Visualisation tools for dependent peptide searches to support the exploration of in vitro protein modifications.

Dependent peptide searching is a method for discovering covalently-modified peptides-and therefore proteins-in mass-spectrometry-based proteomics experiments. Being more permissive than standard search methods, it has the potential to discover novel modifications (e.g., post-translational modifications occurring in vivo, or modifications introduced in vitro). However, few studies have explored dependent peptide search results in an untargeted way. In the present study, we sought to evaluate dependent peptide searching as a means of characterising proteins that have been modified in vitro. We generated a model data set by analysing N-ethylmaleimide-treated bovine serum albumin, and performed dependent peptide searches using the popular MaxQuant software. To facilitate interpretation of the search results (hundreds of dependent peptides), we developed a series of visualisation tools (R scripts). We used the tools to assess the diversity of putative modifications in the albumin, and to pinpoint hypothesised modifications. We went on to explore the tools' generality via analyses of public data from studies of rat and human proteomes. Of 19 expected sites of modification (one in rat cofilin-1 and 18 across six different human plasma proteins), eight were found and correctly localised. Apparently, some sites went undetected because chemical enrichment had depleted necessary analytes (potential 'base' peptides). Our results demonstrate (i) the ability of the tools to provide accurate and informative visualisations, and (ii) the usefulness of dependent peptide searching for characterising in vitro protein modifications. Our model data are available via PRIDE/ProteomeXchange (accession number PXD013040).

OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins.

In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.

The role of GTP binding and hydrolysis at the atToc159 preprotein receptor during protein import into chloroplasts.

The majority of nucleus-encoded chloroplast proteins are targeted to the organelle by direct binding to two membrane-bound GTPase receptors, Toc34 and Toc159. The GTPase activities of the receptors are implicated in two key import activities, preprotein binding and driving membrane translocation, but their precise functions have not been defined. We use a combination of in vivo and in vitro approaches to study the role of the Toc159 receptor in the import reaction. We show that atToc159-A864R, a receptor with reduced GTPase activity, can fully complement a lethal insertion mutation in the ATTOC159 gene. Surprisingly, the atToc159-A864R receptor increases the rate of protein import relative to wild-type receptor in isolated chloroplasts by stabilizing the formation of a GTP-dependent preprotein binding intermediate. These data favor a model in which the atToc159 receptor acts as part of a GTP-regulated switch for preprotein recognition at the TOC translocon.

Detection and characterization of altered conformations of protein pharmaceuticals using complementary mass spectrometry-based approaches.

Unlike small-molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety of factors that are not solely defined by their covalent chemical structure. Since the conformation (or higher order structure) of a protein is a major modulator of its biological activity, the ability to detect changes in both the higher order structure and conformational dynamics of a protein, induced by an array of extrinsic factors, is of central importance in producing, purifying, and formulating a commercial biopharmaceutical with consistent therapeutic properties. In this study we demonstrate that two complementary mass spectrometry-based approaches (analysis of ionic charge-state distribution and hydrogen/deuterium exchange) can be a potent tool in monitoring conformational changes in protein biopharmaceuticals. The utility of these approaches is demonstrated by detecting and characterizing conformational changes in the biopharmaceutical product interferon beta-1a (IFN-beta-1a). The protein degradation process was modeled by inducing a single chemical modification of IFN-beta1a (alkylation of its only free cysteine residue with N-ethylmaleimide), which causes significant reduction in its antiviral activity. Analysis of IFN-beta1a ionic charge-state distributions unequivocally reveals a significant decrease of conformational stability in the degraded protein, while hydrogen/deuterium exchange measurements provide a clear indication that the higher order structure is affected well beyond the covalent modification site. Importantly, neither technique required that the location or indeed the nature of the chemical modification be known prior to or elucidated in the process of the analysis. In contrast, application of the standard armamentarium of biophysical tools, which are commonly employed for quality control of protein pharmaceuticals, met with very limited success in detection and characterization of conformational changes in the modified IFN-beta1a. This work highlights the role mass spectrometry can and should play in the biopharmaceutical industry beyond the presently assigned task of primary structure analysis.

15d-PGJ2 upregulates synthesis of IL-8 in endothelial cells through induction of oxidative stress.

15-Deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) is a cyclopentenone prostaglandin regarded as antiinflammatory mediator, which can act through peroxisome proliferator-activated receptor-gamma (PPARgamma) or through G protein-coupled surface receptors. It has been demonstrated that 15d-PGJ(2) potently increases the generation of interleukin-8 (IL-8) in human microvascular endothelial cells (HMEC-1s); however, the mechanism of this induction is not known. The aim of the study was to find the pathway involved in 15d-PGJ(2)-mediated IL-8 stimulation. Our data confirmed that the effect of 15d-PGJ(2) is independent of PPARgamma. For the first time, we excluded the activation of G proteins and the contribution of G protein-coupled surface receptors in endothelial cells treated with 15d-PGJ(2). Instead, we demonstrated that stimulation of IL-8 involved induction of oxidative stress, activation of p38 kinases, and increase in stability of IL-8 mRNA. Upregulation of IL-8 promoter, although measurable, seemed to play a less-pronounced role. Additionally, our results indicate the involvement of cAMP elevation and may suggest a role for ATF2 transcription factor. Concomitant induction of heme oxygenase-1 in HMEC-1s did not influence the synthesis of IL-8. In summary, we showed that 15d-PGJ(2), acting through oxidative stress, may exert proinflammatory effects. The upregulation of IL-8 is mostly associated with p38-mediated stabilization of mRNA.

Cardiolipin controls the osmotic stress response and the subcellular location of transporter ProP in Escherichia coli.

The phospholipid composition of the membrane and transporter structure control the subcellular location and function of osmosensory transporter ProP in Escherichia coli. Growth in media of increasing osmolality increases, and entry to stationary phase decreases, the proportion of phosphatidate in anionic lipids (phosphatidylglycerol (PG) plus cardiolipin (CL)). Both treatments increase the CL:PG ratio. Transporters ProP and LacY are concentrated with CL (and not PG) near cell poles and septa. The polar concentration of ProP is CL-dependent. Here we show that the polar concentration of LacY is CL-independent. The osmotic activation threshold of ProP was directly proportional to the CL content of wild type bacteria, the PG content of CL-deficient bacteria, and the anionic lipid content of cells and proteoliposomes. CL was effective at a lower concentration in cells than in proteoliposomes, and at a much lower concentration than PG in either system. Thus, in wild type bacteria, osmotic induction of CL synthesis and concentration of ProP with CL at the cell poles adjust the osmotic activation threshold of ProP to match ambient conditions. ProP proteins linked by homodimeric, C-terminal coiled-coils are known to activate at lower osmolalities than those without such structures and coiled-coil disrupting mutations raise the osmotic activation threshold. Here we show that these mutations also prevent polar concentration of ProP. Stabilization of the C-terminal coiled-coil by covalent cross-linking of introduced Cys reverses the impact of increasing CL on the osmotic activation of ProP. Association of ProP C termini with the CL-rich membrane at cell poles may raise the osmotic activation threshold by blocking coiled-coil formation. Mutations that block coiled-coil formation may also block association of the C termini with the CL-rich membrane.

Mapping the cofilin binding site on yeast G-actin by chemical cross-linking.

Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the alpha 3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 A) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.

Structure and function of transmembrane segment XII in osmosensor and osmoprotectant transporter ProP of Escherichia coli.

Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain and linked to TM XII, form an intermolecular, homodimeric alpha-helical coiled-coil that tunes the transporter's response to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged, Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with Oregon Green Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except for V429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior of ProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydrated polar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formed when ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TM XII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C rendered ProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at high osmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lower osmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XII required a higher osmolality to activate. They included elements of a GXXXG motif that are predicted to form the interface of TM XII with TM VII. These studies define the position of ProP TM XII within the membrane, further support the predicted structure of ProP, reveal the dimerization interface, and show that the structure of TM XII influences the osmolality at which ProP activates.

Formation of an antiparallel, intermolecular coiled coil is associated with in vivo dimerization of osmosensor and osmoprotectant transporter ProP in Escherichia coli.

Membrane transporter ProP from Escherichia coli senses extracellular osmolality and responds by mediating the uptake of osmoprotectants such as glycine betaine when osmolality is high. Earlier EPR and NMR studies showed that a peptide replica of the cytoplasmic ProP carboxyl terminus (residues D468-R497) forms a homodimeric, antiparallel, alpha-helical coiled coil in vitro stabilized by electrostatic interactions involving R488. Amino acid replacement R488I disrupted coiled-coil formation by the ProP peptide, elevated the osmolality at which ProP became active, and rendered the osmolality response of ProP transient. In the present study, either E480 or K473 was replaced with cysteine (Cys) in ProP, a Cys-less, fully functional, histidine-tagged ProP variant, to use Cys-specific cross-linking approaches to determine if antiparallel coiled-coil formation and dimerization of the intact protein occur in vivo. The Cys at positions 480 would be closer in an antiparallel dimer than those at positions 473. These replacements did not disrupt coiled-coil formation by the ProP peptide. Partial homodimerization of variant ProP-E480C could be demonstrated in vivo and in membrane preparations via Cys-specific cross-linking with dithiobis(maleimidoethane) or by Cys oxidation to cystine by copper phenanthroline. In contrast, these reagents did not cross-link ProP with Cys at position 133 or 241. Cross-linking of ProP with Cys at position 473 was limited and occurred only if ProP was overexpressed, consistent with an antiparallel orientation of the coiled coil in the intact protein in vivo. Although replacement E480C did not alter transporter activity, replacement K473C reduced the extent and elevated the threshold for osmotic activation. K473 may play a role in ProP structure and function that is not reflected in altered coiled-coil formation by the corresponding peptide. Substitution R488I affected the activities of ProP-(His)(6), ProP-E480C, and ProP-K473C as it affected the activity of ProP. Surprisingly, it did not eliminate cross-linking of Cys at position 480, and it elevated cross-linking at position 473, even when ProP was expressed at physiological levels. This suggested that the R488I substitution may have changed the relative orientation of the C-termini within the dimeric protein from antiparallel to parallel, resulting in only transient osmotic activation. These results suggest that ProP is in monomer-dimer equilibrium in vivo. Dimerization may be mediated by C-terminal coiled-coil formation and/or by interactions between other structural domains, which in turn facilitate C-terminal coiled-coil formation. Antiparallel coiled-coil formation is required for activation of ProP at low osmolality.

Tellurite uptake by cells of the facultative phototroph Rhodobacter capsulatus is a Delta pH-dependent process.

The uptake by light-grown cells of Rhodobacter capsulatus of the highly toxic metalloid oxyanion tellurite (TeO(3)(2-)) was examined. We show that tellurite is rapidly taken up by illuminated cells in a process which is inhibited by the protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) and by the K(+)/H(+) exchanger nigericin. Notably, the light-driven membrane potential (Delta psi) is enhanced by K(2)TeO(3)> or =200 microM. Further, tellurite uptake is largely insensitive to valinomycin, strongly repressed by the sulfhydryl reagent N-ethylethylmaleimide (NEM) and competitively inhibited by phosphate. We conclude that tellurite is transported into cells by a Delta pH-dependent, non-electrogenic process which is likely to involve the phosphate transporter (PiT family).

4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by N-ethylmaleimide-reductase of Yarrowia lipolytica.

We studied the steric course of the reaction catalyzed by the N-ethylmaleimide (NEM) reductase of Yarrowia (Candida) lipolytica (Y. lipolytica), using 4R-[4-2H1]NADPH and 4S-[4-2H1]NADPH as cofactors and N-ethylcitraconimide as substrate. Active substrates and inhibitors of NEM reductase and its subcellular distribution were also investigated to clarify the biochemical properties of this enzyme. NEM reductase catalyzes the reduction of N-ethylmaleimide to N-ethylsuccinimide with NAD(P)H as the cofactor. Several maleimide and cyclopentenone derivatives tested were also active substrates for NEM reductase of Y. lipolytica. Some pyrazolone derivatives, particularly 1-phenyl-5-pyrazolone, were found to be effective inhibitors of NEM reductase. Subcellular localization of NEM reductase was carried out using protoplast formation and differential centrifugation. Ninety-eight percent of the NEM reductase activity was recovered in the cytosolic fraction, indicating that NEM reductase in Y. lipolytica was the cytosolic enzyme. We also determined the stereochemical specificity of the reduction of N-ethylcitraconimide by NEM reductase in Y. lipolytica, showing that 4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by NEM reductase, and two hydrogen atoms from NADPH and H2O added to opposite faces of the double bond of N-ethylcitraconimide.

Characterization of SH groups in porin of bovine heart mitochondria. Porin cysteines are localized in the channel walls.

Porin from bovine heart mitochondria contains probably two cysteines (Cys126 and Cys230 in human porin, Kayser, H., Kratzin, H. D., Thinnes, F. P., Götz, H., Schmidt, W. E., Eckart, K. & Hilschmann, N. (1989) Biol. Chem. Hoppe-Seyler 370, 1265-1278). Reduced and oxidized forms of these cysteines were investigated in purified protein and in intact mitochondria using the agents dithioerythritol, cuprous(II) phenantroline, diamide and performic acid. Furthermore, intact mitochondria were labelled with the sulfhydryl-alkylating agents N-[14C]ethylmaleimide, eosin-5-maleimide and N-(1-pyrenyl)-maleimide. Affinity chromatography of bovine heart porin was performed with cysteine-specific material. The results can be summarized as follows: (1) Porin has one reduced and two oxidized forms of apparent molecular masses between 30 and 35 kDa. The native form of porin is the reduced 33 kDa form. The oxidized forms only appear after denaturation with SDS. (2) The 35-kDa reduced and the 33.5-kDa oxidized forms of porin show the same pore-forming properties after reconstitution of the protein into lipid bilayer membranes. (3) Labelling of cysteines by eosin-5-maleimide and N-(1-pyrenyl)-maleimide suggested their location at a boundary between the water-phase and the lipid-phase. Incubation of intact mitochondria with N-ethylmaleimide prior to eosin-5-maleimide and N-(1-pyrenyl)maleimide treatment resulted in the inhibition of the fluorescent labelling. Among the cysteines present in the primary structure, Cys126 is the most sensitive to N-ethylmaleimide binding. (4) Bovine heart mitochondrial porin covalently bound to Affi-Gel 501 (with a 1.75 nm long spacer), but not to Thiopropyl-Sepharose 6B (with a 0.51 nm spacer). This suggests that at least one of the cysteines is localized between 0.51 nm and 1.75 nm deep in the protein micelle.

Differential inhibition of human T-lymphocyte activation by maleimide probes.

Cellular thiols are known to be involved in lymphocyte activation, differentiation, and growth. In theory, alkylation of selective cellular thiols could be used to regulate specific processes in the activation sequence by inactivating particular enzymes or structural proteins, although to date specific alkylating probes have not been reported. N-Ethylmaleimide (NEM) is a lipophilic sulfhydryl-alkylating agent that is known to block the in vitro proliferative response of T lymphocytes. NEM (10 microM) was found to be fully inhibitory in PHA, Con A, and MLC assays only when added prior to or simultaneously with the mitogens or allogeneic cells; the addition of NEM only 15 sec after stimulating the cells with PHA resulted in a loss of greater than 50% of the inhibitory activity. The addition of 50 microM 2-ME 10 min after treating the cells with NEM failed to block the inhibitory effect. NEM (10-20 microM) had no adverse effect on lymphocyte viability, but completely blocked lymphocyte agglutination in response to mitogens or allogeneic cells. The lymphocytes overcame the inhibitory effects of NEM after 48 hr in both the PHA and MLC experiments. Resumption of the proliferative response was associated with the onset of agglutination in the PHA assay. In experiments using various analogs of NEM, we noted that the presence of a nonpolar N-linked side group was necessary for inhibitory activity. Pretreatment of PBMC with NEM decreased the total cellular thiols by 50% and blocked proliferation by 99%, whereas N-hydroxymaleimide decreased the total cellular thiols by 38% but had no effect on the proliferative response. The additional 12% of the cellular thiols that react with NEM, but not NHM, account for the inhibitory effect of NEM on lymphocyte proliferation. These findings suggest that selective cellular thiols are critical for T-cell activation.

Transport of glutathione by intestinal brush border membrane vesicles.

Intestinal brush border membranes prepared from pig jejunum were shown to transport intact reduced glutathione into an osmotically responsive intravesicular space. At early incubation time (under 1 min.) GSH was poorly hydrolysed in its free amino acids. Primarily glutathione uptake was obtained against a concentration gradient (vesicle greater than medium) and was stimulated by Na+.

[Comparative studies on the DNA synthesis of rat thymus and spleen cells in vitro under the influence of x rays, UV light and radiomimetic compounds]. / Vergleichende Untersuchungen zur DNA-Synthese von Thymus- und Milzzellen der Ratte in vitro unter dem Einfluss von Röntgenstrahlen, UV-Licht und radiomimetisch wirksamen Verbindungen.

In order to further characterize the different repairing behavior of thymus and spleen cells of rats in vitro under the influence of X-rays, UV radiation and methylmethanesulfonate (MMS), the effect of bleomycin (BM), L-cysteine (CY-E), N-ethylmaleimide (NEM), 1-beta-D-arabinofuranosylcytosine (araC), dideoxythymidine (ddT), and novobiocin (NB) on the semiconservative and restorative DNA synthesis as well as on the behavior of DNA under the alkaline elution was studied. The semiconservative DNA synthesis was inhibited by all examined agents except ddT, the restorative DNA synthesis only by NEM, araC, and NB. The stimulation of the restorative DNA synthesis was increased by UV radiation and MMS in spleen cells and by X-rays, BM and CY-E in thymus cells. Under the conditions of alkaline elution, there was a more sensitive reaction of spleen cells than of thymus cells to X-rays, BM and CY-E. The results show that thymus cells are especially qualified for the repair of short chains and spleen cells for the repair of long chains.

Electron spin resonance studies of bovine plasma amine oxidase. Probing of the environment about the substrate-liberated sulfhydryl groups in the active site.

A series of nitroxide spin-labeled reagents have been employed to explore the environment of the cysteine residues in bovine plasma amine oxidase. When the enzyme was reduced by substrate or phenylhydrazine, 1 essential sulfhydryl residue/subunit was liberated. This cysteine residue was reacted then with the spin label 3-(maleimido-methyl)-2,2,5,5-tetramethyl-1-pyrrolinyloxyl. The ESR spectra of the labeled enzyme derivatives suggested that this essential sulfhydryl residue is located in a pocket, whereas the nonessential sulfhydryl residues are probably located near the surface. By varying the length of the nitroxide spin-labeled N-ethylmaleimide derivatives, it was determined that the liberated essential cysteine residues are in a restricted environment. The ESR spectral data also suggested that the nitroxide radical and the essential copper in the enzyme do not interact with one another. The effect of ionic strength, pH, and urea denaturation on the environment of the essential sulfhydryl residue were also investigated.

Inhibition of lactose transport in E. coli by N-ethylmaleimide-beta-galactoside, N-ethylsuccinimide-beta-galactoside and N-ethylmaleimide.

N-ethylmaleimide (NEM) inhibits lactose uptake in E. coli by reacting with the M protein component of the lac permease system. In an attempt to estimate the distance between the NEM reactive site and the substrate binding site, we have synthesized a beta-galactoside with NEM as the aglycon moiety (NEM-gal). NEM-gal was a more effective inhibitor of lactose transport than was NEM. Part of the inhibition by NEM-gal was caused by competition with lactose for the substrate binding site. To estimate this part of the inhibition, we synthesized the saturated and thus the unreactive N-ethylsuccinimide (NES) analog of NEM-gal. Nes-gal was a competitive inhibitor of lactose uptake. The remainder of the inhibition by NEM-gal followed first-order kinetics with the same rate constant as NEM. In addition, the protective effect of thiodigalactoside against the inhibition of transport by NEM was also observed against irreversible inhibition by NEM-gal. We suggest that the reactivity of NEM was unaltered by bringing it near the beta-galactoside binding site by way of covalent attachment to galactose. We conclude that the distance between the NEM reactive site and the position of the glycosidic oxygen of beta-galactosides bound to the lactose site is greater than 8 A.

Reversible uncoupling of photophosphorylation by a new bifunctional maleimide.

A new bifunctional maleimide that contains a disulfide bond has been synthesized. This maleimide, dithiobis-N-ethylmaleimide (DTEM), like another bifunctional maleimide, o-phenylenebismaleimide, is about 500-fold more effective as an inhibitor of photophosphorylation than N-ethylmaleimide. Thylakoids must be illuminated in the presence of DTEM before the assay of phosphorylation for the inhibition to occur. Phosphoryalation in thylakoids treated with DTEM in the light is uncoupled and proton permeability of the treated thylakoids is enhanced. This uncoupling of photophosphorylation in thylakoids treated with DTEM can be reversed by thiol compounds. The addition of 50 mM dithiothreitol restores H+ uptake in thylakoids treated with DTEM in the light to control levels and partially reverses the inhibition of phosphorylation. Evidence is provided to show that DTEM cross-links groups within the gamma subunit of the coupling factor 1, and that the cross-link is broken by high concentrations of thiols. These results suggest that cross-linking is the cause for the increased proton permeability in thylakoids treated with bifunctional maleimides in the light.

Conformational transition of polypeptide chain elongation factor G as determined by electron spin resonance.

The conformational transition of the polypeptide chain elongation factor G (EF-G) induced by interaction with guanine nucleotide has been investigated by means of the spin-labeling technique. Various spin-label probes were attached specifically to the sulfhydryl group of the protein that is essential for binding to ribosomes, and the effects of these ligands on the electron spin resonance (ESR) spectra were examined. It was found that the ESR spectra of EF-G labeled with nitroxide maleimide reagents were modified by the addition of various guanine nucleotides such as GDP, GTP and, to a lesser extent, by Gpp(NH)p and Gpp(CH2)p, indicating that conformational changes accompany the binding of nucleotide ligand. However, the ESR spectra of labeled EF-G-GDP and EF-G-GTP were almost identical. On the other hand, when EF-G was labeled with nitroxide iodoacetamide reagents, a clear difference in the ESR spectra of EF-G-GDP and EF-G-GTP derivatives was observed. In this case, the spectral shape of the spin-labeled EF-G in the presence of GTP or its analogs, Gpp(NH)p or Gpp(CH2)p, was quite similar to that of free, unliganded EF-G derivative. These results, together with those previously obtained using hydrophobic probes (Arai, Arai, & Kaziro (1975) J. Biochem. 78, 243-246) demonstrate the existence of an EF-G-guanine nucleotide binary complex. They also indicate that there is a substantial difference in conformation between free EF-G, EF-G-GDP, and EF-G-GTP near the active site essential for interaction with ribosomes.

N-substituted maleimide inactivation of the response to taste cell stimulation.

N-Ethylmaleimide (NEM) irreversibly inactivates the response of gustatory cells to stimulation by NaCl, sucrose and hydrogen ions. The rate of inactivation can be measured by monitoring the decay of NaCl-stimulated summated electrophysiological activity at the chorda tympani nerve in the presence of NEM. The observed pseudo first-order rate constants are linear with NEM concentration, and the second-order rate constant is 0.38 M-1 sec-1. Other N-substituted maleimides, such as N-methylmaleimide and N-butylmaleimide, which have ether:water partition coefficient and is essentially ineffective as an inactivator of the NaCl response. These results, together with the observation that the inactivation rate is independent of pH between 4.5 and 7.0, indicate the inactivation site is either intracellular or buried within the cell membrane at a locus inaccessible to most extracellular fluids. The rate of inactivation of the sucrose and HCl responses were measured indirectly and found to be comparable to the NaCl-stimulated inactivation rate, indicating the inhibited event is common to the transduction of the response for all of the stimuli examined. Possible sites of inactivation by N-substituted maleimides are considered in the context for and characterizing receptor-specific as well as other classes of taste cell inhibitors.