Ab initio study of the excited-state deactivation pathways of protonated tryptophan and tyrosine

In recent experiments, the excited-state lifetimes of protonated aromatic amino acids (TrpH+ and TyrH+) have been recorded by means of pump-probe photodissociation technique. The lifetime of TyrH+ is much longer than that of TrpH+, which has been initially rationalized on the basis of a simple phenomenological model. Besides, specific photofragments including the formation of radical cation after hydrogen loss are observed for TrpH+ that are not found for TyrH+. The ab initio calculations reported here for TrpH+ and TyrH+ using a coupled-cluster method are meant to track the rich photochemistry of these protonated amino acids following UV excitation.     

Control of bond-cleaving reactions of free protonated tryptophan ion by femtosecond laser pulses

The excited-state dynamics of protonated tryptophan ions is investigated by photoinduced fragmentation in the gas phase. In contrast to the neutral molecule that decays on the nanosecond time scale, the protonated species exhibits an ultrafast decay with two time constants of about 400 fs and 15 ps. In addition, after UV excitation by a pump photon at 266 nm, specific photofragments, and in particular the NH3-loss channel, can be enhanced by the absorption of a probe photon at 800 nm. The bond-cleaving reactions can thus be controlled by a variation of the pump/probe delay.     

SELECTIVE DEGRADATION OF AMINO ACIDS PHOTOSENSITIZED BY TRYPTOPHAN IN POLYPEPTIDE STRUCTURES

Polypeptides containing a basic amino acid close to their single tryptophan residue were irradiated with monochromatic 302 nm radiation. Tryptophan photolysis was monitored by absorption and fluorescence spectroscopy. Amino acid loss was evaluated by amino acid analysis. Only the protonated residues adjacent to tryptophan in the sequence were destroyed upon tryptophan excitation. This reaction is probably due essentially to direct interaction between the excited tryptophan and the neighbouring residue without electron solvation.     

ENERGETICS OF PROTONATION-DEPROTONATION OF THE CHROMOPHORE IN RETINAL PROTEINS

Abstract— We investigate the energetics of protonation and deprotonation of retinylidene Schiff-base (SB) which is realized in the functioning of retinal proteins. We first calculate the energy difference ΔE between the protonated and unprotonated states of the SB by the ab initio molecular orbital method, using two kinds of molecular model; a counter-ion model where a carboxyl group of Glu or Asp is directly hydrogen-bonded to the SB, and a water-bridge model where a water molecule bridges the carboxyl group and the SB. The calculated results indicate that the protonated SB state is unstable compared with the unprotonated SB state in either model. In addition, we find that coordination of some water molecules to the carboxyl group reduces ΔE significantly. The value of AE for the counterion model with two coordinated water molecules is 0.003 eV. Next, we calculate the electrostatic interaction energy between a tryptophan residue and the SB. We find that the protonated state is more stabilized than the unprotonated state by about 0.1 eV with one tryptophan residue. This fact indicates that if some aromatic amino acid residues work cooperatively, they can contribute to significantly reducing ΔE. We also discuss the possible role of amino acid residues which make hydrogen-bond with the carboxyl group of interest.     

Microcalorimetric studies of the mechanism of interaction between designed peptides and hydrophobic adsorbents

To understand the mechanism of interaction between peptides and peptides with hydrophobic ligands, the oligomers (GWG, GWWG, GWWWG) were designed and synthesized to study adsorption behavior with octyl sepharose and CM-octyl sepharose. By batch equilibrium binding analysis and dilution heat of peptide solution measurement, the binding isotherm and adsorption enthalpy were obtained and the binding thermodynamics parameters were calculated and analyzed. In the isotherm analysis, we reveled that the affinity of GWG for both adsorbents is stronger than that of GWWG and GWWWG. The results demonstrate that the cation-pi interaction between the peptides and the buffer molecules is significant for solutions of peptides with tryptophan residues, and the solvation is competitive with the hydrophobic interaction between the peptides and the hydrophobic ligands. From the dilution heat measurements, we observed an endothermic dilution heat for GWG and exothermic for GWWG and GWWWG. All these results indicate that the increased tryptophan chain length can promote the solvation behavior of the peptides by the peptide-buffer interaction in this buffer system. Comparing the types of ligands reveals that the binding affinities of each peptide for the two adsorbents are similar. However, the mechanism of adsorption for peptides with hydrophobic ligands might be quite different with respect to the binding enthalpy between peptides and adsorbents. The adsorption of the peptides on octyl sepharose is an entropy-driven process for all the peptides. In contrast, the adsorption of CM-octyl sepharose with GWG and GWWG is an enthalpy-driven process, whereas that with GWWWG is entropy-driven. These findings indicate that the amount of tryptophan controls the characteristics of the peptides and the interaction mechanism in the binding procedure. This study of the adsorption mechanism of the designed peptide could provide fundamental information for peptide purification and amino acid residue behavior in peptide drug design.     

Excited state dynamics and fragmentation channels of the protonated dipeptide H2N-Leu-Trp-COOH

The excited state dynamics of the isolated and protonated peptide H(2)N-Leu-Trp-COOH are analyzed by fs pump-probe spectroscopy. The peptides are brought into the gas phase by electrospray ionization, and fs pump-probe excitation is detected by fragment ion formation. The pump laser addressed the excited pipi* state of the indole chromophore of the amino acid tryptophan. The subsequent excited state dynamics agreed with a biexponential decay with time constants of 500 fs and 10 ps. This is considerably shorter than the lifetime of neutral tryptophan in solution and in proteins, but similar to isolated, protonated tryptophan. Several models are discussed to explain the experimental results but the detailed quenching mechanism remains unresolved.     

Conformation-specific spectroscopy and photodissociation of cold, protonated tyrosine and phenylalanine

We present here ultraviolet and infrared spectra of protonated aromatic amino acids in a cold, 22-pole ion trap. Ultraviolet photofragmentation spectra of protonated tyrosine and phenylalanine show vibronically resolved bands corresponding to different stable conformers: two for PheH+ and four in the case of TyrH+. We subsequently use the resolved UV spectra to perform conformer-specific infrared depletion spectroscopy. Comparison of the measured infrared spectra to density functional theory calculations helps assign the geometry of the various conformers, all of which exhibit NH...pi hydrogen bonds and NH...O=C interactions, with the COOH group oriented either anti or gauche to the aromatic ring. In both molecules the majority of the observed fragments result from dissociation on an excited electronic state. In TyrH+, different conformers excited with practically the same energy exhibit different fragmentation patterns, suggesting that the excited-state dynamics depend upon conformation.     

细胞松弛素B对葡萄糖/质子共转运蛋白GlcPSe的抑制机理

通过分子动力学对细胞松弛素B与葡萄糖/质子共转运蛋白的两种质子化状态进行了模拟, 发现细胞松弛素B对处于去质子化阶段的葡萄糖转运蛋白具有更好的抑制效果. 结果表明, 357号色氨酸和117号脯氨酸是葡萄糖转运蛋白结合细胞松弛素B的关键氨基酸; 并且当抑制剂与受体蛋白结合时, 位于第10号跨膜螺旋上的357号色氨酸与细胞松弛素B的相对位置对抑制剂的结合有重要意义.     

Recognition of Free Tryptophan in Water by Synthetic Pseudopeptides: Fluorescence and Thermodynamic Studies

Pseudopeptidic receptors containing an acridine unit have been prepared and their fluorescence response to a series of amino acids was measured in water. Free amino acids, not protected either at the C or the N terminus, were used for this purpose. The prepared receptors display a selective response to tryptophan (Trp) versus the other assayed amino acids under acidic conditions. The macrocyclic nature of the receptor is important as the fluorescence quenching is higher for the macrocyclic compound than for the related open‐chain receptor. Notably, under the experimental acidic conditions used, both the receptor and guest are fully protonated and positively charged; thus, the experimental results suggest the formation of supramolecular species that contain two positively charged organic molecular components in proximity stabilized through aromatic–aromatic interactions and a complex set of cation‐anion‐cation interactions. The selectivity towards Trp seems to be based on the existence of a strong association between the indole ring of the monocharged amino acid and the acridinium fragment of the triprotonated form of the receptor, which is established to be assisted by the interaction of the cationic moieties with hydrogen sulfate anions.     

Selective transport of amino acids into the gas phase: driving forces for amino acid solubilization in gas-phase reverse micelles

We report a study on encapsulation of various amino acids into gas-phase sodium bis(2-ethylhexyl) sulfosuccinate (NaAOT) reverse micelles, using electrospray ionization guided-ion-beam tandem mass spectrometry. Collision-induced dissociation of mass-selected reverse micellar ions with Xe was performed to probe structures of gas-phase micellar assemblies, identify solute-surfactant interactions, and determine preferential incorporation sites of amino acids. Integration into gas-phase reverse micelles depends upon amino acid hydrophobicity and charge state. For examples, glycine and protonated amino acids (such as protonated tryptophan) are encapsulated within the micellar core via electrostatic interactions; while neutral tryptophan is adsorbed in the surfactant layer. As verified using model polar hydrophobic compounds, the hydrophobic effect and solute-interface hydrogen-bonding do not provide sufficient driving force needed for interfacial solubilization of neutral tryptophan. Neutral tryptophan, with a zwitterionic structure, is intercalated at the micellar interface between surfactant molecules through complementary effects of electrostatic interactions between tryptophan backbone and AOT polar heads, and hydrophobic interactions between tryptophan side chain and AOT alkyl tails. Protonation of tryptophan could significantly improve its incorporation capacity into gas-phase reverse micelles, and displace its incorporation site from the micellar interfacial zone to the core; protonation of glycine, on the other hand, has little effect on its encapsulation capacity. Another interesting observation is that amino acids of different isoelectric points could be selectively encapsulated into, and transported by, reverse micelles from solution to the gas phase, based upon their competition for protonation and subsequent encapsulation within the micellar core.     

The aromatic sidechains of amino acids as neutral donor groups for alkali metal cations

An aromatic residue that can serve as a pi-donor occurs in all known protein sequences about one out of every 11 amino acids. Benzene, phenol, and indole, the sidechains of phenylalanine, tyrosine, and tryptophan, are particularly important in protein structure. Solid state structures confirm the interactions of these neutral arenes, along with double and triple bonds, with sodium and potassium cations.     

The gas-phase dipeptide analogue acetyl-phenylalanyl-amide: a model for the study of side chain/backbone interactions in proteins

The issue of the influence of the side chain/backbone interaction on the local conformational preferences of a phenylalanine residue in a peptide chain is addressed. A synergetic approach is used, which combines gas-phase UV spectroscopy as well as gas-phase IR/UV double-resonance experiments with DFT and post Hartree-Fock calculations. N-Acetyl-Phe-amide was chosen as a model system for which three different conformers were observed. The most stable conformer has been identified as an extended beta(L) conformation of the peptide backbone. It is stabilized by a weak but significant NH-pi interaction bridging the aromatic ring on the residue (i) with the NH group on residue (i+1), with the aromatic side chain being in an anti conformation. This stable conformation corresponds to the common NH(i+1)-aromatic(i) interaction encountered in proteins for the three aromatic residues (phenylalanine, tyrosine, and tryptophan), which illustrates the relevance of gas-phase investigations to structural biology issues. The two other less abundant conformers have been assigned to two gamma-folded backbone conformations that differ by the orientation of the side chain. In all cases, the IR data provided spectroscopic fingerprints of these interactions. Finally, the strong conformational dependence of the fluorescence yield found for N-acetyl-Phe-amide illustrates the role of the environment on the excited-state dynamics of these species, which is often exploited by biochemists to monitor protein structural changes from tryptophan lifetime measurements.     

Microsolvation effects on the excited-state dynamics of protonated tryptophan

To better understand the complex photophysics of the amino acid tryptophan, which is widely used as a probe of protein structure and dynamics, we have measured electronic spectra of protonated, gas-phase tryptophan solvated with a controlled number of water molecules and cooled to approximately 10 K. We observe that, even at this temperature, the bare molecule exhibits a broad electronic spectrum, implying ultrafast, nonradiative decay of the excited state. Surprisingly, the addition of two water molecules sufficiently lengthens the excited-state lifetime that we obtain a fully vibrationally resolved electronic spectrum. Quantum chemical calculations at the RI-CC2/aug-cc-pVDZ level, together with TDDFT/pw based first-principles MD simulations of the excited-state dynamics, clearly demonstrate how interactions with water destabilize the photodissociative states and increase the excited-state lifetime.     

Molecular mechanics study of the β-spiral conformations of the Phe4, Tyr4, and Trp4 analogs of elastomeric poly(Val1–Pro2–Gly3–Val4–Gly5)

The elastin-derived polypentapeptide, poly(VPGVG), and analogs are becoming model systems for protein as well as forming a new class of biomaterials. The β-spiral of poly(VPGVG) represents a new family of molecular structures in fibrous proteins. In particular for our interests here, aromatic amino acid residue substitutions at position four of poly(VPGVG) give rise to new polypeptides with interesting properties of increased elastic modulus, pressure sensitivity, etc. Molecular mechanics computations were carried out to answer the question, can the polypeptides with Phe, Tyr, and Trp substitution at position four of poly(VPGVG) form a β-spiral structure like poly(VPGVG)? Employed in this study was a combination of conformational search using ECEPP/2 and the build-up strategy and of molecular dynamics calculations using CHARMm. The results indicate that the VPGX Type II β-turn structure appears to be the most prominent secondary structural feature for the three aromatic residues containing polypentapeptides, although there is more evidence for VPGFG and VPGVG to assume the VPGVG-type structure than for VPGWG to do so. The study also shows that the aromatic side chains do not interrupt the β-spiral structure, even in the case of tryptophan.     

Gas-phase reactions of protonated tryptophan

The gas phase reactions of protonated tryptophan have been examined in a quadrupole ion trap using a combination of collision induced dissociation, hydrogen-deuterium exchange, regiospecific deuterium labeling and molecular orbital calculations (at the B3LYP/6-31G* level of theory). The loss of ammonia from protonated tryptophan is observed as the primary fragmentation pathway, with concomitant formation of a [M + H - NH(3)](+) ion by nucleophilic attack from the C3 position of the indole side chain. Hydrogen-deuterium exchange and regiospecific deuterium labeling reveals that scrambling of protons in the C2 and C4 positions of the indole ring, via intramolecular proton transfer from the thermodynamically preferred site of protonation at the amino nitrogen, precedes ammonia loss. Molecular orbital calculations have been employed to demonstrate that the activation barriers to intramolecular proton transfer are lower than that for NH(3) loss.     

Quantitative determination of singlet oxygen generated by excited state aromatic amino acids, proteins, and immunoglobulins

Singlet oxygen quantum yields generated by excited state aromatic amino acids (tryptophan, tyrosine, phenylalanine), N-acetylated amino acids (N-acetyl-tryptophan, N-acetyl-tyrosine, N-acetyl-phenylalanine), and from selected proteins and immunoglobulins have been quantified by time-resolved phosphorescence measurements. A small, but significant, quantum yield found for proteins and immunoglobulins demonstrates that molecular oxygen can diffuse through the polypeptide matrix and can be sensitized by residues buried within the folds of protein structure.     

A comparison of the fragmentation pathways of [Cu(II)(Ma)(Mb)]•2+ complexes where Ma and Mb are peptides containing either a tryptophan or a tyrosine residue

[Cu^II(M_a)(M_b)]^?2+ complexes, where M_a and M_b are dipeptides or tripeptides each containing either a tryptophan (W) or tyrosine (Y) residue, have been examined by means of electrospray tandem mass spectrometry. Collision‐induced dissociations (CIDs) of complexes containing identical peptides having a tryptophan residue generated abundant radical cations of the peptides; by contrast, for complexes containing peptides having a tyrosine residue, the main fragmentation channel is dissociative proton transfer to give [M_a + H]⁺ and [Cu^II(M_b – H)]^?+. When there are two different peptides in the complex, each containing a tryptophan residue, radical cations are again the major products, with their relative abundances depending on the locations of the tryptophan residue in the peptides. In the CIDs of mixed complexes, where one peptide contains a tryptophan residue and the other a tyrosine residue, the main fragmentation channel is formation of the radical cation of the tryptophan‐containing peptide and not proton transfer from the tyrosine‐containing peptide to give a protonated peptide. Copyright © 2010 John Wiley & Sons, Ltd.     

A matrix-assisted laser desorption/ionization compatible reagent for tagging tryptophan residues

Chemical tagging of amino acids is an important tool in proteomics analysis, and has been used to introduce isotope labels and mass defect labels into proteolytic peptides by derivatization of cysteine or lysine residues. Here, we present a new reagent with chemical specificity for tryptophan residues. Previously, 2-nitrobenzenesulfenyl chloride has been used as a highly specific reagent for labeling tryptophan residues. We show that this tag undergoes UV dissociation during matrix assisted laser desorption/ionization (MALDI). The multiplicity of photofragments increases the difficulty of characterizing the derivatization products. To overcome this problem, we have synthesized a new reagent, 2-(trifluoromethyl)benzenesulfenyl chloride, which is shown to react quantitatively with tryptophan in peptides and proteins. Most significantly, it exhibits high photostability in MALDI-Fourier transform mass spectrometry analyses.     

Ultrafast quenching of tryptophan fluorescence in proteins: Interresidue and intrahelical electron transfer

Quenching of tryptophan fluorescence in proteins has been critical to the understanding of protein dynamics and enzyme reactions using tryptophan as a molecular optical probe. We report here our systematic examinations of potential quenching residues with more than 40 proteins. With site-directed mutation, we placed tryptophan to desired positions or altered its neighboring residues to screen quenching groups among 20 amino acid residues and of peptide backbones. With femtosecond resolution, we observed the ultrafast quenching dynamics within 100 ps and identified two ultrafast quenching groups, the carbonyl- and sulfur-containing residues. The former is glutamine and glutamate residues and the later is disulfide bond and cysteine residue. The quenching by the peptide-bond carbonyl group as well as other potential residues mostly occurs in longer than 100 ps. These ultrafast quenching dynamics occur at van der Waals distances through intraprotein electron transfer with high directionality. Following optimal molecular orbital overlap, electron jumps from the benzene ring of the indole moiety in a vertical orientation to the LUMO of acceptor quenching residues. Molecular dynamics simulations were invoked to elucidate various correlations of quenching dynamics with separation distances, relative orientations, local fluctuations and reaction heterogeneity. These unique ultrafast quenching pairs, as recently found to extensively occur in high-resolution protein structures, may have significant biological implications.     

Effect of the basic residue on the energetics, dynamics, and mechanisms of gas-phase fragmentation of protonated peptides

The effect of the basic residue on the energetics, dynamics, and mechanisms of backbone fragmentation of protonated peptides was investigated. Time-resolved and collision energy-resolved surface-induced dissociation (SID) of singly protonated peptides with the N-terminal arginine residue and their analogues, in which arginine is replaced with less basic lysine and histidine residues, was examined using a specially configured Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). SID experiments demonstrated different kinetics of formation of several primary product ions of peptides with and without arginine residue. The energetics and dynamics of these pathways were determined from Rice-Ramsperger-Kassel-Marcus (RRKM) modeling of the experimental data. Comparison between the kinetics and energetics of fragmentation of arginine-containing peptides and the corresponding methyl ester derivatives provides important information on the effect of dissociation pathways involving salt bridge (SB) intermediates on the observed fragmentation behavior. Because pathways involving SB intermediates are characterized by low threshold energies, they efficiently compete with classical oxazolone and imine/enol pathways of arginine-containing peptides on a long time scale of the FTICR instrument. In contrast, fragmentation of histidine- and lysine-containing peptides is largely determined by canonical pathways. Because SB pathways are characterized by negative activation entropies, fragmentation of arginine-containing peptides is kinetically hindered and observed at higher collision energies as compared to their lysine- and histidine-containing analogues.