Infrared-induced conformational isomerization and vibrational relaxation dynamics in melatonin and 5-methoxy-N-acetyl tryptophan methyl amide

The conformational isomerization dynamics of melatonin and 5-methoxy N-acetyltryptophan methyl amide (5-methoxy NATMA) have been studied using the methods of IR-UV hole-filling spectroscopy and IR-induced population transfer spectroscopy. Using these techniques, single conformers of melatonin were excited via a well-defined NH stretch fundamental with an IR pump laser. This excess energy was used to drive conformational isomerization. By carrying out the infrared excitation early in a supersonic expansion, the excited molecules were re-cooled into their zero-point levels, partially re-filling the hole created in the ground state population of the excited conformer, and creating gains in population of the other conformers. These changes in population were detected using laser-induced fluorescence downstream in the expansion via an UV probe laser. The isomerization quantum yields for melatonin show some conformation specificity but no hint of vibrational mode specificity. In 5-methoxy NATMA, no isomerization was observed out of the single conformational well populated in the expansion in the absence of the infrared excitation. In order to study the dependence of the isomerization on the cooling rate, the experimental arrangement was modified so that faster cooling conditions could be studied. In this arrangement, the pump and probe lasers were overlapped in space in the high density region of the expansion, and the time dependence of the zero-point level populations of the conformers was probed following selective excitation of a single conformation. The analysis needed to extract isomerization quantum yields from the timing scans was developed and applied to the melatonin timing scans. Comparison between the frequency and time domain isomerization quantum yields under identical experimental conditions produced similar results. Under fast cooling conditions, the product quantum yields were shifted from their values under standard conditions. The results for melatonin are compared with those for N-acetyl tryptophan methyl amide.     

Two-dimensional infrared investigation of N-acetyl tryptophan methyl amide in solution

The linear infrared and two-dimensional infrared (2D IR) spectra in the amide-I region of N-acetyl tryptophan methyl amide (NATMA) in solvents of varying polarity are reported. The two amide-I transitions have been assigned unambiguously by using 13C isotopic substitution of the carbonyl group. The amide unit at the amino end shows a lower transition frequency in CH2Cl2 and methanol, while the acetyl end has a lower transition frequency in D2O. Multiple conformers exist in CH2Cl2 and methanol, but only one conformer is evident in D2O. The 2D IR cross peaks from the intermode coupling yield off-diagonal anharmonicities 2.5 +/- 0.5, 3.25 +/- 0.5, and 3.0 +/- 0.5 cm(-1) in CH2Cl2, methanol, and D2O, respectively, which by simple matrix diagonalization yield the coupling constants 8.0 +/- 0.5, 8.0 +/- 1.0, and 5.5 +/- 1.0 cm(-1). The major conformer in CH2Cl2 corresponds to a C7 structure, in agreement with that found in the gas phase [Dian, B. C.; Longarte, A.; Mercier, S.; Evans, D. A.; Wales, D. J.; Zwier, T. S. J. Chem. Phys. 2002, 117, 10688-10702] with intramolecular hydrogen bonding between the acetyl end C=O and the amino end N-H. The backbone dihedral angles (phi, psi) are determined to be in the ranges of (-55 +/- 5 degrees , 30 +/- 5 degrees ), (120 +/- 10 degrees , -20 +/- 10 degrees ), and (+/-160 +/- 10 degrees , +/-75 +/- 10 degrees ) in CH2Cl2, methanol, and D2O, respectively.     

Laser probes of conformational isomerization in flexible molecules and complexes

Molecules with several flexible coordinates have potential energy surfaces with a large number of minima and many transition states separating them. A general experimental protocol is described that is capable of studying conformational isomerization in such circumstances, measuring the product quantum yields following conformation-specific infrared excitation, and measuring energy thresholds for isomerization of specific X --> Y reactant-product isomer pairs following excitation via stimulated emission pumping (SEP). These methods have been applied to a series of molecules of varying size and conformational complexity, including 3-indolepropionic acid (IPA), meta-ethynylstyrene, N-acetyltryptophan methyl amide (NATMA), N-acetyltryptophan amide (NATA), and melatonin. Studies of isomerization in solute-solvent complexes are also described, including a measurement of the barrier to isomerization in the IPA-H2O complex, and a unique isomerization reaction in which a single water molecule is shuttled between H-bonding sites on the trans-formanilide (TFA) molecule.     

Hierarchical analysis of conformational dynamics in biomolecules: transition networks of metastable states

Molecular dynamics simulation generates large quantities of data that must be interpreted using physically meaningful analysis. A common approach is to describe the system dynamics in terms of transitions between coarse partitions of conformational space. In contrast to previous work that partitions the space according to geometric proximity, the authors examine here clustering based on kinetics, merging configurational microstates together so as to identify long-lived, i.e., dynamically metastable, states. As test systems microsecond molecular dynamics simulations of the polyalanines Ala(8) and Ala(12) are analyzed. Both systems clearly exhibit metastability, with some kinetically distinct metastable states being geometrically very similar. Using the backbone torsion rotamer pattern to define the microstates, a definition is obtained of metastable states whose lifetimes considerably exceed the memory associated with interstate dynamics, thus allowing the kinetics to be described by a Markov model. This model is shown to be valid by comparison of its predictions with the kinetics obtained directly from the molecular dynamics simulations. In contrast, clustering based on the hydrogen-bonding pattern fails to identify long-lived metastable states or a reliable Markov model. Finally, an approach is proposed to generate a hierarchical model of networks, each having a different number of metastable states. The model hierarchy yields a qualitative understanding of the multiple time and length scales in the dynamics of biomolecules.     

Isomer-specific spectroscopy and conformational isomerization energetics of o-, m-, and p-ethynylstyrenes

The infrared and ultraviolet spectroscopy of o-, m-, and p-ethynylstyrene isomers (oES, mES, and pES) were studied by a combination of methods, including resonance-enhanced two-photon ionization (R2PI), UV-UV hole-burning spectroscopy (UVHB), resonant ion-dip infrared spectroscopy (RIDIRS), and rotationally resolved fluorescence excitation spectroscopy. In addition, the newly developed method of stimulated emission pumping-population transfer spectroscopy (SEP-PTS) was used to determine the energy threshold to conformational isomerization in m-ethynylstyrene. The S(1) <-- S(0) origin transitions of oES and pES occur at 32 369 and 33 407 cm(-1), respectively. In mES, the cis and trans conformations are calculated to be close in energy. In the R2PI spectrum of mES, the two most prominent peaks (32672 and 32926 cm(-1)) were confirmed by UVHB spectroscopy to be S(1) <-- S(0) origins of these two conformers. The red-shifted conformer was identified as the cis structure by least-squares fitting of the rotationally resolved fluorescence excitation spectrum of the origin band. There are also two possible conformations in oES, but transitions due to only one were observed experimentally, as confirmed by UVHB spectroscopy. Density functional theory calculations (B3LYP/6-31+G) predict that the cis-ortho conformer, in which the substituents point toward each other, is about 8 kJ/mol higher in energy than the trans-ortho isomer, and should only be about 5% of the room temperature population of oES. Ground-state infrared spectra in the C-H stretch region (3000-3300 cm(-1)) of each isomer were obtained with RIDIRS. In all three structural isomers, the acetylenic C-H stretch fundamental was split by Fermi resonance. Infrared spectra were also recorded in the excited electronic state, using a UV-IR-UV version of RIDIR spectroscopy. In all three isomers the acetylenic C-H stretch fundamental was unshifted from the ground state, but no Fermi resonance was seen. The first observed and last unobserved transitions in the SEP-PT spectrum were used to place lower and upper bounds on the barrier to cis --> trans isomerization in m-ethynylstyrene of 990-1070 cm(-1). Arguments are given for the lack of a kinetic shift in the measurement. The analogous trans --> cis barrier is in the same range (989-1065 cm(-1)), indicating that the relative energies of the zero-point levels of the two isomers are (E(ZPL)(cis) - E(ZPL)(trans))= -75 to +81 cm(-1). Both the barrier heights and relative energies of the minima are close to those determined by DFT (Becke3LYP/6-31+G) calculations.     

Femtosecond study on the isomerization dynamics of NK88. I. Ground-state dynamics after photoexcitation

Recently, optimal control of a photoisomerization reaction in the liquid phase was demonstrated for the first time on the system 3,3(')-diethyl-2,2(')-thiacyanine (NK88). Additionally, the class of cyanines to which the molecule NK88 belongs draws a lot of attention in different recent theoretical publications. Therefore, a better understanding of the molecular dynamics of this molecular system is of special interest. Experiments using the femtosecond pump-supercontinuum probe technique with an excitation wavelength of 400 nm and a spectral range from 370 to 620 nm for the probe beam have been performed. In order to analyze the dynamics properly the time window has been chosen to comprise the characteristic times of the contributing processes, additionally we have employed two solvents, methanol and ethylene glycol, and have conducted anisotropy measurements. The spectroscopic data have been assigned to different molecular states with the help of density functional theory and second-order Moller-Plesset perturbation theory calculations. The analysis of the data has revealed in the most likely model that three different isomers exist with different lifetimes. On the basis of experimental and theoretical data, a conclusive scheme of the isomerization reaction is presented.     

Simulation of vibrational energy transfer in two-dimensional infrared spectroscopy of amide I and amide II modes in solution

Population transfer between vibrational eigenstates is important for many phenomena in chemistry. In solution, this transfer is induced by fluctuations in molecular conformation as well as in the surrounding solvent. We develop a joint electrostatic density functional theory map that allows us to connect the mixing of and thereby the relaxation between the amide I and amide II modes of the peptide building block N-methyl acetamide. This map enables us to extract a fluctuating vibrational Hamiltonian from molecular dynamics trajectories. The linear absorption spectrum, population transfer, and two-dimensional infrared spectra are then obtained from this Hamiltonian by numerical integration of the Schrodinger equation. We show that the amide I/amide II cross peaks in two-dimensional infrared spectra in principle allow one to follow the vibrational population transfer between these two modes. Our simulations of N-methyl acetamide in heavy water predict an efficient relaxation between the two modes with a time scale of 790 fs. This accounts for most of the relaxation of the amide I band in peptides, which has been observed to take place on a time scale of 450 fs in N-methyl acetamide. We therefore conclude that in polypeptides, energy transfer to the amide II mode offers the main relaxation channel for the amide I vibration.     

Base catalyzed isomerization of epoxides. I. The isomerization of β-chloroepoxides

The base catalyzed isomerizations of epichlorohydrin, 1-chloro-2,3-epoxy-2-methylpropane, 1-chloro-2,3-epoxybutane, and 3-chloro-1,2-epoxybutane have been studied, using lithium ortho-phosphate as the basic catalyst. Chloroketones and dichloro-alcohols are the major products. This is the first example of a compound with an electron withdrawing group attached to the carbon atom adjacent to the oxirane ring which undergoes the α-elimination pathway. A bidirectional mechanism is proposed to explain the experimental results. The stereochemistry of the hydrochlorination of 1-chloro-2,3-epoxybutane and 3-chloro-1,2-epoxybutane has also been studied.     

Synthesis and conformational investigation of methyl 4a-carba-D-arabinofuranosides.

The synthesis of carbasugar analogues of methyl alpha-D-arabinofuranoside and methyl beta-D-arabinofuranoside (3 and 4) is reported. The route developed involves the conversion of D-mannose into a suitably protected diene (13), which is then cyclized via olefin metathesis. The resulting cyclopentene (14) is stereoselectively hydrogenated to provide an intermediate that can be used for the synthesis of both targets. Through the use of NMR spectroscopy, we have probed the ring conformation of 3 and 4, as well as the rotamer populations about the C(4)-C(5) and C(1)-O(1) bonds. These studies have demonstrated that there are differences in ring conformation between these carbasugars and their glycoside parents (1 and 2). However, only minor differences are seen in the rotameric equilibrium about the C(4)-C(5) bond in 3 and 4 relative to 1 and 2. In regard to the C(1)-O(1) bond, NOE data from 3 and 4 suggest that the favored position about this bond is similar to that in the glycosides; that is, the methyl group is anti to C(2). However, confirmation of this preference through measurement of (3)J(C,C) between the methyl group and C(2) or C(4a) was not successful.     

Protonation and conformational dynamics of GFP mutants by two-photon excitation fluorescence correlation spectroscopy

GFP mutants are known to display fluorescence flickering, a process that occurs in a wide time range. Because serine 65, threonine 203, glutamate 222, and histidine 148 have been indicated as key residues in determining the GFP fluorescence photodynamics, we have focused here on the role of histidine 148 and glutamate 222 by studying the fluorescence dynamics of GFPmut2 (S65A, V68L, and S72A GFP) and its H148G (Mut2G) and E222Q (Mut2Q) mutants. Two relaxation components are found in the fluorescence autocorrelation functions of GFPmut2: a 10-100 micros pH-dependent component and a 100-500 micros laser-power-dependent component. The comparison of these three mutants shows that the mutation of histidine 148 to glycine induces a 3-fold increase in the protonation rate, thereby indicating that the protonation-deprotonation of the chromophore occurs via a proton exchange with the solution mediated by the histidine 148 residue. The power-dependent but pH-independent relaxation mode, which is not affected by the E222Q and H148G mutations, is due to an excited-state process that is probably related to conformational rearrangements of the chromophore after the photoexcitation, more than to the chromophore excited-state proton transfer.     

The conformational potential energy surface of IOONO and the isomerization and decomposition processes

The conformational potential energy surface of iodine peroxy nitrite was investigated using high levels of electronic structure theory. Two minimum energy conformers and five energy maxima have been determined. The comparison with other peroxy nitrites shows interesting correlations with the internal rotational barriers of the Cl, Br analogues and with peroxynitrous acid. Distinct transition states for the isomerization to iodine nitrate and the scission of the peroxy bond have been calculated. Finally, the thermochemistry of IOONO and IONO₂ has been reconsidered.     

Ultrafast excited-state isomerization dynamics of 1,1'-diethyl-2,2'-cyanine studied by four-wave mixing spectroscopy

Excited-state dynamics and solvent-solute interactions of 1,1'-diethyl-2,2'-cyanine iodine (1122C) in alcoholic solutions are investigated using time-integrated three-pulse photon-echo spectroscopy. 1122C serves as a model compound for ultrafast photoinduced isomerization-a key process in the light reception of plants, bacteria, and human vision. The photoreaction in 1122C is interrogated in dependence on solvent and excitation wavelength. The wavelength-dependent three-pulse photon-echo peak shift indicates strong alterations of the reaction pathways and points to the existence of a direct internal conversion channel in close proximity to the Franck-Condon point of absorption. The solvent-dependent S1-S0 internal conversion time does not follow conventional sheared viscosity dependence, suggesting that the solvent local friction has to be considered to account for the observed isomerization kinetics. The concerted discussion of transient grating and three-pulse photon-echo peak-shift data allows us to derive a complete picture of the solvent-solute interaction-controlled photoreaction. The results obtained are related to other work on reactive systems and are discussed in the framework of multilevel response functions.     

Multidimensional infrared spectroscopy of water. I. Vibrational dynamics in two-dimensional IR line shapes

In this and the following paper, we describe the ultrafast structural fluctuations and rearrangements of the hydrogen bonding network of water using two-dimensional (2D) infrared spectroscopy. 2D IR spectra covering all the relevant time scales of molecular dynamics of the hydrogen bonding network of water were studied for the OH stretching absorption of HOD in D2O. Time-dependent evolution of the 2D IR line shape serves as a spectroscopic observable that tracks how different hydrogen bonding environments interconvert while changes in spectral intensity result from vibrational relaxation and molecular reorientation of the OH dipole. For waiting times up to the vibrational lifetime of 700 fs, changes in the 2D line shape reflect the spectral evolution of OH oscillators induced by hydrogen bond dynamics. These dynamics, characterized through a set of 2D line shape analysis metrics, show a rapid 60 fs decay, an underdamped oscillation on a 130 fs time scale induced by hydrogen bond stretching, and a long time decay constant of 1.4 ps. 2D surfaces for waiting times larger than 700 fs are dominated by the effects of vibrational relaxation and the thermalization of this excess energy by the solvent bath. Our modeling based on fluctuations with Gaussian statistics is able to reproduce the changes in dispersed pump-probe and 2D IR spectra induced by these relaxation processes, but misses the asymmetry resulting from frequency-dependent spectral diffusion. The dynamical origin of this asymmetry is discussed in the companion paper.     

Folding, conformational changes, and dynamics of cytochromes C probed by NMR spectroscopy

NMR spectroscopy has become a vital tool for studies of protein conformational changes and dynamics. Oxidized Fe(III)cytochromes c are a particularly attractive target for NMR analysis because their paramagnetism (S = (1)/(2)) leads to high (1)H chemical shift dispersion, even for unfolded or otherwise disordered states. In addition, analysis of shifts induced by the hyperfine interaction reveals details of the structure of the heme and its ligands for native and nonnative protein conformational states. The use of NMR spectroscopy to investigate the folding and dynamics of paramagnetic cytochromes c is reviewed here. Studies of nonnative conformations formed by denaturation and by anomalous in vivo maturation (heme attachment) are facilitated by the paramagnetic, low-spin nature of native and nonnative forms of cytochromes c. Investigation of the dynamics of folded cytochromes c also are aided by their paramagnetism. As an example of this analysis, the expression in Escherichia coli of cytochrome c(552) from Nitrosomonas europaea is reported here, along with analysis of its unusual heme hyperfine shifts. The results are suggestive of heme axial methionine fluxion in N. europaea ferricytochrome c(552). The application of NMR spectroscopy to investigate paramagnetic cytochrome c folding and dynamics has advanced our understanding of the structure and dynamics of both native and nonnative states of heme proteins.     

Combination of molecular dynamics method and 3D-RISM theory for conformational sampling of large flexible molecules in solution

We have developed an algorithm for sampling the conformational space of large flexible molecules in solution, which combines the molecular dynamics (MD) method and the three-dimensional reference interaction site model (3D-RISM) theory. The solvent-induced force acting on solute atoms was evaluated as the gradient of the solvation free energy with respect to the solute-atom coordinates. To enhance the computation speed, we have applied a multiple timestep algorithm based on the RESPA (Reversible System Propagator Algorithm) to the combined MD/3D-RISM method. By virtue of the algorithm, one can choose a longer timestep for renewing the solvent-induced force compared with that of the conformational update. To illustrate the present MD/3D-RISM simulation, we applied the method to a model of acetylacetone in aqueous solution. The multiple timestep algorithm succeeded in enhancing the computation speed by 3.4 times for this model case. Acetylacetone possesses an intramolecular hydrogen-bonding capability between the hydroxyl group and the carbonyl oxygen atom, and the molecule is significantly stabilized due to this hydrogen bond, especially in gas phase. The intramolecular hydrogen bond was kept intact during almost entire course of the MD simulation in gas phase, while in the aqueous solutions the bond is disrupted in a significant number of conformations. This result qualitatively agrees with the behavior on a free energy barrier lying upon the process for rotating a torsional degree of freedom of the hydroxyl group, where it is significantly reduced in aqueous solution by a cancellation between the electrostatic interaction and the solvation free energy.     

Classical trajectory simulations of photoionization dynamics of tryptophan: intramolecular energy flow, hydrogen-transfer processes and conformational transitions

One-photon and two-photon ionization dynamics of tryptophan is studied by classical trajectory simulations using the semiempirical parametric method number 3 (PM3) potential surface in "on the fly" calculations. The tryptophan conformer is assumed to be in the vibrational ground state prior to ionization. Initial conditions for the trajectories are weighted according to the Wigner distribution function computed for that state. Vertical ionization in the spirit of the classical Franck-Condon principle is assumed. For the two-photon ionization process the ionization is assumed to go resonantively through the first excited state. Most trajectories are computed, and the analysis is carried out for the first 10 ps. A range of interesting effects are observed. The main findings are as follows: (1) Multiple conformational transitions are observed in most of the trajectories within the ultrafast duration of 10 ps. (2) Hydrogen transfer from the carboxyl group to the amino group and back has been observed. A zwitterion is formed as a transient state. (3) Two new isomers are formed during the dynamics, which have apparently not been previously observed. (4) Fast energy flow between the ring modes and the amino acid backbone is observed for both one- and two-photon ionization. However, the effective vibrational temperatures only approach the same value after 90 ps. The conformation transition dynamics, the proton-transfer processes and the vibrational energy flow are discussed and analyzed.