Critical Sites on Ostreolysin Are Responsible for Interaction with Cytoskeletal Proteins.

We explored the structural features of recombinant ostreolysin A (rOlyA), a protein produced by Pleurotus ostreatus and responsible for binding to α/ß-tubulin. We found that rOlyA cell internalization is essential for the induction of adipocyte-associated activity, which is mediated by the interaction of rOlyA and microtubule proteins. We created different point mutations at conserved tryptophan (W) sites in rOlyA and analyzed their biological activity in HIB-1B preadipocytes. We demonstrated that the protein's cell-internalization ability and the differentiated phenotype induced, such as small lipid-droplet formation and gene expression of mitogenesis activity, were impaired in point-mutated proteins W96A and W28A, where W was converted to alanine (A). We also showed that an rOlyA homologue, OlyA6 complexed with mCherry, cannot bind to ß-tubulin and does not induce mitochondrial biosynthesis-associated markers, suggesting that the OlyA6 region masked by mCherry is involved in ß-tubulin binding. Protein-protein docking simulations were carried out to investigate the binding mode of rOlyA with ß-tubulin. Taken together, we identified functional sites in rOlyA that are essential for its binding to ß-tubulin and its adipocyte-associated biological activity.

Sequence-specific destabilization of azurin by tetramethylguanidinium-dipeptide ionic liquids.

The thermal unfolding of the copper redox protein azurin was studied in the presence of four different dipeptide-based ionic liquids (ILs) utilizing tetramethylguanidinium as the cation. The four dipeptides have different sequences including the amino acids Ser and Asp: TMG-AspAsp, TMG-SerSer, TMG-SerAsp, and TMG-AspSer. Thermal unfolding curves generated from temperature-dependent fluorescence spectroscopy experiments showed that TMG-AspAsp and TMG-SerSer have minor destabilizing effects on the protein while TMG-AspSer and TMG-SerAsp strongly destabilize azurin. Red-shifted fluorescence signatures in the 25 °C correlate with the observed protein destabilization in the solutions with TMG-AspSer and TMG-SerAsp. These signals could correspond to interactions between the Asp residue in the dipeptide and the azurin Trp residue in the unfolded state. These results, supported by appropriate control experiments, suggest that dipeptide sequence-specific interactions lead to selective protein destabilization and motivate further studies of TMG-dipeptide ILs.

Thermodynamic destabilization of azurin by four different tetramethylguanidinium amino acid ionic liquids.

The thermal unfolding of the copper redox protein azurin was studied in the presence of four different amino acid-based ionic liquids (ILs), all of which have tetramethylguanidium as cation. The anionic amino acid includes two with alcohol side chains, serine and threonine, and two with carboxylic acids, aspartate and glutamate. Control experiments showed that amino acids alone do not significantly change protein stability and pH changes anticipated by the amino acid nature have only minor effects on the protein. With the ILs, the protein is destabilized and the melting temperature is decreased. The two ILs with alcohol side chains strongly destabilize the protein while the two ILs with acid side chains have weaker effects. Unfolding enthalpy (ΔHunf°) and entropy (ΔSunf°) values, derived from fits of the unfolding data, show that some ILs increase ΔHunf°while others do not significantly change this value. All ILs, however, increase ΔSunf°. MD simulations of both the folded and unfolded protein conformations in the presence of the ILs provide insight into the different IL-protein interactions and how they affect the ΔHunf° values. The simulations also confirm that the ILs increase the unfolded state entropies which can explain the increased ΔSunf° values.

Effects of Ionic Liquids on Metalloproteins.

In the past decade, innovative protein therapies and bio-similar industries have grown rapidly. Additionally, ionic liquids (ILs) have been an area of great interest and rapid development in industrial processes over a similar timeline. Therefore, there is a pressing need to understand the structure and function of proteins in novel environments with ILs. Understanding the short-term and long-term stability of protein molecules in IL formulations will be key to using ILs for protein technologies. Similarly, ILs have been investigated as part of therapeutic delivery systems and implicated in numerous studies in which ILs impact the activity and/or stability of protein molecules. Notably, many of the proteins used in industrial applications are involved in redox chemistry, and thus often contain metal ions or metal-associated cofactors. In this review article, we focus on the current understanding of protein structure-function relationship in the presence of ILs, specifically focusing on the effect of ILs on metal containing proteins.

Synergistic interactions of ionic liquids and antimicrobials improve drug efficacy.

Combinations of ionic liquids (ILs) with antimicrobial compounds have been shown to produce synergistic activities in model liposomes. In this study, imidazolium chloride-based ILs with alkyl tail length variations are combined with commercially available, small-molecule antimicrobials to examine the potential for combinatorial and synergistic antimicrobial effects on P. aeruginosa, E. coli, S. aureus, and S. cerevisiae. The effects of these treatments in a human cell culture model indicate the cytotoxic limits of ILs paired with antimicrobials. The analysis of these ILs demonstrates that the length of the alkyl chain on the IL molecule is proportional to both antimicrobial activity and cytotoxicity. Moreover, the ILs which exhibit synergy with small-molecule antibiotics appear to be acting in a membrane permeabilizing manner. Collectively, results from these experiments demonstrate an increase in antimicrobial efficacy with specific IL + antimicrobial combinations on microbial cultures while maintaining low cytotoxicity in a mammalian cell culture model.

Effects of Ionic Liquid Alkyl Chain Length on Denaturation of Myoglobin by Anionic, Cationic, and Zwitterionic Detergents.

The unique electrochemical properties of ionic liquids (ILs) have motivated their use as solvents for organic synthesis and green energy applications. More recently, their potential in pharmaceutical chemistry has prompted investigation into their effects on biomolecules. There is evidence that some ILs can destabilize proteins via a detergent-like manner; however, the mechanism still remains unknown. Our hypothesis is that if ILs are denaturing proteins via a detergent-like mechanism, detergent-mediated protein unfolding should be enhanced in the presence of ILs. The properties of myoglobin was examined in the presence of a zwitterionic (N,N-dimethyl-N-dodecylglycine betaine (Empigen BB®, EBB)), cationic (tetradecyltrimethylammonium bromide (TTAB)), and anionic (sodium dodecyl sulfate (SDS)) detergent as well as ILs based on alkylated imidazolium chlorides. Protein structure was measured through a combination of absorbance, fluorescence, and circular dichroism (CD) spectroscopy: absorbance and CD were used to monitor heme complexation to myoglobin, and tryptophan fluorescence quenching was used as an indicator for heme dissociation. Notably, the detergents tested did not fully denature the protein but instead resulted in loss of the heme group. At low IL concentrations, heme dissociation remained a traditional, cooperative process; at high concentrations, ILs with increased detergent-like character exhibited a more complex pattern, which is most likely attributable to micellization of the ionic liquids or direct denaturation or heme dissociation induced by the ILs. These trends were consistent across all species of detergents. 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence was further used to characterize micelle formation in aqueous solutions containing detergent and ionic liquid. The dissociation thermodynamics show that EBB- and TTAB-induced dissociation of heme is not significantly impacted by room temperature ionic liquids (RTILs), whereas SDS-induced dissociation is more dramatically impacted by all RTILs examined. Together, these results indicate a complex interaction of detergents, likely based on headgroup charge, and the active component of RTILs to influence heme dissociation and potentially protein denaturation.

Structural Destabilization of Azurin by Imidazolium Chloride Ionic Liquids in Aqueous Solution.

Alkyl imidazolium chloride ionic liquids (ILs) have been used for numerous biochemical applications. Their hydrophobicity can be tuned by changing the alkyl chain length, and longer-chain ILs can form micelles in aqueous solution. We have investigated the effects of imidazolium chloride ILs on the structure and stability of azurin, which is a very stable Cu2+ redox protein with both α-helix and ß-sheet domains. Temperature-dependent infrared (IR) and vibrational circular dichroism spectroscopy can provide secondary-structure-specific information about how the protein is affected, and temperature-jump transient IR measurements can quantify the IL-influenced unfolding dynamics. Using these techniques, we can quantify how azurin is destabilized by 1.0 M ILs in aqueous solution. The shorter, less hydrophobic ILs, 1-butyl-3-methylimidazolium chloride and 1-hexyl-3-methylimidazolium chloride likely interact with the α-helix domain and decrease protein melting temperature from 82 °C without IL to 55 °C and disturb the overall tertiary structure, resulting in a looser, more open shape. Thermodynamic analysis indicates that protein destabilization is due to increased unfolding entropy. 1-Octyl-3-methylimidazolium chloride [OMIM]Cl, which forms micelles in solution that may partially solvate the protein, has a more significant destabilizing effect, resulting in a melting temperature of 35 °C, larger unfolding entropy, and relaxation kinetics several orders of magnitude faster than with unperturbed azurin. The temperature-independence of the relaxation time constant suggests that in the presence of [OMIM]Cl, the protein folding potential energy surface has become very smooth.

Correlating Lipid Membrane Permeabilities of Imidazolium Ionic Liquids with their Cytotoxicities on Yeast, Bacterial, and Mammalian Cells.

Alkyl-imidazolium chloride ionic liquids (ILs) have been broadly studied for biochemical and biomedical technologies. They can permeabilize lipid bilayer membranes and have cytotoxic effects, which makes them targets for drug delivery biomaterials. We assessed the lipid-membrane permeabilities of ILs with increasing alkyl chain lengths from ethyl to octyl groups on large unilamellar vesicles using a trapped-fluorophore fluorescence lifetime-based leakage experiment. Only the most hydrophobic IL, with the octyl chain, permeabilizes vesicles, and the concentration required for permeabilization corresponds to its critical micelle concentration. To correlate the model vesicle studies with biological cells, we quantified the IL permeabilities and cytotoxicities on different cell lines including bacterial, yeast, and ovine blood cells. The IL permeabilities on vesicles strongly correlate with permeabilities and minimum inhibitory concentrations on biological cells. Despite exhibiting a broad range of lipid compositions, the ILs appear to have similar effects on the vesicles and cell membranes.

Activity and characterization of a pH-sensitive antimicrobial peptide.

Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. Previous work showed that when Histidine was incorporated into the peptide C18G it lost antimicrobial activity. The role of pH on activity and biophysical properties of the peptide was investigated to explain this phenomenon. Minimal inhibitory concentration (MIC) results demonstrated that decreased media pH increased antimicrobial activity. Trichloroethanol (TCE) quenching and red-edge excitation spectroscopy (REES) showed a clear pH dependence on peptide aggregation in solution. Trp fluorescence was used to monitor binding to lipid vesicles and demonstrated the peptide binds to anionic bilayers at all pH values tested, however, binding to zwitterionic bilayers was enhanced at pH 7 and 8 (above the His pKa). Dual Quencher Analysis (DQA) confirmed the peptide inserted more deeply in PC:PG and PE:PG membranes, but could insert into PC bilayers at pH conditions above the His pKa. Bacterial membrane permeabilization assays which showed enhanced membrane permeabilization at pH 5 and 6 but vesicle leakage assays indicate enhanced permeabilization of PC and PC:PG bilayers at neutral pH. The results indicate the ionization of the His side chain affects the aggregation state of the peptide in solution and the conformation the peptide adopts when bound to bilayers, but there are likely more subtle influences of lipid composition and properties that impact the ability of the peptide to form pores in membranes.

Heme Dissociation from Myoglobin in the Presence of the Zwitterionic Detergent N,N-Dimethyl-N-Dodecylglycine Betaine: Effects of Ionic Liquids.

We have investigated myoglobin protein denaturation using the zwitterionic detergent Empigen BB (EBB, N,N-Dimethyl-N-dodecylglycine betaine). A combination of absorbance, fluorescence, and circular dichroism spectroscopic measurements elucidated the protein denaturation and heme dissociation from myoglobin. The results indicated that Empigen BB was not able to fully denature the myoglobin structure, but apparently can induce the dissociation of the heme group from the protein. This provides a way to estimate the heme binding free energy, ΔGdissociation. As ionic liquids (ILs) have been shown to perturb the myoglobin protein, we have investigated the effects of the ILs 1-butyl-3-methylimidazolium chloride (BMICl), 1-ethyl-3-methylimidazolium acetate (EMIAc), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF4) in aqueous solution on the ΔGdissociation values. Absorbance experiments show the ILs had minimal effect on ΔGdissociation values when compared to controls. Fluorescence and circular dichroism data confirm the ILs have no effect on heme dissociation, demonstrating that low concentrations ILs do not impact the heme dissociation from the protein and do not significantly denature myoglobin on their own or in combination with EBB. These results provide important data for future studies of the mechanism of IL-mediated protein stabilization/destabilization and biocompatibility studies.

An activity transition from NADH dehydrogenase to NADH oxidase during protein denaturation.

A decrease in the specific activity of an enzyme is commonly observed when the enzyme is inappropriately handled or is stored over an extended period. Here, we reported a functional transition of an FMN-bound diaphorase (FMN-DI) that happened during the long-term storage process. It was found that FMN-DI did not simply lose its ß-nicotinamide adenine diphosphate (NADH) dehydrogenase activity after a long-time storage, but obtained a new enzyme activity of NADH oxidase. Further mechanistic studies suggested that the alteration of the binding strength of an FMN cofactor with a DI protein could be responsible for this functional switch of the enzyme.

Synergistic effects of polymyxin and ionic liquids on lipid vesicle membrane stability and aggregation.

Ionic liquids (ILs) have been investigated for potential antibacterial and antibiotic applications due to their ability to destabilize and permeabilize the lipid bilayers in cell membranes. Bacterial assays have shown that combining ILs with antibiotics can provide a synergistic enhancement of their antibacterial activities. We have characterized the mechanism by which the conventional ILs 1-butyl-3-methylimidazolium chloride (BMICl) and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIBF4) enhance the lipid membrane permeabilization of the well-known antibiotic polymyxin B (PMB). We studied the sizes and membrane permeabilities of multilamellar and unilamellar lipid bilayer vesicles in the presence of ILs alone in aqueous solution, PMB alone, and ILs combined together with PMB. Light scattering-based experiments show that vesicle sizes dramatically increase when ILs are combined with PMB, which suggests that the materials combine to synergistically enhance lipid membrane disruption leading to vesicle aggregation. Lipid bilayer leakage experiments using tris (2,2'-bipyridyl) ruthenium (II) (Ru(bpy)32+) trapped in lipid vesicles, in which the trapped Ru(bpy)32+ fluorescence lifetime increases when it leaks out of the vesicle, show that combining BMIBF4 and PMB together permeabilize the membrane significantly more than with PMB or the IL alone. This demonstrates that ILs can assist in antibiotic permeabilization of lipid bilayers which could explain the increased antibiotic activities in the presence of ILs in solution.

An Experimental and Molecular Dynamics Study of Red Fluorescent Protein mCherry in Novel Aqueous Amino Acid Ionic Liquids.

The search for biocompatible ionic liquids (ILs) with novel biochemical and biomedical applications has recently gained greater attention. In this report, we characterize the effects of two novel amino acid-based aqueous ILs composed of tetramethylguanidinium (TMG) and amino acids on the structure and stability of a widely used red fluorescent protein (mCherry). Our experimental data shows that while the aspartic acid-based IL (TMGAsp) has effects similar to previously studied conventional ILs (BMIBF4, EMIAc, and TMGAc), the alanine-based IL (TMGAla) has a much stronger destabilization effect on the protein structure. Addition of 0.30 M TMGAla to mCherry decreases the unfolding temperature from 83 to 60 °C. Even at 25 °C, TMGAla results in a blue shift of the mCherry absorbance and fluorescence peaks and an increased Stokes shift. Molecular dynamics simulations show that the chromophore conformation and its interaction with mCherry with TMGAla are changed relative to those with TMGAsp or in the absence of ILs. Protein-ILs contact analysis indicates that the mCherry-Asp interactions are hydrophilic but the (fewer) mCherry-Ala interactions are more hydrophobic and may modulate the TMG interaction with the protein. Hence, the anion hydrophobicity may explain the special TMGAla destabilization of mCherry.

Kinetics and mass spectrometric measurements of myoglobin unfolding in aqueous ionic liquid solutions.

Recent studies have characterized the effects of aqueous ionic liquids on myoglobin unfolding for the broader purposes of understanding their effects on protein structures, stabilities, and ultimately biocompatibilities for future applications. Here, we investigated the effects of four different ionic liquids (ILs) on the thermal stability, unfolding kinetics, and tertiary shape of myoglobin. We compared results for four different ILs: 1-butyl-3-methyl imidazolium tetrafluoroborate (BMIBF4); 1-butyl-3-methyl pyrrolidinium tetrafluoroborate (PyrrBF4); 1-ethyl-3-methyl imidazolium acetate (EMIAc); and tetramethylguanidinium acetate (TMGAc). Results showed that ILs accelerate myoglobin unfolding kinetics both through aqueous solution ionic strength effects and ionic liquid-specific effects. Arrhenius plots of observed rate constants reveal that some ILs lower the energy barrier to unfolding, possibly by destabilizing the native protein state. The magnitude of these ionic liquid effects correlates with their effects on protein thermodynamic stabilities. Hydrogen-deuterium exchange (HDX) experiments using ESI-MS showed that myoglobin exhibits a more open, and presumably less stable, tertiary shape in aqueous IL solutions. Overall, BMIBF4 and TMGAc exhibit the strongest effect on the myoglobin stability, unfolding rate, and tertiary structure while PyrrBF4 and EMIAc have weaker effects under our experimental conditions.

Characterization of the bridged proton structure in HTFSI acid ionic liquid solutions.

Acidic ionic liquid (AIL) solutions were prepared by dissolving bis(trifluoromethanesulfonyl)imide (HTFSI) acid in the ionic liquid (IL) 1-butyl-3-methylpyrrolidinium bis(trifloromethanesulfonyl)imide (PyrrTFSI). The HTFSI/PyrrTFSI solutions were investigated by conductivity measurements, optical spectroscopy, and DFT calculations in order to understand the ionization/solvation mechanism of HTFSI in the solutions. The HTFSI/PyrrTFSI solution conductivities first increased at lower concentrations and then decreased when the concentration of HTFSI is higher than ∼1.5 M. The spectroscopic results indicate that the solvation structure may evolve from lower to higher concentrations to make protonated TFSI(-) motifs. Both spectroscopic and DFT simulation results support the observation of proton-sharing [H(TFSI)2](-) dimers, which may form through a bridged hydrogen in the format of either a N-H-N connection or a N-H-O connection. Both configurations may exist in the AIL solution. The proton-sharing mechanism implied by these structures confirms that the TFSI(-) ion can be a proton acceptor and a Brønsted base as well in IL solutions. However, the IL molecular cations such as imidazolium and (in this work) pyrrolidinium do not contribute significantly to the proton solvation and transportation in the solutions.

Quantitative evaluation of myoglobin unfolding in the presence of guanidinium hydrochloride and ionic liquids in solution.

The use of ionic liquids in biochemical and biophysical applications has increased dramatically in recent years due to their interesting properties. We report results of a thermodynamic characterization of the chaotrope-induced denaturation of equine myoglobin in two different ionic liquid aqueous environments using a combined absorption/fluorescence spectroscopic approach. Denaturation by guanidinium hydrochloride was monitored by loss of heme absorptivity and limited unfolding structural information was obtained from Förster resonance energy transfer experiments. Results show that myoglobin unfolding is generally unchanged in the presence of ethylmethylimidazolium acetate (EMIAc) in aqueous solution up to 150 mM concentration but is facilitated by butylmethylimidazolium boron tetrafluoride (BMIBF4) in solution. The presence of 150 mM BMIBF4 alone does not induce unfolding but destabilizes the structure as observed by a decrease in threshold denaturant concentration for unfolding and an 80% decrease in the magnitude of ΔGunfolding from 44 kJ/mol in the absence of BMIBF4 to 8 kJ/mol in the presence of 150 mM BMIBF4. Thus, the BMIBF4 significantly destabilizes the myoglobin structure while the EMIAc does not, likely due to differences in anion interaction capabilities. This is confirmed with control studies using NaAc and LiBF4 solutions. EMIAc may be chosen as cosolvent additive with minimal effects on protein structure while BMIBF4 may be used as a supplement in protein folding experiments, potentially allowing access to proteins which have been traditionally difficult to denature as well as designing ionic liquids to match protein characteristics.

Complexation between Cu(II) and curcumin in the presence of two different segments of amyloid ß.

The natural product curcumin has been shown to play a role in preventing Aß amyloid fibril formation. This role could include chelation of transition metal ions such as Cu(2+), known to accelerate amyloid aggregation, and/or curcumin-binding directly to the Aß protein. To investigate these different roles, curcumin complexation to Cu(2+) was investigated in the presence and absence of two different segments of the Aß protein including the copper-binding (Aß6-14) and curcumin-binding (Aß14-23) domains. Absorbance and fluorescence spectroscopy in 90% water/10% methanol solutions showed that curcumin can bind Cu(2+) to some extent in the presence of both segments despite strong peptide-ion interactions. Estimated Cu(2+)-curcumin binding affinities in the absence (1.6×10(5)M(-1)) and presence (7.9×10(4)M(-1)) of the peptide provide quantitative support for this Cu(2+) chelation role. With the Aß14-23 segment, the curcumin simultaneously binds to Cu(2+) and the peptide, demonstrating that it can play multiple roles in the prevention of amyloid formation. The stabilities of ternary peptide-Cu(2+)-curcumin complexes were evaluated using ESI mass spectrometry and support the conclusion that curcumin can act as a weak metal ion chelator and also bind directly to the Aß14-23 peptide segment.

Conductivity, spectroscopic, and computational investigation of H3O+ solvation in ionic liquid BMIBF4.

The hydrogen ion is one of the most important species in aqueous solutions, as well as in protic ionic liquids (PILs). PILs are important potential alternatives to H2O for swelling the proton exchange membranes (PEMs) and improving the high-temperature performance of fuel cells. The hydrogen ion (H(+)) or hydronium (H3O(+)) solvation mechanism is not only a fundamental principle of acid/base chemistry in ionic liquids but also key to understanding the charge- and proton-transport properties of the PIL solutions. In this paper, a PIL system was prepared by mixing 1-butyl-3-methyl-imidazolium tetrafluoroborate (BMIBF4) IL with an aqueous solution of a strong acid, HBF4. Water can be partially evaporated from the solution under a vacuum at room temperature. Conductivity and vibrational spectroscopy (IR and Raman) measurements were used in combination with density functional theory (DFT) calculations to characterize the molecular-level solvation of H(+) and H2O in the IL solution. When water is present at high molar fraction, the cations (BMI(+) and H(+)) and anions (BF4(-)) are both solvated by water and the solutions have high conductivity. After water evaporation, the PIL solution has excess H(+) and reduced conductivity, which is still significantly higher than that of pure BMIBF4. Vibrational spectroscopy suggests that the BMI(+) and BF4(-) ions are desolvated from water during the water evaporation. DFT calculations assist the interpretation of the vibrational spectroscopy and show that the remaining water is in the form of H3O(+) solvated by the IL molecular ions. Hence, the species remaining after evaporation is a ternary PIL consisting of BMI(+) cation, BF4(-) anion, and H3O(+) cation. The H3O(+) may be the principle charge carrier in the PIL solution and responsible for the high solution conductivity.

Conductivity and spectroscopic investigation of bis(trifluoromethanesulfonyl)imide solution in ionic liquid 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide.

Protic ionic liquids (PILs) are promising alternatives to water for swelling Nafion as a fuel cell proton exchange membrane (PEM). PILs can significantly improve the high-temperature performance of a PEM. The proton dissociation and solvation mechanisms in a PIL, which are keys to understanding the proton transportation and conductivity, have not been fully explored. In this paper, we used FTIR, Raman, and electronic spectroscopy with computational simulation techniques to explore the spectroscopic properties of bis(trifluoromethanesulfonyl)imide (HTFSI) solutions in 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (BMITFSI) ionic liquid at concentrations from ∼0.1 to as high as ∼1.0 M. Solution conductivities were measured at room temperature and elevated temperatures up to ∼65 °C. The solution structure and properties depend on the concentration of HTFSI. At lower concentration, around 0.1 M, the HTFSI solution has higher conductivity than pure BMITFSI. However, the conductivity decreases when the concentration increases from 0.1 to 1.0 M. Temperature-dependent conductivities followed the Vogel-Fulcher-Tamman equation at all concentrations. Conductivity and spectroscopy results elucidate the complicated ionization and solvation mechanism of HTFSI in BMITFSI solutions. Raman spectroscopy and density functional theory (DFT) calculations are consistent with the complete ionization of HTFSI to generate solvated H(+) at low concentrations. FTIR, Raman, and electronic spectroscopic results as well as DFT computational simulation indicated that when the concentration is as high as 1.0 M, a significant amount of TFSI(-) is protonated, most likely at the imide nitrogen.

Exploring carbohydrate-peptide interactions in the gas phase: structure and selectivity in complexes of pyranosides with N-acetylphenylalanine methylamide.

The physical basis of carbohydrate-peptide interactions has been explored by probing the structures of a series of complexes generated in a solvent-free environment under molecular beam conditions. A combination of double-resonance IR-UV spectroscopy and quantum-chemical calculations has established the structures of complexes of the model, N-acetyl-L-phenylalanine methylamide, bound to the α and ß anomers of methyl D-gluco- and D-galactopyranoside as guests. In all cases, the carbohydrates are bound through hydrogen bonding to the dipeptide chain, although with some differing patterns. The amino acid host "engages" with the most suitable pair of neighboring conjugate sites on each carbohydrate; the specific choice depends on the conformation of the peptide backbone and the configuration and conformation of the carbohydrate ligand. None of the structures is supported by "stacking" interactions with the aromatic ring, despite their common occurrence in bound carbohydrate-protein structures.