Salt-bridge Structure Research Articles

Conformational and zwitterionic preferences of N-amidinoglycine: the effect of microsolvation and metal ion addition

N-Amidinoglycine (NG) is a precursor of creatine, which is the energy source in muscles and plays a vital role in adenosine triphosphate homeostasis. NG possesses the combined structural properties of Gly and Arg. Extensive work on cationic complexation, anionic complexation, microsolvation, excess electron binding, etc. is reported on Gly as well as Arg. Quantum chemical calculations have been performed using B3LYP, BHandHLYP, ωB97XD, M06, M06-2X and MP2 methods, on NG to understand the conformational and zwitterionic preferences under the influence of metal ions (alkali and alkaline earth ions) and water coordination, as well as on the bare structures. NG can have many alternative structures; at least ten structures are possible within 5 kcal mol−1, in the gas phase. NG-1 and NG-2 are highly competitive structures as global minima. The zwitterion (ZNG-1) is about 15 kcal mol−1 less stable than the canonical form (NG-2) in the gas phase although its basicity is high as Arg. This unusual instability of the zwitterionic form of NG may be attributed to the lack of intramolecular self-solvation (multiple hydrogen bonding between the guanidine and carboxylate groups) which is found in Arg. Microsolvation decreases energy differences between canonical and zwitterionic forms of NG, three water molecules are necessary to make them energetically competitive. Computational analysis indicated that the relative stabilities of canonical conformers and zwitterions of NG are very sensitive to their surroundings. Salt bridge structures of NG with alkali and alkaline earth metal cations are stable over charge solvated structures by about 2–8 and 1–14 kcal mol−1, respectively, except in the case of the Be+2 ion.

Exploring salt bridge structures of gas-phase protein ions using multiple stages of electron transfer and collision induced dissociation.

The gas-phase structures of protein ions have been studied by electron transfer dissociation (ETD) and collision-induced dissociation (CID) after electrospraying these proteins from native-like solutions into a quadrupole ion trap mass spectrometer. Because ETD can break covalent bonds while minimally disrupting noncovalent interactions, we have investigated the ability of this dissociation technique together with CID to probe the sites of electrostatic interactions in gas-phase protein ions. By comparing spectra from ETD with spectra from ETD followed by CID, we find that several proteins, including ubiquitin, CRABP I, azurin, and β-2-microglobulin, appear to maintain many of the salt bridge contacts known to exist in solution. To support this conclusion, we also performed calculations to consider all possible salt bridge patterns for each protein, and we find that the native salt bridge pattern explains the experimental ETD data better than nearly all other possible salt bridge patterns. Overall, our data suggest that ETD and ETD/CID of native protein ions can provide some insight into approximate location of salt bridges in the gas phase.

Theoretical studies on complexes of calcium ion with amino acids

The complexes formed by calcium ion and 12 common amino acids were investigated systematically in the gas phase at the level of MP2/6-311++G(d,p)//MP2/6-31G(d,p). The results show that the salt-bridge structure is the most preferred motif for Ca2+ binding aliphatic amino acids without heteroatom in the side chain, while charge-solvated(CS) structure is the most preferred motif for Ca2+ binding other amino acids except for glutamine and lysine. IR spectra of Gln-Ca2+ and Asn-Ca2+ complexes were calculated and compared well with the available experiments. From the study in aqueous solution, the bidentate salt-bridge structure was determined to be the most favorable for all the twenty kinds of amino acids to chelate Ca2+ to both the oxygen atoms of the negatively carboxylate group in the backbone.

A salt-bridge structure in solution revealed by 2D-IR spectroscopy

We determine the spatial structure of a guanidinium:acetate salt bridge in solution from cross peaks in its 2D-IR spectrum.

Improvements in the performance of an incubation-type planar patch clamp biosensor using a salt bridge electrode and a plastic (PMMA) substrate

Incubation-type planar patch clamp biosensors, first described in 2008, are expected to enable the realization of high-throughput neural network screening. Several technical challenges must be overcome before this technique may be implemented. First, it is difficult to achieve a sufficiently high seal resistance (a gigaohm seal), which is an important parameter for patch clamp operation. A low seal resistance produces large baseline fluctuations corresponding to the fluctuation of the solid–liquid and liquid–liquid interface potentials. Second, the sensor cell must be positioned at a micropore over a long incubation time while maintaining the cell viability. Finally, a low-cost high-performance sensor chip suitable for high-throughput screening must be developed. These problems have been resolved to some extent in the present work. A stable electrode with a salt bridge structure was developed here to reduce the baseline fluctuations. The cell positioning technology was improved by forming a novel cell trapping pattern using hot embossing on a plastic (polymethylmethacrylate (PMMA)) substrate for use in a microfluidic sensor chip. The combination of the stable electrode and the cell trapping pattern dramatically increased the device success probability from ∼2% to 60–80% using HEK293 cells as a model system. The utility of the present results was tested by characterizing rat hippocampal neuron networks. The seal resistance was found to be lower than the seal resistance obtained from the HEK293 cells. The effectiveness of the stable electrodes, however, was significantly clear and spontaneous channel currents from the neural network were successfully observed.

Complexation of Ca2+ with selenocysteine and effects on its intrinsic acidity

The interactions of Selenocysteine (Sec) with Ca have been investigated through the use of B3LYP/6-311+G(3df,2p)//B3LYP/6-311+G(d,p) calculations. The global minimum of the [SecCa] potential energy surface is a charge-solvated (CS) species in which the metal dication is bound to the SeH, to the amino groups and to the carbonyl oxygen. Within a gap of 5.0 kJ mol in terms of free energies there are four more complexes three of which are salt-bridged (SB) structures. The interactions between Ca and the amino acid are essentially electrostatic, in contrast with what happens when Ca is replaced by Cu, where the charge transfer from Sec to the metal cation is so large that in the [Sec-Cu] global minimum the amino acid moiety is oxidized. The significant electron density redistribution undergone by the amino acid when interacting with both doubly-charged metal ions is reflected in a significant enhancement of its intrinsic acidity, although this effect is much larger for Cu than for Ca. One of the direct consequences of the oxidation undergone by the amino acid when attached to Cu is that the complexes formed are thermodynamically unstable with respect to a proton loss since the process Sec + Cu → [(Sec-H)Cu] + H is strongly exothermic. Conversely, a similar process when Cu is replaced by Ca is endothermic and therefore the [Sec-Ca] complexes should be stable with respect to a proton loss.

Modeling Interactions between an Amino Acid and a Metal Dication: Cysteine-Calcium(II) Reactions in the Gas Phase.

The gas-phase interactions between Ca2+ and cysteine (Cys) have been investigated through the use of electrospray ionization/mass spectrometry techniques and B3LYP/6-311++ G(3df,2p)//B3LYP/6-311+G(d,p) density functional theory computations. The unimolecular collision-activated decomposition of [Ca(Cys)]2+ is dominated by the loss of ammonia, a Coulomb explosion yielding NH4 + and [CaC3 H3 O2 S]+ , and the loss of H2 S. The detection of lighter [C3 H3 OS]+ monocations indicates that the [CaC3 H4 O2 S]2+ doubly charged species produced by the loss of ammonia undergo a subsequent Coulomb explosion yielding [C3 H3 OS]+ +CaOH+ . This [C3 H3 OS]+ cation finally decomposes into [C2 H3 S]+ +CO. Alternatively, the aforementioned [CaC3 H4 O2 S]2+ dications may also lead to lighter [CaCO2 ]2+ and [CaC2 H4 S]2+ dications by the loss of C2 H4 S and CO2 , respectively. A detailed theoretical exploration of the Ca2+ /Cys potential-energy surface indicates that the salt-bridge structures, in which the metal dication interacts with the carboxylate group of the zwitterionic form of cysteine, are at the origin of the different reaction pathways leading to the observed product ions, even though they lie higher in energy than the charge-solvated adduct in which the metal interacts simultaneously with the carbonyl oxygen, the amino, and the SH group of its canonical form. The interaction between the metal cation and the base is essentially electrostatic, with a calculated binding energy of 560 kJ mol-1 .

Electron Capture Dissociation of Trivalent Metal Ion-Peptide Complexes

With electrospray ionization from aqueous solutions, trivalent metal ions readily adduct to small peptides resulting in formation of predominantly (peptide + M(T) - H)(2+), where M(T) = La, Tm, Lu, Sm, Ho, Yb, Pm, Tb, or Eu, for peptides with molecular weights below ~1000 Da, and predominantly (peptide + M(T))(3+) for larger peptides. ECD of (peptide + M(T) - H)(2+) results in extensive fragmentation from which nearly complete sequence information can be obtained, even for peptides for which only singly protonated ions are formed in the absence of the metal ions. ECD of these doubly charged complexes containing M(T) results in significantly higher electron capture efficiency and sequence coverage than peptide-divalent metal ion complexes that have the same net charge. Formation of salt-bridge structures in which the metal ion coordinates to a carboxylate group are favored even for (peptide + M(T))(3+). ECD of these latter complexes for large peptides results in electron capture by the protonation site located remotely from the metal ion and predominantly c/z fragments for all metals, except Eu(3+), which undergoes a one electron reduction and only loss of small neutral molecules and b/y fragments are formed. These results indicate that solvation of the metal ion in these complexes is extensive, which results in the electrochemical properties of these metal ions being similar in both the peptide environment and in bulk water.

Water Structure at the Potassium Channel Gate: The Importance of the Prolines in the PVPV Sequence, as Shown by Computational Mutations, and the Role of a Histidine in Gating

Conservation of the PVPV sequence at the potassium channel gate has not been well understood. Quantum computations (optimization at HF/4-22GSP level) of the gate of the Kv1.2 channel that are presented here show that the proline-proline distances (ring atoms) are preserved as the ion moves through the gate. This calculation is supplemented by mutating one of the prolines to valine (P407V, 2A79 pdb numbering for all residues in this abstract) in the computation, producing a PVVV sequence. Water structure changed dramatically, as the protein backbone moved inward nearly 3A, effectively closing the channel; the proline ring N-N distance between diagonally opposite prolines dropped from 14.3 A (distance from optimization: X-ray distance = 15.6A) to 11.4 A, too small to allow passage of a hydrated K+. This suggests that the prolines play a key structural role, modulating both protein and water structure. Furthermore, protonation of H418 may close the channel. Protonation of this histidine shows it moving toward the gate by about 3 A, which should close the gate electrostatically, while deprotonation opens the gate. Protonation was calculated with two added protons, the other being accepted by E142 of the T1 intracellular moiety. When deprotonated, the histidine does not move as much as 1A from the X-ray position in the optimization, and is linked to E142; this accounts for the known importance of the T1 segment in gating. E136, also of T1, could accept a third proton. There is a salt bridge structure including these residues similar to the one occupying a key position in the proton pathway in the Hv1 channel.

Site specific interaction between ZnO nanoparticles and tryptophan: a first principles quantum mechanical study

First principles density functional theory calculations are performed on tryptophan-ZnO nanoparticles complex in order to study site specific interactions between tryptophan and ZnO. The calculated results find the salt bridge structure involving the -COOH group and ZnO cluster to be energetically more favorable than other interacting sites, such as indole and amine groups in tryptophan. The interaction between tryptophan and ZnO appears to be mediated by both ionic and hydrogen bonds. The calculated molecular orbital energy levels and charge distributions suggest non-radiative energy transfer from an excited state of tryptophan to states associated with ZnO, which may lead to a reduction in the emission intensity assigned to the π-π* transition of the indole functional group of tryptophan.

Gas-phase structure and fragmentation pathways of singly protonated peptides with N-terminal arginine.

The gas-phase structures and fragmentation pathways of the singly protonated peptide arginylglycylaspartic acid (RGD) are investigated by means of collision-induced-dissociation (CID) and detailed molecular mechanics and density functional theory (DFT) calculations. It is demonstrated that despite the ionizing proton being strongly sequestered at the guanidine group, protonated RGD can easily be fragmented on charge directed fragmentation pathways. This is due to facile mobilization of the C-terminal or aspartic acid COOH protons thereby generating salt-bridge (SB) stabilized structures. These SB intermediates can directly fragment to generate b(2) ions or facilely rearrange to form anhydrides from which both b(2) and b(2)+H(2)O fragments can be formed. The salt-bridge stabilized and anhydride transition structures (TSs) necessary to form b(2) and b(2)+H(2)O are much lower in energy than their traditional charge solvated counterparts. These mechanisms provide compelling evidence of the role of SB and anhydride structures in protonated peptide fragmentation which complements and supports our recent findings for tryptic systems (Bythell, B. J.; Suhai, S.; Somogyi, A.; Paizs, B. J. Am. Chem. Soc. 2009, 131, 14057-14065.). In addition to these findings we also report on the mechanisms for the formation of the b(1) ion, neutral loss (H(2)O, NH(3), guanidine) fragment ions, and the d(3) ion.

Fluorescence Lifetime Probe of Biomolecular Conformations

Methods have been developed to measure the fluorescence lifetime versus temperature of trapped biomolecular ions derivatized with a fluorescent dye. Previous measurements for different sequences of polyproline peptides demonstrated that quenching rates are related to conformations and their spatial fluctuations. This paper presents the results of extending these methods to study the conformational dynamics of larger biomolecules. Vancomycin-peptide noncovalent complexes in the 1+ charge state were studied as a function of temperature for different W-KAA peptide chiralities (L-LDD, D-LDD, L-DLL). Fluorescence-quenching rates, k(q), were found to be stereoselective for these different chiralities with relative magnitudes k(q)(L-LDD) > k(q)(D-LDD) > k(q)(L-DLL). The variation in fluorescent quenching resulting from switching the chirality of the single Trp residue was readily detectable. Molecular dynamics analysis of complexes formed by W-KAA (L-LDD) and W-KAA(L-DLL) indicates that increased flexibility in the (L-DLL) complex is correlated with reduced quenching rates. Fluorescence measurements were also performed for the Trp-cage protein comparing quenching rates in the 1+, 2+, and 3+ charge states for which k(q)(+) >> k(q)(2+) approximately k(q)(3+). Measurements of a sequence including a single-point mutation infer the presence of a salt-bridge structure in the 1+ charge state and its absence in both the 2+ and 3+ states. Molecular dynamics structures of Trp-cage indicate that a salt bridge in the 1+ charge state produces more compact conformations leading to larger quenching rates based on the quenching mechanism. In both these experimental studies the fluorescence-quenching rates were consistent with changes in structure induced by either intermolecular or intramolecular interactions.

Systematic Study of the Structures of Potassiated Tertiary Amino Acids: Salt Bridge Structures Dominate

The gas-phase structures of a series of potassiated tertiary amino acids have been systematically investigated using infrared multiple photon dissociation (IRMPD) spectroscopy utilizing light generated by a free electron laser, ion mobility spectrometry (IMS), and computational modeling. The examined analytes comprise a set of five linear N,N-dimethyl amino acids derived from N,N-dimethyl glycine and three cyclic N-methyl amino acids including N-methyl proline. The number of methylene groups in either the alkyl chain of the linear members or in the ring of the cyclic members of the series is gradually varied. The spectra of the cyclic potassiated molecular ions are similar and well resolved, whereas the clear signals in the respective spectra of the linear analytes increasingly overlap with longer alkyl chains. Measured IRMPD spectra are compared to spectra calculated at the B3LYP/6-311++G(2d,2p) level of theory to identify the structures present in the experimental studies. On the basis of these experiments and calculations, all potassiated molecular ions of this series adopt salt bridge structures in the gas phase, involving bidentate coordination of the potassium cation to the carboxylate moiety. The assigned salt bridge structures are predicted to be the global minima on the potential energy surfaces. IMS cross-section measurements of the potassiated systems show a monotonic increase with growing system size, suggesting that the precursor ions adopt the same type of structure and comparisons between experimental and theoretical cross sections are consistent with salt bridge structures and the IRMPD results.

Structures of Protonated Dipeptides: The Role of Arginine in Stabilizing Salt Bridges

Structures of protonated dipeptides containing N-terminal Gly, Val, Pro, Lys, His, or Arg and C-terminal Arg are investigated with infrared multiple photon dissociation (IRMPD) spectroscopy between 900 and 1850 cm(-1) and theory. The IRMPD spectra clearly indicate that, when Gly, Val, Pro, Lys, or His are N-terminal to Arg, these protonated dipeptides adopt gas-phase structures with a single formal charge site (SCS), whereas ArgArg x H(+) has a salt-bridge (SB) structure in which the C-terminus is deprotonated and two basic sites are protonated. There are only subtle differences in the IRMPD spectra for dipeptides containing Gly, Val, Pro, and Lys. A sharp, intense peak at 1080 cm(-1) is observed for HisArg x H(+) that is attributed to the neutral histidine side chain, an assignment that is confirmed by comparison to the IRMPD spectrum of (HisArg x H(2))(2+). Lowest-energy B3LYP/6-31+G(d,p) structures and energies for the SCS and SB forms of these protonated dipeptides indicate that stability of the SB form relative to the SCS form generally increases with increasing gas-phase basicity of the N-terminal amino acid, but only ArgArg x H(+) is calculated to have a SB ground state at 298 K, in agreement with the results from IRMPD spectroscopy. This is the first direct experimental evidence for a salt-bridge structure in a gaseous protonated peptide, and ArgArg x H(+) is the smallest protonated peptide for which a SB structure has been reported. These results suggest that SB structures should be common for protonated peptides containing at least two arginine residues and may also occur for large protonated peptides or proteins with at least one arginine residue and other basic residues, such as lysine or histidine.

Protonated Arginine and Protonated Lysine: Hydration and Its Effect on the Stability of Salt-Bridge Structures

Using a mass spectrometer equipped with a drift cell, water binding energies of protonated arginine (ArgH+) and protonated lysine (LysH+) were determined in equilibrium experiments and supplementary calculations at the B3LYP/6-311++G** level of theory. The binding energy of the first water molecule was measured to be 10.3 and 10.9 kcal/mol for ArgH+ and LysH+, respectively. Water binding energies decrease with increasing degree of hydration reaching values of 6-7 kcal/mol for the fourth and fifth water molecule. Theory reproduces this trend of decreasing binding energies correctly and theoretical water binding energies agree with experiment quantitatively within 2 kcal/mol. Lowest-energy theoretical structures of ArgH+ and LysH+ are characterized by protonated side chains and neutral alpha-amino and carboxyl groups which form intramolecular hydrogen bonds to the ionic group (charge solvation or CS structures). The salt bridge (SB) structures with two cationic groups (side chain and alpha-amine) and one anionic group (carboxyl) are 13.1 and 9.3 kcal/mol higher in energy for ArgH+ and LysH+, respectively. Theory indicated that the first water molecule binds to the ionic group of the CS structures of ArgH+ and LysH+. With increasing degree of hydration intramolecular interactions are replaced one by one with water bridges with water inserted into the intramolecular hydrogen bonds. Whereas the global minima of ArgH+.(H2O)n and LysH+.(H2O)n, n<7, were calculated to represent CS structures, 7-fold hydrated CS and SB structures, ArgH+.(H2O)7 and LysH+.(H2O)7, are nearly isoenergetic (within <1 kcal/mol).

Kinetic Determination of Potassium Affinities by IRMPD: Elucidation of Precursor Ion Structures

Dissociation kinetics of the K(+) loss reaction of three potassiated tertiary amino acids (Scheme 1) were studied by infrared multiple photon dissociation (IRMPD) in a Fourier transform ion cyclotron resonance (FT ICR)-MS instrument. The aim of the study was to probe if a kinetic study by IRMPD can yield useful information on the ion structure of the precursor ion species. The measured activation energy values determined by IRMPD are related to the potassium affinity, DeltaH(K(+)), of N-methyl proline determined by threshold collision-induced dissociation experiments. By appropriate scaling with this reference value, the experimentally determined activation energy values for the K(+) loss are transformed into respective potassium affinities, DeltaH(K(+))(IRMPD). These values match the calculated potassium affinity values for salt bridge (SB) structures, DeltaH(K(+))(SB), substantially better than those for canonical structures with a single formal charge site (charge solvation (CS)), thereby allowing structure identification. This conclusion is consistent with other spectroscopic data, which yielded unambiguous evidence of these tertiary amino acids adopting SB structures in the gas phase. This study demonstrates that IRMPD can be applied to determine individual ion structures in the gas phase, given that adequate reference values are available for proper scaling.

Role of Sequence in Salt-Bridge Formation for Alkali Metal Cationized GlyArg and ArgGly Investigated with IRMPD Spectroscopy and Theory

The roles of hydrogen bonding, electrostatic interactions, sequence, gas-phase basicity, and molecular geometry in determining the structures of protonated and alkali metal-cationized glycyl-L-arginine (GlyArg) and L-arginylglycine (ArgGly) were investigated using infrared multiple photon dissociation spectroscopy in the spectral range 900-1800 cm(-1) and theory. The IRMPD spectra clearly indicate that GlyArg x M(+), M = Li, Na, and Cs, form similar salt-bridge (SB) structures that do not depend significantly on metal ion size. In striking contrast, ArgGly x Li(+) exists in a charge-solvated (CS) form, whereas ArgGly x M(+), M = K and Cs, form SB structures. SB and CS structures are similarly populated for ArgGly x Na(+). Computed energies of low-energy structures are consistent with these results deduced from the experimental spectra. By comparison to Arg x M(+), GlyArg x M(+) and ArgGly x M(+) have a greater and lesser propensity, respectively, to form SB structures. The greater propensity for GlyArg to adopt SB structures in complexes with smaller metal cations than for ArgGly is due to the ability of alkali metal-cationized GlyArg to adopt a nearly linear arrangement of formal charge sites, a structure unfavorable for ArgGly complexes due to geometric constraints induced by its different amino acid sequence. The amide carbonyl oxygen solvates charge in both the SB and CS form of both dipeptides. ArgGly is calculated to be slightly more basic than GlyArg, indicating that differences in intrinsic basicity do not play a role in the relative SB stabilization of these ions. Loss of a neutral water molecule from complexes in which SB structures are most stable indicates that CS structures are intermediates in the dissociation pathway, but these intermediates do not contribute to the measured IRMPD spectra.

Gas-phase structures of solution-phase zwitterions: Charge solvation or salt bridge?

Sodium and lithium adduct ions of a synthetic guanidiniocarbonylpyrrole-derivative are examined in the gas phase. A wavelength tunable free electron laser (FEL) was used for photo-dissociation spectroscopy experiments in the infrared (1400–1800 cm −1). The photo-dissociation spectra are compared to calculated IR spectra of structures identified by theory. All photo-dissociation spectra acquired are strikingly similar, indicating that all ions adopt analogous gas-phase structures. Additionally, the respective sodium adduct ions were examined with ion mobility mass spectrometry (IMS). Although computational efforts succeeded in finding a charge solvated conformer that matched both the photo-dissociation spectra and the IMS data, the predicted global minimum was a salt-bridge structure.

Infrared Multiple Photon Dissociation Spectroscopy of Potassiated Proline

The structure of proline in [proline + K]+ has been investigated in the gas phase using high level DFT and MP2 calculations and infrared photo dissociation spectroscopy with a free electron laser (FELIX). The respective FELIX spectrum of [proline + K]+ matches convincingly the calculated spectra of two structurally closely related and nearly iso-energetic zwitterionic salt bridge (SB) structures. An additional unresolved band at approximately 1725 cm(-1) matching with the characteristic CO stretching mode of charge solvation (CS) structures points toward the presence of a minor population of these conformers of proline in [proline + K]+. However, theory predicts a significant energy gap of 18.9 kJ mol(-1) (B3LYP/6-311++G(2d,2p)) or 15.6 kJ mol(-1) (MP2) between the lowest CS conformer of proline and the clearly favored SB structure.

Structural stabilities of metalated histidines in gas phase and existence of gaseous zwitterionic histidine conformers

A systematic search of the various structures of metalated histidine bound to Li +, Na +, K +, Mg 2+, and Ca 2+ has been performed. The metal binding energies of the gaseous phase of these histidines have been determined at the B3LYP/6-311 + G(2df,p) level. All computational results indicate that binding energies decrease with increasing size of monovalent and divalent cations and moreover values for divalent metal cations are much higher than for monovalent ones. Also, for all cations except K +, charge solvated (CS) structures of histidine are preferentially more stable compared to their respective salt bridge (SB) structures, even though for potassium cations, the CS structure is 0.9 kcal/mole less stable than the SB structure. The tridenated structures are the most stable for Li +, Na +, Mg 2+ and Ca 2+ metal histidine complexes, while the bidenate structure is the most stable for the potassium histidine complex. Furthermore, some characteristic vibrational assignments for the most stable tridenated and bidenated structures are described, where remarkable shifting of frequencies are noticed. Thermodynamical properties such as enthalpies and the free energy for different metal histidine complexes are also presented. In addition, a systematic search of zwitterionic histidine conformers in the gas phase is performed with the finding of seven locally stable zwitterions in three tautomeric forms.