Search

The intrinsic conformer specific vibrational spectra of two important subunits of the core pentasaccharide of N-linked glycans, the alpha(1,3) and alpha(1,6) dimannosides, have been recorded in the gas phase. Coupling these measurements with a computational exploration of their conformational landscapes has enabled their conformational assignment and has identified characteristic vibrational signatures associated with particular conformational families-including those that do or do not display inter-ring hydrogen bonding across the glycosidic linkage. In addition, the IR spectra of the monosaccharide moieties provide benchmarks, through which the conformational assignments can be refined. This introduces a general concept of modularity and secondary structure in oligosaccharides--essential for the success of similar studies on larger oligosaccharides in the future.

The conformation of phenyl-substituted monosaccharides (mannose, galactose, and glucose) and their singly hydrated complexes has been investigated in the gas phase by means of a combination of mass selected, conformer specific ultraviolet and infrared double resonance hole burning spectroscopy experiments, and ab initio quantum chemistry calculations. In each case, the water molecule inserts into the carbohydrate at a position where it can replace a weak intramolecular interaction by two stronger intermolecular hydrogen bonds. The insertion can produce significant changes in the conformational preferences of the carbohydrates, and there is a clear preference for structures where cooperative effects enhance the stability of the monosaccharide conformers to which the water molecule chooses to bind. The conclusions drawn from the study of monosaccharide-water complexes are extended to the disaccharide lactose and discussed in the light of the underlying mechanisms that may be involved in the binding of carbohydrate assemblies to proteins and the involvement, or not, of key structural water molecules.

The mode of metal ion and water binding to the amino acid valine is investigated using both theory and experiment. Computations indicate that without water, the structure of valine is nonzwitterionic. Both Li(+) and Na(+) are coordinated to the nitrogen and carbonyl oxygen (NO coordination), whereas K(+) coordinates to both oxygens (OO coordination) of nonzwitterionic valine. The addition of a single water molecule does not significantly affect the relative energies calculated for the cationized valine clusters. Experimentally, the rates of water evaporation from clusters of Val.M(+)(H(2)O)(1), M = Li, Na, and K, are measured using blackbody infrared radiative dissociation. The dissociation rate from the valine complex is compared to water evaporation rates from model complexes of known structure. These results indicate that the metal ion in the lithiated and the sodiated clusters is NO-coordinated to nonzwitterionic valine, while that in the potassiated cluster has OO coordination, in full agreement with theory. The zwitterionic vs nonzwitterionic character of valine in the potassiated cluster cannot be distinguished experimentally. Extensive modeling provides strong support for the validity of inferring structural information from the kinetic data.

The evaporation of water from hydrated alkaline earth metal ions, produced by electrospray ionization, was studied in a Fourier transform mass spectrometer. Zero-pressure-limit dissociation rate constants for loss of a single water molecule from the hydrated divalent metal ions, M2+(H2O)n (M = Mg, Ca, and Sr for n = 5–7, and M = Ba for n = 4–7), are measured as a function of temperature using blackbody infrared radiative dissociation. From these values, zero-pressure-limit Arrhenius parameters are obtained. By modeling the dissociation kinetics using a master equation formalism, threshold dissociation energies (Eo) are determined. These reactions should have a negligible reverse activation barrier; therefore, Eo values should be approximately equal to the binding energy or hydration enthalpy at 0 K. For the hepta- and hexahydrated ions at low temperature, binding energies follow the trend expected on the basis of ionic radii: Mg > Ca > Sr > Ba. For the hexahydrated ions at high temperature, binding energies follow the order Ca > Mg > Sr > Ba. The same order is observed for the pentahydrated ions. Collisional dissociation experiments on the tetrahydrated species result in relative dissociation rates that directly correlate with the size of the metals. These results indicate the presence of two isomers for hexahydrated magnesium ions: a low-temperature isomer in which the six water molecules are located in the first solvation shell, and a high-temperature isomer with the most likely structure corresponding to four water molecules in the inner shell and two water molecules in the second shell. These results also indicate that the pentahydrated magnesium ions have a structure with four water molecules in the first solvation shell and one in the outer shell. The dissociation kinetics for the hexa- and pentahydrated clusters of Ca2+, Sr2+, and Ba2+ are consistent with structures in which all the water molecules are located in the first solvation shell.

Blackbody infrared radiative dissociation (BIRD) spectra of singly and doubly protonated bradykinin and its analogues are measured in a Fourier-transform mass spectrometer. Rate constants for dissociation are measured as a function of temperature with reaction delays up to 600 s. From these data, Arrhenius activation parameters in the zero-pressure limit are obtained. The activation parameters and dissociation products for the singly protonated ions are highly sensitive to small changes in ion structure. The Arrhenius activation energy (E(a)) and pre-exponential (or frequency factor, A) of the singly protonated ions investigated here range from 0.6 to 1.4 eV and 10(5) to 10(12) s(-1), respectively. For bradykinin and its analogues differing by modification of the residues between the two arginine groups on either end of the molecule, the singly and doubly protonated ions have average activation energies of 1.2 and 0.8 eV, respectively, and average A values of 10(8) and 10(12) s(-1), respectively, i.e., the presence of a second charge reduces the activation energy by 0.4 eV and decreases the A value by a factor of 10(4). This demonstrates that the presence of a second charge can dramatically influence the dissociation dynamics of these ions. The doubly protonated methyl ester of bradykinin has an E(a) of 0.82 eV, comparable to the value of 0.84 eV for bradykinin itself. However, this value is 0.21 +/- 0.08 eV greater than that of singly protonated methyl ester of bradykinin, indicating that the Coulomb repulsion is not the most significant factor in the activation energy of this ion. Both singly and doubly protonated Lys-bradykinin ions have higher activation energies than the corresponding bradykinin ions indicating that the addition of a basic residue stabilizes these ions with respect to dissociation. Methylation of the carboxylic acid group of the C-terminus reduces the E(a) of bradykinin from 1.3 to 0.6 eV and the A factor from 1012 to 105 s(-1). This modification also dramatically changes the dissociation products. Similar results are observed for [Ala(6)]-bradykinin and its methyl ester. These results, in combination with others presented here, provide experimental evidence that the most stable form of singly protonated bradykinin is a salt-bridge structure.

Despite the many successes of mass spectrometry in the analysis of biological samples, the need to better understand the correlation between condensed-phase properties and those of electrospray species remains. In particular, the link between structures in the condensed phase and in the gaseous environment of the mass spectrometer is still elusive. Here, we show that fluorescence resonance energy transfer (FRET) can be used to probe the conformations of gaseous biopolymers which are formed by electrospray ionization (ESI) and manipulated in a quadrupole ion trap mass spectrometer. A rhodamine dye pair suitable for gas-phase FRET is characterized. Both steady state spectra and lifetime measurements are used to monitor energy transfer in a series of dye-labeled polyproline-based peptides. FRET efficiency is explored as a function of peptide chain length and charge state. For the peptide with eight proline repeats, virtually complete energy transfer is observed. For the peptide with 14 proline repeats, energy transfer decreases as the charge state increases, consistent with Coulomb repulsion induced elongation of the peptide backbone. FRET measurements of the longest peptide examined, which has 20 proline repeats, indicates that the peptide adopts a bent configuration. Evidence for multiple conformations present within the ensemble of trapped ions is provided by fluorescence lifetime measurements. Gas-phase FRET measurements promise to be a new route to probe the conformations of large gaseous ions.

Metal ions play an important role in the function of many metalloenzymes. For example, the interactions of regulatory proteins such as calmodulin with other proteins depend on whether Ca2+ is specifically bound.1 Studies of metal ion hydration provide information not only about the metal ion chemistry in solution, but can lead to an improved understanding of the structure and function of many biomolecules in which metal ion interactions play a role. The hydration enthalpies of singly charged metal ions in the gas phase have been extensively investigated by using a variety of experimental techniques.2 In contrast, gas-phase hydration studies of di- and trivalent metal ions are significantly more limited.3–5 Kebarle and co-workers measured free energies of hydration for a series of divalent metal ions bound to between 7 and 13 water molecules.4b,d From these values, hydration enthalpies for water molecules in the second solvation shell were estimated. Recently, calculations of successive hydration enthalpies of both inner and outer solvent shell water molecules for several divalent metal ions have been reported.6,7 Here, binding energies of water molecules to both divalent calcium and nickel ions are measured by using blackbody infrared radiative dissociation (BIRD) and master equation modeling. These values are considerably lower than MP2 hydration enthalpies reported previously.6 They are in better agreement with recently reported B3LYP values calculated with large basis sets.7 To our knowledge, these are the first experimental measurements of the binding energy of individual inner shell water molecules to divalent metal ions. Experimental measurements are performed with an external electrospray ionization source Fourier transform mass spectrometer. This instrument and the BIRD experiments have been described previously.8 Hydrated metal ions are generated from ~10−4 M chloride salt solutions with nanoelectrospray ionization. The hydrated ions are trapped and thermalized by using a pulse of N2 gas (10−6 Torr). The ion of interest is mass selected and dissociated for times ranging from 10 to 500 s. At the low pressures during the reaction times (

The beta(1--4) glycosidic linkage found in lactose is a prevalent structural motif in many carbohydrates and glycoconjugates. Using UV and IR ion-dip spectroscopies to probe benzyl lactoside isolated in the gas phase, we find that the disaccharide unit adopts only a single, rigid structure. Its fully resolved infrared ion-dip spectrum is in excellent agreement with that of the global minimum structure computed ab initio. This has glycosidic torsion angles of phi(H) (H1-C1-O-C4') approximately 180 degrees and psi(H) (C1-O-C4'-H4') approximately 0 degrees which correspond to a rotation of approximately 150 degrees about the glycosidic bond compared to the accepted solution-phase conformation. We discuss the biological implications of this discovery and the generality of the strategies employed in making it.

The mechanism of fluorescence and fluorescence quenching of the green fluorescent protein (GFP) is not well-understood. To gain insight into the effect of the surrounding protein on the chromophore buried at its center, the intrinsic electronic absorption and deactivation pathways of a gaseous model chromophore, p-hydroxybenzylidene-2,3-dimethylimidazolone (HBDI) were investigated. No fluorescence from photoactivated gaseous HBDI(-) was detected in the range 480-1100 nm, in line with the ultrafast rate of internal conversion of HBDI(-) in solution. Two different gas-phase deactivation pathways were found: photofragmentation and electron photodetachment. Electronic action spectra for each deactivation pathway were constructed by monitoring the disappearance of HBDI(-) and appearance of product ions as a function of excitation wavelength. The action spectra measured for each pathway are distinct, with electron photodetachment being strongly favored at higher photon energies. The combined (total) gas-phase action spectrum has a band origin at 482.5 nm (23340 cm(-1)) and covers a broad spectral range, 390-510 nm. This extended gas-phase action spectrum exhibits vibronic activity that matches well with the results of previous cold condensed-phase experiments and high-level in vacuo computations, with features evident at +550, +1500, and +2800 cm(-1) with respect to the band origin.

One strategy that has commonly been used to gain a more detailed understanding of ion chemistry in bulk solution is to investigate the structure and energetics of solvated ions in the gas phase. While solvation of singly charged metal ions has been studied extensively during the last two decades,1 significantly less information about doubly and triply charged metal ions is known. Solvated divalent metal ions can now be readily produced using electrospray ionization (ESI).2–5 This has led to a significant interest in obtaining thermochemical information about these ions with both experiment3–5 and theory.6–8 Kebarle and co-workers measured the Gibbs free energies of hydration for water molecules located in the second solvation shell of a variety of divalent metal ions using equilibrium experiments.3 More recently, Posey and co-workers combined ESI with laser photofragmentation mass spectrometry to study divalent transition metal–ligand complexes with methanol in the second solvation shell.4 Binding energies of inner solvent shell water molecules around Ni2+ and Ca2+ ions have been determined from blackbody infrared radiative dissociation (BIRD) experiments.5 Here, BIRD kinetics of the hexahydrated alkaline earth metal ions, Mg2+, Ca2+, Sr2+, and Ba2+, are presented. At low temperatures, binding energies obtained from these kinetic data are directly correlated with the radii of the metal ions (Mg2+ > Ca2+ > Sr2+ > Ba2+). In contrast, the binding energies at higher temperatures follow the trend Ca2+ > Mg2+ > Sr2+ > Ba2+. This is the first direct evidence for two distinct gas-phase structures for a hydrated divalent metal ion. Experiments were performed using an external electrospray ionization source Fourier transform mass spectrometer that has been described previously.9 Hydrated alkaline earth M2+(H2O)n ions were generated from ~10−4 M aqueous solutions of the metal chloride salts using nanoelectrospray. Ions are loaded into the cell for 5 s during which time N2 gas (10−6 Torr) is introduced. Dissociation kinetics of the mass-selected ion are investigated at pressures Sr2+ > Ca2+ > Mg2+. This trend is consistent with the expected reactivity based on the ionic radii of the metals (water molecules attached to larger metal ions are expected to be less strongly bound). For the hexahydrated ions, this same ordering of rate constants is observed at low temperatures. In striking contrast, the order is Ba2+ > Sr2+ > Mg2+ > Ca2+ at higher temperatures; the slope of the Arrhenius plot for Mg2+(H2O)6 changes around 80°C! BIRD rate constants for the pentahydrated species have the same trend as that observed for the hexahydrated species at high temperature (Ba2+ > Sr2+ > Mg2+ > Ca2+).13 Thus, these dissociation kinetics do not follow the trend expected based on ionic radii. The change in the slope of the Arrhenius plot for Mg2+-(H2O)6 shows the presence of two distinct gas-phase structuresfor this ion. The first-order behavior (>80% precursor ion depletion) at all temperatures indicates the presence of one structure or several structures that interconvert. Figure 1 Arrhenius plots obtained from blackbody infrared radiative dissociation data of the hexa- and heptahydrated alkaline earth metal ions; Mg2+ (○), Ca2+ (•), Sr2+ (*), and Ba2+ (▵). The measured zero-pressure limit Arrhenius parameters and the binding energies (Eo) obtained from master equation modeling of the kinetic data are given in Table 1. The low-temperature Eo’s consistently increase with decreasing cation size. At high temperatures, the binding energy of the sixth water molecule to Mg2+ (21.1 kcal/mol) is less than that at low temperatures (23.5 kcal/mol). It is also slightly less than that for the larger calcium cation (21.6 kcal/mol). For Mg2+(H2O)6, anomalously large radiative rate constants were used in the modeling in order to fit the kinetic data at low temperature. The reason for this is not clear, but this suggests that the error in Eo at low temperature may be larger than reported. The unexpected ordering of binding energies at high temperature is consistent with ΔH values recently reported by Kebarle and co-workers from equilibrium experiments at 170–225°C.3a Recent density functional theory (DFT) calculations by Pavlov et al.8 have been used to evaluate structures for hydrated divalent magnesium and calcium ions, M2+(H2O)m-(H2O)n, where m and n correspond to the number of water molecules in the first and second shell, respectively. The successive water binding energies (ΔEbinding) reported for these structures are also included in Table 1. Table 1 Measured Zero-Pressure Limit Arrhenius Parameters and EO Values Obtained from Master Equation Modeling for Loss of Water from M2+(H2O)6 Ionsa To further investigate this unexpected reactivity of Mg2+(H2O)6, we also performed DFT calculations on all four hexahydrated divalent metals.14 Three different types of structures were studied in which the six water molecules are either all in the first shell or distributed between the first and second solvation shells around the metals. By the use of the nomenclature of Pavlov et al., the structures investigated were M2+(H2O)6, M2+(H2O)5(H2O),15 and M2+(H2O)4(H2O)2. Calculations indicate that the order of stability is M2+(H2O)6 > M2+(H2O)5(H2O) > M2+(H2O)4(H2O)2 for all of the metals. The energy differences between each of the higher energy structures and the M2+(H2O)6 conformation are reported in Table 2. Also included in the table are the values of Pavlov et al.8 for magnesium and calcium. At this level of theory, all three hexahydrated Mg2+ structures have energies within 5 kcal/mol. Several similar energy structures for hexahydrated barium ions were also found. However, no evidence of multiple gas-phase hexahydrated barium structures was observed over the temperature range of these experiments Table 2 B3LYP Energy Differences between M2+(H2O)5(H2O) and M2+(H2O)4(H2O)2 and the Most Stable M2+(H2O)6 Structurea Although the experimental evidence for two distinct structures for Mg2+(H2O)6 is clear, the exact nature of these isomers is not. One possibility is that the isomers have different number of water molecules in the first and second shell. The results of DFT calculations show that all three hexahydrated Mg2+ structures investigated have 0 K energies within 5 kcal/mol (Table 2). The entropies of Mg2+(H2O)5(H2O) and Mg 2+(H2O)4(H2O)2 under standard conditions are higher than that of Mg2+(H2O)6. The opposite is true for the Ca2+, Sr2+, and Ba2+ hexahydrates. The anomalous entropy for Mg2+(H2O)6 is presumably due to the crowding of the six water molecules around the much smaller metal ion. This would suggest that the higher temperature structure corresponds to a two-shell structure. Another possible explanation is that one of the isomers is a salt-bridge structure (Scheme 1). Such a salt-bridge structure was found to be stable at the semiempirical PM3 level of theory, but not with the density functional levels of theory applied. The proton-transfer process necessary for the formation of a salt-bridge is expected to occur more readily for hydrated Mg than for the larger metal hydrates.3b Scheme 1 In conclusion, evidence for two distinct isomeric structures of Mg2+(H2O)6 is presented. This is the first experimental evidence for two isomeric structures of hydrated metal dications. Binding energies of water to Mg2+(H2O)7 and Mg2+(H2O)6 (low temperature) follow the expected trend in cation size. These values for Mg2+(H2O)6 (high temperature) do not. Calculations suggest that the most stable structure for M2+(H2O)6 is one in which all of the water molecules are in the inner shell. However, further experiments and higher level calculations are necessary to definitively elucidate these structures.

It is well-known that amino acids exist as zwitterions in aqueous solution within a range of pH. Whether zwitterions exist in the absence of solvent has been extensively discussed in the literature.1–6 In the gas phase, experimental evidence that glycine is not a zwitterion has been obtained by millimeter wave spectroscopy1 and by measurements of substituent effects on gas-phase basicity.2 In addition, the potential energy surface of isolated glycine has been thoroughly investigated by ab initio methods.3,4 At the highest levels of theory, the zwitterion is ~16–20 kcal/mol less stable than the neutral with no barrier for proton transfer between the protonated amino and the deprotonated carboxyl groups.4 Calculations indicate that two water molecules complexed with glycine can stabilize the zwitterion; if the two water molecules are hydrogen bonded only to glycine, a barrier to proton transfer occurs although the zwitterion is still higher in energy than the neutral.4d Results of Rizzo et al.5 indicate that electronically excited tryptophan formed in a supersonic jet is a zwitterion. Recently, evidence for salt-bridge interactions in gas-phase peptide ions has been reported.7 For example, the most stable form of protonated bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) is one in which both terminal arginine residues are protonated and interact via the deprotonated carboxyl of the C-terminus.7a Salt-bridge interactions can be significantly stabilized by intramolecular hydrogen bonding to polar groups, such as carbonyl oxygens, which provide the near equivalent of a solvent shell around the charges.8 Here, we present experimental evidence that protonated dimers of arginine are bound by a salt-bridge in the gas phase. Further, our calculations indicate that the most stable form of arginine itself is a zwitterion. We believe that this is the first evidence for a stable ground state zwitterion form of an amino acid in the absence of solvent. The dissociation kinetics of proton-bound dimers of several amino acids were investigated with blackbody infrared radiative dissociation (BIRD) in a 2.7-T Fourier-transform mass spectrometer. These experiments are described elsewhere.9 Proton-bound dimers were formed by electrospray ionization and dissociated at cell temperatures ranging from 37 to 149 °C. Unimolecular rate constants are obtained from the slope of ln-{[D+]/([D+] + [F+])} vs time where [D+] and [F+] refer to the dimer ion abundance and sum of fragment ion abundances, respectively. The resulting Arrhenius plot for dissociation of these dimers is shown in Figure 1. The proton-bound dimer of Gly, (Glyd), dissociates more slowly than both Alad and the methyl ester (ME) of Gly dimer itself (GlyMEd). The increased dissociation rates of the latter two ions are primarily due to their increased molecular size. This results in both an increased rate of blackbody photon absorption and an increased population above the threshold dissociation energy. A small difference in binding energy (