Littérature scientifique sur le sujet « Charged-residue »

To determine whether a charged histidine side chain affects alpha-helix stability only when histidine is close to one end of the helix or also when it is in the central region, we substitute a single histidine residue at many positions in two reference peptides and measure helix stability and histidine pKa. The position of a charged histidine residue has a major effect on helix stability in 0.01 M NaCl: the helix content of a 17-residue peptide is 24% when histidine is at position 3 compared to 76% when it is at position 17. This dependence of helix content on histidine position decreases sharply in 1 M NaCl, as expected for counterion screening of the charge-helix dipole interaction. Results at interior positions indicate that the position of a charged histidine residue affects helix stability at these positions. Unexpectedly high values of the helix content are found when either neutral or charged histidine is at one of the last three C-terminal positions, suggesting that either form can stabilize an isolated helix by hydrogen bonding to a main-chain CO group.

Previous studies using combined techniques of site-directed mutagenesis and electrophysiology of voltage-gated Na+ channels have demonstrated that there are significant overlaps in the regions that are important for the two fundamental properties of the channels, namely gating and permeation. We have previously shown that a pore-lining residue, W402 in S5-S6 region (P loop) in domain I of the μ1 skeletal muscle Na+channel, was important in the gating of the channel. Here, we determined the role of an adjacent pore-lining negatively charged residue (E403) in channel gating. Charge neutralization or substitution with positively charged side chain at this position resulted in a marked delay in the rate of recovery from slow inactivation. Indeed, the fast inactivation process appeared intact. Restoration of the negatively charged side chain with a sulfhydryl modifier, MTS-ethylsulfonate, resulted in a reactivation profile from a slow-inactivated state, which was indistinguishable from that of the wild-type channels. We propose an additional functional role for the negatively charged residue. Assuming no major changes in the pore structure induced by the mutations, the negatively charged residue E403 may work in concert with other pore regions during recovery from slow inactivation of the channel. Our data represent the first report indicating the role of negative charge in the slow inactivation of the voltage-gated Na+ channel.

Rabbit neutral endopeptidase 24.11 (NEP) is a type II membrane protein with a positively charged 27 amino acid residue NH2-terminal cytoplasmic domain, a 20 amino acid residue hydrophobic signal peptide/membrane anchor domain, and a large catalytic COOH-terminal domain exposed on the exoplasmic side of the membrane. To study the role of the cytosolic domain in anchoring NEP in the plasma membrane, we constructed two mutants in which this cytosolic domain was deleted. In the first mutant (NEPΔcyto), a Glu residue was present in NH2-terminus, while a Lys residue was substituted at the same position in the second mutant (NEPΔcyto(K)). To better understand the interaction of these mutants with the rough endoplasmic reticulum membrane, the mutated NEP cDNAs were transcribed and translated in vitro in the presence of microsomal membranes. Our studies showed that deletion of the hydrophillic cytosolic domain affects translocation of the NEP polypeptide chain. Substitution of a positively charged Lys residue for the Glu residue at the NH2-terminus of the deletion mutant only partly restored translocation of the polypeptide chain. Furthermore, carbonate extraction and trypsin digestion of the microsomal membranes indicated that the deletion mutants are inserted in the microsomal membranes as type III membrane proteins with their COOH-terminal domain exposed on the exterior of the microsomes. Thus, efficient translocation is dependent on the presence of a charged cytoplasmic domain.Key words: membrane proteins, translocation, cytosolic domain, neutral endopeptidase 24.11.

Abstract Motivation The number of available membrane protein structures has markedly increased in the last years and, in parallel, the reliability of the methods to detect transmembrane (TM) segments. In the present report, we characterized inter-residue interactions in α-helical membrane proteins using a dataset of 3462 TM helices from 430 proteins. This is by far the largest analysis published to date. Results Our analysis of residue–residue interactions in TM segments of membrane proteins shows that almost all interactions involve aliphatic residues and Phe. There is lack of polar–polar, polar–charged and charged–charged interactions except for those between Thr or Ser sidechains and the backbone carbonyl of aliphatic and Phe residues. The results are discussed in the context of the preferences of amino acids to be in the protein core or exposed to the lipid bilayer and to occupy specific positions along the TM segment. Comparison to datasets of β-barrel membrane proteins and of α-helical globular proteins unveils the specific patterns of interactions and residue composition characteristic of α-helical membrane proteins that are the clue to understanding their structure. Availability and implementation Results data and datasets used are available at http://lmc.uab.cat/TMalphaDB/interactions.php. Supplementary information Supplementary data are available at Bioinformatics online.

Host-Guest complexation of calixarene-bis-hydroxymethylenediphosphonic acid with 17 amino acids in water solution had been studied by the RP HPLC and molecular modelling methods. It had been shown the binding constants of the complexes are depended on the nature of the amino acid residue, log P and pKa of the acids. The complexation is mainly determined by the electrostatic interactions between the positively charged nitrogen atom of the amino acid and the negatively charged oxygen atom of phosphonic acid residue of the calixarene, the Host-Guest p-p, СН-p and solvophobic interactions.

Mnemiopsin, a Ca²⁺-regulated photoprotein isolated fromMnemiopsis leidyi, belongs to the family of ctenophore photoproteins. While there are no charged amino acid residues in the coelenterazine binding cavity of cnidarian photoproteins, ctenophore photoproteins have a positively charged residue (Arg) in this region.

ABSTRACT The fusogenic human immunodeficiency virus type 1 (HIV-1) gp41 core structure is a stable six-helix bundle formed by its N- and C-terminal heptad repeat sequences. Notably, the negatively charged residue Asp632 located at the pocket-binding motif in the C-terminal heptad repeat interacts with the positively charged residue Lys574 in the pocket formation region of the N-terminal heptad repeat to form a salt bridge. We previously demonstrated that the residue Lys574 plays an essential role in six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this study, the functionality of residue Asp632 has been specifically characterized by mutational analysis and biophysical approaches. We show that Asp632 substitutions with positively charged residues (D632K and D632R) or a hydrophobic residue (D632V) could completely abolish Env-mediated viral entry, while a protein with a conserved substitution (D632E) retained its activity. Similar to the Lys574 mutations, nonconserved substitutions of Asp632 also severely impaired the α-helicity, stability, and conformation of six-helix bundles as shown by N36 and C34 peptides as a model system. Furthermore, nonconserved substitutions of Asp632 significantly reduced the potency of C34 to sequestrate six-helix bundle formation and to inhibit HIV-1-mediated cell-cell fusion and infection, suggesting its importance for designing antiviral fusion inhibitors. Taken together, these data suggest that the salt bridge between the N- and C-terminal heptad repeat regions of the fusion-active HIV-1 gp41 core structure is critical for viral entry and inhibition.

Mitochondrial carriers, which transport metabolites, nucleotides, and cofactors across the mitochondrial inner membrane, have six transmembrane α-helices enclosing a translocation pore with a central substrate binding site whose access is controlled by a cytoplasmic and a matrix gate (M-gate). The salt bridges formed by the three PX[DE]XX[RK] motifs located on the odd-numbered transmembrane α-helices greatly contribute to closing the M-gate. We have measured the transport rates of cysteine mutants of the charged residue positions in the PX[DE]XX[RK] motifs of the bovine oxoglutarate carrier, the yeast GTP/GDP carrier, and the yeast NAD+ transporter, which all lack one of these charged residues. Most single substitutions, including those of the non-charged and unpaired charged residues, completely inactivated transport. Double mutations of charged pairs showed that all three carriers contain salt bridges non-essential for activity. Two double substitutions of these non-essential charge pairs exhibited higher transport rates than their corresponding single mutants, whereas swapping the charged residues in these positions did not increase activity. The results demonstrate that some of the residues in the charged residue positions of the PX[DE]XX[KR] motifs are important for reasons other than forming salt bridges, probably for playing specific roles related to the substrate interaction-mediated conformational changes leading to the M-gate opening/closing.

ABSTRACT Human TRIM5α (TRIM5αhu) only modestly inhibits human immunodeficiency virus type 1 (HIV-1) and does not inhibit simian immunodeficiency virus (SIVmac). Alteration of arginine 332 in the TRIM5αhu B30.2 domain to proline, the residue found in rhesus monkey TRIM5α, has been shown to create a potent restricting factor for both HIV-1 and SIVmac. Here we demonstrate that the potentiation of HIV-1 inhibition results from the removal of a positively charged residue at position 332 of TRIM5αhu. The increase in restricting activity correlated with an increase in the ability of TRIM5αhu mutants lacking arginine 332 to bind HIV-1 capsid complexes. A change in the cyclophilin A-binding loop of the HIV-1 capsid decreased TRIM5αhu R332P binding and allowed escape from restriction. The ability of TRIM5αhu to restrict SIVmac could be disrupted by the presence of any charged residue at position 332. Thus, charged residues in the v1 region of the TRIM5αhu B30.2 domain can modulate capsid binding and restriction potency. Therapeutic strategies designed to neutralize arginine 332 of TRIM5αhu might potentiate the innate resistance of human cells to HIV-1 infection.

Intrinsecamente disordinata” viene definita una proteina nativa priva di struttura secondaria o terziaria, non esposta ad agenti denaturanti. Le proteine con queste caratteristiche sono indicate come IDP/IDR, e rappresentano una ampia porzione del proteoma di tutti gli esseri viventi. Le IDP sono coinvolte in molte funzioni fisiologiche e patologiche, come la formazione di organuli cellulari privi di membrane e nei processi di fibrillazione associate ad amiloidosi. Entrambi questi fenomeni appaiono sempre più associati alla capacità delle IDP di formare interazioni intermolecolari. Stati patologici possono essere causati da disfunzioni e cattiva regolazione delle proprietà conformazionali e di aggregazione delle IDP. L’aggregazione e la conformazione delle IDP sono state ascritte a diversi fattori: la lunghezza della sequenza, le interazioni idrofobiche, i legami ad idrogeno e le cariche elettrostatiche. A questa ultima abbiamo rivolto la nostra attenzione dal momento che le IDP sono ricche di amminoacidi carichi. La carica netta per residuo (NCPR), la frazione totale di residui carichi (FCR) e la distribuzione di residui di carica opposta (valore κ) sono stati considerati i principali determinanti della dimensione della catena e delle classi conformazionali delle IDP. La prima parte del piano sperimentale interessa il concetto di NCPR, cioè la carica netta normalizzata per la lunghezza della proteina. Il nostro obiettivo è di descrivere come questo parametro influenzi la risposta delle IDP a cambiamenti di pH, con conseguente perdita di solubilità. Come modello viene utilizzata PNT del virus del morbillo ed a partire da questa si ottiene un array di varianti aventi la stessa idrofobicità e FCR, ma differenti per NCPR ed il punto isoelettrico (pI). Le proteine analizzate mostrano solubilità minima in corrispondenza del loro valore di pI, come atteso, ma tale diminuzione di solubilità non è uniforme, ma guidata dal valore di NCPR di ciascuna variante proteica. I nostri dati suggeriscono che la solubilità complessiva della proteina sia legata al valore di NCPR. La seconda parte del lavoro si è ispirata al concetto di clusterizzazione di cariche ed ha come obiettivo la valutazione di come le proprietà di compattezza delle IDP dipendano dal valore di κ. In questo caso sono state utilizzate due IDP ben caratterizzate, NTAIL dal virus del morbillo e PNT4 da Hendra virus. Grazie alla possibilità di modificare la sequenza amminoacidica delle IDP, senza interferire sul complessivo disordine strutturale, entrambi i geni sono stati riprogettati. Sono stati ottenuti due set di proteine sintetiche, ciascuno contenente una proteina wt e due varianti in cui le cariche sono uniformemente distribuite (low κ) o completamente segregate all’N- ed al C-terminus (high κ). Le proprietà conformazionali della proteina wt e delle corrispondenti varianti sono valutate mediante tecniche biofisiche. Complessivamente i dati sperimentali confermano il trend atteso cioè un aumento del grado di compattezza conformazionale all’aumentare dei valori di κ, secondo una proporzione che è tipica di ciascuna proteina in relazione al suo contenuto di proline. Complessivamente i nostri risultati confermano precedenti dati computazionali e sperimentali suggerendo come residui carichi, attraverso la loro frequenza (NCPR) e la distribuzione (κ) influenzino solubilità e compattezza delle IDP. I due lavori sperimentali sottolineano il contesto, ambientale (ad esempio le condizioni di pH) o di sequenza (la % di proline), in cui NCPR e distribuzione di cariche sono più efficaci nel determinare le caratteristiche di solubilità e compattezza conformazionale delle IDP. La rilevanza di queste informazioni è legata non solo allo studio IDP naturali, ma anche alla progettazione razionale di proteine non naturali con proprietà aggregative e conformazionali ben definite
“Intrinsic disorder” is generally referred to the conformational status of native proteins lacking of secondary and/or tertiary structure, although not exposed to any denaturing agent. These proteins, which are called intrinsically disordered (IDP/IDRs) represent a large class in the proteomes of all living beings, with a remarkable abundance among more complex eukaryotes and viruses. IDPs have been recognized to be involved in many relevant physiological and pathological functions, such as the coacervation of membrane-less organelles or the fibrillation in amyloid bodies. It is becoming clearer that fast and massive intermolecular interactions involving IDPs are governing both kinds of phenomena and that pathologies can arise from dysregulations of conformational properties and aggregation ability. The conformation and aggregation features of IDPs have been ascribed in turn to several factors, such as sequence length, hydrophobic interactions, hydrogen bonds or electrostatic charges. The latter deserve particular attention since charged residues are particularly abundant in IDPs. The net charge per residue (NCPR), the total fraction of charged residues (FCR), and the linear distribution of opposite charges (κ value) have been recently regarded as the primary determinants of IDPs conformational properties. The first part of the experimental work presented in this thesis was inspired by the concept of NCPR, which represents the net charge normalized by the protein length. The aim is to describe how the NCPR influences the ability of IDPs to respond to environment pH changes through loss of solubility. PNT from measles virus was used as a model IDP. Moreover, the wild type (wt) protein was compared with an array of PNT variants sharing the same hydrophobicity and total number of charged residues (FCR), but differing in net charges per residue and isoelectric points (pI). Tested proteins showed a solubility minimum close to their pI, as expected, but the pH-dependent decrease of solubility was not uniform and driven by the NCPR of each variant. Our data suggest that the overall solubility of a protein can be dictated by protein regions endowed with NCPR and, hence, prompter to respond to pH changes. The second part of experimental work was inspired by the concept of charge clustering. The aim was consisting at verifying that the compaction properties of IDPs are tunable by the κ value. We have used two well-characterized IDPs, namely measles virus NTAIL and Hendra virus PNT4, as model systems. Taking advantage of the high sequence designability of IDPs, genes of PNT4 and NTAIL were redesigned to obtain two sets of synthetic proteins each including the wild type (wt) form and two “κ variants”. In low-κ variants, charged amino acids are most evenly distributed, in high-κ variants charges are clustered as much as possible at the N- and C-termini (high κ). κ variants, along with wt forms, were subjected to various biophysical techniques to assess their conformational properties.Overall, experimental data confirm the expected trend, with compactness increasing with κ value. The increase of compactness does not follow a general trend, but it is protein-specific and related to the proline content. All together, these findings confirm previous theoretical and experimental data on the role of charged residues frequency (NCPR) and distribution (κ). The main value of this experimental work is in pinpointing the context, which is the environment – pH – or the amino acid composition – proline % –, where such driving forces of aggregation and compaction are mostly effective. This knowledge is useful not only to describe how the conformational behavior of IDPs is encoded by their amino acid sequence, but also to rationally design non-natural IDPs with desired conformational and aggregation properties

Using hKvl.5 expressed in HEK-293 cells the roles of R487 and H463 in the processes of permeation and inactivation were studied. Mutation of the arginine at position 487 to a valine (R487V) alters the I-Vrelationship through Kvl.5 such that the normalized current at positive voltages, in symmetrical 140 mM K+ , is less than at negative voltages. The magnitude of current reduction at positive voltages was unaffected by extracellular acidification suggesting that the protonation state of H463 does not effect the rate of K + exit or entry at the outer pore mouth. Using an Eyring-rate model we have ascertained that the reduction in the normalized outward currents through R487V occurs through a change at the outer pore mouth that alters the permeation process. In addition to the role of R487V in the process of permeation, it has also been shown that a histidine residue at position 463 in Kvl.5 confers sensitivity to external protons and affects the rate, of inactivation. To further investigate the role these residues, R487 and H463, on Kvl.5 macroscopic current kinetics we engineered a double mutant that swapped the residues at positions 463 and 487. In Kvl.5 H463R/R487H there was a decrease in the time constant for inactivation and an increase in the time constant for recovery from inactivation. Elevation of K + 0 (140 mM) increased the time constant for inactivation and deactivation through a 'foot-in-the-door' mechanism by which elevated K + 0 occupies an outer and inner binding site, respectively, at the selectivity filter and prevents the pore from collapsing. These results support the hypothesis that processes of activation/deactivation and inactivation are not separate entities, but rather, alterations of one parameter can have dramatic effects on the other in Kvl.5.
Medicine, Faculty of
Cellular and Physiological Sciences, Department of
Graduate

Abstract For a recent replacement metal gate (RMG) technology using a SOI substrate, residue from the dummy gate formed a defect that affected the RMG formation. In this FINFET technology, the high aspect ratio of the gate makes removing the dummy gate very difficult. Residue is left behind, especially in multi-fin structures. This residue was poorly detected by existing Broad-Band-Plasma inspection and thus required Electron Beam Inspection. However, this physical inspection is challenging due to high aspect ratio of the gate and an insulating wafer surface. The defect was verified using TEM, and careful sample prep is shown to be critical to verify the defect. The high aspect ratio and insulating sample in a charged-particle inspection is investigated with Monte-Carlo (MC) simulations.

We have previously obtained immunochemical evidence that the von Willebrand factor (vWF)-binding domain of the platelet membrane glycoprotein (GP) Ib is located near the amino terminus of the a subunit (Journal of Biological Chemistry 261: 12579-12585, 1986). We have now determined the complete amino acid sequence of the 45 kDa tryptic fragment of glycocalicin that contains this domain. Purified glycocalicin was subjected to limited digestion with trypsin and the proteolytic fragments were separated by size-exclusion high-pressure liquid chromatography. Two fragments of 45 kDa and 84 kDa, respectively, were obtained under nonreducing conditions. After reduction and S-carboxymethylation, the 84 kDa fragment was unchanged, while the 45 kDa fragment yielded two new fragments, one of 35 kDa and the other of 7 kDa. This finding proves the existence of a trypsin cleavage site within a disulfide loop. Two primary sets of overlapping fragments were obtained by cleavage of the carboxymethylated protein at methionyl and lysyl bonds following treatment with cyanogen bromide and Achromobacter protease I, respectively. Additional fragments were obtained by treatment of glycocalicin with Staphylococcus aureus V8 protease and Serratia marcescens protease. Analysis of all these fragments provided data that allowed determination of the sequence of the amino terminal 299 residues of the GP Ib a-chain. This includes the 45 kDa tryptic fragment containing the vWF-binding domain. This 299-residue sequence, corresponding approximately to two thirds of the α-chain polypeptide, is largely hydrophobic and contains only two N-linked and one O-linked carbohydrate chains. A hydrophilic region exists between residues 215-299, with a cluster of ten negatively charged residues at 269-287. This area is likely to attract positively charged molecules. The hydrophilic, highly glycosylated (at Ser/Thr residues) region corresponding to the previously described "macroglycopeptide" begins at residue 292. The determined sequence of glycocalicin contains a region with seven repeats, indicative of gene duplication, and is highly homologous to human leucine-rich α2-glycoprotein.

The combustion mode transition between spark ignition (SI) and homogeneously charged compression ignition (HCCI) combustions of an internal combustion (IC) engine is challenging due to the distinct engine operational parameters over these two combustion modes and the cycle-to-cycle residue gas dynamics of the HCCI combustion. The control problem becomes even more complicated when multi-cylinder operation is involved. This paper studies the combustion mode transition problem of a multi-cylinder IC engine with dual-stage valve lifts and electrical variable valve timing systems. A control oriented engine model was used to develop a multistep mode transition control strategy via iterative learning for combustion mode transition between SI to HCCI with minimal engine torque fluctuations. The hardware-in-the-loop (HIL) simulations demonstrated the effectiveness of the developed control strategy for the combustion mode transition under both constant load and transient engine operational conditions.

The combustion mode transition between SI (spark ignited) and HCCI (Homogeneously Charged Compression Ignition) of an IC (Internal Combustion) engine is challenge due to the thermo inertia of residue gas; and model-based control becomes a necessity. This paper presents a control oriented two-zone model to describe the hybrid combustion that starts with SI combustion and ends with HCCI combustion. The gas respiration dynamics were modeled using mean-value approach and the combustion process was modeled using crank resolved method. The developed model was validated in an HIL (Hardware-In-the-Loop) simulation environment for both steady-state and transient operations in SI, HCCI, and SI-HCCI hybrid combustion modes through the exhaust valve timing control (recompression). Furthermore, cooled external EGR (exhaust gas re-circulation) was used to suppress engine knock and enhance the fuel efficiency. The simulation results also illustrates that the transient control parameters of hybrid combustion is quite different from these in steady state operation, indicating the need of a control oriented SI-HCCI hybrid combustion model for transient combustion control.

In this paper we report the results of pilot tests of flocculation/precipitation setup for dust-suppressor (DS) and transuranic elements (TUE) removal from wastewaters of Chernobyl Nuclear Power Plant (ChNPP), including those of the Object “Shelter”. Tests were performed on the pilot unit (PU), which included service tank, precipitation tank, and accumulation tank, 300 dm3 each, connected with pipelines with dosing and pumping equipment, and throttle valves providing controlled dosing, mixing, precipitation and mechanical filtration of radioactive wastewaters under different conditions. The reagent compositions used in pilot tests were based on coagulant POLYPACS-30 LF (aluminum polyoxychloride), synthetic cationic flocculants Besfloc K6634, K510CA, K6732 («Kolon Life Science, Inc», South Korea) varying in molecular weight and charge density, and natural cationic flocculant «Chitofloc» (Institute of Chemistry FEBRAS, Russia). The following wastewater parameters were controlled during the pilot tests: pH, dry residue, oxygen consumption, total α- and β-activity, isotope composition, optical density and DS content. The precipitation setup demonstrated lower efficiency DS removal from evaporator concentrates due to high ionic strength suppressing the electrostatic interactions between coagulants/flocculants and oppositely charged colloids of DS and TUE. The residual DS concentration was below 1 mg/L that corresponds to decontamination factor above 300 for the drainage water samples tested. The chitosan-based “Chitofloc” flocculant appeared to be the reagent which was the least sensitive to negative effect of ionic strength; however, the decontamination factor in DS removal was not higher than 5 due to suppressing of electrostatic interactions in high salinity media. Analysis of α-activity of water samples after flocculation/mechanical filtration revealed that TUE were not detected in the drainage water samples with DS content reduced to 2 mg/L that corresponds to TRU decontamination factor above 10000 and confirms immobilization of TUE in DS precipitate.

This study was motivated by the need to develop new means and approaches to the design of future, environmentally-safe, insecticides. Utilization of anti-insect selective toxins from scorpion venoms and clarification of the molecular basis for their specificity, are a major focus in this project and may have an applicative value. Our study concentrated on the highly insecticidal toxin, LqhaIT, and was devoted to: (I) Characterization of the neuropharmacological and electrophysiological features of this toxin. (II) Establishment of a genetic system for studying structure/activity relationships of the toxin. (III) Analysis of the insecticidal efficacy of an entomopathogenic baculovirus engineered and expressing LqhaIT. The results obtained in this project suggest that: 1) The receptor binding site of LqhaIT on insect sodium channels differs most likely from its analogous receptor site 3 on vertebrate sodium channels. 2) The effects of LqhaIT are presynaptic. Hyperexcitation at the neuromuscular results from dramatic slowing of sodium channel inactivation and enhanced peak sodium currents causes by LqhaIT. 3) The putative toxic surface of LqhaIT involves aromatic and charged amino acid residues located around the C-terminal region and five-residue-turn of the toxin (unpublished). 4) The anti-insect/anti-mammalian toxicity ratio can be altered by site-directed mutagenesis (publication 8). This effect was partly shown at the level of sodium channel function. 5) The insecticidal efficacy of AcNPV baculovirus increased to a great extent when infection was accompanied by expression of LqhaIT (publication 5).

The antibotulinal baceriocins, propionicin PLG-1 and jenseniin G., were the first to be identified, purified and characterized for the dairy propionibaceria and are produced by Propionibacterium thoenii P127 and P. thoenii/jensenii P126, respectively. Objectives of this project were to (a) produce polyclonal antibodies for detection, comparison and monitoring of propionicin PLG-1; (b) identify, clone and characterize the propionicin PLG-1 (plg-1) and jenseniin G (jnG) genes; and (3) develop gene transfer systems for dairy propionibacteria using them as models. Polyclonal antibodies for detection, comparison and monitoring of propionicin PLG-1 were produced in rabbits. Anti-PLG-1 antiserum had high titers (256,000 to 512,000), neutralized PLG-1 activity, and detected purified PLG-1 at 0.10 mg/ml (indirect ELISA) and 0.033 mg/ml (competitive indirect ELISA). Thirty-nine of 158 strains (most P. thoenii or P. jensenii) yielded cross-reacting material; four strains of P. thoenii, including two previously unidentified bacteriocin producers, showed biological activity. Eight propionicin-negative P127 mutants produced neither ELISA response nor biological activity. Western blot analyses of supernates detected a PLG-1 band at 9.1 kDa and two additional protein bands with apparent molecular weights of 16.2 and 27.5 kDa. PLG-1 polyclonal antibodies were used for detection of jenseniin G. PLG-1 antibodies neutralized jenseniin G activity and detected a jenseniin G-sized, 3.5 kDa peptide. Preliminary immunoprecipitation of crude preparations with PLG-1 antibodies yielded three proteins including an active 3-4 kDa band. Propionicin PLG-1 antibodies were used to screen a P. jensenii/thoenii P126 genomic expression library. Complete sequencing of a cloned insert identified by PLG-1 antibodies revealed a putative response regulator, transport protein, transmembrane protein and an open reading frame (ORF) potentially encoding jenseniin G. PCR cloning of the putative plg-1 gene yielded a 1,100 bp fragment with a 355 bp ORF encoding 118 amino acids; the deduced N-terminus was similar to the known PLG-1 N-terminus. The 118 amino acid sequence deduced from the putative plg-1 gene was larger than PLG-1 possibly due to post-translational processing. The product of the putative plg-1 gene had a calculated molecular weight of 12.8 kDa, a pI of 11.7, 14 negatively charged residues (Asp+Glu) and 24 positively charged residues (Arg+Lys). The putative plg-1 gene was expressed as an inducible fusion protein with a six-histidine residue tag. Metal affinity chromatography of the fused protein yielded a homogeneous product. The fused purified protein sequence matched the deduced putative plg-1 gene sequence. The data preliminarily suggest that both the plg-1 and jnG genes have been identified and cloned. Demonstrating that antibodies can be produced for propionicin PLG-1 and that those antibodies can be used to detect, monitor and compare activity throughout growth and purification was an important step towards monitoring PLG-1 concentrations in food systems. The unexpected but fortunate cross-reactivity of PLG-1 antibodies with jenseniin G led to selective recovery of jenseniin G by immunoprecipitation. Further refinement of this separation technique could lead to powerful affinity methods for rapid, specific separation of the two bacteriocins and thus facilitate their availability for industrial or pharmaceutical uses. Preliminary identification of genes encoding the two dairy propionibacteria bacteriocins must be confirmed; further analysis will provide means for understanding how they work, for increasing their production and for manipulating the peptides to increase their target species. Further development of these systems would contribute to basic knowledge about dairy propionibacteria and has potential for improving other industrially significant characteristics.