Femtosecond spectroscopic study of the solvation of amphiphilic molecules by water

We use polarization-resolved mid-infrared pump-probe spectroscopy to study the aqueous solvation of proline and N-methylacetamide. These molecules serve as models to study the solvation of proteins. We monitor the orientational dynamics of partly deuterated water molecules (HDO) that are present at a low concentration in the water. We find that the OD vibration of HDO relaxes via an intermediate level, that is characterized by a hydrogen-bond that is stronger than in the ground state. With increasing concentration the lifetime of the excited state increases from 1.8 ps to 2.4 ps and the lifetime of the intermediate level from 0.6 ps to 1.0 ps. Regarding the orientational dynamics we observe biexponential behavior, which finds its origin in the presence of two classes of water molecules. There is a fraction of water molecules that has bulk-like orientational dynamics (τ_rot = 2.5 ps) and a fraction of immobilized water molecules (τ_rot > 10 ps). The relative abundance of the two fractions is determined by the nature and concentration of the solute. We find that the hydrophobic solute groups are responsible for the immobilization of water molecules. Every methyl group causes the immobilization of approximately 4 water OH groups. The hydrophilic solute groups, on the other hand, do not hinder the reorientation and the water molecules solvating them reorient with the same rate as in the bulk liquid.     

Hydration dynamics and time scales of coupled water-protein fluctuations

We report experimental and theoretical studies on water and protein dynamics following photoexcitation of apomyoglobin. Using site-directed mutation and with femtosecond resolution, we experimentally observed relaxation dynamics with a biphasic distribution of time scales, 5 and 87 ps, around the site Trp7. Theoretical studies using both linear response and direct nonequilibrium molecular dynamics (MD) calculations reproduced the biphasic behavior. Further constrained MD simulations with either frozen protein or frozen water revealed the molecular mechanism of slow hydration processes and elucidated the role of protein fluctuations. Observation of slow water dynamics in MD simulations requires protein flexibility, regardless of whether the slow Stokes shift component results from the water or protein contribution. The initial dynamics in a few picoseconds represents fast local motions such as reorientations and translations of hydrating water molecules, followed by slow relaxation involving strongly coupled water-protein motions. We observed a transition from one isomeric protein configuration to another after 10 ns during our 30 ns ground-state simulation. For one isomer, the surface hydration energy dominates the slow component of the total relaxation energy. For the other isomer, the slow component is dominated by protein interactions with the chromophore. In both cases, coupled water-protein motion is shown to be necessary for observation of the slow dynamics. Such biologically important water-protein motions occur on tens of picoseconds. One significant discrepancy exists between theory and experiment, the large inertial relaxation predicted by simulations but clearly absent in experiment. Further improvements required in the theoretical model are discussed.     

Anomalous dielectric relaxation of water molecules at the surface of an aqueous micelle

Dielectric relaxation of aqueous solutions of micelles, proteins, and many complex systems shows an anomalous dispersion at frequencies intermediate between those corresponding to the rotational motion of bulk water and that of the organized assembly or macromolecule. The precise origin of this anomalous dispersion is not well-understood. In this work we employ large scale atomistic molecular dynamics simulations to investigate the dielectric relaxation (DR) of water molecules in an aqueous micellar solution of cesium pentadecafluorooctanoate. The simulations clearly show the presence of a slow component in the moment-moment time correlation function [PhiMW(t)] of water molecules, with a time constant of about 40 ps, in contrast to only 9 ps for bulk water. Interestingly, the orientational time correlation function [Cmu(t)] of individual water molecules at the surface exhibits a component with a time constant of about 19 ps. We show that these two time constants can be related by the well-known micro-macrorelations of statistical mechanics. In addition, the reorientation of surface water molecules exhibits a very slow component that decays with a time constant of about 500 ps. An analysis of hydrogen bond lifetime and of the rotational relaxation in the coordinate frame fixed on the micellar body seems to suggest that the 500 ps component owes its origin to the existence of an extended hydrogen bond network of water molecules at the surface. However, this ultraslow component is not found in the total moment-moment time correlation function of water molecules in the solution. The slow DR of hydration water is found to be well correlated with the slow solvation dynamics of cesium ions at the water-micelle interface.     

Direct Vibrational Energy Transfer in Monomeric Water Probed with Ultrafast Two Dimensional Infrared Spectroscopy

Vibrational relaxation dynamics of monomeric water molecule dissolved in d-chloroform solution were revisited using the two dimensional Infrared (2D IR) spectroscopy. The vibrational lifetime of OH bending in monomeric water shows a bi-exponential decay. The fast component (T₁=(1.2±0.1) ps) is caused by the rapid population equilibration between the vibrational modes of the monomeric water molecule. The slow component (T₂=(26.4±0.2) ps) is mainly caused by the vibrational population decay of OH bending mode. The reorientation of the OH bending in monomeric water is determined with a time constant of τ=(1.2±0.1) ps which is much faster than the rotational dynamics of water molecules in the bulk solution. Furthermore, we are able to reveal the direct vibrational energy transfer from OH stretching to OH bending in monomeric water dissolved in d-chloroform for the first time. The vibrational coupling and relative orientation of transition dipole moment between OH bending and stretching that effect their intra-molecular vibrational energy transfer rates are discussed in detail.     

Ultrafast hydration dynamics in the lipidic cubic phase: Discrete water structures in nanochannels

We report here our studies of hydration dynamics of confined water in aqueous nanochannels (approximately 50 A) of the lipidic cubic phase. By systematically anchoring the hydrocarbon tails of a series of tryptophan-alkyl ester probes into the lipid bilayer, we mapped out with femtosecond resolution the profile of water motions across the nanochannel. Three distinct time scales were observed, revealing discrete channel water structures. The interfacial water at the lipid surface is well-ordered, and the relaxation dynamics occurs in approximately 100-150 ps. These dynamically rigid water molecules are crucial for global structural stability of lipid bilayers and for stabilization of anchored biomolecules in membranes. The adjacent water layers near the lipid interface are hydrogen-bonded networks and the dynamical relaxation takes 10-15 ps. This quasi-bound water motion, similar to the typical protein surface hydration relaxation, facilitates conformation flexibility for biological recognition and function. The water near the channel center is bulklike, and the dynamics is ultrafast in less than 1 ps. These water molecules freely transport biomolecules near the channel center. The corresponding orientational relaxation at these three typical locations is well correlated with the hydration dynamics and local dynamic rigidity. These results reveal unique water structures and dynamical motions in nanoconfinements, which is critical to the understanding of nanoscopic biological activities and nanomaterial properties.     

Strong slowing down of water reorientation in mixtures of water and tetramethylurea

We use mid-infrared pump-probe spectroscopy to study the ultrafast dynamics of HDO molecules in mixtures of tetramethylurea (TMU) and water. The composition of the studied solutions ranges from pure water to an equimolar mixture of water and TMU. We find that the vibrational relaxation of the OD-stretching vibration of HDO proceeds via an intermediate level in which the molecule is more strongly hydrogen bonded than in the ground state. As the TMU concentration is increased, the lifetime of the excited state and of the intermediate increase from 1.8 to 5.2 ps and from 0.7 to 2.2 ps, respectively. The orientational relaxation data indicate that the solutions contain two types of water molecules: bulk-like molecules that have the same reorientation time constant as in the pure liquid (taurot = 2.5 ps) and molecules that are strongly immobilized (taurot > 10 ps). The immobilized water molecules turn out to be involved in the solvation of the methyl groups of the tetramethylurea molecule. The fraction of immobilized water molecules grows with increasing TMU concentration, reaching a limiting value of 60% at very high concentrations.     

Femtosecond time-resolved electronic sum-frequency generation spectroscopy: a new method to investigate ultrafast dynamics at liquid interfaces

We developed a new surface-selective time-resolved nonlinear spectroscopy, femtosecond time-resolved electronic sum-frequency generation (TR-ESFG) spectroscopy, to investigate ultrafast dynamics of molecules at liquid interfaces. Its advantage over conventional time-resolved second harmonic generation spectroscopy is that it can provide spectral information, which is realized by the multiplex detection of the transient electronic sum-frequency signal using a broadband white light continuum and a multichannel detector. We studied the photochemical dynamics of rhodamine 800 (R800) at the air/water interface with the TR-ESFG spectroscopy, and discussed the ultrafast dynamics of the molecule as thoroughly as we do for the bulk molecules with conventional transient absorption spectroscopy. We found that the relaxation dynamics of photoexcited R800 at the air/water interface exhibited three characteristic time constants of 0.32 ps, 6.4 ps, and 0.85 ns. The 0.32 ps time constant was ascribed to the lifetime of dimeric R800 in the lowest excited singlet (S(1)) state (S(1) dimer) that is directly generated by photoexcitation. The S(1) dimer dissociates to a monomer in the S(1) state (S(1) monomer) and a monomer in the ground state with this time constant. This lifetime of the S(1) dimer was ten times shorter than the corresponding lifetime in a bulk aqueous solution. The 6.4 ps and 0.85 ns components were ascribed to the decay of the S(1) monomer (as well as the recovery of the dimer in the ground state). For the 6.4 ps time constant, there is no corresponding component in the dynamics in bulk water, and it is ascribed to an interface-specific deactivation process. The 0.85 ns time constant was ascribed to the intrinsic lifetime of the S(1) monomer at the air/water interface, which is almost the same as the lifetime in bulk water. The present study clearly shows the feasibility and high potential of the TR-ESFG spectroscopy to investigate ultrafast dynamics at the interface.     

Ultrafast Vibrational Dynamics and Local Interactions of Hydrated DNA

Biochemical processes occur mainly in aqueous environments, where interactions with water molecules play a key role for both the structure and function of biomolecules. Deoxyribonucleic acid (DNA), the basic carrier of genetic information, is characterized by an equilibrium double helix structure which is held together by intermolecular hydrogen bonds between base pairs and hydrated by an environment of water molecules with fluctuating hydrogen bonds. Basic vibrational motions of hydrated DNA and the fastest changes in the DNA–water interactions and hydration geometries occur in less than 1 ps. These processes can be accessed by mapping the vibrational dynamics of DNA and water in a time‐resolved way by nonlinear ultrafast vibrational spectroscopy. Recent studies provide a detailed understanding of DNA vibrations and their dynamics, and give insight into nonequilibrium properties and structures of hydrated DNA.     

Comprehensive investigation of vibrational relaxation of non-hydrogen-bonded water molecules in liquids

The vibrational dynamics of isolated water molecules dissolved in the nonpolar organic liquids 1,2-dichloroethane (C(2)H(4)Cl(2)) and d-chloroform (CDCl(3)) have been studied using an IR pump-probe experiment with approximately 2 ps time resolution. Analyzing transient, time, and spectrally resolved data in both the OH bending and the OH stretching region, the anharmonic constants of the bending overtone (v=2) and the bend-stretch combination modes were obtained. Based on this knowledge, the relaxation pathways of single water molecules were disentangled comprehensively, proving that the vibrational energy of H(2)O molecules is relaxing following the scheme OH stretch-->OH bend overtone-->OH bend-->ground state. A lifetime of 4.8+/-0.4 ps is determined for the OH bending mode of H(2)O in 1,2-dichloroethane. For H(2)O in CDCl(3) a numerical analysis based on rate equations suggests a bending overtone lifetime of tau(020)=13+/-5 ps. The work also shows that full 2-dimensional (pump-probe) spectral resolution with access to all vibrational modes of a molecule is required for the comprehensive analysis of vibrational energy relaxation in liquids.     

Effects of concentration on the microwave dielectric spectra of aqueous urea solutions

Several models of relaxation for the dielectric spectra of aqueous urea solutions in the microwave region are compared. The spectra are shown to contain two main Debye components arising from the rotational motions of urea and water molecules. Two essentially different concentration regions in urea solutions are identified. The first is characterized by a small increase in the mobility of water molecules (τ₁ = 7.8 ps) and the existence of hydrated urea molecules (τ₂ = 19 ps). Due to the aggregation of urea molecules, the relaxation times for the latter process grow considerably in highly concentrated solutions. At the same time, faster molecular motions (τ₃ = 6 ps) are observed for water molecules.     

Hydration Dynamics of Water near an Amphiphilic Model Peptide at Low Hydration Levels: A Dielectric Relaxation Study

A dielectric relaxation study of aqueous solutions of the amphiphilic model peptide N‐acetyl‐leucine amide (NALA) at 298 K over a wide range of hydration levels is presented. The experiments range from states where water builds up several hydration layers to states where single water molecules or small water clusters are shared by several NALA molecules. The dielectric spectra reveal two modes on the 10 and 100 ps timescales. These are largely broadened with regard to the Lorentzian shape caused by simple Debye‐type relaxation, and are well described by the Kohlrausch–Williams–Watts stretched exponential function. The fast mode is assigned to water reorientation comprising bulk water as well as hydration water. Even when all water molecules are in contact with the solute, this fast component is dominant, and its mean relaxation time is retarded by less than a factor of two relative to neat water. The amplitude of the slow process is far higher than expected for the dipolar reorientation of the solute. The observations are consistent with results from molecular dynamics simulations for a similar model peptide reported in the literature. They suggest that the slow relaxation mode is mainly founded in peptide–water dipolar couplings, with some additional contribution from slowly reorienting hydration water molecules. The results are discussed with regard to the hydration dynamics of proteins and the interpretation of dielectric spectra of protein solutions.     

Hydrophobic distal pocket affects NO-heme geminate recombination dynamics in dehaloperoxidase and H64V myoglobin

The recombination dynamics of NO with dehaloperoxidase (DHP) from Amphitrite ornata following photolysis were measured by femtosecond time-resolved absorption spectroscopy. Singular value decomposition (SVD) analysis reveals two important basis spectra. The first SVD basis spectrum reports on the population of photolyzed NO molecules and has the appearance of the equilibrium difference spectrum between the deoxy and NO forms of DHP. The first basis time course has two kinetic components with time constants of tau(11) approximately 9 ps and tau(12) approximately 50 ps that correspond to geminate recombination. The fast geminate process tau(11) arises from a contact pair with the heme iron in a bound state with S = 3/2 spin. The slow geminate process tau(12) corresponds to the recombination from a more remote docking site >3 A from the heme iron with the greater barrier corresponding to a S = 5/2 spin state. The second SVD basis spectrum represents a time-dependent Soret band shift indicative of heme photophysical processes and protein relaxation with time constants of tau(21) approximately 3 ps and tau(22) approximately 17 ps, respectively. A comparison between the more rapid rate constant of the slow geminate phase in DHP-NO and horse heart myoglobin (HHMbNO) or sperm whale myoglobin (SWMbNO) suggests that protein interactions with photolyzed NO are weaker in DHP than in the wild-type MbNOs, consistent with the hydrophobic distal pocket of DHP. The slower protein relaxation rate tau(22) in DHP-NO relative to HHMbNO implies less effective trapping in the docking site of the distal pocket and is consistent with a greater yield for the fast geminate process. The trends observed for DHP-NO also hold for the H64V mutant of SWMb (H64V MbNO), consistent with a more hydrophobic distal pocket for that protein as well. We examine the influence of solution viscosity on NO recombination by varying the glycerol content in the range from 0% to 90% (v/v). The dominant effect of increasing viscosity is the increase of the rate of the slow geminate process, tau(12), coupled with a population decrease of the slow geminate component. Both phenomena are similar to the effect of viscosity on wild-type Mb due to slowing of protein relaxation resulting from an increased solution viscosity and protein surface dehydration.     

Molecular view of water dynamics near model peptides

Incoherent quasi-elastic neutron scattering (QENS) has been used to measure the dynamics of water molecules in solutions of a model protein backbone, N-acetyl-glycine-methylamide (NAGMA), as a function of concentration, for comparison with results for water dynamics in aqueous solutions of the N-acetyl-leucine-methylamide (NALMA) hydrophobic peptide at comparable concentrations. From the analysis of the elastic incoherent structure factor, we find significant fractions of elastic intensity at high and low concentrations for both solutes, which corresponds to a greater population of protons with rotational time scales outside the experimental resolution (>13 ps). The higher-concentration solutions show a component of the elastic fraction that we propose is due to water motions that are strongly coupled to the solute motions, while for low-concentration solutions an additional component is activated due to dynamic coupling between inner and outer hydration layers. An important difference between the solute types at the highest concentration studied is found from stretched exponential fits to their experimental intermediate scattering functions, showing more pronounced anomalous diffusion signatures for NALMA, including a smaller stretched exponent beta and a longer structural relaxation time tau than those found for NAGMA. The more normal water diffusion exhibited near the hydrophilic NAGMA provides experimental support for an explanation of the origin of the anomalous diffusion behavior of NALMA as arising from frustrated interactions between water molecules when a chemical interface is formed upon addition of a hydrophobic side chain, inducing spatial heterogeneity in the hydration dynamics in the two types of regions of the NALMA peptide. We place our QENS measurements on model biological solutes in the context of other spectroscopic techniques and provide both confirming as well as complementary dynamic information that attempts to give a unifying molecular view of hydration dynamics signatures near peptides and proteins.     

Ultrafast dynamics of myoglobin probed by time-resolved resonance Raman spectroscopy

Recent experimental work carried out in this laboratory on the ultrafast dynamics of myoglobin (Mb) is summarized with a stress on structural and vibrational energy relaxation. Studies on the structural relaxation of Mb following CO photolysis revealed that the structural change of heme itself, caused by CO photodissociation, is completed within the instrumental response time of the time-resolved resonance Raman apparatus used (approximately 2 ps). In contrast, changes in the intensity and frequency of the iron-histidine (Fe-His) stretching mode upon dissociation of the trans ligand were found to occur in the picosecond regime. The Fe-His band is absent for the CO-bound form, and its appearance upon photodissociation was not instantaneous, in contrast with that observed in the vibrational modes of heme, suggesting appreciable time evolution of the Fe displacement from the heme plane. The band position of the Fe-His stretching mode changed with a time constant of about 100 ps, indicating that tertiary structural changes of the protein occurred in a 100-ps range. Temporal changes of the anti-Stokes Raman intensity of the v4 and v7 bands demonstrated immediate generation of vibrationally excited heme upon the photodissociation and decay of the excited populations, whose time constants were 1.1 +/- 0.6 and 1.9 +/- 0.6 ps, respectively. In addition, the development of the time-resolved resonance Raman apparatus and prospects in this research field are described.     

Influence of hydration on protein dynamics: combining dielectric and neutron scattering spectroscopy data

Combining dielectric spectroscopy and neutron scattering data for hydrated lysozyme powders, we were able to identify several relaxation processes and follow protein dynamics at different hydration levels over a broad frequency and temperature range. We ascribe the main dielectric process to protein's structural relaxation coupled to hydration water and the slowest dielectric process to a larger scale protein's motions. Both relaxations exhibit a smooth, slightly super-Arrhenius temperature dependence between 300 and 180 K. The temperature dependence of the slowest process follows the main dielectric relaxation, emphasizing that the same friction mechanism might control both processes. No signs of a proposed sharp fragile-to-strong crossover at T approximately 220 K are observed in temperature dependences of these processes. Both processes show strong dependence on hydration: the main dielectric process slows down by six orders with a decrease in hydration from h approximately 0.37 (grams of water per grams of protein) to h approximately 0.05. The slowest process shows even stronger dependence on hydration. The third (fastest) dielectric relaxation process has been detected only in samples with high hydration ( h approximately 0.3 and higher). We ascribe it to a secondary relaxation of hydration water. The mechanism of the protein dynamic transition and a general picture of the protein dynamics are discussed.     

The Structural and Dynamical Properties of the Hydration of SNase Based on a Molecular Dynamics Simulation

The dynamics of protein–water fluctuations are of biological significance. Molecular dynamics simulations were performed in order to explore the hydration dynamics of staphylococcal nuclease (SNase) at different temperatures and mutation levels. A dynamical transition in hydration water (at ~210 K) can trigger larger-amplitude fluctuations of protein. The protein–water hydrogen bonds lost about 40% in the total change from 150 K to 210 K, while the Mean Square Displacement increased by little. The protein was activated when the hydration water in local had a comparable trend in making hydrogen bonds with protein– and other waters. The mutations changed the local chemical properties and the hydration exhibited a biphasic distribution, with two time scales. Hydrogen bonding relaxation governed the local protein fluctuations on the picosecond time scale, with the fastest time (24.9 ps) at the hydrophobic site and slowest time (40.4 ps) in the charged environment. The protein dynamic was related to the water’s translational diffusion via the relaxation of the protein–water’s H-bonding. The structural and dynamical properties of protein–water at the molecular level are fundamental to the physiological and functional mechanisms of SNase.     

Ultrafast vibrational energy relaxation of the water bridge

We report the energy relaxation of the OH stretch vibration of HDO molecules contained in an HDO:D(2)O water bridge using femtosecond mid-infrared pump-probe spectroscopy. We found that the vibrational lifetime is shorter (~630 ± 50 fs) than for HDO molecules in bulk HDO:D(2)O (~740 ± 40 fs). In contrast, the thermalization dynamics following the vibrational relaxation are much slower (~1.5 ± 0.4 ps) than in bulk HDO:D(2)O (~250 ± 90 fs). These differences in energy relaxation dynamics strongly indicate that the water bridge and bulk water differ on a molecular scale.     

Multiple time scales in solvation dynamics of DNA in aqueous solution: the role of water, counterions, and cross-correlations

Recent time domain experiments have explored solvation dynamics of a probe located inside a DNA duplex, in an effort to gain information, e.g., on the dynamics of water molecules in the DNA major and minor grooves and their environment. Multiple time constants in the range of a few picoseconds to several nanoseconds were obtained. We have carried out 15 ns long atomistic molecular dynamics simulations to study the solvation dynamics of bases of a 38 base-pair long DNA duplex in an aqueous solution containing counterions. We have computed the energy-energy time correlation function (TCF) of the four individual bases (A, T, G, and C) to characterize the solvation dynamics. All the TCFs display highly nonexponential decay with time. When the trajectories are analyzed with 100 fs time resolution, the TCF of each base shows initial ultrafast decay (with tau1 approximately equal 60-80 fs) followed by two intermediate components (tau2 approximately equal 1 ps, tau3 approximately equal 20-30 ps), in near complete agreement with a recent time domain experiment on DNA solvation. Interestingly, the solvation dynamics of each of the four different nucleotide bases exhibit rather similar time scales. To explore the existence of slow relaxation at longer times reported recently in a series of experiments, we also analyzed the solvation TCFs calculated with longer time trajectories and with a larger time resolution of 1 ps. In this case, an additional slow component with a time constant of the order of 250 ps is observed. Through an analysis of partial solvation TCFs, we find that the slow decay originates mainly from the interaction of the nucleotides with the dipolar water molecules and the counterions. An interesting negative cross-correlation between water and counterions is observed, which makes an important contribution to relaxation at intermediate to longer times.     

Sensitivity of hydrogen bond lifetime dynamics to the presence of ethanol at the interface of a phospholipid bilayer

Atomistic molecular dynamics simulations of a fully hydrated liquid crystalline lamellar phase of a dimyrystoylphosphatidylcholine lipid bilayer containing ethanol at 1:1 composition as well as of the pure lamellar phase of the bilayer have been performed. Detailed analyses have been carried out to investigate the effects of ethanol, if any, on the lifetime dynamics of lipid-water and water-water hydrogen bonds in the hydration layer of the lipid headgroups. The nonexponential hydrogen bond lifetime correlation functions have been analyzed by using the formalism of Luzar and Chandler, which allowed the identification of the bound states at the bilayer interface and the quantification of the dynamic equilibrium between the bound and the free water molecules, in terms of time-dependent relaxation rates. The calculations show that the overall relaxation of phosphate-water hydrogen bonds is faster in the presence of ethanol. Studies of the residence time and the number fluctuation of the hydration layer water molecules reveal that the presence of ethanol molecules decreases the rigidity of the lipid hydration layer.     

Structure and Dynamics of Water/Methanol Mixtures at Hydroxylated Silica Interfaces Relevant to Chromatography

The spectroscopy and dynamics of water/methanol (MeOH) mixtures at hydroxylated silica surfaces is investigated from atomistic simulations. The particular focus is on how the structural dynamics of MeOH changes when comparing surface‐bound and MeOH in the bulk. From analyzing the frequency frequency correlation functions it is found that the dynamics on the picosecond time scale differs by almost a factor of two. While the relaxation time is 2.0 ps for MeOH in the bulk solvent it is considerably slowed‐down to 3.5 ps for surface‐bound MeOH. Surface‐adsorbed MeOH molecules reside there for several nanoseconds and their H‐bonds are strongly oriented towards the surface‐OH groups. These results are of particular relevance for chromatographic systems where the solvent may play a central role in their function. The present simulations suggest that surface‐sensitive spectroscopic techniques should be useful in better characterizing such heterogeneous systems and provide detailed insight into solvent dynamics and structure relevant in chromatographic applications.