In Situ Alkylation of Reconstructed Human Epidermis by Methyl Methanesulfonate: A Quantitative HRMAS NMR Chemical Reactivity Mapping.

Allergic contact dermatitis (ACD) is a reaction of the immune system resulting from skin sensitization to an exogenous hazardous chemical and leading to the activation of antigen-specific T-lymphocytes. The adverse outcome pathway (AOP) for skin sensitization identified four key events (KEs) associated with the mechanisms of this pathology, the first one being the ability of skin chemical sensitizers to modify epidermal proteins to form antigenic structures that will further trigger the immune system. So far, these interactions have been studied in solution using model nucleophiles such as amino acids or peptides. As a part of our efforts to better understand chemistry taking place during the sensitization process, we have developed a method based on the use of high-resolution magic angle spinning (HRMAS) NMR to monitor in situ the reactions of 13C substituted chemical sensitizers with nucleophilic amino acids of epidermal proteins in reconstructed human epidermis. A quantitative approach, developed so far for liquid NMR applications, has not been developed to our knowledge in a context of a semisolid nonanisotropic environment like the epidermis. We now report a quantitative chemical reactivity mapping of methyl methanesulfonate (MMS), a sensitizing methylating agent, in reconstructed human epidermis by quantitative HRMAS (qHRMAS) NMR. First, the haptenation process appeared to be much faster in RHE than in solution with a maximum concentration of adducts reached between 4 and 8 h. Second, it was observed that the concentration of cysteine adducts did not significantly increase with the dose (2.07 nmol/mg at 0.4 M and 2.14 nmol/mg at 1 M) nor with the incubation time (maximum of 2.27 nmol/mg at 4 h) compared to other nucleophiles, indicating a fast reaction and a potential saturation of targets. Third, when increasing the exposure dose, we observed an increase of adducts up to 12.5 nmol/mg of RHE, excluding cysteine adducts, for 3112 µg/cm2 (1 M solution) of (13C)MMS. This methodology applied to other skin sensitizers could allow for better understanding of the potential links between the amount of chemical modifications formed in the epidermis in relation to exposure and the sensitization potency.

Novel applications of modification of thiol enzymes and redox-regulated proteins using S-methyl methanethiosulfonate (MMTS).

S-Methyl methanethiosulfonate (MMTS) is used in experimental biochemistry for alkylating thiol groups of protein cysteines. Its applications include mainly trapping of natural thiol-disulfide states of redox-sensitive proteins and proteins which have undergone S-nitrosylation. The reagent can also be employed as an inhibitor of enzymatic activity, since nucleophilic cysteine thiolates are commonly present at active sites of various enzymes. The advantage of using MMTS for this purpose is the reversibility of the formation of methylthio mixed disulfides, compared to irreversible alkylation using conventional agents. Additional benefits include good accessibility of MMTS to buried protein cysteines due to its small size and the simplicity of the protection and deprotection procedures. In this study we report examples of MMTS application in experiments involving oxidoreductase (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), redox-regulated protein (recoverin) and cysteine protease (triticain-α). We demonstrate that on the one hand MMTS can modify functional cysteines in the thiol enzyme GAPDH, thereby preventing thiol oxidation and reversibly inhibiting the enzyme, while on the other hand it can protect the redox-sensitive thiol group of recoverin from oxidation and such modification produces no impact on the activity of the protein. Furthermore, using the example of the papain-like enzyme triticain-α, we report a novel application of MMTS as a protector of the primary structure of active cysteine protease during long-term purification and refolding procedures. Based on the data, we propose new lines of MMTS employment in research, pharmaceuticals and biotechnology for reversible switching off of undesirable activity and antioxidant protection of proteins with functional thiol groups.

Cruzain structures: apocruzain and cruzain bound to S-methyl thiomethanesulfonate and implications for drug design.

Chagas disease, which is caused by Trypanosoma cruzi, affects more than six million people worldwide. Cruzain is the major cysteine protease involved in the survival of this parasite. Here, the expression, purification and crystallization of this enzyme are reported. The cruzain crystals diffracted to 1.2 Šresolution, yielding two novel cruzain structures: apocruzain and cruzain bound to the reversible covalent inhibitor S-methyl thiomethanesulfonate. Mass-spectrometric experiments confirmed the presence of a methylthiol group attached to the catalytic cysteine. Comparison of these structures with previously published structures indicates the rigidity of the cruzain structure. These results provide further structural information about the enzyme and may help in new in silico studies to identify or optimize novel prototypes of cruzain inhibitors.

Quantification of a Novel DNA-Protein Cross-Link Product Formed by Reacting Apurinic/Apyrimidinic Sites in DNA with Cysteine Residues in Protein by Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.

Emerging evidence suggests that cross-links formed by reacting DNA lesions with proteins may play a significant role in the pathophysiology of human cancer and degenerative diseases. The goal of this study was to develop a method involving liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with the stable isotope-dilution method to quantify DNA-protein cross-link (DPC). A novel type of cross-link involving a S-glycosidic linkage formed by reacting an abasic site in DNA with the cysteine residues in protein was targeted in this study. The method entails hydrolysis of the cross-link to a 2'-deoxyribose-cysteine adduct, addition of isotopically labeled internal standard, and quantitation by LC-MS/MS analysis. The accuracy and precision of the method were evaluated with a synthetic peptide containing the cross-link. The validated method was then applied to quantitate the levels of the DNA-protein cross-link in vitro and in HeLa cells exposed to alkylating agent methylmethanesulfonate (MMS). The analysis detected dosage-dependent formation of the cross-link in both purified DNA (6.0 ± 0.6 DPC per 106 nt µM-1 MMS) and in human cells (7.8 ± 1.2 DPC per 106 nt mM-1 MMS). With the abasic site being one of the most common DNA lesions produced continuously by multiple pathways, the results provide significant new knowledge for better understanding the potential biological implications of its associated DNA-protein cross-link.

Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

BACKGROUND AND PURPOSE: Posttranslational modifications of cysteine residues represent a major aspect of redox biology, and their reliable detection is key in providing mechanistic insights. The metastable character of these modifications and cell lysis-induced artifactual oxidation render current state-of-the-art protocols to rely on alkylation-based stabilization of labile cysteine derivatives before cell/tissue rupture. An untested assumption in these procedures is that for all cysteine derivatives, alkylation rates are faster than their dynamic interchange. However, when the interconversion of cysteine derivatives is not rate limiting, electrophilic labelling is under Curtin-Hammett control; hence, the final alkylated mixture may not represent the speciation that prevailed before alkylation. EXPERIMENTAL APPROACH: Buffered aqueous solutions of inorganic, organic, cysteine, GSH and GAPDH polysulfide species were used. Additional experiments in human plasma and serum revealed that monobromobimane can extract sulfide from the endogenous sulfur pool by shifting speciation equilibria, suggesting caution should be exercised when interpreting experimental results using this tool. KEY RESULTS: In the majority of cases, the speciation of alkylated polysulfide/thiol derivatives depended on the experimental conditions. Alkylation perturbed sulfur speciation in both a concentration- and time-dependent manner and strong alkylating agents cleaved polysulfur chains. Moreover, the labelling of sulfenic acids with dimedone also affected cysteine speciation, suggesting that part of the endogenous pool of products previously believed to represent sulfenic acid species may represent polysulfides. CONCLUSIONS AND IMPLICATIONS: We highlight methodological caveats potentially arising from these pitfalls and conclude that current derivatization strategies often fail to adequately capture physiological speciation of sulfur species. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc.

Surpassing limits of static RNA modification analysis with dynamic NAIL-MS.

Ribonucleic acids (RNA) are extensively modified. These modifications are quantified by mass spectrometry (LC-MS/MS) to determine the abundance of a modification under certain conditions or in various genetic backgrounds. With LC-MS/MS the steady state of modifications is determined, and thus we only have a static view of the dynamics of RNA modifications. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) we overcome this limitation and get access to the dynamics of RNA modifications. We describe labeling techniques for E. coli, S. cerevisiae and human cell culture and the current instrumental limitations. We present the power of NAIL-MS but we also outline validation experiments, which are necessary for correct data interpretation. As an example, we apply NAIL-MS to study the demethylation of adenine and cytidine, which are methylated by the damaging agent methyl-methanesulfonate in E. coli. With NAIL-MS we exclude the concurrent processes for removal of RNA methylation, namely RNA degradation, turnover and dilution. We use our tool to study the speed and efficiency of 1-methyladenosine and 3-methylcytidine demethylation. We further outline current limitations of NAIL-MS but also potential future uses for e.g. relative quantification of tRNA isoacceptor abundances.

Atorvastatin Downregulates In Vitro Methyl Methanesulfonate and Cyclophosphamide Alkylation-Mediated Cellular and DNA Injuries.

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, and this class of drugs has been studied as protective agents against DNA damages. Alkylating agents (AAs) are able to induce alkylation in macromolecules, causing DNA damage, as DNA methylation. Our objective was to evaluate atorvastatin (AVA) antimutagenic, cytoprotective, and antigenotoxic potentials against DNA lesions caused by AA. AVA chemopreventive ability was evaluated using antimutagenicity assays (Salmonella/microsome assay), cytotoxicity, cell cycle, and genotoxicity assays in HepG2 cells. The cells were cotreated with AVA and the AA methyl methanesulfonate (MMS) or cyclophosphamide (CPA). Our datum showed that AVA reduces the alkylation-mediated DNA damage in different in vitro experimental models. Cytoprotection of AVA at low doses (0.1-1.0 µM) was observed after 24 h of cotreatment with MMS or CPA at their LC50, causing an increase in HepG2 survival rates. After all, AVA at 10 µM and 25 µM had decreased effect in micronucleus formation in HepG2 cells and restored cell cycle alterations induced by MMS and CPA. This study supports the hypothesis that statins can be chemopreventive agents, acting as antimutagenic, antigenotoxic, and cytoprotective components, specifically against alkylating agents of DNA.

Assessment of the antioxidant, cytotoxic, and genotoxic potential of the Annona muricata leaves and their influence on genomic stability.

The popular use of Annona muricata L. is based upon a range of medicinal purposes, and the plant exhibits biological activities including antihyperglycemic, antiparasitic, and antitumor activities. The objectives of this study were to examine the antioxidant, cytotoxic, and genotoxic potential of the hydroalcoholic extract of A. muricata leaves (AMEs), as well as its effects on genotoxicity induced by methyl methanesulfonate (MMS) and hydrogen peroxide (H2O2). The results using 2,2-diphenyl-1-picrylhydrazyl assay showed that AME was able to scavenge 44.71% of free radicals. The extract significantly reduced the viability of V79 cells in the clonogenic assay at concentrations ≥8 µg/ml. No significant differences in micronucleus (MN) frequency were observed between V79 cell cultures treated with different concentrations of the extract (0.125, 0.25, 0.5, and 1 µg/ml) and negative control. When AME concentrations were combined with MMS, data revealed no marked differences from mutagen alone. In contrast, significant reductions in the frequencies of MN were noted in cultures treated with AME combined with H2O2 compared to H2O2 alone. In vivo studies found no significant differences in the frequencies of micronucleated polychromatic erythrocytes (MNPCEs) between animals treated with different AME doses compared to control. Animals treated with AME doses of 125 and 250 mg/kg and MMS exhibited significantly higher frequencies of MNPCE compared to mutagen alone. In conclusion, under current experimental conditions, AME was not genotoxic and exerted a modulatory effect on DNA damage depending upon the experimental conditions. The extract did not influence markedly MMS-induced genotoxicity in in vitro test system. However, the extract increased DNA damage induced by mutagen in mice. In V79 cells, AME reduced the genotoxicity produced by H2O2, and this protective effect was attributed in part to the antioxidant activity of AME.

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system.

DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-α and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops) that prime replication in the ribosomal DNA locus. Our results suggest that eukaryotic genomes have developed tools to prevent R-loop-mediated replication events that potentially contribute to copy number variation, particularly relevant to carcinogenesis.

In vitro and in vivo genotoxicity assessment of HI-6 dimethanesulfonate/oxime.

Organophosphate compounds, which induce organophosphate poisoning, were originally used as pesticides. But this type of product has also been used as warfare nerve agent like sarin, soman, Russian VX, or tabun. HI-6-dimethanesulfonate is a salt of the oxime HI-6 used in the treatment of nerve-agent poisoning. It is known to be the best re-activator component of inactivated acetyl cholinesterase. HI-6-dimethanesulfonate has shown a higher level of solubility with similar potency to reactivate acetyl cholinesterase and a similar pharmacokinetics profile compared with HI-6 dichloride. HI-6 dimethanesulfonate was tested for its mutagenic and genotoxic potential by use of the standard ICH S2R (1) battery for the evaluation of pharmaceuticals. HI-6-dimethanesulfonate was mutagenic in the Ames test only in the presence of metabolic activation. In the mutation assay at the Tk locus in L5178Y mouse-lymphoma cells, HI-6-dimethanesulfonate showed mutagenic activity both with and without metabolic activation, with a significant increase in small colonies. The effects were in favour of a clastogenic activity. It was concluded that the compound was mutagenic and possibly clastogenic in vitro. In contrast, the in vivo micronucleus test in rat bone-marrow did not demonstrate any genotoxic activity and the Comet assay performed in rat liver did not show any statistically or biologically significant increases in DNA strand-breaks. The results of both in vivo studies performed on two different organs with two endpoints are sufficient to conclude the absence of a genotoxic hazard in vivo and to consider that there is no genotoxic concern in humans for HI-6-dimethanesulfonate.

Thiol-blocking electrophiles interfere with labeling and detection of protein sulfenic acids.

Cellular exposure to reactive oxygen species induces rapid oxidation of DNA, proteins, lipids and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post-translational process that is implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag-switch techniques. Common to both approaches is chemical blocking of free thiols using reactive electrophiles to prevent post-lysis oxidation or other thiol-mediated cross-reactions. These reagents are used in large excess, and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol-blocking electrophiles, iodoacetamide, N-ethylmaleimide and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine -SOR (product) species are partially or fully susceptible to reduction by dithiothreitol, tris(2-carboxyethyl)phosphine and ascorbate, regenerating protein thiols, or, in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S-nitrosothiols are discussed.

Propofol binding to the resting state of the gloeobacter violaceus ligand-gated ion channel (GLIC) induces structural changes in the inter- and intrasubunit transmembrane domain (TMD) cavities.

General anesthetics exert many of their CNS actions by binding to and modulating membrane-embedded pentameric ligand-gated ion channels (pLGICs). The structural mechanisms underlying how anesthetics modulate pLGIC function remain largely unknown. GLIC, a prokaryotic pLGIC homologue, is inhibited by general anesthetics, suggesting anesthetics stabilize a closed channel state, but in anesthetic-bound GLIC crystal structures the channel appears open. Here, using functional GLIC channels expressed in oocytes, we examined whether propofol induces structural rearrangements in the GLIC transmembrane domain (TMD). Residues in the GLIC TMD that frame intrasubunit and intersubunit water-accessible cavities were individually mutated to cysteine. We measured and compared the rates of modification of the introduced cysteines by sulfhydryl-reactive reagents in the absence and presence of propofol. Propofol slowed the rate of modification of L240C (intersubunit) and increased the rate of modification of T254C (intrasubunit), indicating that propofol binding induces structural rearrangements in these cavities that alter the local environment near these residues. Propofol acceleration of T254C modification suggests that in the resting state propofol does not bind in the TMD intrasubunit cavity as observed in the crystal structure of GLIC with bound propofol (Nury, H., Van Renterghem, C., Weng, Y., Tran, A., Baaden, M., Dufresne, V., Changeux, J. P., Sonner, J. M., Delarue, M., and Corringer, P. J. (2011) Nature 469, 428-431). In silico docking using a GLIC closed channel homology model suggests propofol binds to intersubunit sites in the TMD in the resting state. Propofol-induced motions in the intersubunit cavity were distinct from motions associated with channel activation, indicating propofol stabilizes a novel closed state.

Persulfide reactivity in the detection of protein s-sulfhydration.

Hydrogen sulfide (H2S) has emerged as a new member of the gaseous transmitter family of signaling molecules and appears to play a regulatory role in the cardiovascular and nervous systems. Recent studies suggest that protein cysteine S-sulfhydration may function as a mechanism for transforming the H2S signal into a biological response. However, selective detection of S-sulfhydryl modifications is challenging since the persulfide group (RSSH) exhibits reactivity akin to other sulfur species, especially thiols. A modification of the biotin switch technique, using S-methyl methanethiosulfonate (MMTS) as an alkylating reagent, was recently used to identify a large number of proteins that may undergo S-sulfhydration, but the underlying mechanism of chemical detection was not fully explored. To address this key issue, we have developed a protein persulfide model and analogue of MMTS, S-4-bromobenzyl methanethiosulfonate (BBMTS). Using these new reagents, we investigated the chemistry in the modified biotin switch method and examined the reactivity of protein persulfides toward different electrophile/nucleophile species. Together, our data affirm the nucleophilic properties of the persulfide sulfane sulfur and afford new insights into protein S-sulfhydryl chemistry, which may be exploited in future detection strategies.

Identification of multiple folding pathways of monellin using pulsed thiol labeling and mass spectrometry.

Protein folding reactions often display multiexponential kinetics of changes in intrinsic optical signals, as a manifestation of heterogeneity, either on one folding pathway or on multiple folding pathways. Delineating the origin of this heterogeneity is difficult because different coexisting structural forms of a protein cannot be easily distinguished by optical probes. In this study, the complex folding reaction of single-chain monellin has been investigated using a pulsed thiol labeling (SX) methodology in conjunction with mass spectrometry, which measures the kinetics of burial of a cysteine side chain thiol during folding. Because it can directly distinguish between unfolded and folded molecules and can measure the disappearance of the former during folding, the pulsed SX methodology is an ideal method for investigating whether multiple pathways are operative during folding. The kinetics of burial of the C42 thiol of monellin was observed to follow biexponential kinetics. To determine whether this was because the fast phase leads to the partial protection of the thiol group in all the molecules or to complete protection in only a fraction of the molecules, the duration and intensity of the labeling pulse were varied. The observation that the extent of labeling did not vary with the duration of the pulse cannot be explained by a simple sequential folding mechanism. Two parallel folding pathways are shown to be operative, with one leading to the formation of thiol-protective structure more rapidly than the other.

EMS in Viracept--initial ('traditional') assessment of risk to patients based on linear dose response relations.

Prior to having performed in depth toxicological, genotoxicological and DMPK studies on ethyl methanesulfonate (EMS) providing solid evidence for a thresholded dose response relationship, we had prepared and shared with regulatory authorities a preliminary risk estimate based on standard linear dose-effect projections. We estimated that maximal lifetime cancer risk was in the order of 10(-3) (for lifetime ingestion of the maximally contaminated tablets) or 10(-4) for the exposure lasting for 3 months. This estimate was based on a lifetime cancer study with methyl methanesulfonate (MMS; as insufficient data were available for EMS) in rodents and default linear back extrapolation. Analogous estimates were made specifically for breast cancer based on short term tumorigenicity studies with EMS in rats, for the induction of heritable mutations based on specific locus and dominant lethal tests in mice and for the induction of birth defects based on teratogenicity studies in mice. We concluded that even under worst case assumptions of linear dose relations the chance of experiencing these adverse effects would be very small, comprising at most a minute additional burden among the background incidence of the patients.

Chelator-facilitated chemical modification of IMP-1 metallo-beta-lactamase and its consequences on metal binding.

A method involving the reversible chemical modification of an active site, zinc-binding cysteine residue (Cys221) for the specific removal of one of the two zinc ions in the metallo-beta-lactamase IMP-1 was explored. Covalent modification of Cys221 by 5,5'-dithio-bis(2-nitrobenzoic acid) was greatly enhanced by the presence of dipicolinic acid, and subsequent removal of the modifying group was easily achieved by reduction of the disulfide bond. However, mass spectrometric analyses and an assessment of IMP-1's catalytic competence are consistent with the maintenance of the enzyme's binuclear status. The consequences arising from chemical modification of Cys221 are thus distinct from those reported for Cys-->Ala/Ser mutants of IMP-1 and other metallo-beta-lactamases, which are mononuclear.

Role of DNA mismatch repair and double-strand break repair in genome stability and antifungal drug resistance in Candida albicans.

Drug resistance has become a major problem in the treatment of Candida albicans infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (MSH2 and PMS1) or double-strand break repair (MRE11, RAD50, RAD52, and YKU80). We show that the mre11Delta/mre11Delta, rad50Delta/rad50Delta, and rad52Delta/rad52Delta mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The msh2Delta/msh2Delta, pms1Delta/pms1Delta, and yku80Delta/yku80Delta mutants exhibit none of these phenotypes. We observed an increase in genome instability in mre11Delta/mre11Delta and rad50Delta/rad50Delta mutants by using a GAL1/URA3 marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of mre11Delta/mre11Delta, rad50Delta/rad50Delta, or rad52Delta/rad52Delta leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both msh2Delta/msh2Delta and pms1Delta/pms1Delta (MMR mutants) and rad50Delta/rad50Delta (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in C. albicans strains lacking mismatch repair proteins or proteins central to double-strand break repair.

Gas-phase structure, rotational barrier, and vibrational properties of methyl methanethiosulfonate, CH3SO2SCH3: an experimental and computational study.

The molecular structure of methyl methanethiosulfonate, CH3SO2SCH3, has been determined in the gas phase from electron-diffraction data supplemented by ab initio (HF, MP2) and density functional theory (DFT) calculations using 6-31G(d), 6-311++G(d,p), and 6-311G(3df,3pd) basis sets. Both experimental and theoretical data indicate that although both anti and gauche conformers are possible by rotating about the S-S bond, the preferred conformation is gauche. The barrier to internal rotation in the CSSC skeleton has been calculated using the RHF/6-31G(d), MP2/6-31G(d), and B3LYP/6-31G(d) methods as well as MP2 with a 6-31G(3df) basis set on sulfur and 6-31G(d) on C, H, and O. A 6-fold decomposition of the rotational barrier has been performed in terms of a Fourier-type expansion, enabling us to analyze the nature of the potential function, showing that the coefficients V1 and V2 are the dominant terms; V1 is associated with nonbonding interactions, and V2 is associated with hyperconjugative interactions. A natural bond orbital analysis showed that the lone pair --> sigma* hyperconjugative interactions favor the gauche conformation. Furthermore, the infrared spectra for the liquid and solid phases and the Raman spectrum for the liquid have been recorded, and the observed bands have been assigned to the vibrational normal modes. The experimental vibrational data, along with calculated theoretical force constants, were used to define a scaled quantum mechanical force field for the target system that enabled us to estimate the measured frequencies with a final root-mean-square deviation of 6 cm-1.

Control of inward rectifier K channel activity by lipid tethering of cytoplasmic domains.

Interactions between nontransmembrane domains and the lipid membrane are proposed to modulate activity of many ion channels. In Kir channels, the so-called "slide-helix" is proposed to interact with the lipid headgroups and control channel gating. We examined this possibility directly in a cell-free system consisting of KirBac1.1 reconstituted into pure lipid vesicles. Cysteine substitution of positively charged slide-helix residues (R49C and K57C) leads to loss of channel activity that is rescued by in situ restoration of charge following modification by MTSET(+) or MTSEA(+), but not MTSES(-) or neutral MMTS. Strikingly, activity is also rescued by modification with long-chain alkyl-MTS reagents. Such reagents are expected to partition into, and hence tether the side chain to, the membrane. Systematic scanning reveals additional slide-helix residues that are activated or inhibited following alkyl-MTS modification. A pattern emerges whereby lipid tethering of the N terminus, or C terminus, of the slide-helix, respectively inhibits, or activates, channel activity. This study establishes a critical role of the slide-helix in Kir channel gating, and directly demonstrates that physical interaction of soluble domains with the membrane can control ion channel activity.