effective strategy was developed for the metabolic engineering of cell-surface GPIs and GPI-anchored proteins by using inositol derivatives carrying an azido group. The azide-labeled GPIs and GPI-anchored proteins were then tagged with biotin on live cells through a click reaction, which allows further elaboration with streptavidin-conjugated dyes or other molecules. The strategy can be used to label GPI-anchored proteins with various tags for biological studies.

  11. First results on the incorporation and excretion of 15N from orally administered urea in lactating pony mares.

    PubMed

    Schubert, R; Zander, R; Gruhn, K; Hennig, A

    1991-05-01

    Two lactating pony mares were given oral offers of 20 g 15N urea [95 atom-% 15N-excess (15N')] on 6 subsequent days. About 80% of the consumed 15N' were excreted via urine and faeces, but only about 2% via milk. The 15N' secreted via milk-lysine only amounted to 0.04% of the 15N' intake. The recovery was about 90% in each case. Tissues with active metabolism had an unexpectedly high labelling (greater than 0.3 atom-% 15N'). The low extent of the conversion of oral urea N into milk-lysine speaks against an essential participation of the enteral synthesis in meeting the amino acid requirement of lactating mares. It was already concluded from this results that the determination of the amino acid requirement will be necessary for this group of performance. PMID:1888274

  12. Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation

    PubMed Central

    Berger, Christian; Ho, Jenny T.C.; Kimura, Tomohiro; Hess, Sonja; Gawrisch, Klaus; Yeliseev, Alexei

    2010-01-01

    We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and 15N2-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2 mg of purified, labeled CB2 per liter of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two 15N atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of 15N chemical shifts strongly suggests that the 15N atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotope-labeled G protein-coupled receptor by bacterial fermentation. PMID:20044006

  13. Short peptide tag for covalent protein labeling based on coiled coils.

    PubMed

    Wang, Jianpeng; Yu, Yongsheng; Xia, Jiang

    2014-01-15

    To label proteins covalently, one faces a trade-off between labeling a protein specifically and using a small tag. Often one must compromise one parameter for the other or use additional components, such as an enzyme, to satisfy both requirements. Here, we report a new reaction that covalently labels proteins by using engineered coiled-coil peptides. Harnessing the concept of "proximity-induced reactivity", the 21-amino-acid three-heptad peptides CCE/CCK were modified with a nucleophilic cysteine and an α-chloroacetyl group at selected positions. When pairs of coiled coils associated, an irreversible covalent bond spontaneously formed between the peptides. The specificity of the cross-linking reaction was characterized, the probes were improved by making them bivalent, and the system was used to label a protein in vitro and receptors on the surface of mammalian cells. PMID:24341800

  14. Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label

    PubMed Central

    Yang, Yi; Song, Haiping; He, Dan; Zhang, Shuai; Dai, Shizhong; Lin, Shixian; Meng, Rong; Wang, Chu; Chen, Peng R.

    2016-01-01

    Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein–protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey–bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein–protein interactions and enables simultaneous mapping of the binding interface under living conditions. PMID:27460181

  15. Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

    PubMed

    Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

    2014-01-01

    Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

  16. Saturation Recovery EPR and Nitroxide Spin Labeling for Exploring Structure and Dynamics in Proteins.

    PubMed

    Yang, Zhongyu; Bridges, Michael; Lerch, Michael T; Altenbach, Christian; Hubbell, Wayne L

    2015-01-01

    Experimental techniques capable of determining the structure and dynamics of proteins are continuously being developed in order to understand protein function. Among existing methods, site-directed spin labeling in combination with saturation recovery (SR) electron paramagnetic resonance spectroscopy contributes uniquely to the determination of secondary and tertiary protein structure under physiological conditions, independent of molecular weight and complexity. In addition, SR of spin labeled proteins was recently demonstrated to be sensitive to conformational exchange events with characteristic lifetimes on the order of μs, a time domain that presents a significant challenge to other spectroscopic techniques. In this chapter, we present the theoretical background necessary to understand the capabilities of SR as applied to spin labeled proteins, the instrumental requirements, and practical experimental considerations necessary to obtain interpretable data, and the use of SR to obtain information on protein: (1) secondary structure via solvent accessibility measurements, (2) tertiary structure using interspin distance measurements, and (3) conformational exchange. PMID:26477246

  17. Synthesis and evaluation of radioactive and fluorescent residualizing labels for identifying sites of plasma protein catabolism

    SciTech Connect

    Maxwell, J.L.; Baynes, J.W.; Thorpe, S.R.

    1986-05-01

    Inulin and lactose were each coupled to tyramine by reductive amination with NaBH/sub 3/CN and the tyramine then labeled with /sup 125/I. Dilactitol-/sup 125/I-tyramine (DLT) and inulin-/sup 125/I-tyramine (InTn) were coupled by reductive amination and cyanuric chloride, respectively, to asialofetuin (ASF), fetuin and rat serum albumin (RSA). Attachment of either label had no effect on the circulating half-lives of the proteins. Radioactivity from labeled ASF was recovered in rat liver (> 90%) by 1 h post-injection and remained in liver with half-lives of 2 and 6 days, respectively, for the DLT and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn-labeled RSA were 5 and 6.5 days, respectively, again indicating that the larger glycoconjugate label residualized more efficiently in cells following protein degradation. (Lactitol)/sub 2/-N-CH/sub 2/-CH/sub 2/-NH-fluroescein (DLF) was also coupled to ASF by reductive amination and recovered quantitatively in liver at 1 h post-injection. Native ASF was an effective competitor for clearance of DLF-ASF from the circulation. Fluorescent degradation products were retained in liver with a half-life of 1.2 days. Residualizing fluorescent labels should be useful for identification and sorting of cells active in the degradation of plasma proteins.

  18. Protein domain mapping by internal labeling and single particle electron microscopy.

    PubMed

    Ciferri, Claudio; Lander, Gabriel C; Nogales, Eva

    2015-11-01

    In recent years, electron microscopy (EM) and single particle analysis have emerged as essential tools for investigating the architecture of large biological complexes. When high resolution is achievable, crystal structure docking and de-novo modeling allows for precise assignment of individual protein domain sequences. However, the achievable resolution may limit the ability to do so, especially when small or flexible complexes are under study. In such cases, protein labeling has emerged as an important complementary tool to characterize domain architecture and elucidate functional mechanistic details. All labeling strategies proposed to date are either focused on the identification of the position of protein termini or require multi-step labeling strategies, potentially interfering with the final labeling efficiency. Here we describe a strategy for determining the position of internal protein domains within EM maps using a recombinant one-step labeling approach named Efficient Mapping by Internal Labeling (EMIL). EMIL takes advantage of the close spatial proximity of the GFP's N- and C-termini to generate protein chimeras containing an internal GFP at desired locations along the main protein chain. We apply this method to characterize the subunit domain localization of the human Polycomb Repressive Complex 2. PMID:26431894

  19. Small Molecule-Photoactive Yellow Protein Labeling Technology in Live Cell Imaging.

    PubMed

    Gao, Feng; Gao, Tang; Zhou, Kechao; Zeng, Wenbin

    2016-01-01

    Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag), by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure. PMID:27589715

  20. Patterns of 15N assimilation and growth of methanotrophic ANME-2 archaea and sulfate-reducing bacteria within structured syntrophic consortia revealed by FISH-SIMS.

    PubMed

    Orphan, Victoria J; Turk, Kendra A; Green, Abigail M; House, Christopher H

    2009-07-01

    Methane release from the oceans is controlled in large part by syntrophic interactions between anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (DSS), frequently found as organized consortia. An understanding of the specifics of this symbiotic relationship and the metabolic heterogeneity existing between and within individual methane-oxidizing aggregates is currently lacking. Here, we use the microanalytical method FISH-SIMS (fluorescence in situ hybridization-secondary ion mass spectrometry) to describe the physiological traits and anabolic activity of individual methanotrophic consortia, specifically tracking (15)N-labelled protein synthesis to examine the effects of organization and size on the metabolic activity of the syntrophic partners. Patterns of (15)N distribution within individual aggregates showed enhanced (15)N assimilation in ANME-2 cells relative to the co-associated DSS revealing a decoupling in anabolic activity between the partners. Protein synthesis in ANME-2 cells was sustained throughout the core of individual ANME-2/DSS consortia ranging in size range from 4 to 20 μm. This indicates that metabolic activity of the methane-oxidizing archaea is not limited to, or noticeably enhanced at the ANME-2/DSS boundary. Overall, the metabolic activity of both syntrophic partners within consortia was greater than activity measured in representatives of the ANME-2 and DSS observed alone, with smaller ANME-2/DSS aggregates displaying a tendency for greater (15)N uptake and doubling times ranging from 3 to 5 months. The combination of (15)N-labelling and FISH-SIMS provides an important perspective on the extent of heterogeneity within methanotrophic aggregates and may aid in constraining predictive models of activity and growth by these syntrophic consortia.

  1. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    PubMed

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.

  2. Introduction to Spin Label Electron Paramagnetic Resonance Spectroscopy of Proteins

    ERIC Educational Resources Information Center

    Melanson, Michelle; Sood, Abha; Torok, Fanni; Torok, Marianna

    2013-01-01

    An undergraduate laboratory exercise is described to demonstrate the biochemical applications of electron paramagnetic resonance (EPR) spectroscopy. The beta93 cysteine residue of hemoglobin is labeled by the covalent binding of 3-maleimido-proxyl (5-MSL) and 2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate (MTSL), respectively. The excess…

  3. Ultrastructural localization of extracellular matrix proteins in liver biopsies using ultracryomicrotomy and immuno-gold labelling.

    PubMed

    Burt, A D; Griffiths, M R; Schuppan, D; Voss, B; MacSween, R N

    1990-01-01

    We describe a technique for the localization of extracellular matrix proteins in wedge and needle biopsy specimens of human liver. Using ultra-thin (50-70 nm) sections of puncture perfusion fixed tissue, extracellular matrix proteins were localized using a protein A-gold labelling procedure. We obtained good preservation of ultrastructural detail and specific labelling for collagen types I, III and IV and fibronectin. The method represents a sensitive means of identifying the extracellular distribution and the cellular origin of these proteins in normal and diseased human liver.

  4. Peptide tag/probe pairs based on the coordination chemistry for protein labeling.

    PubMed

    Uchinomiya, Shohei; Ojida, Akio; Hamachi, Itaru

    2014-02-17

    Protein-labeling methods serve as essential tools for analyzing functions of proteins of interest under complicated biological conditions such as in live cells. These labeling methods are useful not only to fluorescently visualize proteins of interest in biological systems but also to conduct protein and cell analyses by harnessing the unique functions of molecular probes. Among the various labeling methods available, an appropriate binding pair consisting of a short peptide and a de novo designed small molecular probe has attracted attention because of its wide utility and versatility. Interestingly, most peptide tag/probe pairs exploit metal-ligand coordination interactions as the main binding force responsible for their association. Herein, we provide an overview of the recent progress of these coordination-chemistry-based protein-labeling methods and their applications for fluorescence imaging and functional analysis of cellular proteins, while highlighting our originally developed labeling methods. These successful examples clearly exemplify the utility and versatility of metal coordination chemistry in protein functional analysis.

  5. Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for Proteome-Wide Analysis

    SciTech Connect

    Goshe, Michael B.; Blonder, Josip; Smith, Richard D.

    2003-03-01

    The ability to identify and quantify integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and derivatize these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified of which 40 were integral membrane proteins containing from 1 to 9 mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g. ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g. pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.

  6. A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin.

    PubMed

    Facemyer, K C; Cremo, C R

    1992-01-01

    We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.

  7. The 15N isotope to evaluate fertilizer nitrogen absorption efficiency by the coffee plant.

    PubMed

    Fenilli, Tatiele A B; Reichart, Klaus; Bacchi, Osny O S; Trivelin, Paulo C O; Dourado-Neto, Durval

    2007-12-01

    The use of the 15N label for agronomic research involving nitrogen (N) cycling and the fate of fertilizer-N is well established, however, in the case of long term experimentation with perennial crops like citrus, coffee and rubber tree, there are still shortcomings mainly due to large plant size, sampling procedures, detection levels and interferences on the system. This report tries to contribute methodologically to the design and development of 15N labeled fertilizer experiments, using as an example a coffee crop fertilized with 15N labeled ammonium sulfate, which was followed for two years. The N of the plant derived from the fertilizer was studied in the different parts of the coffee plant in order to evaluate its distribution within the plant and the agronomic efficiency of the fertilizer application practice. An enrichment of the fertilizer-N of the order of 2% 15N abundance was sufficient to study N absorption rates and to establish fertilizer-N balances after one and two years of coffee cropping. The main source of errors in the estimated values lies in the inherent variability among field replicates and not in the measurements of N contents and 15N enrichments of plant material by mass-spectrometry. PMID:18066442

  8. Stem injection of 15N-NH4NO3 into mature Sitka spruce (Picea sitchensis).

    PubMed

    Nair, Richard; Weatherall, Andrew; Perks, Mike; Mencuccini, Maurizio

    2014-10-01

    Stem injection techniques can be used to introduce (15)N into trees to overcome a low variation in natural abundance and label biomass with a distinct (15)N signature, but have tended to target small and young trees, of a variety of species, with little replication. We injected 98 atom% (15)N ammonium nitrate (NH4NO3) solution into 13 mature, 9- to 13-m tall edge-profile Sitka spruce trees in order to produce a large quantity of labelled litter, examining the distribution of the isotope throughout the canopy after felling in terms of both total abundance of (15)N and relative distribution of the isotope throughout individual trees. Using a simple mass balance of the canopy alone, based on observed total needle biomass and modelled branch biomass, all of the isotope injected was accounted for, evenly split between needles and branches, but with a high degree of variability both within individual trees, and among trees. Both (15)N abundance and relative within-canopy distribution were biased towards the upper and middle crown in foliage. Recovery of the label in branches was much more variable than in needles, possibly due to differences in nitrogen allocation for both growth and storage, which differ seasonally between foliage and woody biomass.

  9. The 15N isotope to evaluate fertilizer nitrogen absorption efficiency by the coffee plant.

    PubMed

    Fenilli, Tatiele A B; Reichart, Klaus; Bacchi, Osny O S; Trivelin, Paulo C O; Dourado-Neto, Durval

    2007-12-01

    The use of the 15N label for agronomic research involving nitrogen (N) cycling and the fate of fertilizer-N is well established, however, in the case of long term experimentation with perennial crops like citrus, coffee and rubber tree, there are still shortcomings mainly due to large plant size, sampling procedures, detection levels and interferences on the system. This report tries to contribute methodologically to the design and development of 15N labeled fertilizer experiments, using as an example a coffee crop fertilized with 15N labeled ammonium sulfate, which was followed for two years. The N of the plant derived from the fertilizer was studied in the different parts of the coffee plant in order to evaluate its distribution within the plant and the agronomic efficiency of the fertilizer application practice. An enrichment of the fertilizer-N of the order of 2% 15N abundance was sufficient to study N absorption rates and to establish fertilizer-N balances after one and two years of coffee cropping. The main source of errors in the estimated values lies in the inherent variability among field replicates and not in the measurements of N contents and 15N enrichments of plant material by mass-spectrometry.

  10. Multi-instance multi-label distance metric learning for genome-wide protein function prediction.

    PubMed

    Xu, Yonghui; Min, Huaqing; Song, Hengjie; Wu, Qingyao

    2016-08-01

    Multi-instance multi-label (MIML) learning has been proven to be effective for the genome-wide protein function prediction problems where each training example is associated with not only multiple instances but also multiple class labels. To find an appropriate MIML learning method for genome-wide protein function prediction, many studies in the literature attempted to optimize objective functions in which dissimilarity between instances is measured using the Euclidean distance. But in many real applications, Euclidean distance may be unable to capture the intrinsic similarity/dissimilarity in feature space and label space. Unlike other previous approaches, in this paper, we propose to learn a multi-instance multi-label distance metric learning framework (MIMLDML) for genome-wide protein function prediction. Specifically, we learn a Mahalanobis distance to preserve and utilize the intrinsic geometric information of both feature space and label space for MIML learning. In addition, we try to deal with the sparsely labeled data by giving weight to the labeled data. Extensive experiments on seven real-world organisms covering the biological three-domain system (i.e., archaea, bacteria, and eukaryote; Woese et al., 1990) show that the MIMLDML algorithm is superior to most state-of-the-art MIML learning algorithms.

  11. Surface-Enhanced Raman Scattering Nanoparticles as Optical Labels for Imaging Cell Surface Proteins

    NASA Astrophysics Data System (ADS)

    MacLaughlin, Christina M.

    Assaying the expression of cell surface proteins has widespread application for characterizing cell type, developmental stage, and monitoring disease transformation. Immunophenotyping is conducted by treating cells with labelled targeting moieties that have high affinity for relevant surface protein(s). The sensitivity and specificity of immunophenotyping is defined by the choice of contrast agent and therefore, the number of resolvable signals that can be used to simultaneously label cells. Narrow band width surface-enhanced Raman scattering (SERS) nanoparticles are proposed as optical labels for multiplexed immunophenotying. Two types of surface coatings were investigated to passivate the gold nanoparticles, incorporate SERS functionality, and to facilitate attachment of targeting antibodies. Thiolated poly(ethylene glycol) forms dative bonds with the gold surface and is compatible with multiple physisorbed Raman-active reporter molecules. Ternary lipid bilayers are used to encapsulate the gold nanoparticles particles, and incorporate three different classes of Raman reporters. TEM, UV-Visible absorbance spectroscopy, DLS, and electrophoretic light scattering were used characterize the particle coating. Colourimetric protein assay, and secondary antibody labelling were used to quantify the antibody conjugation. Three different in vitromodels were used to investigate the binding efficacy and specificity of SERS labels for their biomarker targets. Primary human CLL cells, LY10 B lymphoma, and A549 adenocarcinoma lines were targeted. Dark field imaging was used to visualize the colocalization of SERS labels with cells, and evidence of receptor clustering was obtained based on colour shifts of the particles' Rayleigh scattering. Widefield, and spatially-resolved Raman spectra were used to detect labels singly, and in combination from labelled cells. Fluorescence flow cytometry was used to test the particles' binding specificity, and SERS from labelled cells was also

  12. Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

    NASA Astrophysics Data System (ADS)

    Zhou, Yuping; Vachet, Richard W.

    2012-04-01

    Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for

  13. Quantification of intact covalently metal labeled proteins using ESI-MS/MS.

    PubMed

    Benda, David; Schwarz, Gunnar; Beck, Sebastian; Linscheid, Michael W

    2014-01-01

    Mass spectrometric methods matured from the successful qualitative characterization of proteins in complex mixtures into methods for quantitative proteomics often based on chemical tags with stable isotope labeling. In the study presented here, we extended the application of lanthanide-ion-based tags from the quantification using inductively coupled plasma-MS into the quantification of labeled intact proteins using electrospray ionization (ESI)-MS and ESI-MS/MS. We applied the metal chelate tag MeCAT-iodoacetamide (IA) (1,4,7,10-tetraazacyclododecane N,N',N″,N″ '-tetra acetic acid with a IA reactive site). Labeled proteins were separated using C3-reversed phase-high-performance liquid chromatography interfaced to ESI-MS. We could prove that even large proteins were completely labeled at all available cysteine residues using MeCAT-IA with only a small excess of reagent. Fragmentation of labeled proteins either using infrared multiphoton dissociation in Fourier transform ion cyclotron resonance-MS or higher-energy collision dissociation with an Orbitrap gave characteristic fragments. We used these fragments to quantify several intact proteins avoiding digestion. To demonstrate the applicability, human serum albumin was quantified in blood serum. The high-performance liquid chromatography/ESI-MS/MS quantification data were validated using inductively coupled plasma-MS. Because the metal within the tag may be any of the lanthanides, multiplexing capabilities are inherent.

  14. Multilabel learning via random label selection for protein subcellular multilocations prediction.

    PubMed

    Wang, Xiao; Li, Guo-Zheng

    2013-01-01

    Prediction of protein subcellular localization is an important but challenging problem, particularly when proteins may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular localization methods are only used to deal with the single-location proteins. In the past few years, only a few methods have been proposed to tackle proteins with multiple locations. However, they only adopt a simple strategy, that is, transforming the multilocation proteins to multiple proteins with single location, which does not take correlations among different subcellular locations into account. In this paper, a novel method named random label selection (RALS) (multilabel learning via RALS), which extends the simple binary relevance (BR) method, is proposed to learn from multilocation proteins in an effective and efficient way. RALS does not explicitly find the correlations among labels, but rather implicitly attempts to learn the label correlations from data by augmenting original feature space with randomly selected labels as its additional input features. Through the fivefold cross-validation test on a benchmark data set, we demonstrate our proposed method with consideration of label correlations obviously outperforms the baseline BR method without consideration of label correlations, indicating correlations among different subcellular locations really exist and contribute to improvement of prediction performance. Experimental results on two benchmark data sets also show that our proposed methods achieve significantly higher performance than some other state-of-the-art methods in predicting subcellular multilocations of proteins. The prediction web server is available at >http://levis.tongji.edu.cn:8080/bioinfo/MLPred-Euk/ for the public usage. PMID:23929867

  15. Selective labeling of proteins in biological systems by photosensitization of 5-iodonaphthalene-1-azide

    SciTech Connect

    Raviv, Y.; Salomon, Y.; Gitler, C.; Bercovici, T.

    1987-09-01

    The apolar azide of 5-iodonaphthalene-1-azide (Ina) partitions into the lipid bilayer of biological membranes. Upon photolysis at 314 nm, it is rapidly converted into the reactive nitrene, which efficiently attaches covalently to lipid-embedded domains of proteins and, to a lesser extent, to membrane phospholipids. Above 370 nm, Ina absorption is negligible and photolysis at these wavelengths does not occur. However, on addition of the photosensitizing molecule 3-aminopyrene, trifluoperazine, or 8-anilinonaphthalene-1-sulfonate, followed by irradiation at 380 nm, efficient conversion of Ina to reactive species was observed, as measured by (/sup 125/I)Ina-labeling of membrane proteins and inactivation of the hormonal response of adenylate cyclase. Irradiation at 480 nm in the presence of a fluorescein derivative of n-undecylamine also resulted in a pattern of (/sup 125/I)Ina-labeled membrane proteins and hormone uncoupling indistinguishable from that obtained following direct photolysis at 314 nm. Photosensitization of the azide molecules is confined to the vicinity of the photosensitizer chromophore. This allowed selective labeling of chromophore-bearing proteins in solution or in membranes. Bovine serum albumin-fluorescein conjugate, in the presence of nonderivatized soluble proteins, was exclusively labeled by (/sup 125/I)Ina when irradiated at 480 nm, but random labeling occurred on photolysis at 314 nm. Likewise, rhodopsin in rod outer segment membranes from frog retina was exclusively labeled by (/sup 125/I)Ina upon photosensitization at 380 nm. Random labeling again occurred on direct irradiation at 314 nm. The results suggest that selective labeling in complex biological systems may be achieved by photosensitized activation of azides.

  16. Bifunctional Spin Labeling of Muscle Proteins: Accurate Rotational Dynamics, Orientation, and Distance by EPR.

    PubMed

    Thompson, Andrew R; Binder, Benjamin P; McCaffrey, Jesse E; Svensson, Bengt; Thomas, David D

    2015-01-01

    While EPR allows for the characterization of protein structure and function due to its exquisite sensitivity to spin label dynamics, orientation, and distance, these measurements are often limited in sensitivity due to the use of labels that are attached via flexible monofunctional bonds, incurring additional disorder and nanosecond dynamics. In this chapter, we present methods for using a bifunctional spin label (BSL) to measure muscle protein structure and dynamics. We demonstrate that bifunctional attachment eliminates nanosecond internal rotation of the spin label, thereby allowing the accurate measurement of protein backbone rotational dynamics, including microsecond-to-millisecond motions by saturation transfer EPR. BSL also allows for accurate determination of helix orientation and disorder in mechanically and magnetically aligned systems, due to the label's stereospecific attachment. Similarly, labeling with a pair of BSL greatly enhances the resolution and accuracy of distance measurements measured by double electron-electron resonance (DEER). Finally, when BSL is applied to a protein with high helical content in an assembly with high orientational order (e.g., muscle fiber or membrane), two-probe DEER experiments can be combined with single-probe EPR experiments on an oriented sample in a process we call BEER, which has the potential for ab initio high-resolution structure determination. PMID:26477249

  17. Labeling proteins via hole burning of their aromatic amino acids: pressure tuning spectroscopy of BPTI.

    PubMed Central

    Stübner, Markus; Hecht, Christoph; Friedrich, Josef

    2002-01-01

    We demonstrate hole burning on a protein by using an intrinsic aromatic amino acid as a probe. The protein is bovine pancreatic trypsin inhibitor (BPTI), the labeled amino acid is tyrosine. Only one of the four tyrosines could be burned. As an application we present pressure tuning experiments from which the local compressibility around the burned tyrosine probe is determined. PMID:12496122

  18. Independent valine and leucine isotope labeling in Escherichia coli protein overexpression systems.

    PubMed

    Lichtenecker, Roman J; Weinhäupl, Katharina; Reuther, Lukas; Schörghuber, Julia; Schmid, Walther; Konrat, Robert

    2013-11-01

    The addition of labeled α-ketoisovalerate to the growth medium of a protein-expressing host organism has evolved into a versatile tool to achieve concomitant incorporation of specific isotopes into valine- and leucine- residues. The resulting target proteins represent excellent probes for protein NMR analysis. However, as the sidechain resonances of these residues emerge in a narrow spectral range, signal overlap represents a severe limitation in the case of high-molecular-weight NMR probes. We present a protocol to eliminate leucine labeling by supplying the medium with unlabeled α-ketoisocaproate. The resulting spectra of a model protein exclusively feature valine signals of increased intensity, confirming the method to be a first example of independent valine and leucine labeling employing α-ketoacid precursor compounds.

  19. Fluorescence labeling of carbon nanotubes and visualization of a nanotube-protein hybrid under fluorescence microscope.

    PubMed

    Yoshimura, Shige H; Khan, Shahbaz; Maruyama, Hiroyuki; Nakayama, Yoshikazu; Takeyasu, Kunio

    2011-04-11

    Biological applications of carbon nanotubes have been hampered by the inability to visualize them using conventional optical microscope, which is the most common tool for the observation and measurement of biological processes. Recently, a number of fluorescence labeling methods for biomolecules and various fluorescence probes have been developed and widely utilized in biological fields. Therefore, labeling carbon nanotubes with such fluorophores under physiological conditions will be highly useful in their biological applications. In this Article, we present a method to fluorescently label nanotubes by combining a detergent and a fluorophore commonly used in biological experiments. Fluorophores carrying an amino group (Texas Red hydrazide or BODIPY FL-hydrazide) were covalently attached to the hydroxyl groups of Tween 20 using carbonyldiimidazole. Fluorescence microscopy demonstrated that nanotubes were efficiently solubilized and labeled by this fluorescently labeled detergent. By using this technique, we also demonstrated multicolor fluorescence imaging of a nanotube-protein hybrid.

  20. Hydrogen doppler spectroscopy using 15N ions

    NASA Astrophysics Data System (ADS)

    Borucki, L.; Becker, H. W.; Gorris, F.; Kubsky, S.; Schulte, W. H.; Rolfs, C.

    The energy spread of atomic and molecular ion beams from the 4 MV Dynamitron tandem accelerator at the Ruhr-Universität Bochum has been studied and in part minimized. Using the ER= 6.40 MeV narrow resonance in 1H(15N,αγ)12C with an 15N energy spread of 4.55 keV, the Doppler broadening for several hydrogen-bearing gases was found to be in good agreement with expectation: e.g. for NH3 gas a rotational-vibrational Doppler width of 10.41 +/- 0.25 keV was observed (theory = 10.4 keV). Studies of the vibrational Doppler widths of H-bonds on a Si <100> surface were performed using a 4πγ-ray detection system together with UHV-chambers for sample preparation, transport, and analysis. The results showed that further improvements in the experimental set-ups are needed for such investigations.

  1. A multi-label classifier for prediction membrane protein functional types in animal.

    PubMed

    Zou, Hong-Liang

    2014-11-01

    Membrane protein is an important composition of cell membrane. Given a membrane protein sequence, how can we identify its type(s) is very important because the type keeps a close correlation with its functions. According to previous studies, membrane protein can be divided into the following eight types: single-pass type I, single-pass type II, single-pass type III, single-pass type IV, multipass, lipid-anchor, GPI-anchor, peripheral membrane protein. With the avalanche of newly found protein sequences in the post-genomic age, it is urgent to develop an automatic and effective computational method to rapid and reliable prediction of the types of membrane proteins. At present, most of the existing methods were based on the assumption that one membrane protein only belongs to one type. Actually, a membrane protein may simultaneously exist at two or more different functional types. In this study, a new method by hybridizing the pseudo amino acid composition with multi-label algorithm called LIFT (multi-label learning with label-specific features) was proposed to predict the functional types both singleplex and multiplex animal membrane proteins. Experimental result on a stringent benchmark dataset of membrane proteins by jackknife test show that the absolute-true obtained was 0.6342, indicating that our approach is quite promising. It may become a useful high-through tool, or at least play a complementary role to the existing predictors in identifying functional types of membrane proteins.

  2. Heterogeneous distribution of dye-labelled biomineralizaiton proteins in calcite crystals

    PubMed Central

    Liu, Chuang; Xie, Liping; Zhang, Rongqing

    2015-01-01

    Biominerals are highly ordered crystals mediated by organic matters especially proteins in organisms. However, how specific proteins are distributed inside biominerals are not well understood. In the present study, we use fluorescein isothiocyanate (FITC) to label extracted proteins from the shells of bivalve Pinctada fucata. By confocal laser scanning microscopy (CLSM), we observe a heterogeneous distribution of dye-labelled proteins inside synthetic calcite at the microscale. Proteins from the prismatic calcite layers accumulate at the edge of crystals while proteins from the nacreous aragonite layers accumulate at the center of crystals. Raman and X-ray powder diffraction show that both the proteins cannot alter the crystal phase. Scanning electron microscope demonstrates both proteins are able to affect the crystal morphology. This study may provide a direct approach for the visualization of protein distributions in crystals by small-molecule dye-labelled proteins as the additives in the crystallization process and improve our understanding of intracrystalline proteins distribution in biogenic calcites. PMID:26675363

  3. Live-cell protein labelling with nanometre precision by cell squeezing

    PubMed Central

    Kollmannsperger, Alina; Sharei, Armon; Raulf, Anika; Heilemann, Mike; Langer, Robert; Jensen, Klavs F.; Wieneke, Ralph; Tampé, Robert

    2016-01-01

    Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy. PMID:26822409

  4. Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein.

    PubMed

    Yang, Sung-Tae; Lim, Sung In; Kiessling, Volker; Kwon, Inchan; Tamm, Lukas K

    2016-01-01

    Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein's folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca(2+)-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches. PMID:27605302

  5. Selective labelling of cell-surface proteins using CyDye DIGE Fluor minimal dyes.

    PubMed

    Hagner-McWhirter, Asa; Winkvist, Maria; Bourin, Stephanie; Marouga, Rita

    2008-01-01

    Surface proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan DIGE technology and analysis of protein expression changes using DeCyder 2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

  6. Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

    PubMed Central

    Meyer, Virginia; Swanson, Michael A.; Clouston, Laura J.; Boratyński, Przemysław J.; Stein, Richard A.; Mchaourab, Hassane S.; Rajca, Andrzej; Eaton, Sandra S.; Eaton, Gareth R.

    2015-01-01

    Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label. PMID:25762332

  7. A general method for the covalent labeling of fusion proteins with small molecules in vivo.

    PubMed

    Keppler, Antje; Gendreizig, Susanne; Gronemeyer, Thomas; Pick, Horst; Vogel, Horst; Johnsson, Kai

    2003-01-01

    Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.

  8. Hydrophilic trans-Cyclooctenylated Noncanonical Amino Acids for Fast Intracellular Protein Labeling.

    PubMed

    Kozma, Eszter; Nikić, Ivana; Varga, Balázs R; Aramburu, Iker Valle; Kang, Jun Hee; Fackler, Oliver T; Lemke, Edward A; Kele, Péter

    2016-08-17

    Introduction of bioorthogonal functionalities (e.g., trans-cyclooctene-TCO) into a protein of interest by site-specific genetic encoding of non-canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine-functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours-long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO-ncAAs. One derivative, DOTCO-lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.

  9. Continuous field measurement of N2O isotopologues using FTIR spectroscopy following 15N addition

    NASA Astrophysics Data System (ADS)

    Phillips, R. L.; Griffith, D. W.; Dijkstra, F. A.; Lugg, G.; Lawrie, R.; Macdonald, B.

    2012-12-01

    Anthropogenic additions of fertilizer nitrogen (N) have significantly increased the mole fraction of nitrous oxide (N2O) in the troposphere. Tracking the fate of fertilizer N and its transformation to N2O is important to advance knowledge of greenhouse gas emissions from soils. Transport and transformations are frequently studied using 15N labeling experiments, but instruments capable of continuous measurements of 15N-N2O at the surface of soil have only recently come to the fore. Our primary aim was to quantify emissions of N2O and the fraction of 15N emitted as N2O from an agricultural soil following 15N addition using a mobile Fourier Transform Infrared (FTIR) spectrometer. We set up a short-term field experiment on a coastal floodplain site near Nowra, New South Wales. We deployed an automated chamber system connected to a multi-pass cell (optical pathlength 24 m) and low resolution FTIR spectrometer to measure fluxes of all N2O isotopologues collected from five 0.25 m2 chambers every three hours. We measured N2O fluxes pre and post-application of 15N-labeled substrate as potassium nitrate (KNO3) or urea [CO(NH2)2] to the soil surface. Root mean square uncertainties for all isotopologue measurements were less than 0.3 nmol mol-1 for 1 minute average concentration measurements, and minimum detectable fluxes for each isotopologue were <0.1 ng N m-2 s-1. Emissions of all N2O isotopologues were evident immediately following 15N addition. Emissions of 14N15NO, 15N14NO and 15N15NO isotopologues subsided within 10 d, but 14N14NO fluxes were evident over the entire experiment. The figure provides an overview of the emissions. Cumulative 15N-N2O fluxes (sum of the three 15N isotopologues) per chamber for the 14 days following 15N addition ranged from 1.5 to 10.3 mg 15N-N2O m-2. The chambers were destructively sampled after 2 weeks and 15N analyzed in soil and plant material using isotope ratio mass spectrometry. Approximately 1% (range 0.7 - 1.9%) of the total amount of

  10. Site-specific fluorescent labeling to visualize membrane translocation of a myristoyl switch protein

    PubMed Central

    Yang, Sung-Tae; Lim, Sung In; Kiessling, Volker; Kwon, Inchan; Tamm, Lukas K.

    2016-01-01

    Fluorescence approaches have been widely used for elucidating the dynamics of protein-membrane interactions in cells and model systems. However, non-specific multi-site fluorescent labeling often results in a loss of native structure and function, and single cysteine labeling is not feasible when native cysteines are required to support a protein’s folding or catalytic activity. Here, we develop a method using genetic incorporation of non-natural amino acids and bio-orthogonal chemistry to site-specifically label with a single fluorescent small molecule or protein the myristoyl-switch protein recoverin, which is involved in rhodopsin-mediated signaling in mammalian visual sensory neurons. We demonstrate reversible Ca2+-responsive translocation of labeled recoverin to membranes and show that recoverin favors membranes with negative curvature and high lipid fluidity in complex heterogeneous membranes, which confers spatio-temporal control over down-stream signaling events. The site-specific orthogonal labeling technique is promising for structural, dynamical, and functional studies of many lipid-anchored membrane protein switches. PMID:27605302

  11. An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore

    PubMed Central

    Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M.; Shoffner, Matthew; Albers, Aaron E.; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

    2015-01-01

    Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins. PMID:26582263

  12. An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.

    PubMed

    Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M; Shoffner, Matthew; Albers, Aaron E; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

    2015-01-01

    Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins. PMID:26582263

  13. Detection of an azido-(/sup 14/C)-atrazine labeled protein transferred to nitrocellulose paper

    SciTech Connect

    Ivey, S.; Metz, J.G.; Berg, S.P.

    1986-04-01

    An electrophoretically similar protein in spinach and maize can be covalently labeled with azido-(/sup 14/C)-atrazine and separated by 10-18% gradient LDS-PAGE. The protein profile can be transferred to nitrocellulose paper (ncp) by western blotting. The ncp containing the protein profile is sliced into 2 mm slices and counted with liquid scintillation. The labeled protein migrates as a diffuse band with a Mr of 34 kD. This band migrates at a higher Mr (40 kD) under different gel conditions. The ncp dissolves in the organic scintillation cocktail thus providing a more sensitive and quantitative detection of the /sup 14/C. This technique allows the simultaneous immunological and radiochemical identification of many electrophoretically separable proteins.

  14. 13N,15N isotope and kinetic evidence against hyponitrite as an intermediate in dentrification.

    PubMed

    Hollocher, T C; Garber, E; Cooper, A J; Reiman, R E

    1980-06-10

    13N- and 15N-labeling experiments were carried out with Paracoccus denitrificans, grown anaerobically on nitrate, to determine whether hyponitrite might be an obligatory intermediate in denitrification and a precursor of nitrous oxide. From experiments designed to trap [13N]- or [15N,15N]hyponitrite by dilution into authentic hyponitrite it was calculated that the intracellular concentration of a presumptive hyponitrite pool must be less than 0.4 mM. In order for a pool of this size to turn over rapidly enough to handle the flux of nitrogen during dentrifucation, the spontaneous rate of hyponitrite dehydration must be enhanced by a factor of several thousand through enzyme catalysis. Cell extracts failed to catalyze this reaction under a variety of conditions. It is concluded that hyponitrite cannot be an intermediate in dentrification. In addition, the assimilation of inorganic nitrogen was studied in P. denitrificans using 13N as tracer. At low concentrations (less than 10(-8) M) of labeled nitrate and nitrite 5 to 10% of the label was assimilated into non-volatile metabolites and 90 to 95% was reduced to N2. Similarly, with 15 mM [13N]nitrate, 5% of the label went into metabolites and 95% to N2. High pressure liquid chromatography analysis of the labeled metabolites indicated that the major pathway for assimilation of inorganic nitrogen in P. denitrificans under these conditions is through ammonia incorporation via the aspartase reaction. PMID:7372623

  15. Synthesis and evaluation of radioactive and fluorescent residualizing labels for monitoring protein degradation in vivo and in vitro

    SciTech Connect

    Maxwell, J.L.

    1988-01-01

    Residualizing labels for proteins, such as dilactitol-{sup 125}I-tyramine, are tracers which have been used to identify the tissue and cellular sites of catabolism of long-lived plasma proteins, such as albumin. The radioactive degradation products formed from labeled proteins are relatively large and hydrophilic. These tracers accumulate in lysosomes following uptake and catabolism of the carrier protein. However, the gradual loss of the catabolites from cells has limited the usefulness of these radioactive labels in studies on longer-lived proteins. The objective of this dissertation was to design a radioactive residualizing label, Inulin-{sup 125}I-tyramine ({sup 125}I-InTn), that would be retained more efficiently in cells than existing labels and to develop and evaluate the first fluorescent residualizing label, N,N-dilactitol-N{prime}-fluoresceinyl-ethylenediamine (DLF).

  16. Marked by association: techniques for proximity-dependent labeling of proteins in eukaryotic cells.

    PubMed

    Roux, Kyle J

    2013-10-01

    Various methods have been established for the purpose of identifying and characterizing protein-protein interactions (PPIs). This diverse toolbox provides researchers with options to overcome challenges specific to the nature of the proteins under investigation. Among these techniques is a category based on proximity-dependent labeling of proteins in living cells. These can be further partitioned into either hypothesis-based or unbiased screening methods, each with its own advantages and limitations. Approaches in which proteins of interest are fused to either modifying enzymes or receptor sequences allow for hypothesis-based testing of protein proximity. Protein crosslinking and BioID (proximity-dependent biotin identification) permit unbiased screening of protein proximity for a protein of interest. Here, we evaluate these approaches and their applications in living eukaryotic cells.

  17. Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling.

    PubMed

    Kramer, W; Burger, H J; Arion, W J; Corsiero, D; Girbig, F; Weyland, C; Hemmerle, H; Petry, S; Habermann, P; Herling, A

    1999-05-01

    The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver. PMID:10215602

  18. Protistan grazing analysis by flow cytometry using prey labeled by in vivo expression of fluorescent proteins.

    PubMed

    Fu, Yutao; O'Kelly, Charles; Sieracki, Michael; Distel, Daniel L

    2003-11-01

    Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.

  19. Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR

    PubMed Central

    Reginsson, Gunnar W.; Shelke, Sandip A.; Rouillon, Christophe; White, Malcolm F.; Sigurdsson, Snorri Th.; Schiemann, Olav

    2013-01-01

    Site-directed spin labeling and pulsed electron–electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands. PMID:22941643

  20. Intracellular imaging of targeted proteins labeled with quantum dots

    SciTech Connect

    Yoo, Jungwoo; Kambara, Taketoshi; Gonda, Kohsuke; Higuchi, Hideo

    2008-11-15

    We developed a new method for imaging the movement of targeted proteins in living cancer cells with photostable and bright quantum dots (QDs). QDs were conjugated with various molecules and proteins, such as phalloidin, anti-tubulin antibody and kinesin. These bioconjugated QDs were mixed with a transfection reagent and successfully internalized into living cells. The movements of individual QDs were tracked for long periods of time. Phalloidin conjugated QDs bound to actin filaments and showed almost no movement. In contrast, anti-tubulin antibody conjugated QDs bound to microtubules and revealed dynamic movement of microtubules. Kinesin showed an interesting behavior whereby kinesin came to be almost paused briefly for a few seconds and then moved once again. This is in direct contrast to the smoothly continuous movement of kinesin in an in vitro assay. The maximum velocity of kinesin in cells was faster than that in the in vitro assay. These results suggest that intracellular movement of kinesin is different from that in the in vitro assay. This newly described method will be a powerful tool for investigating the functions of proteins in living cells.

  1. Microspectroscopic imaging tracks the intracellular processing of a signal transduction protein: fluorescent-labeled protein kinase C beta I.

    PubMed Central

    Bastiaens, P I; Jovin, T M

    1996-01-01

    We have devised a microspectroscopic strategy for assessing the intracellular (re)distribution and the integrity of the primary structure of proteins involved in signal transduction. The purified proteins are fluorescent-labeled in vitro and reintroduced into the living cell. The localization and molecular state of fluorescent-labeled protein kinase C beta I isozyme were assessed by a combination of quantitative confocal laser scanning microscopy, fluorescence lifetime imaging microscopy, and novel determinations of fluorescence resonance energy transfer based on photobleaching digital imaging microscopy. The intensity and fluorescence resonance energy transfer efficiency images demonstrate the rapid nuclear translocation and ensuing fragmentation of protein kinase C beta I in BALB/c3T3 fibroblasts upon phorbol ester stimulation, and suggest distinct, compartmentalized roles for the regulatory and catalytic fragments. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8710884

  2. The First in Vivo Observation of 13C- 15N Coupling in Mammalian Brain

    NASA Astrophysics Data System (ADS)

    Kanamori, Keiko; Ross, Brian D.

    2001-12-01

    [5-13C,15N]Glutamine, with 1J(13C-15N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by 13C MRS at 4.7 T. The brain [5-13C]glutamine peak consisted of the doublet from [5-13C,15N]glutamine and the center [5-13C,14N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-13C,15N]glutamine was monitored in vivo with a time resolution of 20-35 min. This [5-13C,15N]glutamine was formed by glial uptake of released neurotransmitter [5-13C]glutamate and its reaction with 15NH3 catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively13C-enriched by intravenous [2,5-13C]glucose infusion to 13C-label whole-brain glutamate C5, followed by [12C]glucose infusion to chase 13C from the small and rapidly turning-over glial glutamate pool, leaving 13C mainly in the neurotransmitter [5-13C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-13C,15N]glutamine arises from a coupling between 13C of neuronal origin and 15N of glial origin. Measurement of the rate of brain [5-13C,15N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.

  3. Evaluation of chemical labeling methods for identifying functional arginine residues of proteins by mass spectrometry.

    PubMed

    Wanigasekara, Maheshika S K; Chowdhury, Saiful M

    2016-09-01

    Arginine residues undergo several kinds of post-translational modifications (PTMs). These PTMs are associated with several inflammatory diseases, such as rheumatoid arthritis, atherosclerosis, and diabetes. Mass spectrometric studies of arginine modified proteins and peptides are very important, not only to identify the reactive arginine residues but also to understand the tandem mass spectrometry behavior of these peptides for assigning the sequences unambiguously. Herein, we utilize tandem mass spectrometry to report the performance of two widely used arginine labeling reagents, 1,2-cyclohexanedione (CHD) and phenylglyoxal (PG) with several arginine containing peptides and proteins. Time course labeling studies were performed to demonstrate the selectivity of the reagents in proteins or protein digests. Structural studies on the proteins were also explored to better understand the reaction sites and position of arginine residues. We found CHD showed better labeling efficiencies compared to phenylglyoxal. Reactive arginine profiling on a purified albumin protein clearly pointed out the cellular glycation modification site for this protein with high confidence. We believe these detailed mass-spectrometric studies will provide significant input to profile reactive arginine residues in large-scale studies; therefore, targeted proteomics can be performed to the short listed reactive sites for cellular arginine modifications. PMID:27543028

  4. iMem-Seq: A Multi-label Learning Classifier for Predicting Membrane Proteins Types.

    PubMed

    Xiao, Xuan; Zou, Hong-Liang; Lin, Wei-Zhong

    2015-08-01

    Predicting membrane protein type is a challenging problem, particularly when the query proteins may simultaneously have two or more different types. Most of the existing methods can only be used to deal with the single-label proteins. Actually, multiple-label proteins should not be ignored because they usually bear some special functions worthy of in-depth studies. By introducing the "multi-labeled learning" and hybridizing evolution information through Grey-PSSM, a novel predictor called iMem-Seq is developed that can be used to deal with the systems containing both single and multiple types of membrane proteins. As a demonstration, the jackknife cross-validation was performed with iMem-Seq on a benchmark dataset of membrane proteins classified into the eight types, where some proteins belong to two or there types, but none has ≥25% pairwise sequence identity to any other in a same subset. It was demonstrated via the rigorous cross-validations that the new predictor remarkably outperformed all its counterparts. As a user-friendly web-server, iMem-Seq is freely accessible to the public at the website http://www.jci-bioinfo.cn/iMem-Seq .

  5. Optimized orthogonal translation of unnatural amino acids enables spontaneous protein double-labelling and FRET

    PubMed Central

    Cox, Daniel J.; Wilf, Nabil M.; Lang, Kathrin; Wallace, Stephen; Mehl, Ryan A.; Chin, Jason W.

    2015-01-01

    The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer. PMID:24755590

  6. A readily synthesized cyclic pyrrolysine analogue for site-specific protein "click" labeling.

    PubMed

    Hao, Ziyang; Song, Yanqun; Lin, Shixian; Yang, Maiyun; Liang, Yujie; Wang, Jing; Chen, Peng R

    2011-04-21

    A concise route was developed for the facile synthesis of a cyclic pyrrolysine analogue bearing an azide handle. Directed evolution enabled the encoding of this non-natural amino acid in both prokaryotic and eukaryotic cells, which offers a highly efficient approach for the site-specific protein labeling using click chemistry.

  7. Enhanced sample multiplexing for nitrotyrosine-modified proteins using combined precursor isotopic labeling and isobaric tagging.

    PubMed

    Robinson, Renã A S; Evans, Adam R

    2012-06-01

    Current strategies for identification and quantification of 3-nitrotyrosine (3NT) post-translationally modified proteins (PTM) generally rely on biotin/avidin enrichment. Quantitative approaches have been demonstrated which employ isotopic labeling or isobaric tagging in order to quantify differences in the relative abundances of 3NT-modified proteins in two or potentially eight samples, respectively. Here, we present a novel strategy which uses combined precursor isotopic labeling and isobaric tagging (cPILOT) to increase the multiplexing capability of quantifying 3NT-modified proteins to 12 or 16 samples using commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), respectively. This strategy employs "light" and "heavy" labeled acetyl groups to block both N-termini and lysine residues of tryptic peptides. Next, 3NT is reduced to 3-aminotyrosine (3AT) using sodium dithionite followed by derivatization of light and heavy labeled 3AT-peptides with either TMT or iTRAQ multiplex reagents. We demonstrate the proof-of-principle utility of cPILOT with in vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins using TMT(0), TMT(6), and iTRAQ(8) reagents and discuss limitations of the strategy. PMID:22509719

  8. Uniformly 13C-labeled algal protein used to determine amino acid essentiality in vivo.

    PubMed Central

    Berthold, H K; Hachey, D L; Reeds, P J; Thomas, O P; Hoeksema, S; Klein, P D

    1991-01-01

    The edible alga Spirulina platensis was uniformly labeled with 13C by growth in an atmosphere of pure 13CO2. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly 13C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo. Images PMID:11607211

  9. Probing adenosine nucleotide-binding proteins with an affinity-labeled nucleotide probe and mass spectrometry.

    PubMed

    Qiu, Haibo; Wang, Yinsheng

    2007-08-01

    Mass spectrometry combined with chemical labeling strategies has become very important in biological analysis. Herein, we described the application of a biotin-conjugated acyl nucleotide for probing adenosine nucleotide-binding proteins. We demonstrated that the probe reacted specifically with the lysine residue at the nucleotide-binding site of two purified adenosine nucleotide-binding proteins, Escherichia coli recombinase A (RecA) and Saccharomyces cerevisiae alcohol dehydrogenase-I (YADH-I). A single conjugate peptide with a specifically labeled lysine residue was identified, by using LC-MS/MS, from the tryptic digestion mixture of the reaction products of the nucleotide analogue with RecA or YADH-I. The strategy, which involved labeling reaction, enzymatic digestion, affinity purification, and LC-MS/MS analysis, was relatively simple, fast, and straightforward. The method should be generally applicable for the identification of lysine residues at the nucleotide-binding site of other proteins. The biotin-conjugated acyl nucleotide probe also allowed for the enrichment and identification of nucleotide-binding proteins from complex protein mixtures; we showed that more than 50 adenosine nucleotide-binding proteins could be identified from the whole-cell lysates of HeLa-S3 and WM-266-4 cells.

  10. Probing adenosine nucleotide-binding proteins with an affinity labeled-nucleotide probe and mass spectrometry

    PubMed Central

    Qiu, Haibo; Wang, Yinsheng

    2008-01-01

    Mass spectrometry combined with chemical labeling strategies has become very important in biological analysis. Herein, we described the application of a biotin-conjugated acyl nucleotide for probing adenosine nucleotide-binding proteins. We demonstrated that the probe reacted specifically with the lysine residue at the nucleotide-binding site of two purified adenosine nucleotide-binding proteins, Escherichia coli RecA and Saccharomyces cerevisiae alcohol dehydrogenase-I (YADH-I). A single conjugate peptide with a specifically labeled lysine residue was identified, by using LC-MS/MS, from the tryptic digestion mixture of the reaction products of the nucleotide analog with RecA or YADH-I. The strategy, which involved labeling reaction, enzymatic digestion, affinity purification and LC-MS/MS analysis, was relatively simple, fast and straightforward. The method should be generally applicable for the identification of lysine residues at the nucleotide-binding site of other proteins. The biotin-conjugated acyl nucleotide probe also allowed for the enrichment and identification of nucleotide-binding proteins from complex protein mixtures; we showed that more than 50 adenosine nucleotide-binding proteins could be identified from the whole cell lysates of HeLa-S3 and WM-266-4 cells. PMID:17602667

  11. The Effect of Peptide Identification Search Algorithms on MS2-Based Label-Free Protein Quantification

    PubMed Central

    Degroeve, Sven; Staes, An; De Bock, Pieter-Jan

    2012-01-01

    Abstract Several approaches exist for the quantification of proteins in complex samples processed by liquid chromatography-mass spectrometry followed by fragmentation analysis (MS2). One of these approaches is label-free MS2-based quantification, which takes advantage of the information computed from MS2 spectrum observations to estimate the abundance of a protein in a sample. As a first step in this approach, fragmentation spectra are typically matched to the peptides that generated them by a search algorithm. Because different search algorithms identify overlapping but non-identical sets of peptides, here we investigate whether these differences in peptide identification have an impact on the quantification of the proteins in the sample. We therefore evaluated the effect of using different search algorithms by examining the reproducibility of protein quantification in technical repeat measurements of the same sample. From our results, it is clear that a search engine effect does exist for MS2-based label-free protein quantification methods. As a general conclusion, it is recommended to address the overall possibility of search engine-induced bias in the protein quantification results of label-free MS2-based methods by performing the analysis with two or more distinct search engines. PMID:22804230

  12. (/sup 59/Fe)Ferrous bathophenanthroline sulfonate: a radioactive stain for labeling proteins in situ in polyacrylamide gels

    SciTech Connect

    Zapolski, E.J.; Gersten, D.M.; Ledley, R.S.

    1982-07-01

    The use of a protein stain, (/sup 59/Fe)ferrous bathophenanthroline, to radioactively label proteins in polyacrylamide gels after electrophoresis using simple staining and destaining procedures is described.

  13. Structural Characterization of Membrane-Curving Proteins: Site-Directed Spin Labeling, EPR, and Computational Refinement.

    PubMed

    Ambroso, Mark R; Haworth, Ian S; Langen, Ralf

    2015-01-01

    Endocytosis and other membrane remodeling processes require the coordinated generation of different membrane shapes. Proteins capable of manipulating lipid bilayers mediate these events using mechanisms that are not fully understood. Progress is limited by the small number of structures solved for proteins bound to different membrane shapes and tools capable of resolving such information. However, recent studies have shown site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) to be capable of obtaining high-resolution structural information for proteins bound to different membrane shapes. This technique can be applied to proteins with no known structure or proteins with structures known in solution. By refining the data obtained by EPR with computational modeling, 3D structures or structural models of membrane-bound proteins can be generated. In this chapter, we highlight the basic considerations and steps required to investigate the structures of membrane-bound proteins using SDSL, EPR, and computational refinement. PMID:26477254

  14. Label-free single-cell protein quantification using a drop-based mix-and-read system

    PubMed Central

    Abbaspourrad, Alireza; Zhang, Huidan; Tao, Ye; Cui, Naiwen; Asahara, Haruichi; Zhou, Ying; Yue, Dongxian; Koehler, Stephan A.; Ung, Lloyd W.; Heyman, John; Ren, Yukun; Ziblat, Roy; Chong, Shaorong; Weitz, David A.

    2015-01-01

    Quantitative protein analysis of single cells is rarely achieved due to technical difficulties of detecting minute amounts of proteins present in one cell. We develop a mix-and-read assay for drop-based label-free protein analysis of single cells. This high-throughput method quantifies absolute, rather than relative, amounts of proteins and does not involve antibody labeling or mass spectrometry. PMID:26234416

  15. Diffusion technique for 15N and inorganic N analysis of low-N aqueous solutions and Kjeldahl digests.

    PubMed

    Chen, Rui Rui; Dittert, Klaus

    2008-06-01

    Diffusion of ammonia is a common sample preparation method for the stable isotope analysis of inorganic nitrogen in aqueous solution. Classical diffusion methods usually require 6-12 days of diffusion and often focus on (15)N/(14)N analysis only. More recent studies have discussed whether complete N recovery was necessary for the precise analysis of stable N isotope ratios. In this paper we present a newly revised diffusion technique that allows correct and simultaneous determination of total N and (15)N at% from aqueous solutions and Kjeldahl digests, with N concentrations down to sub-0.5-mg N L(-1) levels, and it is tested under different conditions of (15)N isotope labelling. With the modification described, the diffusion time was reduced to 72 h, while the ratios of measured and expected (15)N at% were greater than 99% and the simultaneous recovery of total N was >95%. Analysis of soil microbial biomass N and its (15)N/(14)N ratio is one of the most important applications of this diffusion technique. An experiment with soil extracts spiked with (15)N-labelled yeast showed that predigestion was necessary to prevent serious N loss during Kjeldahl digestion of aqueous samples (i.e. soil extracts). The whole method of soil microbial biomass N preparation for (15)N/(14)N analysis included chloroform fumigation, predigestion, Kjeldahl digestion and diffusion. An experiment with soil spiked with (15)N-labelled yeast was carried out to evaluate the method. Results showed a highly significant correlation of recovered and added N, with the same recovery rate (0.21) of both total N and (15)N. A k(N) value of 0.25 was obtained based on the data. In conclusion, the diffusion method works for soil extracts and microbial biomass N determination and hence could be useful in many types of soil/water studies.

  16. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction

    PubMed Central

    Torres-Kolbus, Jessica; Chou, Chungjung; Deiters, Alexander; Chin, Jason W.

    2013-01-01

    The site-specific incorporation of bioorthogonal groups via genetic code expansion provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl tRNA synthetase/tRNACUA pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit `turn-on' fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and demonstrate the rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface. PMID:22437715

  17. EPR Distance Measurements in Native Proteins with Genetically Encoded Spin Labels.

    PubMed

    Schmidt, Moritz J; Fedoseev, Artem; Bücker, Dennis; Borbas, Julia; Peter, Christine; Drescher, Malte; Summerer, Daniel

    2015-12-18

    The genetic encoding of nitroxide amino acids in combination with electron paramagnetic resonance (EPR) distance measurements enables precise structural studies of native proteins, i.e. without the need for mutations to create unique reactive sites for chemical labeling and thus with minimal structural perturbation. We here report on in vitro DEER measurements in native E. coli thioredoxin (TRX) that establish the nitroxide amino acid SLK-1 as a spectroscopic probe that reports distances and conformational flexibilities in the enzyme with nonmutated catalytic centers that are not accessible by the use of the traditional methanethiosulfonate spin label (MTSSL). We generated a rotamer library for SLK-1 that in combination with molecular dynamics (MD) simulation enables predictions of distance distributions between two SLK-1 labels incorporated into a target protein. Toward a routine use of SLK-1 for EPR distance measurements in proteins and the advancement of the approach to intracellular environments, we study the stability of SLK-1 in E. coli cultures and lysates and establish guidelines for protein expression and purification that offer maximal nitroxide stability. These advancements and insights provide new perspectives for facile structural studies of native, endogenous proteins by EPR distance measurements.

  18. Identification of Asp isomerization in proteins by ¹⁸O labeling and tandem mass spectrometry.

    PubMed

    Zhang, Jennifer; Katta, Viswanatham

    2012-01-01

    Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) via succinimide intermediate is a common route of degradation for proteins that can affect their structural integrity. As Asp/isoAsp is isobaric in mass, it is difficult to identify the site of modification by LC-MS/MS peptide mapping. Here, we describe an approach to label the Asp residue involved in isomerization at the protein level by hydrolyzing the succinimide intermediate in H₂¹⁸O. Tryptic digestion of this labeled protein will result in peptides containing the site of isomerization being 2 Da heavier than the ¹⁶O-containing counterparts, due to ¹⁸O incorporation during the hydrolysis process. Comparison of tandem mass spectra of isomerized peptides with and without ¹⁸O incorporation allows easy identification of the Asp residue involved. This method proved to be especially useful in identifying the sites when isomerization occurs in Asp-Asp motifs.

  19. Structural analysis of alpha-helical proteins from wool using cysteine labelling and mass spectrometry.

    PubMed

    O'Cualain, R D M; Sims, P F G; Carr, C M

    2011-10-01

    A simple reduction/labelling/extraction protocol has been developed to fractionate cortical matrix proteins from filament proteins in wool. Through differential labelling of cysteine residues their relative accessibility in the wool fibre has been investigated. This has allowed the preliminary development of a map of the chemical functionality that is accessible within wool fibres under native conditions. Protein analyses of wool subjected to mechanical action, wet chemical permonosulphate/sulphite treatment and dry argon plasma treatment revealed that none of these detectably improved the accessibility of functional groups at the wool cortex. It is anticipated that this analytical method can be extended to improve the sensitivity and scope with which chemical functionality within native fibres can be mapped and lead to a better understanding of the potential limits/opportunities for fibre modification.

  20. Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

    NASA Technical Reports Server (NTRS)

    Mednieks, M. I.; Popova, I.; Grindeland, R. E.

    1992-01-01

    Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

  1. A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and Cell-Cell Induced Protein Secretion.

    PubMed

    Stiess, Michael; Wegehingel, Sabine; Nguyen, Chuong; Nickel, Walter; Bradke, Frank; Cambridge, Sidney B

    2015-08-01

    Recent evidence suggests that the extracellular protein milieu is much more complex than previously assumed as various secretome analyses from different cell types described the release of hundreds to thousands of proteins. The extracellular function of many of these proteins has yet to be determined particularly in the context of three-dimensional tissues with abundant cell-cell contacts. Toward this goal, we developed a strategy of dual SILAC labeling astrocytic cultures for in silico exclusion of unlabeled proteins from serum or neurons used for stimulation. For constitutive secretion, this strategy allowed the precise quantification of the extra-to-intracellular protein ratio of more than 2000 identified proteins. Ratios covered 4 orders of magnitude indicating that the intracellular vs extracellular contributions of different proteins can be variable. Functionally, the secretome of labeled forebrain astrocytic cultures specifically changed within hours after adding unlabeled, "physiological" forebrain neurons. "Nonphysiological" cerebellar hindbrain neurons, however, elicited a different, highly repulsive secretory response. Our data also suggest a significant association of constitutive secretion with the classical secretion pathway and regulated secretion with unconventional pathways. We conclude that quantitative proteomics can help to elucidate general principles of cellular secretion and provide functional insight into the abundant extracellular presence of proteins.

  2. Genetically engineered, biarsenically labeled influenza virus allows visualization of viral NS1 protein in living cells.

    PubMed

    Li, Yang; Lu, Xinya; Li, Junwei; Bérubé, Nathalie; Giest, Kerri-Lane; Liu, Qiang; Anderson, Deborah H; Zhou, Yan

    2010-07-01

    Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication-competent fluorescent influenza A virus is that the segmented genome does not allow fusion of a fluorescent protein gene to any viral gene. Here, we introduced the tetracysteine (TC) biarsenical labeling system into influenza virus in order to fluorescently label viral protein in the virus life cycle. We generated infectious influenza A viruses bearing a small TC tag (CCPGCC) in the loop/linker regions of the NS1 proteins. In the background of A/Puerto Rico/8/34 (H1N1) (PR8) virus, the TC tag can be inserted into NS1 after amino acid 52 (AA52) (PR8-410), AA79 (PR8-412), or AA102 (PR8-413) or the TC tag can be inserted and replace amino acids 79 to 84 (AA79-84) (PR8-411). Although PR8-410, PR8-411, and PR8-412 viruses are attenuated than the wild-type (WT) virus to some extent in multiple-cycle infection, their growth potential is similar to that of the WT virus during a single cycle of infection, and their NS1 subcellular localization and viral protein synthesis rate are quite similar to those of the WT virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in fluorescent NS1 protein in the context of virus infection. We could exploit this strategy on NS1 protein of A/Texas/36/91 (H1N1) (Tx91) by successfully rescuing a TC-tagged virus, Tx91-445, which carries the TC tag replacement of AA79-84. The infectivity of Tx91-445 virus was similar to that of WT Tx91 during multiple cycles of replication and a single cycle of replication. The NS1 protein derived from Tx91-445 can be fluorescently labeled in living cells. Finally, with biarsenical labeling, the engineered replication-competent virus allowed us to visualize NS1 protein nuclear import in virus-infected cells in real time.

  3. Spin labeling studies of membrane proteins in erythrocyte ghosts from patients with Huntington's disease.

    PubMed

    Butterfield, D A; Markesbery, W R

    1983-02-21

    Previous electron spin resonance (ESR) studies employing a protein-specific spin label have suggested an alteration in membrane proteins in erythrocytes in Huntington's disease (HD). Recently, Beverstock and Pearson [4] have published the results of an ESR study not confirming our findings. In the present communication, we show that with correct use of the statistical technique of the Dixon test to determine if extreme values can be eliminated, one of the data points of these authors can be rejected. Recalculation of their results suggests a significant difference in the physical state of membrane proteins in HD erythrocytes, confirming our findings.

  4. Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins.

    PubMed

    Uttamapinant, Chayasith; Howe, Jonathan D; Lang, Kathrin; Beránek, Václav; Davis, Lloyd; Mahesh, Mohan; Barry, Nicholas P; Chin, Jason W

    2015-04-15

    Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

  5. Genetic Code Expansion Enables Live-Cell and Super-Resolution Imaging of Site-Specifically Labeled Cellular Proteins

    PubMed Central

    2015-01-01

    Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (β-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells. PMID:25831022

  6. Label-free quantitative analysis for studying the interactions between nanoparticles and plasma proteins.

    PubMed

    Capriotti, Anna Laura; Caracciolo, Giulio; Caruso, Giuseppe; Cavaliere, Chiara; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2013-01-01

    A shotgun proteomics approach was used to compare human plasma protein binding capability with cationic liposomes, DNA-cationic lipid complexes (lipoplexes), and lipid-polycation-DNA (LPD) complexes. Nano-high-performance liquid chromatography coupled with a high-resolution LTQ Orbitrap XL mass spectrometer was used to characterize and compare their protein corona. Spectral counting and area under curve methods were used to perform label-free quantification. Substantial qualitative and quantitative differences were found among proteins bound to the three different systems investigated. Protein variety found on lipoplexes and LPD complexes was richer than that found on cationic liposomes. There were also significant differences between the amounts of protein. Such results could help in the design of gene-delivery systems, because some proteins could be more selectively bound rather than others, and their bio-distribution could be driven in vivo for more efficient and effective gene therapy.

  7. Intein Applications: From Protein Purification and Labeling to Metabolic Control Methods*

    PubMed Central

    Wood, David W.; Camarero, Julio A.

    2014-01-01

    The discovery of inteins in the early 1990s opened the door to a wide variety of new technologies. Early engineered inteins from various sources allowed the development of self-cleaving affinity tags and new methods for joining protein segments through expressed protein ligation. Some applications were developed around native and engineered split inteins, which allow protein segments expressed separately to be spliced together in vitro. More recently, these early applications have been expanded and optimized through the discovery of highly efficient trans-splicing and trans-cleaving inteins. These new inteins have enabled a wide variety of applications in metabolic engineering, protein labeling, biomaterials construction, protein cyclization, and protein purification. PMID:24700459

  8. In situ immunogold labeling analysis of the rice hoja blanca virus nucleoprotein and major noncapsid protein.

    PubMed

    Espinoza, A M; Hernández, M; Pereira, R; Falk, B; Medina, V

    1992-12-01

    Ribonucleoprotein particles (RNPs) of rice hoja blanca virus (RHBV) were purified and used for electron microscopic analysis and antibody production. Antibodies made to RNPs specifically decorated purified RNPs. The RNPs typically showed characteristic tenuivirus morphologies. They were approximately 8 nm in diameter, mostly circular in nature, and exhibited branching and a high degree of superhelicity. When the RNP antibodies were used for in situ immunogold labeling analysis of RHBV-infected tissues, no specific structures were identified, but gold particles were distributed throughout the cytosol of RHBV-infected but not healthy plants. However, amorphous semi-electron opaque inclusion bodies (ASO-IBs) were abundant in cells of RHBV-infected plants. While the ASO-IBs were not labeled with the anti-RNP antiserum, they were specifically labeled with antibodies to the RHBV major noncapsid protein (NCP) and with antibodies to the NCP of another tenuivirus, maize stripe virus. PMID:1448918

  9. Effects of trichloroacetic acid on the nitrogen metabolism of Pinus sylvestris--a 13C/15N tracer study.

    PubMed

    Hafner, Christoph; Jung, Klaus; Schüürmann, Gerrit

    2002-01-01

    Trichloroacetic acid (TCA) can be found in various environmental compartments like air, rain and plants all over the world. It is assumed that TCA is an atmospheric degradation product of volatile chloroorganic hydrocarbons. The herbicide effect of TCA in higher concentrations is well known, but not much is known about the phytotoxic effects in environmentally relevant concentrations. It can be shown in this study by using the 13C/15N stable isotope tracer technique that [13C]TCA is taken up by roots of two-year-old seedlings of Pinus sylvestris L. and transported into the needles. At the same time the effect of the substance on nitrogen metabolism can be analyzed by measuring the incorporation of 15NO3- into different nitrogen fractions of the plant. The more [13C]TCA incorporation, the higher the synthesis of 15N labelled amino acids and proteins is. These effects on the nitrogen metabolism are probably based on the activation of stress- and detoxification metabolism. It has to be assumed that there is an influence on N metabolism of Pinus sylvestris caused by the deposition of environmentally relevant TCA concentrations.

  10. Labelling of endogenous target protein via N-S acyl transfer-mediated activation of N-sulfanylethylanilide.

    PubMed

    Denda, Masaya; Morisaki, Takuya; Kohiki, Taiki; Yamamoto, Jun; Sato, Kohei; Sagawa, Ikuko; Inokuma, Tsubasa; Sato, Youichi; Yamauchi, Aiko; Shigenaga, Akira; Otaka, Akira

    2016-07-14

    The ligand-dependent incorporation of a reporter molecule (e.g., fluorescence dye or biotin) onto a endogenous target protein has emerged as an important strategy for elucidating protein function using various affinity-based labelling reagents consisting of reporter, ligand and reactive units. Conventional labelling reagents generally use a weakly activated reactive unit, which can result in the non-specific labelling of proteins in a ligand-independent manner. In this context, the activation of a labelling reagent through a targeted protein-ligand interaction could potentially overcome the problems associated with conventional affinity-based labelling reagents. We hypothesized that this type of protein-ligand-interaction-mediated activation could be accomplished using N-sulfanylethylanilide (SEAlide) as the reactive unit in the labelling reagent. Electrophilically unreactive amide-type SEAlide can be activated by its conversion to the corresponding active thioester in the presence of a phosphate salt, which can act as an acid-base catalyst. It has been suggested that protein surfaces consisting of hydrophilic residues such as amino, carboxyl and imidazole groups could function as acid-base catalysts. We therefore envisioned that a SEAlide-based labelling reagent (SEAL) bearing SEAlide as a reactive unit could be activated through the binding of the SEAL with a target protein. Several SEALs were readily prepared in this study using standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase protocols. These SEAL systems were subsequently applied to the ligand-dependent labelling of human carbonic anhydrase (hCA) and cyclooxyganese 1. Although we have not yet obtained any direct evidence for the target protein-mediated activation of the SEAlide unit, our results for the reaction of these SEALs with hCA1 or butylamine indirectly support our hypothesis. The SEALs reported in this study represent valuable new entries to the field of affinity-based labelling reagents

  11. Why do total-body decay curves of iodine-labeled proteins begin with a delay

    SciTech Connect

    Regoeczi, E.

    1987-09-01

    The initial delay that occurs in total-body radiation curves reaching their single-exponential slopes was analyzed from 106 experiments involving several mammalian species (guinea pig, mouse, rabbit, and rat) and plasma proteins (alpha 1-acid glycoprotein, antithrombin III, fibrinogen, immunoglobulin G, and transferrin) in 14 different combinations. The time interval (Td) between injection and the intercept of the slope with the full-dose value was adopted as a measure of curve nonideality. The overall mean Td was 6.6 h, but individual values showed a significant correlation to protein half-lives, whereby proteins of unequal metabolic properties exhibited different mean Td values. Targeting protein to the liver abolished delay. Choice of the isotope (/sup 125/I or /sup 131/I) and size of the labeled protein had no influence on the magnitude of delay. Whole-body radiation curves of animals that received (/sup 125/I)iodotyrosines, Na/sup 131/I, or /sup 131/I-polyvinylpyrrolidone exhibited no initial delays. These results do not support the earlier notion that delay is caused by a redistribution of the labeled protein in the body to radiometrically more favorable sites. However, they are compatible with the assumption that delayed passage of a protein dose through the extracellular matrix and/or retarded transfer of proteolytic products from extravascular catabolic sites to plasma may be responsible for the phenomenon.

  12. Sparse (13)C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins.

    PubMed

    Liu, Jing; Liu, Chang; Fan, Ying; Munro, Rachel A; Ladizhansky, Vladimir; Brown, Leonid S; Wang, Shenlin

    2016-05-01

    We demonstrate a novel sparse (13)C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically (13)C labelled glycerol as carbon sources in the expression medium. This strategy improves the spectral resolution by 1.5 fold, displays site-specific labelling patterns, and has advantages for collecting long-range distance restraints for structure determination of large eukaryotic membrane proteins by solid-state NMR.

  13. Micromorphological characterization and label-free quantitation of small rubber particle protein in natural rubber latex.

    PubMed

    Wang, Sai; Liu, Jiahui; Wu, Yanxia; You, Yawen; He, Jingyi; Zhang, Jichuan; Zhang, Liqun; Dong, Yiyang

    2016-04-15

    Commercial natural rubber is traditionally supplied by Hevea brasiliensis, but now there is a big energy problem because of the limited resource and increasing demand. Intensive study of key rubber-related substances is urgently needed for further research of in vitro biosynthesis of natural rubber. Natural rubber is biosynthesized on the surface of rubber particles. A membrane protein called small rubber particle protein (SRPP) is a key protein associated closely with rubber biosynthesis; however, SRPP in different plants has been only qualitatively studied, and there are no quantitative reports so far. In this work, H. brasiliensis was chosen as a model plant. The microscopic distribution of SRPP on the rubber particles during the washing process was investigated by transmission electron microscopy-immunogold labeling. A label-free surface plasmon resonance (SPR) immunosensor was developed to quantify SRPP in H. brasiliensis for the first time. The immunosensor was then used to rapidly detect and analyze SRPP in dandelions and prickly lettuce latex samples. The label-free SPR immunosensor can be a desirable tool for rapid quantitation of the membrane protein SRPP, with excellent assay efficiency, high sensitivity, and high specificity. The method lays the foundation for further study of the functional relationship between SRPP and natural rubber content. PMID:26844871

  14. Dual-Quantum-Dots-Labeled Lateral Flow Strip Rapidly Quantifies Procalcitonin and C-reactive Protein

    NASA Astrophysics Data System (ADS)

    Qi, XiaoPing; Huang, YunYe; Lin, ZhongShi; Xu, Liang; Yu, Hao

    2016-03-01

    In the article, a dual-quantum-dots-labeled (dual-QDs-labeled) lateral flow strip (LFS) method was developed for the simultaneous and rapid quantitative detection of procalcitonin (PCT) and C-reactive protein (CRP) in the blood. Two QD-antibody conjugates with different fluorescence emission spectra were produced and sprayed on the LFS to capture PCT and CRP in the blood. Furthermore, a double antibody sandwich method for PCT and, meanwhile, a competitive inhibition method for CRP were employed in the LFS. For PCT and CRP in serum assayed by the dual-QDs-labeled LFS, their detection sensitivities reached 0.1 and 1 ng/mL, respectively, and their linear quantitative detection ranges were from 0.3 to 200 ng/mL and from 50 to 250 μg/mL, respectively. There was little evidence that the PCT and CRP assays would be interfered with each other. The correlations for testing CRP and PCT in clinical samples were 99.75 and 97.02 %, respectively, between the dual-QDs-labeled LFS we developed and commercial methods. The rapid quantification of PCT and CRP on dual-QDs-labeled LFS is of great clinical value to distinguish inflammation, bacterial infection, or viral infection and to provide guidance for the use of antibiotics or other medicines.

  15. Covalent binding of reduced metabolites of [{sup 15}N{sub 3}]TNT to soil organic matter during a bioremediation process analyzed by {sup 15}N NMR spectroscopy

    SciTech Connect

    Achtnich, C.; Fernandes, E.; Bollag, J.M.; Knackmuss, H.J.; Lenke, H.

    1999-12-15

    Evidence is presented for the covalent binding of biologically reduced metabolites of 2,4,6-{sup 15}N{sub 3}-trinitrotoluene (TNT) to different soil fractions, using liquid {sup 15}N NMR spectroscopy. A silylation procedure was used to release soil organic matter from humin and whole soil for spectroscopic measurements. TNT-contaminated soil was spiked with 2,4,6-{sup 15}N{sub 3}-trinitrotoluene and {sup 14}C-ring labeled TNT, before treatment in a soil slurry reactor. During the anaerobic/aerobic incubation the amount of radioactivity detected in the fulvic and humic acid fractions did not change significantly whereas the radioactivity bound to humin increased to 71%. The {sup 15}N NMR spectra of the fulvic acid samples were dominated by a large peak that corresponded to aliphatic amines or ammonia. In the early stages of incubation, {sup 15}N NMR analysis of the humic acids indicated bound azoxy compounds. The signals arising from nitro and azoxy groups disappeared with further anaerobic treatment. At the end of incubation, the NMR shifts showed that nitrogen was covalently bound to humic acid as substituted amines and amides. The NMR spectra of the silylated humin suggest formation of azoxy compounds and imine linkages. Bound metabolites possessing nitro groups were also detected. Primary amines formed during the anaerobic incubation disappeared during the aerobic treatment. Simultaneously, the amount of amides and tertiary amines increased. Nitro and azoxy groups of bound molecules were still present in humin at the end of the incubation period. Formation of azoxy compounds from partially reduced TNT followed by binding and further reduction appears to be an important mechanism for the immobilization of metabolites of TNT to soil.

  16. Solid-state /sup 15/N NMR of oriented lipid bilayer bound gramicidin A'

    SciTech Connect

    Nicholson, L.K.; Moll, F.; Mixon, T.E.; LoGrasso, P.V.; Lay, J.C.; Cross, T.A.

    1987-10-20

    Highly oriented samples of lipid and gramicidin A' (8:1 molar ratio) have been prepared with the samples extensively hydrated (approximately 70% water v/w). These preparations have been shown to be completely in a bilayer phase with a transition temperature of 28/sup 0/C, and evidence is presented indicating that the gramicidin is in the channel conformation. An estimate of the disorder in the alignment of the bilayers parallel with the glass plates used to align the bilayers can be made from the asymmetry of the nuclear magnetic resonances (NMR). Such an analysis indicates a maximal range of disorder of +-3/sup 0/. Uniformly /sup 15/N-labeled gramicidin has been biosynthesized by Bacillus brevis grown in a media containing /sup 15/N-labeled Escherichia coli cells as the only nitrogen source. When prepared with labeled gramicidin, the oriented samples result in high-resolution /sup 15/N NMR spectra showing 12 resonances for the 20 nitrogen sites of the polypeptide. The frequency of the three major multiple resonance peaks has been interpreted to yield the approximate orientation of the N-H bonds in the peptide linkages with respect to the magnetic field. The bond orientations are only partially consistent with the extant structural models of gramicidin.

  17. Radioactive labeling of proteins in cultured postimplantation mouse embryos. II. Dose and time dependency

    SciTech Connect

    Nowak, J.; Klose, J. )

    1989-07-01

    The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode's solution. Incubation time and concentration of ({sup 3}H (or {sup 14}C))amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 microCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions.

  18. Plasma protein binding of (99m)Tc-labeled hydrazino nicotinamide derivatized polypeptides and peptides.

    PubMed

    Ono, M; Arano, Y; Mukai, T; Uehara, T; Fujioka, Y; Ogawa, K; Namba, S; Nakayama, M; Saga, T; Konishi, J; Horiuchi, K; Yokoyama, A; Saji, H

    2001-02-01

    6-Hydrazinopyridine-3-carboxylic acid (HYNIC) constitutes one of the most attractive reagents to prepare (99m)Tc-labeled polypeptides and peptides of various molecular weights in combination with two tricine molecules as coligands. Indeed, (99m)Tc-HYNIC-conjugated IgG showed biodistribution of radioactivity similar to that of (111)In-DTPA-conjugated IgG. However, recent studies indicated significant plasma protein binding when the (99m)Tc labeling procedure was expanded to low molecular weight peptides. In this study, pharmacokinetics of (99m)Tc-HYNIC-conjugated IgG, Fab and RC160 using tricine were compared with their radioiodinated counterparts to evaluate this (99m)Tc-labeling method. In mice, [(99m)Tc](HYNIC-IgG)(tricine)(2) and [(99m)Tc](HYNIC-Fab)(tricine)(2) showed persistent localization of radioactivity in tissues when compared with their (125)I-labeled counterparts. [(99m)Tc](HYNIC-IgG)(tricine)(2) eliminated from the blood at a rate similar to that of (125)I-labeled IgG, while [(99m)Tc](HYNIC-Fab)(tricine)(2) showed significantly slower clearance of the radioactivity than (125)I-labeled Fab. On size-exclusion HPLC analyses, little changes were observed in radiochromatograms after incubation of [(99m)Tc](HYNIC-IgG)(tricine)(2) in murine plasma. However, [(99m)Tc](HYNIC-Fab)(tricine)(2) and [(99m)Tc](HYNIC-RC160)(tricine)(2) demonstrated significant increases in the radioactivity in higher molecular weight fractions in plasma. Formation of higher molecular weight species was reduced when [(99m)Tc](HYNIC-RC160)(tricine)(2) was stabilized with nicotinic acid (NIC) to generate [(99m)Tc](HYNIC-RC160)(tricine)(NIC). [(99m)Tc](HYNIC-RC160)(tricine)(NIC) also demonstrated significantly faster clearance of the radioactivity from the blood than [(99m)Tc](HYNIC-RC160)(tricine)(2). These findings suggested that one of the tricine coligands in (99m)Tc-HYNIC-labeled (poly)peptides would be replaced with plasma proteins to generate higher molecular weight species that

  19. A New Strategy of Using O18-Labeled Iodoacetic Acid for Mass Spectrometry-Based Protein Quantitation

    NASA Astrophysics Data System (ADS)

    Wang, Shunhai; Kaltashov, Igor A.

    2012-07-01

    A new O18 labeling protocol is designed to assist quantitation of cysteine-containing proteins using LC/MS. Unlike other O18 labeling strategies, the labeling is carried out at the intact protein level (prior to its digestion) during reduction/alkylation of cysteine side chains using O18-labeled iodoacetic acid (IAA). The latter can be easily prepared by exchanging carboxylic oxygen atoms of commercially available IAA in O18-enriched water at low pH. Since incorporation of the O18 label in the protein occurs at the whole protein, rather than peptide level, the quantitation results are not peptide-dependent. The excellent stability of the label in mild pH conditions provides flexibility and robustness needed of sample processing steps following the labeling. In contrast to generally costly isotope labeling reagents, this approach uses only two relatively inexpensive commercially available reagents (IAA and H2O18). The feasibility of the new method is demonstrated using an 80 kDa human serum transferrin (hTf) as a model, where linear quantitation is achieved across a dynamic range spanning three orders of magnitude. The new approach can be used in quantitative proteomics applications and is particularly suitable for a variety of tasks in the biopharmaceutical sector, ranging from pharmacokinetic studies to quality control of protein therapeutics.

  20. Polyglutaraldehyde - A new reagent for coupling proteins to microspheres and for labeling cell-surface receptors

    NASA Technical Reports Server (NTRS)

    Rembaum, A.; Levy, J.; Margel, S.

    1978-01-01

    Glutaraldehyde polymerized in basic aqueous solutions was found to react with low molecular weight amines, immunoglobulins and hemoglobin. The polyglutaraldehyde was covalently bound to hydrophilic microspheres. The rate of addition of proteins to the polyglutaraldehyde-derivatized microspheres was investigated spectrophotometrically as a function of pH and temperature. The reaction of polyglutaraldehyde was found to be faster than that of the monomer. The findings led to successful labeling of human lymphocyte subpopulations.

  1. Label-free detection of differential protein expression by LC/MALDI mass spectrometry.

    PubMed

    Neubert, Hendrik; Bonnert, Timothy P; Rumpel, Klaus; Hunt, Brandon T; Henle, Ernst S; James, Ian T

    2008-06-01

    Protein abundance changes during disease or experimental perturbation are increasingly analyzed by label-free LC/MS approaches. Here we demonstrate the use of LC/MALDI MS for label-free detection of protein expression differences using Escherichia coli cultures grown on arabinose, fructose or glucose as a carbon source. The advantages of MALDI, such as detection of only singly charged ions, and MALDI plate archiving to facilitate retrospective MS/MS data collection are illustrated. MALDI spectra from RP chromatography of tryptic digests of the E. coli lysates were aligned and quantitated using the Rosetta Elucidator system. Approximately 5000 peptide signals were detected in all LC/MALDI runs spanning over 3 orders of magnitude of signal intensity. The average coefficients of variation for all signals across the entire intensity range in all technical replicates were found to be <25%. Pearson correlation coefficients from 0.93 to 0.98 for pairwise comparisons illustrate high replicate reproducibility. Expression differences determined by Analysis of Variance highlighted over 500 isotope clusters ( p < 0.01), which represented candidates for targeted peptide identification using MS/MS. Biologically interpretable protein identifications that could be derived underpin the general utility of this label-free LC/MALDI strategy.

  2. Pulsed EPR Distance Measurements in Soluble Proteins by Site-directed Spin-labeling (SDSL)

    PubMed Central

    de Vera, Ian Mitchelle S.; Blackburn, Mandy E.; Galiano, Luis; Fanucci, Gail E.

    2015-01-01

    The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electron-electron resonance (DEER) technique. This unit focuses on EPR-based distance measurements by site-directed spin-labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV-1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance distribution profiles. Experimental considerations, sample preparation and error analysis for artifact suppression are also outlined here. PMID:24510645

  3. Lipid-protein interactions with cardiac phospholamban studied by spin-label electron spin resonance.

    PubMed

    Arora, Ashish; Williamson, Ian M; Lee, Anthony G; Marsh, Derek

    2003-05-01

    Phospholamban is a cardiac regulatory protein that, in its monomeric form, inhibits the Ca(2+)-ATPase. Lipid-protein interactions with a synthetic variant of phospholamban, in which all cysteine residues are replaced with alanine, have been studied by spin-label electron spin resonance (ESR) in different lipid host membranes. Both the stoichiometry and selectivity of lipid interactions were determined from the two-component ESR spectra of phospholipid species spin-labeled on the 14 C atom of the sn-2 chain. The lipid stoichiometry is determined by the oligomeric state of the protein and the selectivity by the membrane disposition of the positively charged residues in the N-terminal section of the protein. In dimyristoylphosphatidylcholine (DMPC) membranes, the stoichiometry (N(b)) is 7 lipids/monomer for the full-length protein and 4 for the transmembrane section (residues 26-52). These stoichiometries correspond to the dimeric and pentameric forms, respectively. In palmitoyloleoylphosphatidylcholine, N(b) = 4 for both the whole protein and the transmembrane peptide. In negatively charged membranes of dimyristoylphosphatidylglycerol (DMPG), the lipid stoichiometry is N(b) = 10-11 per monomer for both the full-length protein and the transmembrane peptide. This stoichiometry corresponds to monomeric dispersion of the protein in the negatively charged lipid. The sequence of lipid selectivity is as follows: stearic acid > phosphatidic acid > phosphatidylserine = phosphatidylglycerol = phosphatidylcholine > phosphatidylethanolamine for both the full-length protein and the transmembrane peptide in DMPC. Absolute selectivities are, however, lower for the transmembrane peptide. A similar pattern of lipid selectivity is obtained in DMPG, but the absolute selectivities are reduced considerably. The results are discussed in terms of the integration of the regulatory species in the lipid membrane. PMID:12718559

  4. Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells.

    PubMed

    Naganbabu, Matharishwan; Perkins, Lydia A; Wang, Yi; Kurish, Jeffery; Schmidt, Brigitte F; Bruchez, Marcel P

    2016-06-15

    Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics. PMID:27159569

  5. Stable isotope probing with 15N achieved by disentangling the effects of genome G+C content and isotope enrichment on DNA density.

    PubMed

    Buckley, Daniel H; Huangyutitham, Varisa; Hsu, Shi-Fang; Nelson, Tyrrell A

    2007-05-01

    Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with 15N, nearly all applications of this technique to date have used 13C. Successful application of SIP using 15N-DNA (15N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml-1 for 15N-labeled DNA, relative to 0.036 g ml-1 for 13C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml-1. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of 15N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate 15N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of "heavy" DNA from primary CsCl density gradients followed by purification of 15N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use 15N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in 13C-DNA-SIP applications.

  6. Stable Isotope Probing with 15N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density▿ †

    PubMed Central

    Buckley, Daniel H.; Huangyutitham, Varisa; Hsu, Shi-Fang; Nelson, Tyrrell A.

    2007-01-01

    Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with 15N, nearly all applications of this technique to date have used 13C. Successful application of SIP using 15N-DNA (15N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml−1 for 15N-labeled DNA, relative to 0.036 g ml−1 for 13C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml−1. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of 15N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate 15N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of 15N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use 15N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in 13C-DNA-SIP applications. PMID:17369331

  7. Label-Free Quantitative Mass Spectrometry Reveals a Panel of Differentially Expressed Proteins in Colorectal Cancer

    PubMed Central

    Fan, Nai-Jun; Gao, Jiang-Ling; Liu, Yan; Song, Wei; Zhang, Zhan-Yang; Gao, Chun-Fang

    2015-01-01

    To identify potential biomarkers involved in CRC, a shotgun proteomic method was applied to identify soluble proteins in three CRCs and matched normal mucosal tissues using high-performance liquid chromatography and mass spectrometry. Label-free protein profiling of three CRCs and matched normal mucosal tissues were then conducted to quantify and compare proteins. Results showed that 67 of the 784 identified proteins were linked to CRC (28 upregulated and 39 downregulated). Gene Ontology and DAVID databases were searched to identify the location and function of differential proteins that were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, and so on. Among the differentially expressed proteins, tropomyosin-3 (TPM3), endoplasmic reticulum resident protein 29 (ERp29), 18 kDa cationic antimicrobial protein (CAMP), and heat shock 70 kDa protein 8 (HSPA8) were verified to be upregulated in CRC tissue and seven cell lines through western blot analysis. Furthermore, the upregulation of TPM3, ERp29, CAMP, and HSPA8 was validated in 69 CRCs byimmunohistochemistry (IHC) analysis. Combination of TPM3, ERp29, CAMP, and HSPA8 can identify CRC from matched normal mucosal achieving an accuracy of 73.2% using IHC score. These results suggest that TPM3, ERp29, CAMP, and HSPA8 are great potential IHC diagnostic biomarkers for CRC. PMID:25699276

  8. Juvenile hormone-binding proteins of Melanoplus bivittatus identified by EFDA photoaffinity labeling

    SciTech Connect

    Winder, B.S.

    1988-01-01

    Proteins that bind juvenile hormone in the hemolymph and fat body of the grasshopper, Melanoplus bivittatus were identified by photoaffinity labeling with radiolabeled epoxyfarnesyl diazoacetate ({sup 3}H-EFDA), and were characterized by electrophoretic analysis. A protocol was developed which allowed detection of {sup 3}H-EFDA that was covalently linked to proteins upon exposure to ultraviolet light at 254 nm. Quantification of protein-linked {sup 3}H-EFDA by liquid scintillation spectrometry took advantage of the differential solubility of unlinked {sup 3}H-EFDA in toluene alone, and of the protein-linked {sup 3}H-EFDA in toluene plus the detergent, Triton X-100. Competition between EFDA and juvenile hormone (JH) for binding to JH-specific binding sites was measured by hydroxyapatite protein binding assays in the presence of radiolabeled JH or EFDA and competing non-radiolabeled hormone. The protein-linked EFDA was detected on fluorograms of SDS or nondenaturing polyacrylamide gels (PAGE), and by liquid scintillation spectrometry of membranes to which the proteins had been electrophoretically transferred. Proteins which specifically bound JH were identified by photolabeling proteins in the presence and absence of nonlabeled JH-III.

  9. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier.

    PubMed

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-01-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics. PMID:27323846

  10. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier

    NASA Astrophysics Data System (ADS)

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-06-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics.

  11. The whispering gallery mode biosensor: label-free detection from virus to single protein

    NASA Astrophysics Data System (ADS)

    Holler, S.; Dantham, V. R.; Keng, D.; Kolchenko, V.; Arnold, S.; Mulroe, Brigid; Paspaley-Grbavac, M.

    2014-08-01

    The whispering gallery mode (WGM) biosensor is a micro-optical platform capable of sensitive label-free detection of biological particles. Described by the reactive sensing principle (RSP), this analytic formulation quantifies the response of the system to the adsorption of bioparticles. Guided by the RSP, the WGM biosensor enabling from detection of virus (e.g., Human Papillomavirus, HPV) to the ultimate goal of single protein detection. The latter was derived from insights into the RSP, which resulted in the development of a hybrid plasmonic WGM biosensor, which has recently demonstrated detection of individual protein cancer markers. Enhancements from bound gold nanoparticles provide the sensitivity to detect single protein molecules (66 kDa) with good signal-to-noise (S/N > 10), and project that detection of proteins as small as 5 kDa.

  12. Retrieving nitrogen isotopic signatures from fresh leaf reflectance spectra: disentangling δ(15)N from biochemical and structural leaf properties.

    PubMed

    Hellmann, Christine; Große-Stoltenberg, André; Lauströ, Verena; Oldeland, Jens; Werner, Christiane

    2015-01-01

    Linking remote sensing methodology to stable isotope ecology provides a promising approach to study ecological processes from small to large spatial scales. Here, we show that δ(15)N can be detected in fresh leaf reflectance spectra of field samples along a spatial gradient of increasing nitrogen input from an N2-fixing invasive species. However, in field data it is unclear whether δ(15)N directly influences leaf reflectance spectra or if the relationship is based on covariation between δ(15)N and foliar nitrogen content or other leaf properties. Using a (15)N-labeling approach, we experimentally varied δ(15)N independently of any other leaf properties in three plant species across different leaf developmental and physiological states. δ(15)N could successfully be modeled by means of partial least squares (PLSs) regressions, using leaf reflectance spectra as predictor variables. PLS models explained 53-73% of the variation in δ(15)N within species. Several wavelength regions important for predicting δ(15)N were consistent across species and could furthermore be related to known absorption features of N-containing molecular bonds. By eliminating covariation with other leaf properties as an explanation for the relationship between reflectance and δ(15)N, our results demonstrate that (15)N itself has an inherent effect on leaf reflectance spectra. Thus, our study substantiates the use of spectroscopic measurements to retrieve isotopic signatures for ecological studies and encourages future development. Furthermore, our results highlight the great potential of optical measurements for up-scaling isotope ecology to larger spatial scales.

  13. Retrieving nitrogen isotopic signatures from fresh leaf reflectance spectra: disentangling δ15N from biochemical and structural leaf properties

    PubMed Central

    Hellmann, Christine; Große-Stoltenberg, André; Lauströ, Verena; Oldeland, Jens; Werner, Christiane

    2015-01-01

    Linking remote sensing methodology to stable isotope ecology provides a promising approach to study ecological processes from small to large spatial scales. Here, we show that δ15N can be detected in fresh leaf reflectance spectra of field samples along a spatial gradient of increasing nitrogen input from an N2-fixing invasive species. However, in field data it is unclear whether δ15N directly influences leaf reflectance spectra or if the relationship is based on covariation between δ15N and foliar nitrogen content or other leaf properties. Using a 15N-labeling approach, we experimentally varied δ15N independently of any other leaf properties in three plant species across different leaf developmental and physiological states. δ15N could successfully be modeled by means of partial least squares (PLSs) regressions, using leaf reflectance spectra as predictor variables. PLS models explained 53–73% of the variation in δ15N within species. Several wavelength regions important for predicting δ15N were consistent across species and could furthermore be related to known absorption features of N-containing molecular bonds. By eliminating covariation with other leaf properties as an explanation for the relationship between reflectance and δ15N, our results demonstrate that 15N itself has an inherent effect on leaf reflectance spectra. Thus, our study substantiates the use of spectroscopic measurements to retrieve isotopic signatures for ecological studies and encourages future development. Furthermore, our results highlight the great potential of optical measurements for up-scaling isotope ecology to larger spatial scales. PMID:25983740

  14. Nitrate removal in two relict oxbow urban wetlands: a 15N mass-balance approach

    NASA Astrophysics Data System (ADS)

    Harrison, M. D.; Groffman, P. M.; Mayer, P. M.; Kaushal, S.

    2012-12-01

    A mass-balance approach was used to directly determine the flow of 15NO3- to plants, algae, and sediments,with unaccounted for 15N assumed to be denitrified. During the summer, plant and algal uptake accounted for 42%, of the added 15NO3 - in oxbow 1, less than 1% remained in the water column and 57% was unaccounted for. In oxbow 2 during the summer, plant and algal uptake accounted for 63% of the added 15NO3 -, with 1% remaining in the water column and 38% unaccounted for. During the early spring, plant and algal uptake were much lower in both oxbows, ranging from 0.05 to 13.3% of the 15N added, with 97 and 87% was unaccounted for in oxbow 1 and 2, respectively. The amount of unaccounted for 15N was equivalent to estimated areal denitrification rates of 12 and 6 mg N m-2 d-1 in the summer and 78 and 15 mg N m-2 d-1 in the spring, in oxbow 1 and oxbow 2, respectively. However, the uncertainty of these estimates is high as it was difficult to detect accumulation of 15N in the sediments which could have accounted for a very large percentage of the added 15N. Our results suggest that the two relict oxbow wetlands are sinks for NO3 - during both summer and spring. Plane view of Ox1 (A) and Ox2 (B) wetlands with closed contour intervals (color scale) and surrounding stream and upland elevations (labeled in black) located at Minebank Run, near Glen Arms, MD. 15N enrichment (atom %) of measured N pools prior to (Day 0) and after (Day 5) the end of the experiment in July 2009 and April 2010 for Ox1 and Ox2. Values are mean atom % (n = 2 algae, macrophytes and sediment; n = 6 for water samples).

  15. Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence

    SciTech Connect

    Coleman, Bradley M.; Nisbet, Rebecca M.; Han, Sen; Cappai, Roberto; Hatters, Danny M.; Hill, Andrew F.

    2009-03-13

    Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP{sup C}) into a disease associated form (PrP{sup Sc}). Recombinant PrP can be refolded into either an {alpha}-helical rich conformation ({alpha}-PrP) resembling PrP{sup C} or a {beta}-sheet rich, protease resistant form similar to PrP{sup Sc}. Here, we generated tetracysteine tagged recombinant PrP, folded this into {alpha}- or {beta}-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished {beta}-PrP from {alpha}-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the {alpha}-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the {beta}-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP{sup Sc} from PrP{sup C}. This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer's, Huntington's and Parkinson's diseases.

  16. Sequence-specific labeling of nucleic acids and proteins with methyltransferases and cofactor analogues.

    PubMed

    Hanz, Gisela Maria; Jung, Britta; Giesbertz, Anna; Juhasz, Matyas; Weinhold, Elmar

    2014-11-22

    S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5'-ATCGAT-3' sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as

  17. Accurate measurements of {sup 13}C-{sup 13}C distances in uniformly {sup 13}C-labeled proteins using multi-dimensional four-oscillating field solid-state NMR spectroscopy

    SciTech Connect

    Straasø, Lasse Arnt; Nielsen, Jakob Toudahl; Bjerring, Morten; Nielsen, Niels Chr.; Khaneja, Navin

    2014-09-21

    Application of sets of {sup 13}C-{sup 13}C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important {sup 13}C-{sup 13}C distances in uniformly {sup 13}C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl ({sup 13}C′) and aliphatic ({sup 13}C{sub aliphatic}) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys. 130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly {sup 13}C,{sup 15}N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of {sup 13}C′-{sup 13}C{sub aliphatic} distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform {sup 13}C,{sup 15}N-labeling on the FGAIL fragment.

  18. Emerging tools for real-time label-free detection of interactions on functional protein microarrays.

    PubMed

    Ramachandran, Niroshan; Larson, Dale N; Stark, Peter R H; Hainsworth, Eugenie; LaBaer, Joshua

    2005-11-01

    The availability of extensive genomic information and content has spawned an era of high-throughput screening that is generating large sets of functional genomic data. In particular, the need to understand the biochemical wiring within a cell has introduced novel approaches to map the intricate networks of biological interactions arising from the interactions of proteins. The current technologies for assaying protein interactions--yeast two-hybrid and immunoprecipitation with mass spectrometric detection--have met with considerable success. However, the parallel use of these approaches has identified only a small fraction of physiologically relevant interactions among proteins, neglecting all nonprotein interactions, such as with metabolites, lipids, DNA and small molecules. This highlights the need for further development of proteome scale technologies that enable the study of protein function. Here we discuss recent advances in high-throughput technologies for displaying proteins on functional protein microarrays and the real-time label-free detection of interactions using probes of the local index of refraction, carbon nanotubes and nanowires, or microelectromechanical systems cantilevers. The combination of these technologies will facilitate the large-scale study of protein interactions with proteins as well as with other biomolecules.

  19. Meet the neighbors: Mapping local protein interactomes by proximity‐dependent labeling with BioID

    PubMed Central

    Varnaitė, Renata

    2016-01-01

    Proximity‐dependent biotin identification (BioID) is a recently developed method that allows the identification of proteins in the close vicinity of a protein of interest in living cells. BioID relies on fusion of the protein of interest with a mutant form of the biotin ligase enzyme BirA (BirA*) that is capable of promiscuously biotinylating proximal proteins irrespective of whether these interact directly or indirectly with the fusion protein or are merely located in the same subcellular neighborhood. The covalent addition of biotin allows the labeled proteins to be purified from cell extracts on the basis of their affinity for streptavidin and identified by mass spectrometry. To date, BioID has been successfully applied to study a variety of proteins and processes in mammalian cells and unicellular eukaryotes and has been shown to be particularly suited to the study of insoluble or inaccessible cellular structures and for detecting weak or transient protein associations. Here, we provide an introduction to BioID, together with a detailed summary of where and how the method has been applied to date, and briefly discuss technical aspects involved in the planning and execution of a BioID study. PMID:27329485

  20. Isotope labeling for NMR studies of macromolecular structure and interactions

    SciTech Connect

    Wright, P.E.

    1994-12-01

    Implementation of biosynthetic methods for uniform or specific isotope labeling of proteins, coupled with the recent development of powerful heteronuclear multidimensional NMR methods, has led to a dramatic increase in the size and complexity of macromolecular systems that are now amenable to NMR structural analysis. In recent years, a new technology has emerged that combines uniform {sup 13}C, {sup 15}N labeling with heteronuclear multidimensional NMR methods to allow NMR structural studies of systems approaching 25 to 30 kDa in molecular weight. In addition, with the introduction of specific {sup 13}C and {sup 15}N labels into ligands, meaningful NMR studies of complexes of even higher molecular weight have become feasible. These advances usher in a new era in which the earlier, rather stringent molecular weight limitations have been greatly surpassed and NMR can begin to address many central biological problems that involve macromolecular structure, dynamics, and interactions.

  1. Selected reaction monitoring (SRM) mass spectrometry without isotope labeling can be used for rapid protein quantification.

    PubMed

    Zhi, Wenbo; Wang, Meiyao; She, Jin-Xiong

    2011-06-15

    The validation of putative biomarker candidates has become the major bottle-neck in protein biomarker development. Conventional immunoaffinity methods are limited by the availability of antibodies and kits. Here we demonstrate the feasibility of using selected reaction monitoring (SRM) without isotope labeling to achieve fast and reproducible quantification of serum proteins. The SRM/MRM assays for three standard serum proteins, including ceruloplasmin (CP), serum aymloid A (SAA) and sex hormone binding globulin (SHBG), have good linear ranges, generally 10(3) to 10(4) . There are almost perfect correlations between SRM intensities and the loaded peptide amounts (R(2) is usually ~0.99). Our data suggest that SRM/MRM is able to quantify proteins within the range of 0.2-2 fmol, which is comparable to the commercial ELISA/LUMINEX kits for these proteins. Excellent correlations between SRM/MRM and ELISA/LUMINEX assays were observed for SAA and SHBG (R(2)=0.928 and 0.851, respectively). However, the correlation between SRM/MRM and ELISA for CP is less desirable (R(2)=0.565). The reproducibility for SRM/MRM assays is generally very good but may depend on the proteins/peptides being analyzed (R(2)=0.931 and 0.882 for SAA and SHBG, and 0.723 for CP). The SRM/MRM assay without isotope labeling is a rapid and useful method for protein biomarker validation in a modest number of samples and is especially useful when other assays such as ELISA or LUMINEX are not available. PMID:21594933

  2. Analysis of saturation transfer electron paramagnetic resonance spectra of a spin-labeled integral membrane protein, band 3, in terms of the uniaxial rotational diffusion model.

    PubMed Central

    Hustedt, E J; Beth, A H

    1995-01-01

    Algorithms have been developed for the calculation of saturation transfer electron paramagnetic resonance (ST-EPR) spectra of a nitroxide spin-label assuming uniaxial rotational diffusion, a model that is frequently used to describe the global rotational dynamics of large integral membrane proteins. One algorithm explicitly includes terms describing Zeeman overmodulation effects, whereas the second more rapid algorithm treats these effects approximately using modified electron spin-lattice and spin-spin relaxation times. Simulations are presented to demonstrate the sensitivity of X-band ST-EPR spectra to the rate of uniaxial rotational diffusion and the orientation of the nitroxide probe with respect to the diffusion axis. Results obtained by using the algorithms presented, which are based on the transition-rate formalism, are in close agreement with those obtained by using an eigenfunction expansion approach. The effects of various approximations used in the simulation algorithms are considered in detail. Optimizing the transition-rate formalism to model uniaxial rotational diffusion results in over an order of magnitude reduction in computation time while allowing treatment of nonaxial A- and g-tensors. The algorithms presented here are used to perform nonlinear least-squares analyses of ST-EPR spectra of the anion exchange protein of the human erythrocyte membrane, band 3, which has been affinity spin-labeled with a recently developed dihydrostilbene disulfonate derivative, [15N,2H13]-SL-H2DADS-MAL. These results suggest that all copies of band 3 present in intact erythrocytes undergo rotational diffusion about the membrane normal axis at a rate consistent with a band 3 dimer. PMID:8534811

  3. Homonuclear dipolar recoupling techniques for structure determination in uniformly 13C-labeled proteins.

    PubMed

    Ladizhansky, Vladimir

    2009-11-01

    In solid-state NMR magic angle spinning is often used to remove line broadening associated with anisotropic interactions, such as chemical shift anisotropy and dipolar couplings. Dipolar recoupling refers to sequences of pulses designed to reintroduce dipolar interactions that are otherwise averaged by magic angle spinning. One of the key applications of homonuclear (and heteronuclear) dipolar recoupling is for the purpose of protein structure determination. Recoupling experiments, originally designed for applications in spin-pair labeled samples, have been revised in recent years for applications in samples with extensive or uniform incorporation of isotopic labels. In these samples multiple internuclear distances can in principle be probed simultaneously, but the dipolar truncation effects (i.e. attenuation of the effects of weak couplings by strong ones) circumvent such measurements. In this article we review some of the recent developments in homonuclear recoupling methods that allow overcoming this problem.

  4. (15)N- and (2)H proteomic stable isotope probing links nitrogen flow to archaeal heterotrophic activity.

    PubMed

    Justice, Nicholas B; Li, Zhou; Wang, Yingfeng; Spaudling, Susan E; Mosier, Annika C; Hettich, Robert L; Pan, Chongle; Banfield, Jillian F

    2014-10-01

    Understanding how individual species contribute to nutrient transformations in a microbial community is critical to prediction of overall ecosystem function. We conducted microcosm experiments in which floating acid mine drainage (AMD) microbial biofilms were submerged - recapitulating the final stage in a natural biofilm life cycle. Biofilms were amended with either (15)NH4(+) or deuterium oxide ((2)H2O) and proteomic stable isotope probing (SIP) was used to track the extent to which different members of the community used these molecules in protein synthesis across anaerobic iron-reducing, aerobic iron-reducing and aerobic iron-oxidizing environments. Sulfobacillus spp. synthesized (15)N-enriched protein almost exclusively under iron-reducing conditions whereas the Leptospirillum spp. synthesized (15)N-enriched protein in all conditions. There were relatively few (15)N-enriched archaeal proteins, and all showed low atom% enrichment, consistent with Archaea synthesizing protein using the predominantly (14)N biomass derived from recycled biomolecules. In parallel experiments using (2)H2O, extensive archaeal protein synthesis was detected in all conditions. In contrast, the bacterial species showed little protein synthesis using (2)H2O. The nearly exclusive ability of Archaea to synthesize proteins using (2)H2O may be due to archaeal heterotrophy, whereby Archaea offset deleterious effects of (2)H by accessing (1)H generated by respiration of organic compounds.

  5. Nanowire biosensors for label-free, real-time, ultrasensitive protein detection.

    PubMed

    Zheng, Gengfeng; Lieber, Charles M

    2011-01-01

    Sensitive and quantitative analysis of proteins is central to disease diagnosis, drug screening, and proteomic studies. Among recent research advances exploiting new nanomaterials for biomolecule analysis, silicon nanowires (SiNWs), which are configured as field-effect transistors (FETs), have emerged as one of the most promising and powerful platforms for label-free, real-time, and highly sensitive electrical detection of proteins as well as many other biological species. Here, we describe a detailed protocol for realizing SiNW biosensors for protein detection that includes SiNW synthesis, FET device fabrication, surface receptor functionalization, and electrical sensing measurements. Moreover, incorporating both p-type and n-type SiNWs in the same sensor array provides a unique means of internal control for sensing signal verification. PMID:21948419

  6. Label-free protein assay based on a nanomechanical cantilever array

    NASA Astrophysics Data System (ADS)

    Arntz, Y.; Seelig, J. D.; Lang, H. P.; Zhang, J.; Hunziker, P.; Ramseyer, J. P.; Meyer, E.; Hegner, M.; Gerber, Ch

    2003-01-01

    We demonstrate continuous label-free detection of two cardiac biomarker proteins (creatin kinase and myoglobin) using an array of microfabricated cantilevers functionalized with covalently anchored anti-creatin kinase and anti-myoglobin antibodies. This method allows biomarker proteins to be detected via measurement of surface stress generated by antigen-antibody molecular recognition. Reference cantilevers are used to eliminate thermal drifts, undesired chemical reactions and turbulences from injections of liquids by calculating differential deflection signals with respect to sensor cantilevers. The sensitivity achieved for myoglobin detection is below 20 µg ml-1. Both myoglobin and creatin kinase could be detected independently using cantilevers functionalized with the corresponding antibodies, in unspecific protein background. This approach permits the use of up to seven different antigen-antibody reactions simultaneously, including an additional thermomechanical and chemical in situ reference. Applications lie in the field of early and rapid diagnosis of acute myocardial infarction.

  7. Recognition-induced covalent capturing and labeling as a general strategy for protein detection.

    PubMed

    Li, Hao; Huang, Yue; Yu, Yue; Wang, Yao; Li, Genxi

    2016-06-15

    In this work we have developed a peptide-based method for protein detection, termed as "Recognition-induced Covalent Capturing and Labeling" (RCCL). In this method, upon binding of the peptide with the target protein, electrochemically controlled and metal catalyzed oxidative cross-linking can be induced between the peptide and the target protein. Specifically, the peptide and the target protein are cross-linked by the formation of dityrosine between tyrosine moieties of the two molecules. Meanwhile, the dityrosine formed in this manner also has fluorescent signal readout. Therefore, the proposed method needs only one probe for the target protein, and the initial non-covalent molecular recognition can be finalized by cross-linking between the peptide and the target, while the dityrosine formed between peptide and protein can also act as a signal reporter, thereby greatly simplifying the design. Moreover, the robust covalent capturing via RCCL also enables detection in complex biological and clinical samples. These results point to the prospect of using RCCL as a promising method in protein detection in the future.

  8. Topography of Lipid Droplet-Associated Proteins: Insights from Freeze-Fracture Replica Immunogold Labeling

    PubMed Central

    Robenek, Horst; Buers, Insa; Robenek, Mirko J.; Hofnagel, Oliver; Ruebel, Anneke; Troyer, David; Severs, Nicholas J.

    2011-01-01

    Lipid droplets are not merely storage depots for superfluous intracellular lipids in times of hyperlipidemic stress, but metabolically active organelles involved in cellular homeostasis. Our concepts on the metabolic functions of lipid droplets have come from studies on lipid droplet-associated proteins. This realization has made the study of proteins, such as PAT family proteins, caveolins, and several others that are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry. Our existing understanding of the structure, protein organization, and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. Freeze-fracture replica immunogold labeling overcomes these disadvantages and can be used to define at high resolution the precise location of lipid droplet-associated proteins. In this paper illustrative examples of how freeze-fracture immunocytochemistry has contributed to our understanding of the spatial organization in the membrane plane and function of PAT family proteins and caveolin-1 are presented. By revisiting the lipid droplet with freeze-fracture immunocytochemistry, new perspectives have emerged which challenge prevailing concepts of lipid droplet biology and may hopefully provide a timely impulse for many ongoing studies. PMID:21490801

  9. Low microwave-amplitude ESR spectroscopy: measuring spin-relaxation interactions of moderately immobilized spin labels in proteins.

    PubMed

    Hedin, Eva M K; Hult, Karl; Mouritsen, Ole G; Høyrup, Pernille

    2004-08-31

    Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool for determining protein structure, dynamics and interactions. We report here a method for determining interactions between spin labels and paramagnetic relaxation agents, which is performed under subsaturating conditions. The low microwave-field amplitude employed (h(1)<0.36 G) only requires standard, commercially available ESR equipment. The effect of relaxation enhancement on the spin-spin-relaxation time, T(2e), is measured by this method, and compared to classical progressive power saturation performed on a free spin label, (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl)methanethiosulfonate (MTSL), and a spin-labeled protein (Thermomyces lanuginosa lipase, TLL-I252C), employing the water-soluble relaxation agent chromium(III) oxalate (Crox) in concentrations between 0-10 mM. The low-amplitude theory showed excellent agreement with that of classical power saturation in quantifying Crox-induced relaxation enhancement. Low-amplitude measurements were then performed using a standard resonator, with Crox, on 11 spin-labeled TLL mutants displaying rotational correlation times in the motional narrowing regime. All spin-labeled proteins exhibited significant changes in T(2e). We postulate that this novel method is especially suitable for studying moderately immobilized spin labels, such as those positioned at exposed sites in a protein. This method should prove useful for research groups with access to any ESR instrumentation.

  10. Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins.

    PubMed

    Mas, Guillaume; Crublet, Elodie; Hamelin, Olivier; Gans, Pierre; Boisbouvier, Jérôme

    2013-11-01

    The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D2O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d10. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.

  11. Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers.

    PubMed

    Parkinson, Erika; Boyd, Pete; Aleksic, Maja; Cubberley, Richard; O'Connor, David; Skipp, Paul

    2014-11-01

    The risk of contact sensitization is a major consideration in the development of new formulations for personal care products. However, developing a mechanistic approach for non-animal risk assessment requires further understanding of haptenation of skin proteins by sensitizing chemicals, which is the molecular initiating event causative of skin sensitization. The non-stoichiometric nature of protein haptenation results in relatively low levels of modification, often of low abundant proteins, presenting a major challenge for their assignment in complex biological matrices such as skin. Instrumental advances over the last few years have led to a considerable increase in sensitivity of mass spectrometry (MS) techniques. We have combined these advancements with a novel dual-labeling/LC-MS(E) approach to provide an in-depth direct comparison of human serum albumin (HSA), 2,4-dinitro-1-chlorobenzene (DNCB), 5-chloro-2-methyl-4-isothiazolin-3-one (MCI), trans-cinnamaldehyde, and 6-methyl coumarin. These data have revealed novel insights into the differences in protein haptenation between sensitizers with different reaction mechanisms and sensitizing potency; the extreme sensitizers DNCB and MCI were shown to modify a greater number of nucleophilic sites than the moderate sensitizer cinnamaldehyde; and the weak/non-sensitizer 6-methyl coumarin was restricted to only a single nucleophilic residue within HSA. The evaluation of this dual labeling/LC-MS(E) approach using HSA as a model protein has also demonstrated that this strategy could be applied to studying global haptenation in complex mixtures of skin-related proteins by different chemicals.

  12. Characterization of [3H]palmitate- and [3H]ethanolamine-labelled proteins in the multicellular parasitic trematode Schistosoma mansoni.

    PubMed Central

    Wiest, P M; Tisdale, E J; Roberts, W L; Rosenberry, T L; Mahmoud, A A; Tartakoff, A M

    1988-01-01

    Biosynthetic labelling experiments with cercariae and schistosomula of the multicellular parasitic trematode Schistosoma mansoni were performed to determine whether [3H]palmitate or [3H]ethanolamine was incorporated into proteins. Parasites incorporated [3H]palmitate into numerous proteins, as judged by SDS/polyacrylamide-gel electrophoresis and fluorography. The radiolabel was resistant to extraction with chloroform, but sensitive to alkaline hydrolysis, indicating the presence of an ester bond. Further investigation of the major 22 kDa [3H]palmitate-labelled species showed that the label could be recovered in a Pronase fragment which bound detergent and had an apparent molecular mass of 1200 Da as determined by gel filtration on Sephadex LH-20. Schistosomula incubated with [3H]ethanolamine for up to 24 h incorporated this precursor into several proteins; labelled Pronase fragments recovered from the three most intensely labelled proteins were hydrophilic and had a molecular mass of approx. 200 Da. Furthermore, reductive methylation of such fragments showed that the [3H]ethanolamine bears a free amino group, indicating the lack of an amide linkage. We also evaluated the effect of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: [3H]palmitate-labelled proteins of schistosomula and surface-iodinated proteins were resistant to hydrolysis with this enzyme. In conclusion, [3H]palmitate and [3H]ethanolamine are incorporated into distinct proteins of cercariae and schistosomula which do not bear glycophospholipid anchors. The [3H]ethanolamine-labelled proteins represent a novel variety of protein modification. Images Fig. 1. Fig. 3. Fig. 5. PMID:3178767

  13. Free-Solution, Label-Free Protein-Protein Interactions Characterized by Dynamic Light Scattering

    PubMed Central

    Hanlon, Amy D.; Larkin, Michael I.; Reddick, Ryan M.

    2010-01-01

    We report a free-solution, label-free method for quantitative characterization of macromolecular interactions using dynamic light scattering, a temperature controlled plate reader, and a multiwell concentration gradient. This nondestructive technique enabled determination of stoichiometry of binding, equilibrium dissociation constant, and thermodynamic parameters, as well as the impact of temperature, buffer salinity, and a small-molecule inhibitor. The low volume capability of dynamic light scattering reduced the required sample to 426 pmol/experiment, with detection limits for 150-kDa proteins anticipated to be in the low femtomole range. PMID:20338851

  14. Development of indole chemistry to label tryptophan residues in protein for determination of tryptophan surface accessibility.

    PubMed

    Ladner, Carol L; Turner, Raymond J; Edwards, Robert A

    2007-06-01

    Solvent accessibility can be used to evaluate protein structural models, identify binding sites, and characterize protein conformational changes. The differential modification of amino acids at specific sites enables the accessible surface residues to be identified by mass spectrometry. Tryptophan residues within proteins can be differentially labeled with halocompounds by a photochemical reaction. In this study, tryptophan residues of carbonic anhydrase are reacted with chloroform, 2,2,2-trichloroethanol (TCE), 2,2,2-trichloroacetate (TCA), or 3-bromo-1-propanol (BP) under UV irradiation at 280 nm. The light-driven reactions with chloroform, TCE, TCA, and BP attach a formyl, hydroxyethanone, carboxylic acid, and propanol group, respectively, onto the indole ring of tryptophan. Trypsin and chymotrypsin digests of the modified carbonic anhydrase are used to map accessible tryptophan residues using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tryptophan reactivity is determined by identifying peptides with tryptophan residues modified with the appropriate label. The reactivity is calculated from the frequency that the modification is identified and a semiquantitative measure of the amount of products formed. Both of these measures of tryptophan reactivity correlate significantly with the accessible surface area of tryptophan residues in carbonic anhydrase determined from the X-ray crystal structure. Therefore the photochemical reaction of halocompounds with tryptophan residues in carbonic anhydrase indicates the degree of solvent accessibility of these residues. PMID:17525468

  15. F-18 Labeled Diabody-Luciferase Fusion Proteins for Optical-ImmunoPET

    SciTech Connect

    Wu, Anna M

    2013-01-18

    The goal of the proposed work is to develop novel dual-labeled molecular imaging probes for multimodality imaging. Based on small, engineered antibodies called diabodies, these probes will be radioactively tagged with Fluorine-18 for PET imaging, and fused to luciferases for optical (bioluminescence) detection. Performance will be evaluated and validated using a prototype integrated optical-PET imaging system, OPET. Multimodality probes for optical-PET imaging will be based on diabodies that are dually labeled with 18F for PET detection and fused to luciferases for optical imaging. 1) Two sets of fusion proteins will be built, targeting the cell surface markers CEA or HER2. Coelenterazine-based luciferases and variant forms will be evaluated in combination with native substrate and analogs, in order to obtain two distinct probes recognizing different targets with different spectral signatures. 2) Diabody-luciferase fusion proteins will be labeled with 18F using amine reactive [18F]-SFB produced using a novel microwave-assisted, one-pot method. 3) Sitespecific, chemoselective radiolabeling methods will be devised, to reduce the chance that radiolabeling will inactivate either the target-binding properties or the bioluminescence properties of the diabody-luciferase fusion proteins. 4) Combined optical and PET imaging of these dual modality probes will be evaluated and validated in vitro and in vivo using a prototype integrated optical-PET imaging system, OPET. Each imaging modality has its strengths and weaknesses. Development and use of dual modality probes allows optical imaging to benefit from the localization and quantitation offered by the PET mode, and enhances the PET imaging by enabling simultaneous detection of more than one probe.

  16. Assays for the measurement of tissue transglutaminase (type II) mediated protein crosslinking via epsilon-(gamma-glutamyl) lysine and N',N'-bis (gamma-glutamyl) polyamine linkages using biotin labelled casein.

    PubMed

    Lilley, G R; Griffin, M; Bonner, P L

    1997-02-01

    Two colorimetric assays for tissue transglutaminase (type II) activity involving the crosslinking of proteins have been developed. In one assay, biotin labelled casein is crosslinked into chemically modified casein bound to a microtiter plate by tissue transglutaminase and the biotin labelled reaction product is detected by conjugation to Extravidin peroxidase. The assay can detect activity in 10 ng of commercially available purified guinea pig liver transglutaminase and in the crude homogenate derived from 400 human endothelial cells (cell line ECV 304). A correlation (r2 = 0.977) was shown between this assay and the radiolabeled putrescine incorporation assay for the detection of transglutaminase activity. This assay measures the protein crosslinking activity of tissue transglutaminase as opposed to polyamine incorporation and offers a rapid, non-radiometric method for screening large sample numbers. Typical inter-assay variability is 13.9 +/- 1.5% (n = 8). In a second assay, the ability of tissue transglutaminase to catalyze the formation of N',N'-bis (gamma-glutamyl) polyamine bridges is measured. N',N'-dimethylcasein is bound to a microtiter plate and modified enzymatically using commercially available purified guinea pig liver transglutaminase to incorporate polyamines into glutamine residues. Biotin labelled casein is then crosslinked into the immobilized polyamines by tissue transglutaminase resulting in the formation of N',N'-bis (gamma-glutamyl) polyamine linkages. PMID:9089382

  17. Proteomic Analysis of Protein Turnover by Metabolic Whole Rodent Pulse-Chase Isotopic Labeling and Shotgun Mass Spectrometry Analysis.

    PubMed

    Savas, Jeffrey N; Park, Sung Kyu; Yates, John R

    2016-01-01

    The analysis of protein half-life and degradation dynamics has proven critically important to our understanding of a broad and diverse set of biological conditions ranging from cancer to neurodegeneration. Historically these protein turnover measures have been performed in cells by monitoring protein levels after "pulse" labeling of newly synthesized proteins and subsequent chase periods. Comparing the level of labeled protein remaining as a function of time to the initial level reveals the protein's half-life. In this method we provide a detailed description of the workflow required for the determination of protein turnover rates on a whole proteome scale in vivo. Our approach starts with the metabolic labeling of whole rodents by restricting all the nitrogen in their diet to exclusively nitrogen-15 in the form of spirulina algae. After near complete organismal labeling with nitrogen-15, the rodents are then switched to a normal nitrogen-14 rich diet for time periods of days to years. Tissues are harvested, the extracts are fractionated, and the proteins are digested to peptides. Peptides are separated by multidimensional liquid chromatography and analyzed by high resolution orbitrap mass spectrometry (MS). The nitrogen-15 containing proteins are then identified and measured by the bioinformatic proteome analysis tools Sequest, DTASelect2, and Census. In this way, our metabolic pulse-chase approach reveals in vivo protein decay rates proteome-wide. PMID:26867752

  18. Luminescent Alkyne-Bearing Terbium(III) Complexes and Their Application to Bioorthogonal Protein Labeling.

    PubMed

    O'Malley, William I; Abdelkader, Elwy H; Aulsebrook, Margaret L; Rubbiani, Riccardo; Loh, Choy-Theng; Grace, Michael R; Spiccia, Leone; Gasser, Gilles; Otting, Gottfried; Tuck, Kellie L; Graham, Bim

    2016-02-15

    Two new bifunctional macrocyclic chelate ligands that form luminescent terbium(III) complexes featuring an alkyne group for conjugation to (bio)molecules via the Cu(I)-catalyzed "click" reaction were synthesized. Upon ligation, the complexes exhibit a significant luminescent enhancement when excited at the λ(max) of the "clicked" products. To demonstrate the utility of the complexes for luminescent labeling, they were conjugated in vitro to E. coli aspartate/glutamate-binding protein incorporating a genetically encoded p-azido-L-phenylalanine or p-(azidomethyl)-L-phenylalanine residue. The complexes may prove useful for time-gated assay applications.

  19. The detection of tritium-labeled ligands and their carrier proteins using a multiwire proportional counter

    SciTech Connect

    Scott, B.J.; Bateman, J.E.; Bradwell, A.R.

    1982-06-01

    Two-dimensional immunoelectrophoresis combined with autoradiography is a powerful technique for studying the binding of radiolabeled ligands to their carrier proteins. Tritium-labeled compounds are difficult to detect by autoradiography, yet this isotope is often the radiolabel of choice, because it is possible to achieve high specific activity with no loss of biological function. Therefore an electronic detection system called a multiwire proportional counter has been investigated. This has resulted in an increase in detection speed for tritium of several thousand fold over conventional autoradiography and furthermore the results are potentially quantitative.

  20. Correlating labeling chemistry and in-vitro test results with the biological behavior of radiolabeled proteins

    SciTech Connect

    Srivastava, S.C.; Meinken, G.E.

    1985-01-01

    Monoclonal antibodies possess enormous potential for delivery of therapeutic amounts of radionuclides to target antigens in vivo, in particular for tumor imaging and therapy. Translation of this concept into practice has encountered numerous problems. Specifically whereas general protein radiolabeling methods are applicable to antibodies, immunological properties of the antibodies are often compromised resulting in reduced in-vivo specificity for the target antigens. The bifunctional chelating agent approach shows the most promise, however, development of other agents will be necessary for widespread usefulness of this technique. The effects of labeling chemistry on the in-vivo behavior of several monoclonal antibodies are described. 30 refs., 4 figs., 10 tabs.

  1. Purification and stable isotope labeling of the calcium- and integrin-binding protein 1 for structural and functional NMR studies.

    PubMed

    Huang, Hao; Vogel, Hans J

    2013-01-01

    The Calcium- and Integrin-Binding protein 1 (CIB1) has been identified as an important regulatory Ca(2+)-binding protein that is involved in various cellular functions. Nuclear Magnetic Resonance (NMR) spectroscopy provides a powerful approach to study the structure, dynamics, and interactions of CIB1 and related proteins. Multidimensional NMR spectroscopy combined with various selective isotope labeling strategies has proven to be successful in the structure determination of CIB1. Moreover, the same approach allowed the detection of conformational changes when the protein binds different metal ions, and it facilitated the study of the interaction of CIB1 with the cytoplasmic domain of the human integrin αIIb subunit. In this protocol, we describe the purification and isotope labeling strategies for productive NMR studies of CIB1. The same isotope labeling strategies can be implemented to study numerous related regulatory calcium-binding proteins.

  2. Resolution and measurement of heteronuclear dipolar couplings of a noncrystalline protein immobilized in a biological supramolecular assembly by proton-detected MAS solid-state NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Park, Sang Ho; Yang, Chen; Opella, Stanley J.; Mueller, Leonard J.

    2013-12-01

    Two-dimensional 15N chemical shift/1H chemical shift and three-dimensional 1H-15N dipolar coupling/15N chemical shift/1H chemical shift MAS solid-state NMR correlation spectra of the filamentous bacteriophage Pf1 major coat protein show single-site resolution in noncrystalline, intact-phage preparations. The high sensitivity and resolution result from 1H detection at 600 MHz under 50 kHz magic angle spinning using ∼0.5 mg of perdeuterated and uniformly 15N-labeled protein in which the exchangeable amide sites are partially or completely back-exchanged (reprotonated). Notably, the heteronuclear 1H-15N dipolar coupling frequency dimension is shown to select among 15N resonances, which will be useful in structural studies of larger proteins where the resonances exhibit a high degree of overlap in multidimensional chemical shift correlation spectra.

  3. High-Pressure EPR and Site-Directed Spin Labeling for Mapping Molecular Flexibility in Proteins.

    PubMed

    Lerch, Michael T; Yang, Zhongyu; Altenbach, Christian; Hubbell, Wayne L

    2015-01-01

    High hydrostatic pressure is a powerful probe of protein conformational flexibility. Pressurization reveals regions of elevated compressibility, and thus flexibility, within individual conformational states, but also shifts conformational equilibria such that "invisible" excited states become accessible for spectroscopic characterization. The central aim of this chapter is to describe recently developed instrumentation and methodologies that enable high-pressure site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) experiments on proteins and to demonstrate the information content of these experiments by highlighting specific recent applications. A brief introduction to the thermodynamics of proteins under pressure is presented first, followed by a discussion of the principles underlying SDSL-EPR detection of pressure effects in proteins, and the suitability of SDSL-EPR for this purpose in terms of timescale and ability to characterize conformational heterogeneity. Instrumentation and practical considerations for variable-pressure continuous wave EPR and pressure-resolved double electron-electron resonance (PR DEER) experiments are reviewed, and finally illustrations of data analysis using recent applications are presented. Although high-pressure SDSL-EPR is in its infancy, the recent applications presented highlight the considerable potential of the method to (1) identify compressible (flexible) regions in a folded protein; (2) determine thermodynamic parameters that relate conformational states in equilibrium; (3) populate and characterize excited states of proteins undetected at atmospheric pressure; (4) reveal the structural heterogeneity of conformational ensembles and provide distance constraints on the global structure of pressure-populated states with PR DEER.

  4. Metal-tag labeling coupled with multiple reaction monitoring-mass spectrometry for absolute quantitation of proteins.

    PubMed

    Wang, Xueying; Wang, Xin; Qin, Weijie; Lin, Hongjun; Wang, Jifeng; Wei, Junying; Zhang, Yangjun; Qian, Xiaohong

    2013-09-21

    Mass spectrometry-based quantitative proteomics, consisting of relative and absolute parts, has been used to discover and validate proteins with key functions related to physiological and pathological processes. Currently, stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) is the most commonly used method for the absolute determination of proteins in a biological sample. A prerequisite for this method is obtaining internal standards with isotope labels. Although many approaches have been developed for the labeling and preparation of internal peptides, expensive stable isotope labeling coupled with SID-MRM-MS has limited the application and development of an absolute quantitative method. Recently, a low-cost strategy using metal-tag labeling and MS has been developed for relative quantification of peptides or proteins. The introduction of labeling using metal tags has the merits of allowing multiple labeling and enlarging the mass shift to overcome the overlap of adjacent isotope clusters. However, most papers described MRM-MS for protein absolute quantification based on the metal in its peptides labelled with metal by inductively coupled plasma mass spectrometry (ICP MS) but not on its peptides labelled with metal. In this work, a novel approach based on metal-tag labeling coupled with MRM-MS was established for the absolute quantification of peptides or proteins. The principle of the method is that a bifunctional chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid bearing an N-hydroxysuccinimide ester (DOTA-NHS ester), is used to modify the N-termini of signature peptides from a target protein, and the modified peptides then chelate a certain metal, such as thulium, to form metal-tagged peptides (Tm-DOTA-P). Internal peptides are chemically synthesized and labeled with another metal, such as terbium (Tb-DOTA-P), as the internal standard. Both the Tb-DOTA- and Tm-DOTA-labeled peptides in samples can be analysed via

  5. Some quantitative aspects of the labelling of proteins with 125 I by the iodine monochloride method.

    PubMed

    Ceska, M; Sjödin, A V; Grossmüller, F

    1971-01-01

    The labelling of proteins by the iodine monochloride method was studied by using a mathematical model. The equations used were primarily derived from the mass law equation of the isotopic exchange reaction between [(125)I]iodide and iodine monochloride. For convenient application, all equations were programmed into a computing desk-top calculator. To support the validity of the theoretical model, a series of iodinations of insulin were performed under various labelling conditions. The results of these experiments compare well with the theoretically derived values. Deviations from the theoretical values occurring at molar ratios of [(125)I]iodide to iodine monochloride < 0.1 and > 4.0 are explained and suggestions made about how to prevent them. The mathematical model was used to simulate the isotopic exchange, and the iodination reaction under various conditions, to study (a) the influence of the amount of [(125)I]iodide on the amount of [(125)I]iodine monochloride formed, (b) the influence of the specific radioactivity of [(125)I]iodide on the amount of [(125)I]iodine monochloride formed, and (c) the influence of the specific radioactivity of [(125)I]iodide on the number of millicuries needed for labelling to a desired extent.

  6. Inulin-125I-tyramine, an improved residualizing label for studies on sites of catabolism of circulating proteins

    SciTech Connect

    Maxwell, J.L.; Baynes, J.W.; Thorpe, S.R.

    1988-10-05

    Residualizing labels for protein, such as dilactitol-125I-tyramine (125I-DLT) and cellobiitol-125I-tyramine, have been used to identify the tissue and cellular sites of catabolism of long-lived plasma proteins, such as albumin, immunoglobulins, and lipoproteins. The radioactive degradation products formed from labeled proteins are relatively large, hydrophilic, resistant to lysosomal hydrolases, and accumulate in lysosomes in the cells involved in degradation of the carrier protein. However, the gradual loss of the catabolites from cells (t1/2 approximately 2 days) has limited the usefulness of residualizing labels in studies on longer lived proteins. We describe here a higher molecular weight (Mr approximately 5000), more efficient residualizing glycoconjugate label, inulin-125I-tyramine (125I-InTn). Attachment of 125I-InTn had no effect on the plasma half-life or tissue sites of catabolism of asialofetuin, fetuin, or rat serum albumin in the rat. The half-life for hepatic retention of degradation products from 125I-InTn-labeled asialofetuin was 5 days, compared to 2.3 days for 125I-DLT-labeled asialofetuin. The whole body half-lives for radioactivity from 125I-InTn-, 125I-DLT-, and 125I-labeled rat serum albumin were 7.5, 4.3, and 2.2 days, respectively. The tissue distribution of degradation products from 125I-InTn-labeled proteins agreed with results of previous studies using 125I-DLT, except that a greater fraction of total degradation products was recovered in tissues. Kinetic analyses indicated that the average half-life for retention of 125I-InTn degradation products in tissues is approximately 5 days and suggested that in vivo there are both slow and rapid routes for release of degradation products from cells.

  7. Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures.

    PubMed

    Doll, Andrin; Bordignon, Enrica; Joseph, Benesh; Tschaggelar, René; Jeschke, Gunnar

    2012-09-01

    We demonstrate the application of continuous wave dynamic nuclear polarization (DNP) at 0.35 T for site-specific water accessibility studies on spin-labeled membrane proteins at concentrations in the 10-100 μM range. The DNP effects at such low concentrations are weak and the experimentally achievable dynamic nuclear polarizations can be below the equilibrium polarization. This sensitivity problem is solved with an optimized home-built DNP probe head consisting of a dielectric microwave resonator and a saddle coil as close as possible to the sample. The performance of the probe head is demonstrated with both a modified pulsed EPR spectrometer and a dedicated CW EPR spectrometer equipped with a commercial NMR console. In comparison to a commercial pulsed ENDOR resonator, the home-built resonator has an FID detection sensitivity improvement of 2.15 and an electron spin excitation field improvement of 1.2. The reproducibility of the DNP results is tested on the water soluble maltose binding protein MalE of the ABC maltose importer, where we determine a net standard deviation of 9% in the primary DNP data in the concentration range between 10 and 100 μM. DNP parameters are measured in a spin-labeled membrane protein, namely the vitamin B(12) importer BtuCD in both detergent-solubilized and reconstituted states. The data obtained in different nucleotide states in the presence and absence of binding protein BtuF reveal the applicability of this technique to qualitatively extract water accessibility changes between different conformations by the ratio of primary DNP parameters ϵ. The ϵ-ratio unveils the physiologically relevant transmembrane communication in the transporter in terms of changes in water accessibility at the cytoplasmic gate of the protein induced by both BtuF binding at the periplasmic region of the transporter and ATP binding at the cytoplasmic nucleotide binding domains.

  8. Liquid state DNP for water accessibility measurements on spin-labeled membrane proteins at physiological temperatures

    NASA Astrophysics Data System (ADS)

    Doll, Andrin; Bordignon, Enrica; Joseph, Benesh; Tschaggelar, René; Jeschke, Gunnar

    2012-09-01

    We demonstrate the application of continuous wave dynamic nuclear polarization (DNP) at 0.35 T for site-specific water accessibility studies on spin-labeled membrane proteins at concentrations in the 10-100 μM range. The DNP effects at such low concentrations are weak and the experimentally achievable dynamic nuclear polarizations can be below the equilibrium polarization. This sensitivity problem is solved with an optimized home-built DNP probe head consisting of a dielectric microwave resonator and a saddle coil as close as possible to the sample. The performance of the probe head is demonstrated with both a modified pulsed EPR spectrometer and a dedicated CW EPR spectrometer equipped with a commercial NMR console. In comparison to a commercial pulsed ENDOR resonator, the home-built resonator has an FID detection sensitivity improvement of 2.15 and an electron spin excitation field improvement of 1.2. The reproducibility of the DNP results is tested on the water soluble maltose binding protein MalE of the ABC maltose importer, where we determine a net standard deviation of 9% in the primary DNP data in the concentration range between 10 and 100 μM. DNP parameters are measured in a spin-labeled membrane protein, namely the vitamin B12 importer BtuCD in both detergent-solubilized and reconstituted states. The data obtained in different nucleotide states in the presence and absence of binding protein BtuF reveal the applicability of this technique to qualitatively extract water accessibility changes between different conformations by the ratio of primary DNP parameters ɛ. The ɛ-ratio unveils the physiologically relevant transmembrane communication in the transporter in terms of changes in water accessibility at the cytoplasmic gate of the protein induced by both BtuF binding at the periplasmic region of the transporter and ATP binding at the cytoplasmic nucleotide binding domains.

  9. (15)N Heteronuclear Chemical Exchange Saturation Transfer MRI.

    PubMed

    Zeng, Haifeng; Xu, Jiadi; Yadav, Nirbhay N; McMahon, Michael T; Harden, Bradley; Frueh, Dominique; van Zijl, Peter C M

    2016-09-01

    A two-step heteronuclear enhancement approach was combined with chemical exchange saturation transfer (CEST) to magnify (15)N MRI signal of molecules through indirect detection via water protons. Previous CEST studies have been limited to radiofrequency (rf) saturation transfer or excitation transfer employing protons. Here, the signal of (15)N is detected indirectly through the water signal by first inverting selectively protons that are scalar-coupled to (15)N in the urea molecule, followed by chemical exchange of the amide proton to bulk water. In addition to providing a small sensitivity enhancement, this approach can be used to monitor the exchange rates and thus the pH sensitivity of the participating (15)N-bound protons. PMID:27548755

  10. Interrogation of in vivo protein-protein interactions using transgenic mouse models and stable isotope labeling.

    PubMed

    Dey, Anwesha; Wu, Jiansheng; Kirkpatrick, Donald S

    2014-01-01

    Methods in mass spectrometry have evolved in recent years, facilitating proteomic analyses that were previously beyond the limits of the technology. Transgenic mouse models, coupled with mass spectrometry proteomics, have served as valuable platform for elucidating the in vivo function of individual genes and proteins. Here we discuss the methods we have recently employed to characterize protein-protein interactions and posttranslational modifications in tagged knock-in mouse models. These methods can be broadly applied to other systems for various applications in both basic and translational science.

  11. Labeling of recombinant protein for NMR spectroscopy: global and specific labeling of the rat liver fructose 2,6-bisphosphatase domain.

    PubMed

    Okar, D A; Felicia, N D; Gui, L; Lange, A J

    1997-10-01

    Methods for the efficient use of the 13C-labeled nutrients, glucose and histidine, in the production of recombinant protein were developed to provide the large amount of sample required for NMR studies. The nutrient requirements were reduced by determining the minimum amount of these metabolites needed during both the growth and the induction phases of the BL21(DE3) and newly constructed BL21(DE3) histidine auxotrophic Escherichia coli cultures. These methods were developed using the separate bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase, which is expressed to high levels in the pET3a/BL21 (DE3) bacterial system. Use of the optimized expression methods reduced the requirements for the labeled nutrients, glucose and histidine, by 90 and 93.8%, respectively. The savings realized by use of the minimized media and modified induction protocols were obtained without significant reduction of the yield of purified protein. Comprehensive study of the bisphosphatase domain by NMR spectroscopy requires large amounts of protein because of its low solubility and the short lifetime (2-3 days) of the NMR samples. The significant reduction in the costs of labeled protein samples realized by the optimized expression methods can meet these sample requirements in a cost-effective way, and thereby, allow NMR studies of the bisphosphatase domain to proceed.

  12. Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology.

    PubMed

    Armenta, Jenny M; Hoeschele, Ina; Lazar, Iulia M

    2009-07-01

    An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17beta-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of <4%. The reproducibility of protein identifications was approximately 60%-67% between duplicate, and approximately 50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P < 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate approximately 2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, approximately 16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up- or down-regulated. PMID:19345114

  13. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain

    PubMed Central

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-01-01

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23–230) as detected by [1H, 15N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn2+-binding to the octarepeat motif. PMID:27341298

  14. Structural transitions in full-length human prion protein detected by xenon as probe and spin labeling of the N-terminal domain.

    PubMed

    Narayanan, Sunilkumar Puthenpurackal; Nair, Divya Gopalakrishnan; Schaal, Daniel; Barbosa de Aguiar, Marisa; Wenzel, Sabine; Kremer, Werner; Schwarzinger, Stephan; Kalbitzer, Hans Robert

    2016-01-01

    Fatal neurodegenerative disorders termed transmissible spongiform encephalopathies (TSEs) are associated with the accumulation of fibrils of misfolded prion protein PrP. The noble gas xenon accommodates into four transiently enlarged hydrophobic cavities located in the well-folded core of human PrP(23-230) as detected by [(1)H, (15)N]-HSQC spectroscopy. In thermal equilibrium a fifth xenon binding site is formed transiently by amino acids A120 to L125 of the presumably disordered N-terminal domain and by amino acids K185 to T193 of the well-folded domain. Xenon bound PrP was modelled by restraint molecular dynamics. The individual microscopic and macroscopic dissociation constants could be derived by fitting the data to a model including a dynamic opening and closing of the cavities. As observed earlier by high pressure NMR spectroscopy xenon binding influences also other amino acids all over the N-terminal domain including residues of the AGAAAAGA motif indicating a structural coupling between the N-terminal domain and the core domain. This is in agreement with spin labelling experiments at positions 93 or 107 that show a transient interaction between the N-terminus and the start of helix 2 and the end of helix 3 of the core domain similar to that observed earlier by Zn(2+)-binding to the octarepeat motif. PMID:27341298

  15. Peptides and proteins

    SciTech Connect

    Bachovchin, W.W.; Unkefer, C.J.

    1994-12-01

    Advances in magnetic resonance and vibrational spectroscopy make it possible to derive detailed structural information about biomolecular structures in solution. These techniques are critically dependent on the availability of labeled compounds. For example, NMR techniques used today to derive peptide and protein structures require uniformity {sup 13}C-and {sup 15}N-labeled samples that are derived biosynthetically from (U-6-{sup 13}C) glucose. These experiments are possible now because, during the 1970s, the National Stable Isotope Resource developed algal methods for producing (U-6-{sup 13}C) glucose. If NMR techniques are to be used to study larger proteins, we will need sophisticated labelling patterns in amino acids that employ a combination of {sup 2}H, {sup 13}C, and {sup 15}N labeling. The availability of these specifically labeled amino acids requires a renewed investment in new methods for chemical synthesis of labeled amino acids. The development of new magnetic resonance or vibrational techniques to elucidate biomolecular structure will be seriously impeded if we do not see rapid progress in labeling technology. Investment in labeling chemistry is as important as investment in the development of advanced spectroscopic tools.

  16. Natural-abundance 15N NMR studies of Turkey ovomucoid third domain. Assignment of peptide 15N resonances to the residues at the reactive site region via proton-detected multiple-quantum coherence

    NASA Astrophysics Data System (ADS)

    Ortiz-Polo, Gilberto; Krishnamoorthi, R.; Markley, John L.; Live, David H.; Davis, Donald G.; Cowburn, David

    Heteronuclear two-dimensional 1H{ 15N} multiple-quantum (MQ) spectroscopy has been applied to a protein sample at natural abundance: ovomucoid third domain from turkey ( Meleagris gallopavo), a serine proteinase inhibitor of 56 amino acid residues. Peptide amide 1H NMR assignments obtained by two-dimensional 1H{ 1H} NMR methods (R. Krishnamoorthi and J. L. Markley, unpublished data) led to identification of the corresponding 1H{ 15N} MQ coherence cross peaks. From these, 15N NMR chemical shifts were determined for several specific backbone amide groups of amino acid residues located around the reactive site region of the inhibitor. The results suggest that amide 15N chemical shifts, which are readily obtained in this way, may serve as sensitive probes for conformational studies of proteins.

  17. Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide.

    PubMed

    Nguyen, Giang K T; Cao, Yuan; Wang, Wei; Liu, Chuan Fa; Tam, James P

    2015-12-21

    An efficient ligase with exquisite site-specificity is highly desirable for protein modification. Recently, we discovered the fastest known ligase called butelase 1 from Clitoria ternatea for intramolecular cyclization. For intermolecular ligation, butelase 1 requires an excess amount of a substrate to suppress the reverse reaction, a feature similar to other ligases. Herein, we describe the use of thiodepsipeptide substrates with a thiol as a leaving group and an unacceptable nucleophile to render the butelase-mediated ligation reactions irreversible and in high yields. Butelase 1 also accepted depsipeptides as substrates, but unlike a thiodesipeptide, the desipeptide ligation was partially reversible as butelase 1 can tolerate an alcohol group as a poor nucleophile. The thiodesipeptide method was successfully applied in N-terminal labeling of ubiquitin and green fluorescent protein using substrates with or without a biotin group in high yields. PMID:26563575

  18. Transferrin protein nanospheres: a nanoplatform for receptor-mediated cancer cell labeling and gene delivery

    NASA Astrophysics Data System (ADS)

    McDonald, Michael A.; Spurlin, Tighe A.; Tona, Alessandro; Elliott, John T.; Halter, Michael; Plant, Anne L.

    2010-02-01

    This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for targeting cancer cells in vitro. Protein nanospheres represent an easily prepared and modifiable nanoplatform for receptor-specific targeting, molecular imaging and gene delivery. Rhodamine B isothiocyanate conjugated TfpNS (RBITC-TfpNS) show significantly enhanced uptake in vitro in SK-MEL-28 human malignant melanoma cells known to overexpress transferrin receptors compared to controls. RBITCTfpNS labeling of the cancer cells is due to transferrin receptor-mediated uptake, as demonstrated by competitive inhibition with native transferrin. Initial fluorescence microscopy studies indicate GFP plasmid can be transfected into melanoma cells via GFP plasmid encapsulated by TfpNS.

  19. Monitoring Protein O-GlcNAc Status via Metabolic Labeling and Copper-free Click Chemistry

    PubMed Central

    Teo, Chin Fen; Wells, Lance

    2014-01-01

    O-GlcNAc modification found on the serine and threonine residues of intracellular proteins is an inducible post-translational modification that regulates numerous biological processes. In combination with other cell biological and biochemical approaches, a robust and streamlined strategy for detecting the number and stoichiometry of O-GlcNAc modification can provide valuable insights for decoding the functions of O-GlcNAc at the molecular level. Herein, we report an optimized workflow for evaluating the O-GlcNAc status of proteins using a combination of metabolic labeling and click chemistry based mass tagging. This method is strategically complementary to the chemoenzymatic-based mass-tagging method. PMID:24995865

  20. Rapid Covalent Fluorescence Labeling of Membrane Proteins on Live Cells via Coiled-Coil Templated Acyl Transfer.

    PubMed

    Reinhardt, Ulrike; Lotze, Jonathan; Mörl, Karin; Beck-Sickinger, Annette G; Seitz, Oliver

    2015-10-21

    Fluorescently labeled proteins enable the microscopic imaging of protein localization and function in live cells. In labeling reactions targeted against specific tag sequences, the size of the fluorophore-tag is of major concern. The tag should be small to prevent interference with protein function. Furthermore, rapid and covalent labeling methods are desired to enable the analysis of fast biological processes. Herein, we describe the development of a method in which the formation of a parallel coiled coil triggers the transfer of a fluorescence dye from a thioester-linked coil peptide conjugate onto a cysteine-modified coil peptide. This labeling method requires only small tag sequences (max 23 aa) and occurs with high tag specificity. We show that size matching of the coil peptides and a suitable thioester reactivity allow the acyl transfer reaction to proceed within minutes (rather than hours). We demonstrate the versatility of this method by applying it to the labeling of different G-protein coupled membrane receptors including the human neuropeptide Y receptors 1, 2, 4, 5, the neuropeptide FF receptors 1 and 2, and the dopamine receptor 1. The labeled receptors are fully functional and able to bind the respective ligand with high affinity. Activity is not impaired as demonstrated by activation, internalization, and recycling experiments. PMID:26367072

  1. Visualizing Quantum Dot Labeled ORAI1 Proteins in Intact Cells Via Correlative Light and Electron Microscopy.

    PubMed

    Peckys, Diana B; Alansary, Dalia; Niemeyer, Barbara A; de Jonge, Niels

    2016-08-01

    ORAI1 proteins are ion channel subunits and the essential pore-forming units of the calcium release-activated calcium channel complex essential for T-cell activation and many other cellular processes. In this study, we used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to image plasma membrane expressed ORAI1 proteins in whole Jurkat T cells in the liquid state. Utilizing a stably transfected Jurkat T cell clone expressing human ORAI1 with an extracellular human influenza hemagglutinin (HA) tag we investigated if liquid-phase STEM can be applied to detect recombinant surface expressed protein. Streptavidin coated quantum dots were coupled in a one-to-one stoichiometry to ORAI1 proteins detected by biotinylated anti-HA fragmented antibody fragments. High-resolution electron microscopic images revealed the individual label locations from which protein pair distances were determined. These data were analyzed using the pair correlation function and, in addition, an analysis of cluster size and frequency was performed. ORAI1 was found to be present in hexamers in a small fraction only, and ORAI1 resided mostly in monomers and dimers.

  2. Traceless labeling of glycoproteins and its application to the study of glycoprotein-protein interactions.

    PubMed

    Yang, Yung-Lin; Lee, Yen-Pin; Yang, Yen-Ling; Lin, Po-Chiao

    2014-02-21

    A new chemical method for the traceless labeling of glycoproteins with synthetic boronic acid (BA)-tosyl probes was successfully developed. The BA moiety acts as an affinity head to direct the formation of a cyclic boronate diester with the diol groups of glycans. Following this step, the electrophilic tosyl group is displaced by an SN2 reaction with a nucleophilic residue of the boronated glycoprotein, and finally, a reporter group is tagged onto the glycoprotein via an ether linkage. In the presence of polyols, a competition reaction recovers the native glycan of the tagged glycoprotein, conserving its biological significance. The BA-tosyl probes were used successfully for the specific labeling of glycosylated fetuins in a mixed protein pool and from crude Escherichia coli (E. coli) lysate. Further, a BA-tosyl-functionalized glass slide was used to fabricate glycoprotein microarrays with highly conserved glycans. By interacting with various lectins (carbohydrate-binding proteins), such as Concanavalin A (Con A) and wheat germ agglutinin (WGA), the types of carbohydrates and specific linkages of glycoproteins (α or β) could be systematically monitored. It is believed that the newly developed method will greatly accelerate the understanding of glycoproteins.

  3. Acetylene inhibition of N2O reduction in laboratory soil and groundwater denitrification assays: evaluation by 15N tracer and 15N site preference of N2O

    NASA Astrophysics Data System (ADS)

    Weymann, Daniel; Well, Reinhard; Lewicka-Szczebak, Dominika; Lena, Rohe

    2013-04-01

    The measurement of denitrification in soils and aquifers is still challenging and often enough associated with considerable experimental effort and high costs. Against this background, the acetylene inhibition technique (AIT) applied in laboratory soil and groundwater denitrification assays is by far the most effective approach. However, this method has been largely criticized, as it is susceptible to underestimate denitrification rates and adds an additional carbon source to the substrates to be investigated. Here we provide evidence that the AIT is not necessarily an inappropriate approach to measure denitrification, that its reliability depends on the drivers governing the process, and that the 15N site preference of N2O (SP) may serve as a tool to assess this reliability. Two laboratory batch experiments were conducted, where sandy aquifer material and a peat soil were incubated as slurries. We established (i) a standard anaerobic treatment by adding KNO3 (10 mg N L-1), (ii) an oxygen treatment by adding KNO3 and O2 (5 mg L-1), and (iii) a glucose treatment by adding KNO3 supplemented with glucose (200 mg C L-1). Both experiments were run under 10 % (v/v) acetylene atmosphere and as 15N tracer treatments using labeled K15NO3 (60 atom % 15N). In the case of the standard anaerobic treatments, we found a very good agreement of denitrification potential obtained by the AIT and 15N tracer methods. SP of N2O of the AIT samples from this treatment ranged between -4.8 and 2.6 ‰ which is indicative for N2O production during bacterial denitrification but not for N2O reduction to N2. In contrast, we observed substantial underestimation of denitrification by AIT for the glucose treatments compared to the 15N method, i.e. denitrification was underestimated by 36 % (sandy aquifer material) and 47 % (peat soil). SP of N2O of the AIT samples from this treatment ranged between 4.5 and 9.6 ‰, which suggests occurrence of bacterial N2O reduction. In the case of the oxygen

  4. Selective cell-surface labeling of the molecular motor protein prestin

    SciTech Connect

    McGuire, Ryan M.; Silberg, Jonathan J.; Pereira, Fred A.; Raphael, Robert M.

    2011-06-24

    Highlights: {yields} Trafficking to the plasma membrane is required for prestin function. {yields} Biotin acceptor peptide (BAP) was fused to prestin through a transmembrane domain. {yields} BAP-prestin can be metabolically labeled with biotin in HEK293 cells. {yields} Biotin-BAP-prestin allows for selective imaging of fully trafficked prestin. {yields} The biotin-BAP-prestin displays voltage-sensitive activity. -- Abstract: Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

  5. Phenylalanine δ15N in Paleo Archives as a New Proxy for δ15N of Exported Primary Production

    NASA Astrophysics Data System (ADS)

    McCarthy, M.; Batista, F. C.; Vokhshoori, N. L.; Brown, J. T.; Guilderson, T. P.; Ravelo, A. C.; Sherwood, O.

    2012-12-01

    Compound-specific isotope analysis of individual amino acids (CSI-AA) is emerging as a powerful new tool for studying the paleo nitrogen cycle. Because most detrital organic nitrogen is composed of amino acids, CSI-AA can reveal the mechanistic basis for organic nitrogen diagenesis, preserve a record of past food web structure, and potentially reconstruct the δ15N values of past nitrate and primary production. Within the commonly measured amino acids, the δ15N value of phenylalanine (Phe) appears uniquely promising as a new proxy that reflects the nitrogen isotopic value of the original source. Phe δ15N values remain almost unchanged with trophic transfer through food webs, and also during at least the initial stages of organic matter degradation. Here we synthesize results from both bio-archives and recent sediments, which together suggest that at least in Holocene archives the Phe δ15N value does in fact record the average inorganic nitrogen δ15N value at the base of planktonic food webs. However, several important unknowns also remain. These include the extent of variation in amino acid isotopic fractionation patterns in phylogenetically distinct algal groups. The stability of Phe δ15N values in older sediments where organic matter has undergone extensive diagenesis is also an important research area, which may ultimately establish the temporal limit for application of this approach to study past geological epochs. Together, however, results to date suggest that of Phe δ15N values in paleo archives represent a novel molecular-level proxy which is not tied to any specific organism or group, but rather can provide an integrated estimate of δ15N value of exported primary production.

  6. Measurement of (15)N enrichment of glutamine and urea cycle amino acids derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using liquid chromatography-tandem quadrupole mass spectrometry.

    PubMed

    Nakamura, Hidehiro; Karakawa, Sachise; Watanabe, Akiko; Kawamata, Yasuko; Kuwahara, Tomomi; Shimbo, Kazutaka; Sakai, Ryosei

    2015-05-01

    6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is an amino acid-specific derivatizing reagent that has been used for sensitive amino acid quantification by liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS). In this study, we aimed to evaluate the ability of this method to measure the isotopic enrichment of amino acids and to determine the positional (15)N enrichment of urea cycle amino acids (i.e., arginine, ornithine, and citrulline) and glutamine. The distribution of the M and M+1 isotopomers of each natural AQC-amino acid was nearly identical to the theoretical distribution. The standard deviation of the (M+1)/M ratio for each amino acid in repeated measurements was approximately 0.1%, and the ratios were stable regardless of the injected amounts. Linearity in the measurements of (15)N enrichment was confirmed by measuring a series of (15)N-labeled arginine standards. The positional (15)N enrichment of urea cycle amino acids and glutamine was estimated from the isotopic distribution of unique fragment ions generated at different collision energies. This method was able to identify their positional (15)N enrichment in the plasma of rats fed (15)N-labeled glutamine. These results suggest the utility of LC-MS/MS detection of AQC-amino acids for the measurement of isotopic enrichment in (15)N-labeled amino acids and indicate that this method is useful for the study of nitrogen metabolism in living organisms.

  7. Multilayer polymer microchip capillary array electrophoresis devices with integrated on-chip labeling for high-throughput protein analysis

    PubMed Central

    Yu, Ming; Wang, Qingsong; Patterson, James E.; Woolley, Adam T.

    2011-01-01

    It is desirable to have inexpensive, high-throughput systems that integrate multiple sample analysis processes and procedures, for applications in biology, chemical analysis, drug discovery, and disease screening. In this paper, we demonstrate multilayer polymer microfluidic devices with integrated on-chip labeling and parallel electrophoretic separation of up to 8 samples. Microchannels were distributed in two different layers and connected through interlayer through-holes in the middle layer. A single set of electrophoresis reservoirs and one fluorescent label reservoir address parallel analysis units for up to 8 samples. Individual proteins and a mixture of cancer biomarkers have been successfully labeled on-chip and separated in parallel with this system. A detection limit of 600 ng/mL was obtained for heat shock protein 90. Our integrated on-chip labeling microdevices show great potential for low-cost, simplified, rapid and high-throughput analysis. PMID:21449615

  8. 15N Hyperpolarization by Reversible Exchange Using SABRE-SHEATH

    PubMed Central

    2016-01-01

    NMR signal amplification by reversible exchange (SABRE) is a NMR hyperpolarization technique that enables nuclear spin polarization enhancement of molecules via concurrent chemical exchange of a target substrate and parahydrogen (the source of spin order) on an iridium catalyst. Recently, we demonstrated that conducting SABRE in microtesla fields provided by a magnetic shield enables up to 10% 15N-polarization (Theis, T.; et al. J. Am. Chem. Soc.2015, 137, 1404). Hyperpolarization on 15N (and heteronuclei in general) may be advantageous because of the long-lived nature of the hyperpolarization on 15N relative to the short-lived hyperpolarization of protons conventionally hyperpolarized by SABRE, in addition to wider chemical shift dispersion and absence of background signal. Here we show that these unprecedented polarization levels enable 15N magnetic resonance imaging. We also present a theoretical model for the hyperpolarization transfer to heteronuclei, and detail key parameters that should be optimized for efficient 15N-hyperpolarization. The effects of parahydrogen pressure, flow rate, sample temperature, catalyst-to-substrate ratio, relaxation time (T1), and reversible oxygen quenching are studied on a test system of 15N-pyridine in methanol-d4. Moreover, we demonstrate the first proof-of-principle 13C-hyperpolarization using this method. This simple hyperpolarization scheme only requires access to parahydrogen and a magnetic shield, and it provides large enough signal gains to enable one of the first 15N images (2 × 2 mm2 resolution). Importantly, this method enables hyperpolarization of molecular sites with NMR T1 relaxation times suitable for biomedical imaging and spectroscopy. PMID:25960823

  9. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

    PubMed Central

    Evans, Eric G.B.; Millhauser, Glenn L.

    2016-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain “K1,” and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron–electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite

  10. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling.

    PubMed

    Evans, Eric G B; Millhauser, Glenn L

    2015-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain "K1," and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron-electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite reagents

  11. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling.

    PubMed

    Evans, Eric G B; Millhauser, Glenn L

    2015-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain "K1," and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron-electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite reagents

  12. Uptake of stormwater nitrogen in bioretention systems demonstrated from 15N tracer techniques

    NASA Astrophysics Data System (ADS)

    Houdeshel, D.; Hultine, K. R.; Pomeroy, C. A.

    2012-12-01

    Bioretention stormwater management systems are engineered ecosystems that capture urban stormwater in order to reduce the harmful effects of stormwater pollution on receiving waters. Bioretention systems have been shown to be effective at reducing the volume of runoff, and thereby reduce the nutrient loading to receiving waters from urban areas. However, little work has been done to evaluate the treatment processes that are responsible for reductions in effluent nitrogen (N). We hypothesize that the pulses of inorganic nitrogen associated with urban runoff events are captured in the plat tissues within these systems and not adsorbed to the soil media, thus creating a long-term, sustainable treatment approach to reducing the total nutrient loading to receiving waters. Nitrogen treatment performance was tested on two bioretention systems in Salt Lake City, UT: 1) an upland native community that does not require irrigation in semi-arid climates, and 2) a wetland community that requires 250 l of daily irrigation to offset the relatively high evaporative demand in the region. Each cell is sized to treat a 2.5 cm storm from a 140 m2 impervious surface: the area of the bioretention system is 10 m2. To test the N removal performance of each system, runoff events were simulated to represent an average precipitation regime using a synthetic stormwater blend starting in January, 2012. Effluent was collected from an underdrain and analyzed for total nitrogen (TN); mass removal was calculated for each month by subtracting the TN mass added to the garden minus the TN mass that flowed out of the garden. To test the hypothesis that plants assimilate stormwater N, 4 g of 100 atom% 15N NH4NO3 tracer was used as the N source in the synthetic stormwater during the first 2,000 l synthetic storm event in May. This isotopic label was calculated to enrich the total N pool of each garden to 100‰ 15N/14Nair. New growth was harvested from each plant in both cells and analyzed for 15N

  13. Is seeing believing? An assessment of the impact of fluorescent labelling on protein structure and interaction potential

    NASA Astrophysics Data System (ADS)

    Quinn, Michelle K.; James, Susan; McNamara, Ruth; McManus, Jennifer J.

    2014-03-01

    Fluorescent labelling is extensively used in conjunction with spectroscopy and microscopy for the in-vivo and in-vitro study of proteins. However, there is little data quantifying how this impacts on the protein in terms of its net interaction potential and its structure. Human ?D-crystallin (HGD), a protein found in the eye lens at high concentrations, undergoes liquid-liquid phase separation (LLPS) and has a well-studied phase diagram. LLPS is indicative of short-ranged attractive interactions between the proteins and the conditions this occurs under are sensitive to changes in the protein itself (e.g. mutations, dimer formation) and its environmental conditions (e.g. pH, salt concentration). HGD is produced recombinantly in E. coli and fluorescently labelled via covalent attachment after purification. Comparison of the coexistence curves for labelled and unlabelled protein indicates if there has been a change in the net interaction potential and various spectroscopic techniques are used to elucidate structural changes between the labelled and unlabelled protein. These studies are important for understanding the relationship between in-vitro phase diagram experiments and those conducted in complex biological fluids, such as plasma or cells where fluorescent tagging is required. The authors acknowledge Science Foundation Ireland (SFI) (grant number 11/RFP.1/PHY3165). J.J. McManus acknowledges SFI Stokes Lectureship.

  14. Selective cell-surface labeling of the molecular motor protein prestin

    PubMed Central

    McGuire, Ryan M.; Silberg, Jonathan J.; Pereira, Fred A.; Raphael, Robert M.

    2011-01-01

    Prestin, a multipass transmembrane protein whose N- an C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity. PMID:21651892

  15. Accurate determination of protein methionine oxidation by stable isotope labeling and LC-MS analysis.

    PubMed

    Liu, Hongcheng; Ponniah, Gomathinayagam; Neill, Alyssa; Patel, Rekha; Andrien, Bruce

    2013-12-17

    Methionine (Met) oxidation is a major modification of proteins, which converts Met to Met sulfoxide as the common product. It is challenging to determine the level of Met sulfoxide, because it can be generated during sample preparation and analysis as an artifact. To determine the level of Met sulfoxide in proteins accurately, an isotope labeling and LC-MS peptide mapping method was developed. Met residues in proteins were fully oxidized using hydrogen peroxide enriched with (18)O atoms before sample preparation. Therefore, it was impossible to generate Met sulfoxide as an artifact during sample preparation. The molecular weight difference of 2 Da between Met sulfoxide with the (16)O atom and Met sulfoxide with the (18)O atom was used to differentiate and calculate the level of Met sulfoxide in the sample originally. Using a recombinant monoclonal antibody as a model protein, much lower levels of Met sulfoxide were detected for the two susceptible Met residues with this new method compared to a typical peptide mapping procedure. The results demonstrated efficient elimination of the analytical artifact during LC-MS peptide mapping for the measurement of Met sulfoxide. This method can thus be used when accurate determination of the level of Met sulfoxide is critical.

  16. 15N2 Fixation by Bacteria Associated with Maize Roots at a Low Partial O2 Pressure

    PubMed Central

    Alexander, David B.; Zuberer, David A.

    1989-01-01

    Nitrogen fixation by bacteria associated with roots of intact maize plants was measured by exposing the roots to 15N2 at a partial O2 pressure (pO2) of 2 or 10 kPa. The plants were grown in a mixture of Weswood soil and sand and then transferred to plastic cylinders containing an N-free plant nutrient solution. The solution was sparged continuously with a mixture of air and N2 at a pO2 of 2 or 10 kPa. Acetylene reduction was measured after the roots were exposed to the low pO2 overnight. The air-N2 atmosphere in the cylinders was then replaced with an O2-He atmosphere at the same pO2, and the roots were exposed to 20 kPa of 15N2 for 20 to 22 h. Incorporation of 15N into the roots was 200 times greater at 2 kPa of O2 than at 10 kPa of O2. Adding l-malate (1 g of C liter−1) to the nutrient solution increased root-associated nitrogenase activity, producing a strong 15N label which could be traced into the shoots. Fixed 15N was detected in the shoots within 5 days after the plants were returned to unfertilized soil. In a similar experiment with undisturbed plants grown in fritted clay, movement of fixed 15N into the shoots was evident within 4 days after the roots were exposed to 15N2 at 2 kPa of O2. Inoculation with Azospirillum lipoferum yielded no significant differences in shoot dry weight, total nitrogen content, percent nitrogen, or 15N enrichment of plant tissues. Inoculated plants did exhibit greater root dry weight than uninoculated plants, however. PMID:16347968

  17. Subcellular localization of proteins in the anaerobic sulfate reducer Desulfovibrio vulgaris via SNAP-tag labeling and photoconversion

    SciTech Connect

    Gorur, A.; Leung, C. M.; Jorgens, D.; Tauscher, A.; Remis, J. P.; Ball, D. A.; Chhabra, S.; Fok, V.; Geller, J. T.; Singer, M.; Hazen, T. C.; Juba, T.; Elias, D.; Wall, J.; Biggin, M.; Downing, K. H.; Auer, M.

    2010-06-01

    Systems Biology studies the temporal and spatial 3D distribution of macromolecular complexes with the aim that such knowledge will allow more accurate modeling of biological function and will allow mathematical prediction of cellular behavior. However, in order to accomplish accurate modeling precise knowledge of spatial 3D organization and distribution inside cells is necessary. And while a number of macromolecular complexes may be identified by its 3D structure and molecular characteristics alone, the overwhelming number of proteins will need to be localized using a reporter tag. GFP and its derivatives (XFPs) have been traditionally employed for subcelllar localization using photoconversion approaches, but this approach cannot be taken for obligate anaerobic bacteria, where the intolerance towards oxygen prevents XFP approaches. As part of the GTL-funded PCAP project (now ENIGMA) genetic tools have been developed for the anaerobe sulfate reducer Desulfovibrio vulgaris that allow the high-throughput generation of tagged-protein mutant strains, with a focus on the commercially available SNAP-tag cell system (New England Biolabs, Ipswich, MA), which is based on a modified O6-alkylguanine-DNA alkyltransferase (AGT) tag, that has a dead-end reaction with a modified O6-benzylguanine (BG) derivative and has been shown to function under anaerobic conditions. After initial challenges with respect to variability, robustness and specificity of the labeling signal we have optimized the labeling. Over the last year, as a result of the optimized labeling protocol, we now obtain robust labeling of 20 out of 31 SNAP strains. Labeling for 13 strains were confirmed at least five times. We have also successfully performed photoconversion on 5 of these 13 strains, with distinct labeling patterns for different strains. For example, DsrC robustly localizes to the periplasmic portion of the inner membrane, where as a DNA-binding protein localizes to the center of the cell, where the

  18. Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by /sup 15/N NMR using magnetization transfer and indirect detection via protons

    SciTech Connect

    Live, D.H.; Cowburn, D.

    1987-10-06

    NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, /sup 15/N labeling being used to identify specific backbone /sup 15/N and /sup 1/H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence for hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neutrophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of /sup 15/N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone. The results suggest significant conformational alteration in neurophysin-hormone complexes at low pH possibly associated with protonation of the carboxyl group of the hormone-protein salt bridge.

  19. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling

    PubMed Central

    Zhang, Haizhen; Brown, Roslyn N.; Qian, Wei-Jun; Monroe, Matthew E.; Purvine, Samuel O.; Moore, Ronald J.; Gritsenko, Marina A.; Shi, Liang; Romine, Margaret F; Fredrickson, James K.; Paša-Tolić, Ljiljana; Smith, Richard D.; Lipton, Mary S.

    2010-01-01

    We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope 18O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level 16O and 18O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in ΔgspD mutant cells of many outer membrane proteins including the outer membrane c-cype cytochromes OmcA and MtrC, in agreement with previously investigation demonstrating that these proteins are substrates of the type II secretion system. PMID:20380418

  20. Effects of ancillary ligands on selectivity of protein labeling with platinum(II) chloro complexes

    SciTech Connect

    Zhou, Xia-Ying.

    1990-02-01

    Potassium (2,6-pyridinedicarboxylato)chloroplatinate(II) was synthesized. The molecular structure of the complex in (n-Bu){sub 4}N(Pt(dipic)Cl){center dot}0.5H{sub 2}O was determined by x-ray crystallography. The (Pt(dipic)Cl){sup {minus}} is essentially planar and contains a Pt(II) atom, a tridentate dipicolinate dianion ligand, and a unidentate Cl{sup {minus}} ligand. The bis(bidentate) complex trans-(Pt(dipic){sub 2}){sup 2{minus}} was also observed by {sup 1}H NMR. A red gel-like substance was observed when the yellow aqueous solution of K(Pt(dipic)Cl) was cooled or concentrated. The K(Pt(dipic)Cl) molecules form stacks in the solid state and gel-like substance but remain monomeric over a wide range of concentrations and temperatures. The reactivity and selectivity of(Pt(dipic)Cl){sup {minus}} toward cytochromes c from horse and tuna were studied. The new transition-metal reagent is specific for methionine residues. Di(2-pyridyl-{beta}-ethyl)sulfidochloroplatinum(II) chloride dihydrate was also synthesized. This complex labels histidine and methionine residues in cytochrome c. The ancillary ligands in these platinum(II) complexes clearly determine the selectivity of protein labeling. 106 refs., 10 figs., 11 tabs.

  1. High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies

    PubMed Central

    Anthony, Kelsey C.; You, Changjiang; Piehler, Jacob; Pomeranz Krummel, Daniel A.

    2014-01-01

    SUMMARY There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle (AuNPtris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of AuNPtris-NTA labeled proteins by electron microscopy is further ensured by the reagent’s short conformationally restricted linker. We have employed AuNPtris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. AuNPtris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our new labeling reagent should find broad application in non-covalent site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies. PMID:24560806

  2. High-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies.

    PubMed

    Anthony, Kelsey C; You, Changjiang; Piehler, Jacob; Pomeranz Krummel, Daniel A

    2014-04-01

    There is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. We report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-NTA) conjugated with a monofunctional gold nanoparticle ((AuNP)tris-NTA) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (His-tag). Multivalent binding of tris-NTA to a His-tag via complexed Ni(II) ions results in subnanomolar affinity and a defined 1:1 stoichiometry. Precise localization of (AuNP)tris-NTA labeled proteins by electron microscopy is further ensured by the reagent's short conformationally restricted linker. We used (AuNP)tris-NTA to localize His-tagged proteins in an oligomeric ATPase and in the bacterial 50S ribosomal subunit. (AuNP)tris-NTA can specifically bind to the target proteins in these assemblies and is clearly discernible. Our labeling reagent should find broad application in noncovalent, site-specific labeling of protein termini to pinpoint their location in macromolecular assemblies.

  3. Immunological comparison of the in vitro and in vivo labeled victorin binding protein from susceptible oats. [Avena sativa L

    SciTech Connect

    Wolpert, T.J.; Macko, V. )

    1991-03-01

    The fungus Cochliobolus victoriae causes victoria blight of oats and produces the host-specific toxin victorin. The reaction of oats to the fungus and its toxin is controlled by a single dominant gene whose product has been hypothesized to function as the site of action (receptor) of the toxin in susceptible oat genotypes. Previously, using a biologically active {sup 125}I derivative of the toxin, the authors identified a 100 kilodalton victorin-binding protein (VBP) which binds victorin in a ligand-specific manner and binds in vivo only in susceptible oat genotypes. However, a VBP in both the susceptible and resistant oat genotypes was identified by in vitro binding experiments. One interpretation of the lack of genotype-specific binding in vitro is that the 100 kilodalton protein detected in vitro is not the same 100 kilodalton protein detected in vivo. To clarify the relationship between the 100 kilodalton protein(s) labeled in vivo and in vitro, they developed antisera to the in vitro-labeled VBP from the susceptible genotype and demonstrated that these preparations react with the in vivo-labeled VBP from the susceptible genotype. This finding coupled with previous observations strongly suggest that the VBP observed in vivo is the same protein detected in vitro. Furthermore, the results support our previous observations which suggest that the VBPs labeled in vitro in susceptible and resistant genotypes are closely related or identical.

  4. A novel method for trapping and analyzing 15N in NO for tracing NO sources

    NASA Astrophysics Data System (ADS)

    Kang, Ronghua; Mulder, Jan; Dörsch, Peter

    2016-04-01

    15N isotope tracing is an effective and direct approach to investigate the biological and chemical sources of nitric oxide (NO) in soil. However, NO is highly reactive and rapidly converted to nitrogen dioxide (NO2) in the presence of ozone. Various chemical conversions of NO to the more stable solutes nitrite (NO2-) and nitrate (NO3-) have been proposed, which allow analysing the 15N abundance without major fractionation. However, NO emissions from soils are usually small, posing major challenges to conversion efficiency and background contamination. Here we present a novel method in which NO is oxidized to NO2- by chromium trioxide (CrO3) prior to conversion to NO2- and NO3- in an alkaline hydrogen peroxide (H2O2) solution. Immediately following trapping, manganese dioxide (MnO2) and 5M HCl are added to remove excess H2O2, and to adjust the pH to around 6.0-7.0, respectively. The resulting solution can be stored until analysis and is none-toxic, allowing to use a modified denitrifier method (Zhu et al., submitted), where NO2- and NO3- are reduced quantitatively to nitrous oxide (N2O). Optimum NO conversion rates of > 90% even at extremely low initial NO concentration were obtained with 4% H2O2, 0.5 M NaOH, and 0.5 L min-1 gas flow rate. In a laboratory test, using NO gas with different 15N signals produced from unlabelled and labelled NO2-, we found an overall precision of 0.4‰ for unlabelled and 49.7‰ for NO enriched with 1.0 atom% 15N, respectively. This indicates that this method can be used for both natural abundance studies of NO, as well as in labelling studies tracing NO sources. Zhu J, Yu L, Bakken LR, Mørkved PT, Mulder J, Dörsch P. Controlled induction of denitrification in Pseudomonas aureofaciens: a modified denitrifier method for 15N and 18O analysis in NO3- from natural water samples by IRMS. Submitted.

  5. Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immunoperoxidase labeling.

    PubMed

    Schachner, M; Hedley-Whyte, E T; Hsu, D W; Schoonmaker, G; Bignami, A

    1977-10-01

    Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirmation of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia. Astrocytic processes were stained in white matter, in the granular layet surrounding synaptic glomerular complexes, and in the molecular layer in the form of radially oriented fibers and of sheaths surrounding Purkinje cell dendrites. Astrocytic endfeet impinging on meninges and perivascular membranes were also antigen positive. In astrocytic perikarya and processes, the immunohistochemical reaction product appears both as a diffuse cytoplasmic label and as elongated strands, which by their distribution and frequency could be considered glial filaments.

  6. Efficient 18F labeling of cysteine-containing peptides and proteins using tetrazine-trans-cyclooctene ligation.

    PubMed

    Liu, Shuanglong; Hassink, Matthew; Selvaraj, Ramajeyam; Yap, Li-Peng; Park, Ryan; Wang, Hui; Chen, Xiaoyuan; Fox, Joseph M; Li, Zibo; Conti, Peter S

    2013-01-01

    18F positron emission tomography (PET) has a number of attributes that make it clinically attractive, including nearly 100% positron efficiency, very high specific radioactivity, and a short half-life of ≈ 110 minutes. However, the short half-life of 18F and the poor nucleophilicity of fluoride introduce challenges for the incorporation of 18F into complex molecules. Recently, the tetrazine-trans-cyclooctene ligation was introduced as a novel 18F labeling method that proceeds with fast reaction rates without catalysis. Herein we report an efficient method for 18F labeling of free cysteines of peptides and proteins based on sequential ligation with a bifunctional tetrazinyl-maleimide and an 18F-labeled trans-cyclooctene. The newly developed method was tested for site-specific labeling of both c(RGDyC) peptide and vascular endothelial growth factor (VEGF)-SH protein. Starting with 4 mCi of 18F-trans-cyclooctene and only 10 μg of tetrazine-RGD (80-100 μM) or 15 μg of tetrazine-VEGF (6.0 μM), 18F-labeled RGD peptide and VEGF protein could be obtained within 5 minutes in 95% yield and 75% yield, respectively. The obtained tracers were then evaluated in mice. In conclusion, a highly efficient method has been developed for site-specific 18F labeling of cysteine-containing peptides and proteins. The special characteristics of the tetrazine-trans-cyclooctene ligation provide unprecedented opportunities to synthesize 18F-labeled probes with high specific activity for PET applications.

  7. Exploring Structure, Dynamics, and Topology of Nitroxide Spin-Labeled Proteins Using Continuous-Wave Electron Paramagnetic Resonance Spectroscopy.

    PubMed

    Altenbach, Christian; López, Carlos J; Hideg, Kálmán; Hubbell, Wayne L

    2015-01-01

    Structural and dynamical characterization of proteins is of central importance in understanding the mechanisms underlying their biological functions. Site-directed spin labeling (SDSL) combined with continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy has shown the capability of providing this information with site-specific resolution under physiological conditions for proteins of any degree of complexity, including those associated with membranes. This chapter introduces methods commonly employed for SDSL and describes selected CW EPR-based methods that can be applied to (1) map secondary and tertiary protein structure, (2) determine membrane protein topology, (3) measure protein backbone flexibility, and (4) reveal the existence of conformational exchange at equilibrium. PMID:26477248

  8. Application of meta- and para- phenylenediamine as enhanced oxime ligation catalysts for protein labeling, PEGylation, immobilization and release

    PubMed Central

    Mahmoodi, Mohammad M.; Rashidian, Mohammad; Zhang, Yi; Distefano, Mark D.

    2015-01-01

    Meta- and para- phenylenediamines have recently been shown to catalyze oxime and hydrazone ligation reactions at rates much faster than aniline, a commonly used catalyst. Here, it is demonstrated how these new catalysts can be used in a generally applicable procedure for fluorescent labeling, PEGylation, immobilization and release of aldehyde and ketone functionalized proteins. The chemical orthogonality of phenylenediamine-catalyzed oxime ligation versus copper catalyzed click reaction has also been harnessed for simultaneous dual labeling of bifunctional proteins containing both aldehyde and alkyne groups in high yield. PMID:25640893

  9. Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry.

    PubMed

    Heudi, Olivier; Barteau, Samuel; Zimmer, Dieter; Schmidt, Joerg; Bill, Kurt; Lehmann, Natalie; Bauer, Christian; Kretz, Olivier

    2008-06-01

    Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC

  10. Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

    NASA Astrophysics Data System (ADS)

    Yang, Jun; Parkanzky, Paul D.; Bodner, Michele L.; Duskin, Craig A.; Weliky, David P.

    2002-12-01

    Clean MAS observation of 13C-labeled carbons in membrane-bound HIV-1 and influenza fusion peptides was made by using a rotational-echo double-resonance spectroscopy (REDOR) filter of directly bonded 13C- 15N pairs. The clean filtering achieved with the REDOR approach is superior to filtering done with sample difference spectroscopy. In one labeling approach, the peptide had labels at a single 13C carbonyl and its directly bonded 15N. The resulting chemical shift distribution of the filtered signal is used to assess the distribution of local secondary structures at the labeled carbonyl. For the influenza peptide, the Leu-2 carbonyl chemical shift distribution is shown to vary markedly with lipid and detergent composition, as well as peptide:lipid ratio, suggesting that the local peptide structure also has a strong dependence on these factors. Because most carboxylic- and amino-labeled amino acids are commercially available, this REDOR approach should have broad applicability to chemically synthesized peptides as well as bacterially synthesized proteins. In a second labeling approach, the HIV-1 fusion peptide had U- 13C, 15N labeling over three sequential residues. When a 1.6 ms REDOR dephasing time is used, only backbone 13C signals are observed. The resulting spectra are used to determine spectral linewidths and to assess feasibility of assignment of uniformly labeled peptide.

  11. Label-acquired magnetorotation as a signal transduction method for protein detection: aptamer-based detection of thrombin

    PubMed Central

    Hecht, Ariel; Akshay Kumar, Anand; Kopelman, Raoul

    2011-01-01

    This paper presents a new signal transduction method, called Label-Acquired Magnetorotation (LAM), for the measurement of proteins in solution. We demonstrate the use of LAM to detect the protein thrombin using aptamers, with an LOD (limit of detection) of 300 pM. LAM is modeled after a sandwich assay, with a 10 µm nonmagnetic “mother” sphere as the capture component, and with 1 µm magnetic “daughter” beads as the labels. The protein-mediated attachment of daughter beads to the mother sphere forms a rotating sandwich complex. In a rotating magnetic field, the rotational frequency of a sandwich complex scales with the number of attached magnetic beads, which scales with the concentration of the protein present in solution. This paper represents the first instance of the detection of a protein using LAM. PMID:21805996

  12. Potential probe for examining opiate-receptor interactions: model compound study of dynamic effects on /sup 15/N INEPT enhancements

    SciTech Connect

    Schilling, K.H.; Mikita, M.A.

    1987-10-01

    Model systems were chosen in an attempt to mimic the proton exchange environment of an agonist nitrogen in an opiate-receptor interaction. The two model systems studied were an ammonium: 18-crown-6 ether complex and a quinuclidine-trifluoroacetic acid ion pair. Each system was examined for their effects on /sup 15/N NMR INEPT enhancements. Both models were found to retard proton exchange dynamics, as observed by increased enhancements relative to free ions in neutral aqueous solutions. These results suggest that the confinement of a protonated nitrogen, such as that expected in receptor binding, may alter exchange dynamics to favor INEPT enhancements, while unbound agonists would remain unenhanced. As a result, /sup 15/N NMR INEPT enhancements from a solution of receptor subtypes with an appropriate /sup 15/N-labeled agonist may present a means of exploring the dynamics of direct opiate-receptor interactions.

  13. Detection of organic sulfur by {sup 15}N and {sup 19}F NMR via formation of iminosulfuranes

    SciTech Connect

    Franz, J.A.; Linehan, J.C.; Lamb, C.N.

    1992-08-01

    We have synthesized new iminosulfuranes from a variety of diaryl-and dialkyl sulfides and dibenzothiophene. The pattern of {sup 15}N chemical shifts indicates that functional groups attached to sulfur are not simply resolved into aryl and alkyl groups. Thus, resolution of sulfur functional groups using {sup 15}N NMR via iminosulfurane does not appear practicable. However, iminosulfurane formation, together with the N-haloamide reaction and the Pummerer rearrangement, provides pathways for chemical discrimination of different sulfur substituents using unique {sup 15}N- or, {sup 19}F-labelled fragments for different categories of sulfur functional groups. In efforts currently underway, we are applying these reactions to methylated extracts and conversion products of the high-organic-sulfur containing Yugoslavian Rasa and Spanish Mequinenza lignites. 1 tab, 14 refs.

  14. Detection of organic sulfur by [sup 15]N and [sup 19]F NMR via formation of iminosulfuranes

    SciTech Connect

    Franz, J.A.; Linehan, J.C.; Lamb, C.N.

    1992-08-01

    We have synthesized new iminosulfuranes from a variety of diaryl-and dialkyl sulfides and dibenzothiophene. The pattern of [sup 15]N chemical shifts indicates that functional groups attached to sulfur are not simply resolved into aryl and alkyl groups. Thus, resolution of sulfur functional groups using [sup 15]N NMR via iminosulfurane does not appear practicable. However, iminosulfurane formation, together with the N-haloamide reaction and the Pummerer rearrangement, provides pathways for chemical discrimination of different sulfur substituents using unique [sup 15]N- or, [sup 19]F-labelled fragments for different categories of sulfur functional groups. In efforts currently underway, we are applying these reactions to methylated extracts and conversion products of the high-organic-sulfur containing Yugoslavian Rasa and Spanish Mequinenza lignites. 1 tab, 14 refs.

  15. 1H, 13C, and 15N backbone, side-chain, and heme chemical shift assignments for oxidized and reduced forms of the monoheme c-type cytochrome ApcA isolated from the acidophilic metal-reducing bacterium Acidiphilium cryptum.

    SciTech Connect

    Cort, John R.; Swenson, Michael; Magnuson, Timothy S.

    2011-03-04

    We report the 1H, 13C, and 15N chemical shift assignments of both oxidized and reduced forms of an abundant periplasmic c-type cytochrome, designated ApcA, from the acidophilic gram-negative facultatively anaerobic metal-reducing alpha-proteobacterium Acidiphilium cryptum. These resonance assignments prove that ApcA is a monoheme cytochrome c2 and the product of the Acry_2099 gene. An absence of resonance peaks in the NMR spectra for the 21 N-terminal residues suggests that a predicted N-terminal signal sequence is cleaved. We also describe the preparation and purification of the protein in labeled form from laboratory cultures of A. cryptum growing on 13C- and 15N- labeled substrates.

  16. Analysis of side chain rotational restrictions of membrane-embedded proteins by spin-label ESR spectroscopy

    NASA Astrophysics Data System (ADS)

    Štrancar, Janez; Kavalenka, Aleh; Ziherl, Primož; Stopar, David; Hemminga, Marcus A.

    2009-04-01

    Site-directed spin-labeling electron spin resonance (SDSL-ESR) is a promising tool for membrane protein structure determination. Here we propose a novel way to translate the local structural constraints gained by SDSL-ESR data into a low-resolution structure of a protein by simulating the restrictions of the local conformational spaces of the spin label attached at different protein sites along the primary structure of the membrane-embedded protein. We test the sensitivity of this approach for membrane-embedded M13 major coat protein decorated with a limited number of strategically placed spin labels employing high-throughput site-directed mutagenesis. We find a reasonably good agreement of the simulated and the experimental data taking a protein conformation close to the one determined by fluorescence resonance energy transfer analysis [P.V. Nazarov, R.B.M. Koehorst, W.L. Vos, V.V. Apanasovich, M.A. Hemminga, FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein, Biophys. J. 92 (2007) 1296-1305].

  17. Analysis of side chain rotational restrictions of membrane-embedded proteins by spin-label ESR spectroscopy.

    PubMed

    Strancar, Janez; Kavalenka, Aleh; Ziherl, Primoz; Stopar, David; Hemminga, Marcus A

    2009-04-01

    Site-directed spin-labeling electron spin resonance (SDSL-ESR) is a promising tool for membrane protein structure determination. Here we propose a novel way to translate the local structural constraints gained by SDSL-ESR data into a low-resolution structure of a protein by simulating the restrictions of the local conformational spaces of the spin label attached at different protein sites along the primary structure of the membrane-embedded protein. We test the sensitivity of this approach for membrane-embedded M13 major coat protein decorated with a limited number of strategically placed spin labels employing high-throughput site-directed mutagenesis. We find a reasonably good agreement of the simulated and the experimental data taking a protein conformation close to the one determined by fluorescence resonance energy transfer analysis [P.V. Nazarov, R.B.M. Koehorst, W.L. Vos, V.V. Apanasovich, M.A. Hemminga, FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein, Biophys. J. 92 (2007) 1296-1305]. PMID:19138542

  18. Calcium translocation mechanism in sarcoplasmic reticulum vesicles, deduced from location studies of protein-bound spin labels.

    PubMed Central

    Champeil, P; Rigaud, J L; Gary-Bobo, C M

    1980-01-01

    Sarcoplasmic reticulum vesicles were exposed to various thiol-directed spin labels, and the position of the label on the inner or outer vesicle surface was investigated as a function of the ATPase (adenosinetriphosphatase; ATP phosphohydrolase, EC 3.6.1.3) chemical state. Previous measurements of label accessibility to externally added ascorbate had been considered to suggest an external-internal transition of protein-bound labels, coupled with ion translocation [Tonomura, Y. & Morales, M.F. (1974) Proc. Natl. Acad. Sci. USA 71, 3687-3691]. We show that these ascorbate studies do not lead to convincing conclusions. We demonstrate, on the contrary, that transition ions (nickel and ferricyanide) can be used as selective line-broadening agents for the signals arising from external labels. No significant difference in nickel- or ferricyanide-label interaction can be attributed to a different orientation of the label in any of the enzyme chemical states tested. Our results therefore contradict the current interpretation of ascorbate quenching experiments in terms of calcium ATPase rotatory motion; rather they are consistent with ion transport models involving only limited conformational rearrangements of the pump. PMID:6446710

  19. Aliphatic (1)H, (13)C and (15)N chemical shift assignments of dihydrofolate reductase from the psychropiezophile Moritella profunda in complex with NADP(+) and folate.

    PubMed

    Loveridge, E Joel; Matthews, Stella M; Williams, Christopher; Whittaker, Sara B-M; Günther, Ulrich L; Evans, Rhiannon M; Dawson, William M; Crump, Matthew P; Allemann, Rudolf K

    2013-04-01

    Dihydrofolate reductase from the deep-sea bacterium Moritella profunda (MpDHFR) has been (13)C/(15)N isotopically labelled and purified. Here, we report the aliphatic (1)H, (13)C and (15)N resonance assignments of MpDHFR in complex with NADP(+) and folate. The spectra of MpDHFR suggest considerably greater conformational heterogeneity than is seen in the closely related DHFR from Escherichia coli.

  20. Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

    PubMed

    Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

    2014-01-01

    Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment.

  1. Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study

    PubMed Central

    Shotton, D.; Thompson, K.; Wofsy, L.; Branton, D.

    1978-01-01

    We have used freeze-etching, before and after immunoferritin labeling, to visualize spectrin molecules and other surface proteins of the human erythrocyte membrane. After intramembrane particle aggregation was induced, spectrin molecules, identified by labeling with ferritin-conjugated antispectrin, were clustered on the cytoplasmic surface of the membrane in patches directly underlying the particle clusters. This labeling pattern confirms the involvement of spectrin in such particle aggregates, as previously inferred from indirect evidence. Ferritin-conjugated antihapten molecules, directed against external and cytoplasmic surface proteins of the erythrocyte membrane which had been covalently labeled nonspecifically with the hapten p-diazoniumphenyl-beta-D-lactoside, were similarly found in direct association with such intramembrane particle aggregates. This indicates that when spectrin and the intramembrane particles are aggregated, all the major proteins of the erythrocyte membrane are constrained to coaggregate with them. Although giving no direct information concerning the freedom of translational movement of proteins in the unperturbed erythrocyte membrane, these experiments suggest that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others. PMID:10605454

  2. Metabolic labeling of cellular glycoproteins with glucosamine: potential for erroneous interpretations due to nonenzymatic radiolabeling of proteins

    SciTech Connect

    Briles, E.I.B.; Updyke, T.V.

    1986-05-01

    Proteins, including serum proteins of culture media, become nonenzymatically radiolabeled under conditions used for metabolic labeling of cultured cells with glucosamine. This occurs even under sterile conditions in the absence of cells. Various commercial lots of /sup 3/H or /sup 14/C glcN gave similar results: approx. 0.7% of total label was incorporated into 20% serum (14 mg/ml protein) in 48 h at 37/sup 0/C. By SDS-PAGE fluorography, labeled serum bands correspond to Coomassie stained bands. Incorporation is linear with protein concentration and label input, shows biphasic kinetics (initial rapid rate within first 3 hr, followed by slower linear rate with no sign of saturation through 120 hr), and is temperature-dependent (no reaction at 0/sup 0/C; incorporation at 20/sup 0/C is approx. 45% of that at 37/sup 0/C). Poly-D-lysine is a better acceptor than protein: 0.5 mg/ml PL accepts as much label as 7 mg/ml protein. Incorporation is inhibited by excess unlabeled glcN and ethanolamine, but not by man, gal or glucose. However, when proteins were incubated with 160 mM glcN, SDS-PAGE bands were yellow-brown, suggesting the occurrence of Maillard-type reactions. Although the chemical mechanism(s) responsible for nonmetabolic radiolabeling by glcN are not clear at this point, the fact that it occurs represents a serious artifact which may lead to erroneous interpretation of data.

  3. Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics.

    PubMed

    Smits, Arne H; Jansen, Pascal W T C; Poser, Ina; Hyman, Anthony A; Vermeulen, Michiel

    2013-01-01

    Many cellular proteins assemble into macromolecular protein complexes. The identification of protein-protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not suitable for high-throughput screenings. Here, we describe a robust and easy to implement label-free relative quantification approach that combines the detection of high-confidence protein-protein interactions with an accurate determination of the stoichiometry of the identified protein-protein interactions in a single experiment. We applied this method to two chromatin-associated protein complexes for which the stoichiometry thus far remained elusive: the MBD3/NuRD and PRC2 complex. For each of these complexes, we accurately determined the stoichiometry of the core subunits while at the same time identifying novel interactors and their stoichiometry.

  4. Changes in diacylglycerol labeling, cell shape, and protein phosphorylation distinguish triggering from activation of human neutrophils

    SciTech Connect

    Reibman, J.; Korchak, H.M.; Vosshall, L.B.; Haines, K.A.; Rich, A.M.; Weissmann, G.

    1988-05-05

    Upon activation neutrophils release reactive oxygen intermediates such as superoxide anion (O/sub 2//sup -/) which are potent mediators of inflammation. Various agents elicit different responses. In contrast, phorbol myristate acetate (PMA, 1.6 ..mu..M) acting directly via protein kinase C is a potent stimulus for O/sub 2//sup -/. The authors compared the kinetics of appearance of various second messengers with the capacity of these ligands to elicit O/sub 2//sup -/ generation. Kinetic analysis showed a two-phase response to membrane ligands; both an early (greater than or equal to 15 s) and a late (>15 s) increase in (/sup 3/H)- and (/sup 14/C)diacylglycerol (DG) was noted in response to fMLP. In contrast, LTB/sub 4/ elicited only a rapid early increase in DG. The rise in DG evoked by PMA was late. Moreover, comparison of increases in (/sup 3/H)DG versus those of (/sup 14/C)DG at early and late time points suggested that DG was not formed exclusively from the hydrolysis of polyphosphoinositides. Kinetic analysis of protein phosphorylation was compared to the early and late increments of DG labeling. A 47,000 M/sub r/ protein was phosphorylated with kinetics consistent with the production of O/sub 2//sup -/ and DG in response to fMLP and PMA. The temporal pattern of the formation of diacylglycerol and the phosphorylation of proteins describe a dual signal. The data suggest that neutrophils require not only triggering (the rapid generation of a signal) but also activation (the maintenance of a signal) to sustain responses.

  5. Sparse Markov chain-based semi-supervised multi-instance multi-label method for protein function prediction.

    PubMed

    Han, Chao; Chen, Jian; Wu, Qingyao; Mu, Shuai; Min, Huaqing

    2015-10-01

    Automated assignment of protein function has received considerable attention in recent years for genome-wide study. With the rapid accumulation of genome sequencing data produced by high-throughput experimental techniques, the process of manually predicting functional properties of proteins has become increasingly cumbersome. Such large genomics data sets can only be annotated computationally. However, automated assignment of functions to unknown protein is challenging due to its inherent difficulty and complexity. Previous studies have revealed that solving problems involving complicated objects with multiple semantic meanings using the multi-instance multi-label (MIML) framework is effective. For the protein function prediction problems, each protein object in nature may associate with distinct structural units (instances) and multiple functional properties (class labels) where each unit is described by an instance and each functional property is considered as a class label. Thus, it is convenient and natural to tackle the protein function prediction problem by using the MIML framework. In this paper, we propose a sparse Markov chain-based semi-supervised MIML method, called Sparse-Markov. A sparse transductive probability graph is constructed to encode the affinity information of the data based on ensemble of Hausdorff distance metrics. Our goal is to exploit the affinity between protein objects in the sparse transductive probability graph to seek a sparse steady state probability of the Markov chain model to do protein function prediction, such that two proteins are given similar functional labels if they are close to each other in terms of an ensemble Hausdorff distance in the graph. Experimental results on seven real-world organism data sets covering three biological domains show that our proposed Sparse-Markov method is able to achieve better performance than four state-of-the-art MIML learning algorithms.

  6. Ligand-free palladium-mediated site-specific protein labeling inside gram-negative bacterial pathogens.

    PubMed

    Li, Jie; Lin, Shixian; Wang, Jie; Jia, Shang; Yang, Maiyun; Hao, Ziyang; Zhang, Xiaoyu; Chen, Peng R

    2013-05-15

    Palladium, a key transition metal in advancing modern organic synthesis, mediates diverse chemical conversions including many carbon-carbon bond formation reactions between organic compounds. However, expanding palladium chemistry for conjugation of biomolecules such as proteins, particularly within their native cellular context, is still in its infancy. Here we report the site-specific protein labeling inside pathogenic Gram-negative bacterial cells via a ligand-free palladium-mediated cross-coupling reaction. Two rationally designed pyrrolysine analogues bearing an aliphatic alkyne or an iodophenyl handle were first encoded in different enteric bacteria, which offered two facial handles for palladium-mediated Sonogashira coupling reaction on proteins within these pathogens. A GFP-based bioorthogonal reaction screening system was then developed, allowing evaluation of both the efficiency and the biocompatibilty of various palladium reagents in promoting protein-small molecule conjugation. The identified simple compound-Pd(NO3)2 exhibited high efficiency and biocompatibility for site-specific labeling of proteins in vitro and inside living E. coli cells. This Pd-mediated protein coupling method was further utilized to label and visualize a Type-III Secretion (T3S) toxin-OspF in Shigella cells. Our strategy may be generally applicable for imaging and tracking various virulence proteins within Gram-negative bacterial pathogens.

  7. Cerebral glutamine metabolism under hyperammonemia determined in vivo by localized 1H and 15N NMR spectroscopy

    PubMed Central

    Cudalbu, Cristina; Lanz, Bernard; Duarte, João MN; Morgenthaler, Florence D; Pilloud, Yves; Mlynárik, Vladimir; Gruetter, Rolf

    2012-01-01

    Brain glutamine synthetase (GS) is an integral part of the glutamate–glutamine cycle and occurs in the glial compartment. In vivo Magnetic Resonance Spectroscopy (MRS) allows noninvasive measurements of the concentrations and synthesis rates of metabolites. 15N MRS is an alternative approach to 13C MRS. Incorporation of labeled 15N from ammonia in cerebral glutamine allows to measure several metabolic reactions related to nitrogen metabolism, including the glutamate–glutamine cycle. To measure 15N incorporation into the position 5N of glutamine and position 2N of glutamate and glutamine, we developed a novel 15N pulse sequence to simultaneously detect, for the first time, [5-15N]Gln and [2-15N]Gln+Glu in vivo in the rat brain. In addition, we also measured for the first time in the same experiment localized 1H spectra for a direct measurement of the net glutamine accumulation. Mathematical modeling of 1H and 15N MRS data allowed to reduce the number of assumptions and provided reliable determination of GS (0.30±0.050 μmol/g per minute), apparent neurotransmission (0.26±0.030 μmol/g per minute), glutamate dehydrogenase (0.029±0.002 μmol/g per minute), and net glutamine accumulation (0.033±0.001 μmol/g per minute). These results showed an increase of GS and net glutamine accumulation under hyperammonemia, supporting the concept of their implication in cerebral ammonia detoxification. PMID:22167234

  8. Resolving Conformational and Rotameric Exchange in Spin-Labeled Proteins Using Saturation Recovery EPR

    PubMed Central

    Bridges, Michael D.; Hideg, Kálmán

    2010-01-01

    The function of many proteins involves equilibria between conformational substates, and to elucidate mechanisms of function it is essential to have experimental tools to detect the presence of conformational substates and to determine the time scale of exchange between them. Site-directed spin labeling (SDSL) has the potential to serve this purpose. In proteins containing a nitroxide side chain (R1), multicomponent electron paramagnetic resonance (EPR) spectra can arise either from equilibria involving different conformational substates or rotamers of R1. To employ SDSL to uniquely identify conformational equilibria, it is thus essential to distinguish between these origins of multicomponent spectra. Here we show that this is possible based on the time scale for exchange of the nitroxide between distinct environments that give rise to multicomponent EPR spectra; rotamer exchange for R1 lies in the ≈0.1–1 μs range, while conformational exchange is at least an order of magnitude slower. The time scales of exchange events are determined by saturation recovery EPR, and in favorable cases, the exchange rate constants between substates with lifetimes of approximately 1–70 μs can be estimated by the approach. PMID:20157634

  9. Stepwise enrichment of 15N along food chains: Further evidence and the relation between δ 15N and animal age

    NASA Astrophysics Data System (ADS)

    Minagawa, Masao; Wada, Eitaro

    1984-05-01

    The isotopic composition of nitrogen was measured in marine and fresh-water animals from the East China Sea, The Bering Sea, Lake Ashinoko and Usujiri intertidal zone. Primary producers, showed average δ15Nversus atmospheric nitrogen of +5.0%. (+3.4 to +7.5) in the Bering Sea and Lake Ashinoko, and +6.8%. (+6.0 to +7.6) in Usujiri intertidal zone. Blue green algae from the East China Sea show an average -0.55%. (-0.8 to +1.2). All consumers, Zooplankton, fish and bird exhibited Stepwise enrichment of 15N with increasing trophic level. The 15N enrichment at a single feeding process ranged from +1.3 to +5.3 averaging +3.4 ± 1.1%.. This isotopic fractionation seems to be independent of habitat. The effect of age in animals was obtained by analyzing two marine mussels. The soft tissue nitrogen showed +2.0%. enrichment relative to that of primary producers, and the magnitude was almost constant with shell ages ranging from 0 to 8 years. A similar 15N enrichment occurs in all Molluscs, Crustaceans, Insecta, Amphibia, Fish, Ave and Mammal species regardless of the difference in the form of excreted nitrogen and in laboratory cultured fish, brine shrimp and mice (+2.9 to +4.9%.). The excreted ammonia from guppy was sufficiently light to balance the concentration of 15N to animal body.

  10. The effects of sex, tissue type, and dietary components on stable isotope discrimination factors (Δ13C and Δ15N) in mammalian omnivores.

    PubMed

    Kurle, Carolyn M; Koch, Paul L; Tershy, Bernie R; Croll, Donald A

    2014-01-01

    We tested the effects of sex, tissue, and diet on stable isotope discrimination factors (Δ(13)C and Δ(15)N) for six tissues from rats fed four diets with varied C and N sources, but comparable protein quality and quantity. The Δ(13)C and Δ(15)N values ranged from 1.7-4.1‰ and 0.4-4.3‰, respectively. Females had higher Δ(15)N values than males because males grew larger, whereas Δ(13)C values did not differ between sexes. Differences in Δ(13)C values among tissue types increased with increasing variability in dietary carbon sources. The Δ(15)N values increased with increasing dietary δ(15)N values for all tissues except liver and serum, which have fast stable isotope turnover times, and differences in Δ(15)N values among tissue types decreased with increasing dietary animal protein. Our results demonstrate that variability in dietary sources can affect Δ(13)C values, protein source affects Δ(15)N values even when protein quality and quantity are controlled, and the isotope turnover rate of a tissue can influence the degree to which diet affects Δ(15)N values.

  11. Solution NMR Structure Determination of Polytopic α-Helical Membrane Proteins: A Guide to Spin Label Paramagnetic Relaxation Enhancement Restraints.

    PubMed

    Columbus, Linda; Kroncke, Brett

    2015-01-01

    Solution nuclear magnetic resonance structures of polytopic α-helical membrane proteins require additional restraints beyond the traditional Nuclear Overhauser Effect (NOE) restraints. Several methods have been developed and this review focuses on paramagnetic relaxation enhancement (PRE). Important aspects of spin labeling, PRE measurements, structure calculations, and structural quality are discussed.

  12. Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as antibody labels to increase the fluorescence signal and sensitivity. Ep...

  13. Biological characterization of a new radioactive labeling reagent for bacterial penicillin-binding proteins

    SciTech Connect

    Preston, D.A.; Wu, C.Y.; Blaszczak, L.C.; Seitz, D.E.; Halligan, N.G. )

    1990-05-01

    Radiolabeled penicillin G is widely used as the imaging agent in penicillin-binding protein (PBP) assays. The disadvantages of most forms of labeled penicillin G are instability on storage and the long exposure times usually required for autoradiography or fluorography of electrophoretic gels. We investigated the utility of radioiodinated penicillin V as an alternative reagent. Radioiodination of p-(trimethylstannyl)penicillin V with ({sup 125}I)Na, using a modification of the chloramine-T method, is simple, high yielding, and site specific. We demonstrated the general equivalence of commercially obtained ({sup 3}H)penicillin G and locally synthesized ({sup 125}I)penicillin V (IPV) in their recognition of bacterial PBPs. Profiles of PBPs in membranes from Bacteroides fragilis, Escherichia coli, Providencia rettgeri, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, and Enterococcus faecium labeled with IPV or (3H)penicillin G were virtually identical. Use of IPV as the imaging agent in competition experiments for determination of the affinities of various beta-lactam antibiotics for the PBPs of E. coli yielded results similar to those obtained in experiments with ({sup 3}H)penicillin G. Dried electrophoretic gels from typical PBP experiments, using IPV at 37.3 Ci/mmol and 30 micrograms/ml, exposed X-ray film in 8 to 24 h. The stability of IPV on storage at 4{degrees}C was inversely proportional to specific activity. At 37.3 Ci/mmol and 60 micrograms/ml, IPV retained useful activity for at least 60 days at 4{degrees}C. IPV represents a practical and stable reagent for rapid PBP assays.

  14. Quantification of (15)N-nitrate in urine with gas chromatography combustion isotope ratio mass spectrometry to estimate endogenous NO production.

    PubMed

    Houben, Els; Hamer, Henrike M; Luypaerts, Anja; De Preter, Vicky; Evenepoel, Pieter; Rutgeerts, Paul; Verbeke, Kristin

    2010-01-15

    The use of stable isotope labeled substrates and subsequent analysis of urinary nitrate, forms a noninvasive test for evaluation of the in vivo NO metabolism. The present paper describes a new method for simultaneous quantification of (15)N-nitrate and total nitrate with gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS). Nitrate, isolated from urine with a nitrate selective resin, was reduced to nitrite using copperized cadmium. Subsequently, Sudan I was formed by diazotation. Sudan II was added as internal standard, and both molecules were analyzed with GC-C-IRMS as tert-butyldimethylsilyl derivatives. The accuracy was determined during a recovery study of two different known nitrate concentrations and two (15)N-enrichments. A recovery of 101.6% and 103.9% for total nitrate and 107.6% and 91.2% for (15)N-nitrate was obtained, respectively. The validated method was applied on complete 72 h urine collections after intravenous administration of (15)N-nitrate and (15)N-arginine in humans. On average, 51.8% (47.0-71.0%) of administered (15)N-nitrate was excreted, while 0.68% (0.44-1.17%) of (15)N-arginine was metabolized to nitrate. In conclusion, this method can be used for accurate simultaneous determination of (15)N-nitrate and total nitrate concentrations in urine and can be applied in clinical studies for noninvasive evaluation of NO metabolism in vivo.

  15. Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

    SciTech Connect

    Link, R.P.; Kutner, A.; Schnoes, H.K.; DeLuca, H.F.

    1987-06-30

    3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3.

  16. Site-Specific Labeling of Protein Kinase CK2: Combining Surface Display and Click Chemistry for Drug Discovery Applications.

    PubMed

    Nienberg, Christian; Retterath, Anika; Becher, Kira-Sophie; Saenger, Thorsten; Mootz, Henning D; Jose, Joachim

    2016-01-01

    Human CK2 is a heterotetrameric constitutively active serine/threonine protein kinase and is an emerging target in current anti-cancer drug discovery. The kinase is composed of two catalytic CK2α subunits and two regulatory CK2β subunits. In order to establish an assay to identify protein-protein-interaction inhibitors (PPI) of the CK2α/CK2β interface, a bioorthogonal click reaction was used to modify the protein kinase α-subunit with a fluorophore. By expanding the genetic code, the unnatural amino acid para azidophenylalanine (pAzF) could be incorporated into CK2α. Performing the SPAAC click reaction (Strain-Promoted Azide-Alkyne Cycloaddition) by the use of a dibenzylcyclooctyne-fluorophore (DBCO-fluorophore) led to a specifically labeled human protein kinase CK2α. This site-specific labeling does not impair the phosphorylation activity of CK2, which was evaluated by capillary electrophoresis. Furthermore a dissociation constant (KD) of 631 ± 86.2 nM was determined for the substrate αS1-casein towards CK2α. This labeling strategy was also applied to CK2β subunit on Escherichia coli, indicating the site-specific modifications of proteins on the bacterial cell surface when displayed by Autodisplay. PMID:27355959

  17. Metabolically sup 35 S-labeled recombinant calmodulin as a ligand for the detection of calmodulin-binding proteins

    SciTech Connect

    Asselin, J.; Phaneuf, S.; Watterson, D.M.; Haiech, J. )

    1989-04-01

    We have developed a simplified procedure for the production of metabolically labeled calmodulin. We used bacterial clones (Escherichia coli) that were found to express VU-1 calmodulin, a calmodulin that is fully active with a variety of calmodulin-regulated enzymes. VU-1 calmodulin was labeled with sulfur-35 in bacteria maintained in a sulfur-free medium. Calmodulin was then purified by chromatography on phenyl-Sepharose. Under these conditions, the specific activity of the proteins was 150 to 400 cpm/fmol of calmodulin. To demonstrate the utility of this labeled VU-1 calmodulin, we examined the calmodulin-binding proteins in aortic myocyte preparation from Day 0 and Day 15 cultures by using both the gel and the nitrocellulose overlay protocols. The results showed that calmodulin-binding proteins are easily detected by the two procedures and that the profile of these target proteins changed in myocyte with time in culture. While most of these calmodulin-binding proteins have not been identified, the relative mobility on SDS-PAGE gels suggests that myosin light chain kinase (Mr approximately 137,000) was detected by these methods. We demonstrated here that the nitrocellulose overlay was faster than the gel overlay and that this technique can be useful for the study of calmodulin-binding proteins.