Noncovalent Adduct Research Articles

Protein–Metal Interactions of Calmodulin and α-Synuclein Monitored by Selective Noncovalent Adduct Protein Probing Mass Spectrometry

The metal binding properties of proteins are biologically significant, particularly in relationship to the molecular origins of disease and the discovery of therapeutic pharmaceutical treatments. Herein, we demonstrate that selective noncovalent adduct protein probing mass spectrometry (SNAPP-MS) is a sensitive technique to investigate the structural effects of protein-metal interactions. We utilize specific, noncovalent interactions between 18-crown-6 ether (18C6) and lysine to probe protein structure in the presence and absence of metal ions. Application of SNAPP-MS to the calmodulin-Ca2+ system demonstrates that changes in protein structure are reflected in a substantial change in the number and intensity of 18C6s, which bind to the protein as observed by MS. In this manner, SNAPP is demonstrated to be a sensitive technique for monitoring ligand-induced conformational rearrangements in proteins. In addition, SNAPP is well-suited to examine the properties of natively unfolded proteins, where structural changes are more difficult to detect by other methods. For example, alpha-synuclein is a protein associated in the pathology of Parkinson's disease, which is known to aggregate more rapidly in the presence of Al3+ and Cu2+. The 18C6 SNAPP distributions for alpha-synuclein change dramatically in the presence of 3 microM Al3+, revealing that Al3+ binding causes a significant change in the conformational dynamics of the monomeric form of this disordered protein. In contrast, binding of Cu2+ does not induce a significant shift in 18C6 binding, suggesting that noteworthy structural reorganizations at the monomeric level are minimal. These results are consistent with the idea that the metal-induced aggregation caused by Al3+ and Cu2+ proceed by independent pathways.

In vitro stimulatory effect of anti-apoptotic seminal vesicle protein 4 on purified peroxidase enzymes

The enzymatic activities of purified horseradish peroxidase, selenium-dependent glutathione peroxidase, thyroid peroxidase and myeloperoxidase, but not that of lactoperoxidase, were markedly enhanced when added into a reaction mixture containing 5 mum native seminal vesicle protein 4, a major protein secreted from rat seminal vesicle epithelium. A further increase of horseradish peroxidase activity was obtained using Ser58-phosphorylated or acetylated seminal vesicle protein 4. The activating effect of native seminal vesicle protein 4 was highest (about 60-fold) on horseradish peroxidase when 4-chloro-1-naphtol was used as the electron donor substrate. The main kinetics parameters of the stimulatory effect on horseradish peroxidase were evaluated and the enzyme-electron donor substrate interaction was investigated by HPLC and electrospray-MS. A native seminal vesicle protein 4/4-chloro-1-naphtol noncovalent adduct was detected when the protein and 4-chloro-1-naphtol were present in the appropriate molar ratio in the horseradish peroxidase-catalyzed reaction. By contrast, no adducts were formed between native seminal vesicle protein 4 and horseradish peroxidase. This native seminal vesicle protein 4/4-chloro-1-naphtol interaction might underlie the native seminal vesicle protein 4-induced horseradish peroxidase stimulation. Furthermore, native seminal vesicle protein 4 was shown by spectrophotometric and electrospray-MS analysis to interact with NADPH, an electron donor substrate of the selenium-dependent glutathione peroxidase/glutathione reductase redox system, with formation of an adduct between them. Although further investigation is required to elucidate the mechanism of adduct formation, this interaction, probably by promoting the release of the NADPH electrons required for glutathione disulphide reduction, could explain the stimulatory effect of seminal vesicle protein 4 on mammalian peroxidases possibly involved in its physiological function on the selenium-dependent glutathione peroxidase/glutathione reductase system. The biological significance of these properties of native seminal vesicle protein 4 might be related to its ability to downregulate reactive oxygen species and oxidative stress-induced apoptosis.

Surveying Ubiquitin Structure by Noncovalent Attachment of Distance Constrained Bis(crown) Ethers

Selective noncovalent adduct protein probing (SNAPP) mass spectrometry was recently developed to study solution-phase conformations of proteins by exploiting the specific affinity between 18-crown-6 ether (18C6) and lysine side chains. To obtain more detailed information about protein tertiary structure, a novel noncovalent cross-linking reagent with two 18C6 molecules bridged by a covalent phenyl linker (called PBC for phenyl bis-crown) was synthesized. PBC introduces a distance constraint into SNAPP experiments where pairs of lysine side chains that are held in proximity by tertiary structure should be the most favored binding sites. Application of this method to ubiquitin reveals that PBC can bind to one lysine in a monodentate fashion or bind to two lysines via a bidentate interaction. Comparison with 18C6 can be used to reveal the mode of binding. For the native state of ubiquitin, bidentate binding of PBC is not observed. The partially denatured A-state, however, contains a single pair of lysines that are both chemically available and spaced by less than approximately 19 A (the maximum distance spanning the crown ether binding sites in PBC). Collision-induced dissociation and site-directed mutagenesis reveal that the bidentate PBC attaches to K29 and K33, which is in agreement with previous structural data on the A-state of ubiquitin. PBC is shown to be an effective probe of protein structure in SNAPP experiments, although assigning the specific residues to which PBC is attached can be experimentally challenging.

High‐Relaxivity Magnetic Resonance Imaging (MRI) Contrast Agent Based on Supramolecular Assembly between a Gadolinium Chelate, a Modified Dextran, and Poly‐β‐Cyclodextrin

Nanosized contrast agents have great potential in magnetic resonance molecular imaging applications for clinical diagnosis. This study proposes new nanoparticles spontaneously formed under mild conditions and composed of a noncovalent adduct between a gadolinium complex, a polymer of beta-cyclodextrin (pbetaCD: MW 1.5 x 10(6) g mol(-1)) and a dextran grafted with alkyl chains (MD). The formation of this supramolecular nanoassembly is based upon a "lock-and-key" recognition process in which the hydrophobic alkyl chains of MD and the adamantyl moieties of macrocyclic Gd(III) chelates are included in the cavities of pbetaCD. The large number of betaCDs contained in the pbetaCD resulted in the formation of 200 nm diameter nanoparticles, each entrapping 1.8 x 10(5) molecules of a low-molecular-weight Gd complex. This system, which exhibits a great relaxivity enhancement (48.4 mM(-1) s(-1), at 20 MHz and 37 degrees C) compared to the Gd(III) chelate itself (5.2 mM(-1) s(-1)), appears to be a promising strategy for the in vivo targeted delivery of Gd(III) complexes. The mechanisms of particle formation, conjugation strategies, and relaxometric characterizations in the field of contrast-enhanced magnetic resonance imaging are discussed.

Exploring the Mechanism of Selective Noncovalent Adduct Protein Probing Mass Spectrometry Utilizing Site-Directed Mutagenesis To Examine Ubiquitin

Mass spectrometry (MS) is emerging as an additional tool for examining protein structure by way of experiments where structurally related mass changes induced in solution are subsequently detected in the gas phase. Selective noncovalent adduct protein probing (SNAPP) is a recent addition to this type of experiment. SNAPP utilizes noncovalent recognition of lysine residues with 18-crown-6 (18C6) to monitor changes in protein structure. It has been observed that the number of 18C6 adducts that attach to a protein is a function of the structure of the protein. The present work seeks to examine the underlying chemistry which controls the differential attachment of 18C6 to lysine by using ubiquitin as a model system. Ubiquitin is a small protein with a structure that has been well characterized by multiple techniques. Site-directed mutagenesis was used to create a series of ubiquitin mutants where the lysine residues were exchanged for asparagine one at a time. These mutants were then evaluated by SNAPP-MS to determine the relative contribution of each lysine as a binding site for 18C6. It was found that attachment of 18C6 is largely controlled by the strength of intramolecular interactions involving lysine residues. Salt bridges provide the greatest interference, followed by hydrogen bonds. In addition to determining the mechanism for SNAPP, insights are provided about the structure of ubiquitin including confirmation of the existence of two dynamic states for the native structure. These results are discussed in relation to the biological functions of ubiquitin.

Relaxometric and Modelling Studies of the Binding of a Lipophilic Gd‐AAZTA Complex to Fatted and Defatted Human Serum Albumin

A new lipophilic gadolinium chelate consisting of a long aliphatic chain bound to the AAZTA coordination cage (Gd-AAZTAC17) has been synthesised. It possesses two coordinated water molecules (q=2) in fast exchange with the solvent (tau298(M) = 67 ns), which yields a relaxivity of 10.2 mM(-1) s(-1). At concentrations greater than 0.1 mM, it forms micelles (average diameter 5.5 nm) characterised by a relaxivity of approximately 30 mM(-1) s(-1) at 20 MHz and 298 K. The latter value appears to be "quenched" by magnetic interactions among the Gd(III) ions on the surface of the micelle that cause a decrease in the electronic relaxation time. A relaxivity of 41 mM(-1) s(-1) was recorded for this micellar system when 98 % of the Gd(III) ions were replaced by diamagnetic Y(III). Gd-AAZTAC17 exhibits a better affinity for fatted human serum albumin (HSA) than for defatted HSA, whereas the relaxivities of the supramolecular adducts are reversed. The relaxivity shown by Gd-AAZTAC17/defatted HSA ({r b(1) (20 MHz, 298 K)=84 mM(-1) s(-1)) is by far the highest relaxivity reported so far for non-covalent paramagnetic adducts with slow-moving substrates. As shown by molecular docking calculations, the gadolinium complex enters a hydrophobic pocket present in fatted HSA more extensively than the corresponding adduct with defatted HSA. Interestingly, no marked difference was observed in either the relaxation enhancement or the binding affinity between fatted and defatted HSA when the binding titrations were carried out at a Gd-AAZTAC17 concentration higher than its critical micellar concentration (cmc). This behaviour has been attributed to the formation of an association between the negatively charged micelle of the lipophilic metal complexes and the positive residues on the surface of the protein.

Naturally occurring glucosinolates in plant extracts of rocket salad (Eruca sativa L.) identified by liquid chromatography coupled with negative ion electrospray ionization and quadrupole ion‐trap mass spectrometry

A method for the comprehensive profiling of intact glucosinolates (GLSs), major and minor, occurring in leaves and seeds of rocket salad (Eruca sativa L.) is presented using optimized reversed-phase liquid chromatography (RP-LC) with electrospray ionization (ESI) ion trap mass spectrometry (ITMS). ESI-ITMS in the negative mode was confirmed to be very suitable to analyze these compounds in crude extracts. After extraction from the plant material with methanol/water (70:30 v/v) at 70 degrees C, the analytes of interest were separated on a C18 column using an eluent acidified with formic acid (0.1%) and modified with acetonitrile. All the GLSs found in leaves of rocket salad gave good signals corresponding to the deprotonated precursor ion, [M-H]-. Although the mass spectra also exhibited an analytically important non-covalent adduct ion at [2M-H]-, the structures of glucosinolates were confirmed by extensive sequential MS analysis, thereby substantially improving the identification of unknown compounds. The results obtained not only revealed in leaves of E. sativa at least twelve species of GLSs including seven aliphatic compounds (glucoraphanin with [M-H]- at m/z ratio of 436, glucoerucin at m/z 420, 4-mercaptobutyl-GLS at m/z 406, progoitrin/epiprogoitrin at m/z 388, sinigrin at m/z 358, 4-methylpentyl- and n-hexyl-GLS at m/z 402) and three indole glucosinolates (i.e., three N-heterocyclic compounds: 4-hydroxyglucobrassicin and 5-hydroxyglucobrassicin at m/z 463, and 4-methoxy-glucobrassicin at m/z 477), but also two structurally related compounds containing one intermolecular disulfide linkage (4-(beta-D-glucopyranosyldisulfanyl)butyl-GLS at m/z 600 and a dimeric 4-mercaptobutyl-GLS at m/z 811). This latter symmetric disulfide was previously considered as an artefact formed during extraction of GLSs from vegetative tissues. Glucosinolates were detected in the leaves with a wide range of contents (10-200 micromol/g) and a great variation in the composition. Only three GLSs were identified in seeds of rocket salad, namely glucoraphanin, glucoerucin and 4-methoxyglucobrassicin. As expected, the most abundant GLS in seeds is glucoerucin. The feasibility of the strategy was also demonstrated using a rapeseed extract of certified reference material (BCR367R). The results indicated the usefulness of this method for a rapid, sensitive and comprehensive profiling of the GLS family naturally occurring in extracts of crude plant matter.

Gas-Phase Diastereoselectivity of Secondary 5-Substituted (X)-Adamant-2-yl (X = F, Si(CH3)3) Cations

Secondary 5-X-adamant-2-yl cations IX (X = F, Si(CH3)3) have been generated in the gas phase (total pressure = 760 Torr) from protonation-induced defluorination of epimeric 2-F-5-X-adamantanes 1X and their kinetic diastereoselectivity toward CH318OH investigated in the 40-160 degrees C range. The experimental results indicate that the facial selectivity of IX is insensitive to the composition of the starting 1X epimers as well as to the presence and the concentration of a powerful base (N(C2H5)3). This kinetic picture, supported by B3LYP/6-31G* calculations, is consistent with a single stable pyramidalized structure for IX, that is, (Z)-5-F-adamant-2-yl (I(Z)F) and (E)-5-Si(CH3)3-adamant-2-yl cations (I(E)Si). The temperature dependence of the IX diastereoselectivity lends support to the intermediacy of noncovalent adducts [IX*CH318OH], characterized by a specific C2-H+...O18(H)CH3 hydrogen bonding interaction. Their conversion to the covalently bonded O-methylated (Z)- (II(Z)X) and (E)-5-X-adamantan-2-ols (II(E)X; X = F, Si(CH3)3) is governed by activation parameters, whose magnitude depends on the specific IX face accommodating CH318OH. The gas-phase diastereoselectivity of IX toward CH318OH is compared to that exhibited in related gas-phase and solution processes. The emerging picture indicates that the factors determining the diastereoselectivity of IX toward simple nucleophiles in the gaseous and condensed media are completely different.

Non-covalent nano-adducts of co-poly(ester amide) and poly(ethylene glycol): preparation, characterization and model drug-release studies

Biodegradable, biocompatible poly(ester amide)s (co-PEAs), composed of amino acids, fatty diols and carboxylic acids, have been synthesized. To improve the performance of co-PEAs in Federal Drug Administration-approved solvents such as water and ethanol, these polymers were complexed with poly(ethylene glycol) (PEG) of 10 kDa molecular mass have been prepared by solution blending. The non-covalent adducts were purified by precipitation into hexanes. Co-PEAs are soluble in organic solvents but are insoluble in water and ethanol; however, the co-PEA/PEG (0.8:1, w/w) adducts are soluble in ethanol and slightly soluble in water. 2D-NOESY NMR spectroscopy suggests that the non-covalent adducts are held together by multiple non-covalent interactions between the –CH2– groups of the two polymers (co-PEA and PEG). Differential scanning calorimetry studies indicate that the two polymers are interacting in the non-covalent adducts; the thermal properties of the adducts are different from those of the pure polymers. The solid-state adduct structures have been determined by atomic force microscopy (AFM). By one sample preparation method, nanoscale pancake-like structures were observed with an average diameter of 260 nm and an average height of 16 nm. Films of co-PEAs and (co-PEA)/PEG adducts containing Rhodamine B Base (RhBB), a model hydrophobic drug, were prepared. From the adduct/RhBB film containing 3% RhBB, 20% of the total RhBB was released within the first 2 h. Film and adduct composition may be varied to obtain different release profiles. The studies reported here demonstrate that non-covalent conjugation is a relatively easy and effective approach in developing new materials for application as biomaterials.

π-Complex Structure of Gaseous Benzene−NO Cations Assayed by IR Multiple Photon Dissociation Spectroscopy

[C(6)H(6)NO](+) ions, in two isomeric forms involved as key intermediates in the aromatic nitrosation reaction, have been produced in the gas phase and analyzed by IR multiple photon dissociation (IRMPD) spectroscopy in the 800-2200 cm(-)(1) fingerprint wavenumber range, exploiting the high fluence and wide tunability of a free electron laser (FEL) source. The IRMPD spectra were compared with the IR absorption spectra calculated for the optimized structures of potential isomers, thus allowing structural information on the absorbing species. [C(6)H(6)NO](+) ions were obtained by two routes, taking advantage of the FEL coupling to two different ion traps. In the first one, an FT-ICR mass spectrometer, a sequence of ion-molecule reactions was allowed to occur, ultimately leading to an NO(+) transfer process to benzene. The so-formed ions displayed IRMPD features characteristic of a [benzene,NO](+) pi-complex structure, including a prominent band at 1963 cm(-)(1), within the range for the N-O bond stretching vibration of NO (1876 cm(-)(1)) and NO(+) (2344 cm(-)(1)). A quite distinct species is formed by electrospray ionization (ESI) of a methanol solution of nitrosobenzene. The ions transferred and stored in a Paul ion trap showed the IRMPD features of substituent protonated nitrosobenzene, the most stable among conceivable [C(6)H(6)NO](+) isomers according to computations. It is noteworthy that IRMPD is successful in allowing a discrimination between isomeric [C(6)H(6)NO](+) species, whereas high-energy collision-induced dissociation fails in this task. The [benzene,NO](+) pi-complex is characterized by IRMPD spectroscopy as an exemplary noncovalent ionic adduct between two important biomolecular moieties.

Sterically Locked Synthetic Bilin Derivatives and Phytochrome Agp1 from Agrobacterium tumefaciens Form Photoinsensitive Pr- and Pfr-like Adducts

Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15=C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15=C16 double bond is in either the Z or E configuration and the C14-C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15=C16 Z configuration and a C14-C15 anti conformation in the Pr form and a C15=C16 E configuration and a C14-C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.

Agrobacterium Phytochrome as an Enzyme for the Production of ZZE Bilins

Photoconversion of phytochrome from the red-absorbing form Pr to the far-red-absorbing form Pfr is initiated by a Z to E isomerization around the ring C-ring D connecting double bond; the chromophore undergoes a ZZZ to ZZE isomerization. In vivo, phytochrome chromophores are covalently bound to the protein, but several examples of noncovalent in vitro adducts have been reported which also undergo Pr to Pfr photoconversion. We show that free biliverdin or phycocyanobilin, highly enriched in the ZZE isomer, can easily be obtained from chromophores bound in a noncovalent manner to Agrobacterium phytochrome Agp1, and used for spectral assays. Photoconversion of free biliverdin in a methanol/HCl solution from ZZE to ZZZ proceeded with a quantum yield of 1.8%, but was negligible in neutral methanol solution, indicating that this process is proton-dependent. The ZZE form of biliverdin and phycocyanobilin were tested for their ability to assemble with Agp1 and cyanobacterial phytochrome Cph1, respectively. In both cases, a Pfr-like adduct was formed but the chromophore was bound in a noncovalent manner to the protein. Agp1 Pfr undergoes dark reversion to Pr; the same feature was found for the noncovalent ZZE adduct. After dark reversion, the chromophore became covalently bound to the protein. In analogy, the PCB chromophore became covalently bound to Cph1 upon irradiation with strong far-red light which initiated ZZE to ZZZ isomerization. Agrobacterium Agp2 belongs to a yet small group of phytochromes which also assemble in the Pr form but convert from Pr to Pfr in darkness. When the Agp2 apoprotein was assembled with the ZZE form of biliverdin, the formation of the final adduct was accelerated compared to the formation of the ZZZ control, indicating that the ZZE chromophore fits directly into the chromophore pocket of Agp2.

Analysis of nucleotides and oligonucleotides immobilized as self-assembled monolayers by static secondary ion mass spectrometry.

Nucleic acid constituents can be bound to a metal surface in the form of self-assembled monolayers. Binding is achieved either through ionic interactions with a self-assembled 2-aminoethanethiol monolayer or by direct covalent binding of a dithiophosphate oligonucleotide to a metal surface through a sulfur-metal bond. Nucleotides, polynucleotides (both normal and a dithiophosphate analog) and double-stranded DNA have all been bound to surfaces. When the surfaces are interrogated using static secondary ion mass spectrometry (SIMS), the surface-bound nucleic acid constituents are observed in the form of the characteristic protonated nucleic acid base ions (BH2+). While a silver foil substrate was found to provide the highest absolute signal, vapor-deposited gold yields the best signal-to-noise ratio for ionically bound deoxyguanosine monophosphate. Under comparable conditions, a Cs+ projectile produces a 10-fold increase in the secondary ion signal relative to a Ga+ projectile. The experiment has been extended to a triple-quadrupole instrument where tandem mass spectrometric experiments on ionically immobilized dGMP showed the characteristic loss of ammonia from the released BH2+ ion. When a 'biomimetic' surface formed by ionically immobilizing double-stranded DNA is exposed to a solution containing ethidium bromide, ions corresponding to the non-covalent adduct are readily detectable using SIMS. This adduct and the nucleic acid constituents can be monitored at levels below 10 fmol.

Hydroxynaphthaldehyde Phosphate Derivatives as Potent Covalent Schiff Base Inhibitors of Fructose-1,6-bisphosphate Aldolase

Interactions of phosphate derivatives of 2,6-dihydroxynaphthalene (NA-P(2)) and 1,6-dihydroxy-2-naphthaldehyde (HNA-P, phosphate at position 6) with fructose-1,6-bisphosphate aldolase from rabbit muscle were analyzed by enzyme kinetics, difference spectroscopy, site-directed mutagenesis, mass spectrometry, and molecular dynamics. Enzyme activity was competitively inhibited by NA-P(2), whereas HNA-P exhibited slow-binding inhibition with an overall inhibition constant of approximately 24 nM. HNA-P inactivation was very slowly reversed with t(1/2) approximately 10 days. Mass spectrometry and spectrophotometric absorption indicated that HNA-P inactivation occurs by Schiff base formation. Rates of enzyme inactivation and Schiff base formation by HNA-P were identical and corresponded to approximately 4 HNA-P molecules bound par aldolase tetramer at maximal inhibition. Site-directed mutagenesis of conserved active site lysine residues 107, 146, and 229 and Asp-33 indicated that Schiff base formation by HNA-P involved Lys-107 and was promoted by Lys-146. Titration of Lys-107 by pyridoxal 5-phosphate yielded a microscopic pK(a) approximately 8 for Lys-107, corroborating a role as nucleophile at pH 7.6. Site-directed mutagenesis of Ser-271, an active site residue that binds the C(1)-phosphate of dihydroxyacetone phosphate, diminished HNA-P binding and enabled modeling of HNA-P in the active site. Molecular dynamics showed persistent HNA-P phosphate interactions with the C(1)-phosphate binding site in the noncovalent adduct. The naphthaldehyde hydroxyl, ortho to the HNA-P aldehyde, was essential for promoting carbinolamine precursor formation by intramolecular catalysis. The simulations indicate a slow rate of enzyme inactivation due to competitive inhibition by the phenate form of HNA-P, infrequent nucleophilic attack in the phenol form, and significant conformational barrier to bond formation as well as electrostatic destabilization of protonated ketimine intermediates. Solvent accessibility by Lys-107 Nz was reduced in the covalent Schiff base complex, and in those instances where water molecules interacted with Lys-107 in the simulations, Schiff base hydrolysis was not mechanistically favorable. The findings at the molecular level corroborate the observed mechanism of slow-binding tight inhibition by HNA-P of muscle aldolase and should serve as a blueprint for future aldolase inhibitor design.

Observation of an Intact Noncovalent Homotrimer of Detergent-solubilized Rat Microsomal Glutathione Transferase-1 by Electrospray Mass Spectrometry

Microsomal glutathione transferase-1 (MGST1) is a membrane-bound enzyme involved in the detoxification of xenobiotics and the protection of cells against oxidative stress. The proposed active form of the enzyme is a noncovalently associated homotrimer that binds one substrate glutathione molecule/trimer. In this study, this complex has been directly observed by electrospray mass spectrometry analysis of active rat liver MGST1 reconstituted in a minimum amount of detergent. The measured mass of the homotrimer is 53 kDa, allowing for the mass of three MGST molecules in complex with one glutathione molecule. Collision-induced dissociation of the trimer complex resulted in the formation of monomer and homodimer ion species. Two distinct species of homodimer were observed, one unliganded and one identified as a homodimer.glutathione complex. Activation of the enzyme by N-ethylmaleimide through modification of Cys(49) (Svensson, R., Rinaldi, R., Swedmark, S., and Morgenstern, R. (2000) Biochemistry 39, 15144-15149) was monitored by the observation of an appropriate increase in mass in both the denatured monomeric and native trimeric forms of MGST1. Together, the data correspond well with the proposed functional organization of MGST1. These results also represent the first example of direct electrospray mass spectrometry analysis of a detergent-solubilized multimeric membrane protein complex in its native state.

Application of NOE and PGSE NMR Methodologies to Investigate Non-Covalent Intimate Inorganic Adducts in Solution

NOE and PGSE NMR experiments provide crucial information for the structural characterization of non-covalent intimate adducts in solution. The possible presence and the favorite relative orientation of the interacting units can be deduced from NOE results, while the size of the non-covalent adducts can be estimated through PGSE measurements. The complementarity of the two methodologies has been successfully used to investigate transition metal complex ion pairs and, to a lesser extent, intermolecular adducts. The main results concerning the solution structures of non-covalent inorganic adducts are reported and compared with those in the solid state and those from theoretical calculations.

The unusually high proton affinity of aza-18-crown-6 ether: implications for the molecular recognition of lysine in peptides by lariat crown ethers

Recent studies have shown that 18-crown-6 ether (18C6) will selectively form adducts in the gas phase with small, lysine containing peptides. The present study extends this work by investigating the ability of aza-18-crown-6 ether (A18C6) and L1 (a simple lariat crown ether derivative of A18C6) to form similar noncovalent adducts with the side chain of lysine in model peptides in the gas phase. The substitution of nitrogen for oxygen greatly increases the proton affinity of A18C6 relative to 18C6 and inhibits the formation of noncovalent adducts with small lysine containing peptides. The proton affinity of A18C6 is determined by the kinetic method to be 250 ± 1 kcal/mol. This value is much higher than that for diethanolamine (228 kcal/mol) or for 18C6 (231 kcal/mol). This unusually high basicity is rationalized by semi-empirical calculations that suggest a highly symmetrical structure for protonated A18C6 in which the three most distant oxygens are able to fold back and hydrogen bond with the protonated nitrogen. In the case of L1, the lariat side chain is attached by an amide bond, lowering the proton affinity of L1 relative to that of A18C6. This allows L1 to form noncovalent adducts with lysine despite the fact that steric repulsion within the cavity of the crown is increased to some extent. The relative ammonium ion affinities of these various crown ethers are shown to serve as qualitative predictors for the molecular recognition of lysine. The order of the relative ammonium ion affinities is 18C6≫L1>A18C6 as determined by the kinetic method. These results suggest that the substitution of nitrogen for oxygen in the crown ether is not beneficial for the molecular recognition of lysine.

Non-Covalent Adducts of Sodium Poly(α,L-glutamate) with Poly(N-vinyl pyrrolidone): Methods of Preparation and Characterization of Structure

Non-covalent adducts of poly(N-vinyl pyrrolidone) (PVP) (mol. wt. 10 K & 29 K) with sodium poly(α,L-glutamate)(PGNA) (mol. wt. 32 K) are prepared by evaporation of aqueous mixtures (EAM), ultra-centrifugation (UC) and dehydration of reverse micelles (DRM). The EAM and UC adducts contain nearly equal amounts of PVP while the DRM adduct has lower amounts. Higher-molecular-weight PVP favored greater PVP content in the adducts regardless of the method of preparation. DSC thermograms, and FT-IR and CD spectra of the three adducts in the solid state revealed that PVP and PGNA are intimately mixed and the PGNA is in a random conformation. Hydrophobic interactions between PGNA and PVP are evident in dilute aqueous solutions of all three adducts, while Na+ ions of PGNA remain as free ions. 2D-NOESY 1H NMR spectra of the EAM and UC adducts are very similar and show a strong correlation between the α-proton of PGNA with a pyrrolidone ring (no. 3 and no. 4 protons) and β-protons of PGNA with a pyrrolidone ring (no. 5 proton). In contrast, regarding the DRM adduct, only the α-proton of PGNA interacts with the pyrrolidone ring (no. 3 and no. 4 protons), presumably due to the orientation of the pyrrolidone ring at the organic phase–water interface of the reverse micelle, which causes the proton in position 5 of the ring to be buried in the organic phase. All three adducts dissociate in water to form free PVP and PGNA. However, the DRM adduct dissociates faster than other two, presumably due to reduced hydrophobic interactions. Differences in composition and properties observed for the non-covalent adducts may be attributed to the differences in intermolecular (hydrophobic) interactions imposed on the two components, PGNA and PVP, during each method of preparation.

A Theoretical Investigation of the Kinetic Energy of Ions Trapped in a Radio-Frequency Hexapole Ion Trap

The kinetic energy dependence of ions trapped in a radio-frequency (RF) hexapole ion trap has been calculated as a function of space charge, mean free path, mass, RF potential and charge. The average kinetic energy of the ions was found to increase with increasing space charge, mean free path and the ion charge state. For a trapped ion in a given coulombic field, the mass of the ion and the amplitude of the applied RF potential did not affect the average kinetic energy. The consequences for multipole-storage-assisted dissociation (MSAD), in which ions are accumulated for prolonged periods of time in the multipole ion trap of an electrospray ion source, are discussed. As a result of radial stratification inside the ion trap, MSAD can lead to the preferential excitation of ions with larger m/z values. Such discrimination would have negative consequences for the detection of labile non-covalent adducts, which are normally detected at higher m/z values than their constituent species.

Crystallographic characterization of the structure of the endohedral fullerene [Er2@C82 isomer I] with C(s) cage symmetry and multiple sites for erbium along a band of ten contiguous hexagons.

The structure of one of the three previously separated isomers of {Er2@C82} has been determined through a single-crystal X-ray structure determination of the noncovalent adduct, {Er2@C82 Isomer I}.{CoII(OEP)}.1.4(C6H6).0.3(CHCl3). The C82 cage is identified specificlly as the Cs(82:6) isomer (one of nine possible isolated pentagon isomers) from the crystallographic data. The carbon atoms of the C82 cage were individually identified and refined with only a constraint that required the two halves of the cage to possess similar bond lengths. Although the carbon cage is well ordered at 113 K, the erbium atoms are disordered. The electron density within the cage of {Er2@C82 Isomer I} has been modeled with two major sites with occupancies of 0.35 and 21 other individual erbium sites with occupancies ranging from 0.138 to 0.011. These erbium sites all reside near the walls of the fullerence and cluster near a band of ten contiguous hexagons that encircles the carbon cage. Since two other isomers of C82 (C3v(82:8) and C2v(82:9)) have a similar band of ten contiguous hexagons, it is tempting to speculate that the other two known isomers of {Er2@C82} have these cage structures.