Ultrafast dynamics of myoglobin without the distal histidine: stimulated vibrational echo experiments and molecular dynamics simulations

Ultrafast protein dynamics of the CO adduct of a myoglobin mutant with the polar distal histidine replaced by a nonpolar valine (H64V) have been investigated by spectrally resolved infrared stimulated vibrational echo experiments and molecular dynamics (MD) simulations. In aqueous solution at room temperature, the vibrational dephasing rate of CO in the mutant is reduced by approximately 50% relative to the native protein. This finding confirms that the dephasing of the CO vibration in the native protein is sensitive to the interaction between the ligand and the distal histidine. The stimulated vibrational echo observable is calculated from MD simulations of H64V within a model in which vibrational dephasing is driven by electrostatic forces. In agreement with experiment, calculated vibrational echoes show slower dephasing for the mutant than for the native protein. However, vibrational echoes calculated for H64V do not show the quantitative agreement with measurements demonstrated previously for the native protein.     

Temperature dependent equilibrium native to unfolded protein dynamics and properties observed with IR absorption and 2D IR vibrational echo experiments

Dynamic and structural properties of carbonmonoxy (CO)-coordinated cytochrome c(552) from Hydrogenobacter thermophilus (Ht-M61A) at different temperatures under thermal equilibrium conditions were studied with infrared absorption spectroscopy and ultrafast two-dimensional infrared (2D IR) vibrational echo experiments using the heme-bound CO as the vibrational probe. Depending on the temperature, the stretching mode of CO shows two distinct bands corresponding to the native and unfolded proteins. As the temperature is increased from low temperature, a new absorption band for the unfolded protein grows in and the native band decreases in amplitude. Both the temperature-dependent circular dichroism and the IR absorption area ratio R(A)(T), defined as the ratio of the area under the unfolded band to the sum of the areas of the native and unfolded bands, suggest a two-state transition from the native to the unfolded protein. However, it is found that the absorption spectrum of the unfolded protein increases its inhomogeneous line width and the center frequency shifts as the temperature is increased. The changes in line width and center frequency demonstrate that the unfolding does not follow simple two-state behavior. The temperature-dependent 2D IR vibrational echo experiments show that the fast dynamics of the native protein are virtually temperature independent. In contrast, the fast dynamics of the unfolded protein are slower than those of the native protein, and the unfolded protein fast dynamics and at least a portion of the slower dynamics of the unfolded protein change significantly, becoming faster as the temperature is raised. The temperature dependence of the absorption spectrum and the changes in dynamics measured with the 2D IR experiments confirm that the unfolded ensemble of conformers continuously changes its nature as unfolding proceeds, in contrast to the native state, which displays a temperature-independent distribution of structures.     

Time-dependent fifth-order bands in nominally third-order 2D IR vibrational echo spectra

Progress in the field of 2D IR vibrational spectroscopy has been bolstered by the production of intense mid-IR laser pulses. As higher-energy pulses are employed, a concomitant increase occurs in the likelihood of fifth-order contributions to the 2D IR spectra. We report the appearance of fifth-order signals in 2D IR spectra of CO bound to the active site of the enzyme cytochrome P450(cam) with the substrate norcamphor. Two bands with novel time dependences, one on the diagonal and one off-diagonal, are not accounted for by normal third-order interactions. These bands are associated with a ν = 1-2 vibrational transition frequency. Both bands decay to 0 and then grow back in with opposite sign. The diagonal band is positive at short time, decays to 0, reappears with negative sign, before eventually decaying to 0. The off-diagonal band is negative at short time, decays to 0, reappears positive, and then decays to 0. The appearance and time dependence of these bands are characterized. Understanding these fifth-order bands is useful because they may be misidentified with time-dependent bands that arise from other processes, such as chemical exchange, vibrational coupling, or energy transfer. The presence and unusual time dependences of the fifth-order bands are reproduced with model calculations that account for the fact that vibrational relaxation from the ν = 2 to 1 level is approximately a factor of 2 faster than that from the ν = 1 to 0 level.     

Probing dynamics of complex molecular systems with ultrafast 2D IR vibrational echo spectroscopy

Ultrafast 2D IR vibrational echo spectroscopy is described and a number of experimental examples are given. Details of the experimental method including the pulse sequence, heterodyne detection, and determination of the absorptive component of the 2D spectrum are outlined. As an initial example, the 2D spectrum of the stretching mode of CO bound to the protein myoglobin (MbCO) is presented. The time dependence of the 2D spectrum of MbCO, which is caused by protein structural evolution, is presented and its relationship to the frequency-frequency correlation function is described and used to make protein structural assignments based on comparisons to molecular dynamics simulations. The 2D vibrational echo experiments on the protein horseradish peroxidase are presented. The time dependence of the 2D spectra of the enzyme in the free form and with a substrate bound at the active site are compared and used to examine the influence of substrate binding on the protein's structural dynamics. The application of 2D vibrational echo spectroscopy to the study of chemical exchange under thermal equilibrium conditions is described. 2D vibrational echo chemical exchange spectroscopy is applied to the study of formation and dissociation of organic solute-solvent complexes and to the isomerization around a carbon-carbon single bond of an ethane derivative.     

Dynamics of proteins encapsulated in silica sol-gel glasses studied with IR vibrational echo spectroscopy

Spectrally resolved infrared stimulated vibrational echo spectroscopy is used to measure the fast dynamics of heme-bound CO in carbonmonoxy-myoglobin (MbCO) and -hemoglobin (HbCO) embedded in silica sol-gel glasses. On the time scale of approximately 100 fs to several picoseconds, the vibrational dephasing of the heme-bound CO is measurably slower for both MbCO and HbCO relative to that of aqueous protein solutions. The fast structural dynamics of MbCO, as sensed by the heme-bound CO, are influenced more by the sol-gel environment than those of HbCO. Longer time scale structural dynamics (tens of picoseconds), as measured by the extent of spectral diffusion, are the same for both proteins encapsulated in sol-gel glasses compared to that in aqueous solutions. A comparison of the sol-gel experimental results to viscosity-dependent vibrational echo data taken on various mixtures of water and fructose shows that the sol-gel-encapsulated MbCO exhibits dynamics that are the equivalent of the protein in a solution that is nearly 20 times more viscous than bulk water. In contrast, the HbCO dephasing in the sol-gel reflects only a 2-fold increase in viscosity. Attempts to alter the encapsulating pore size by varying the molar ratio of silane precursor to water (R value) used to prepare the sol-gel glasses were found to have no effect on the fast or steady-state spectroscopic results. The vibrational echo data are discussed in the context of solvent confinement and protein-pore wall interactions to provide insights into the influence of a confined environment on the fast structural dynamics experienced by a biomolecule.     

The influence of aqueous versus glassy solvents on protein dynamics: vibrational echo experiments and molecular dynamics simulations

Spectrally resolved infrared stimulated vibrational echo measurements are used to measure the vibrational dephasing of the CO stretching mode of carbonmonoxy-hemoglobin (HbCO), a myoglobin mutant (H64V), and a bacterial cytochrome c(552) mutant (Ht-M61A) in aqueous solution and trehalose glasses. The vibrational dephasing of the heme-bound CO is significantly slower for all three proteins embedded in trehalose glasses compared to that of aqueous protein solutions. All three proteins exhibit persistent but notably slower spectral diffusion when the protein surface is fixed by the glassy solvent. Frequency-frequency correlation functions (FFCFs) of the CO are extracted from the vibrational echo data to reveal that the structural dynamics, as sensed by the CO, of the three proteins in trehalose and aqueous solution are dominated by fast (tens of femtoseconds), motionally narrowed fluctuations. MD simulations of H64V in dynamic and "static" water are presented as models of the aqueous and glassy environments. FFCFs are calculated from the H64V simulations and qualitatively reproduce the important features of the experimentally extracted FFCFs. The suppression of long time scale (picoseconds to tens of picoseconds) frequency fluctuations (spectral diffusion) in the glassy solvent is the result of a damping of atomic displacements throughout the protein structure and is not limited to structural dynamics that occur only at the protein surface. The analysis provides evidence that some dynamics are coupled to the hydration shell of water, supporting the idea that the bioprotection offered by trehalose is due to its ability to immobilize the protein surface through a thin, constrained layer of water.     

Protein dynamics in cytochrome P450 molecular recognition and substrate specificity using 2D IR vibrational echo spectroscopy

Cytochrome (cyt) P450s hydroxylate a variety of substrates that can differ widely in their chemical structure. The importance of these enzymes in drug metabolism and other biological processes has motivated the study of the factors that enable their activity on diverse classes of molecules. Protein dynamics have been implicated in cyt P450 substrate specificity. Here, 2D IR vibrational echo spectroscopy is employed to measure the dynamics of cyt P450(cam) from Pseudomonas putida on fast time scales using CO bound at the active site as a vibrational probe. The substrate-free enzyme and the enzyme bound to both its natural substrate, camphor, and a series of related substrates are investigated to explicate the role of dynamics in molecular recognition in cyt P450(cam) and to delineate how the motions may contribute to hydroxylation specificity. In substrate-free cyt P450(cam), three conformational states are populated, and the structural fluctuations within a conformational state are relatively slow. Substrate binding selectively stabilizes one conformational state, and the dynamics become faster. Correlations in the observed dynamics with the specificity of hydroxylation of the substrates, the binding affinity, and the substrates' molecular volume suggest that motions on the hundreds of picosecond time scale contribute to the variation in activity of cyt P450(cam) toward different substrates.     

Dynamics of hemoglobin in human erythrocytes and in solution: influence of viscosity studied by ultrafast vibrational echo experiments

Ultrafast spectrally resolved stimulated vibrational echo experiments are used to measure the vibrational dephasing of the CO stretching mode of hemoglobin-CO (HbCO) inside living human erythrocytes (red blood cells), in liquid solutions, and in a glassy matrix. A method is presented to overcome the adverse impact on the vibrational echo signal from the strong light scattering caused by the cells. The results from the cytoplasmic HbCO are compared to experiments on aqueous HbCO samples prepared in different buffers, solutions containing low and high concentrations of glycerol, and in a solid trehalose matrix. Measurements are also presented that provide an accurate determination of the viscosity at the very high Hb concentration that is found inside the cells. It is demonstrated that the dynamics of the protein, as sensed by the CO ligand, are the same inside the erythrocytes and in aqueous solution and are independent of the viscosity. In solutions that are predominantly glycerol, the dynamics are modified somewhat but are still independent of viscosity. The experiments in trehalose give the dynamics at infinite viscosity and are used to separate the viscosity-dependent dynamics from the viscosity-independent dynamics. Although the HbCO dynamics are the same in the red blood cell and in the equivalent aqueous solutions, differences in the absorption spectra show that the distribution of a protein's equilibrium substates is sensitive to small pH differences.     

2D IR photon echo of azido-probes for biomolecular dynamics

The vibrations in the azido-, N(3), asymmetric stretching region of 2'-azido-2'-deoxyuridine (N(3)dU) are examined by two-dimensional infrared spectroscopy. In water and tetrahydrofuran (THF), the spectra display a single sharp diagonal peak that shows solvent sensitivity. The frequency-frequency correlation time in water is 1.5 ps, consistent with H-bond making and breaking dynamics. The 2D IR spectrum is reproduced for N(3)dU in water based on a model correlation function and known linear response functions. Its large extinction coefficient, vibrational frequency outside the protein and nucleic acid IR absorption, and sensitivity to water dynamics render -N(3) a very useful probe for 2D IR and other nonlinear IR studies: its signal is ca. 100 times that of nitriles.     

Ultrafast two-dimensional infrared vibrational echo chemical exchange experiments and theory

Ultrafast two-dimensional (2D) infrared vibrational echo experiments and theory are used to examine chemical exchange between solute-solvent complexes and the free solute for the solute phenol and three solvent complex partners, p-xylene, benzene, and bromobenzene, in mixed solvents of the partner and CCl4. The experiments measure the time evolution of the 2D spectra of the hydroxyl (OD) stretching mode of the phenol. The time-dependent 2D spectra are analyzed using time-dependent diagrammatic perturbation theory with a model that includes the chemical exchange (formation and dissociation of the complexes), spectral diffusion of both the complex and the free phenol, orientational relaxation of the complexes and free phenol, and the vibrational lifetimes. The detailed calculations are able to reproduce the experimental results and demonstrate that a method employed previously that used a kinetic model for the volumes of the peaks is adequate to extract the exchange kinetics. The current analysis also yields the spectral diffusion (time evolution of the dynamic line widths) and shows that the spectral diffusion is significantly different for phenol complexes and free phenol.     

Dynamics of water probed with vibrational echo correlation spectroscopy

Vibrational echo correlation spectroscopy experiments on the OD stretch of dilute HOD in H(2)O are used to probe the structural dynamics of water. A method is demonstrated for combining correlation spectra taken with different infrared pulse bandwidths (pulse durations), making it possible to use data collected from many experiments in which the laser pulse properties are not identical. Accurate measurements of the OD stretch anharmonicity (162 cm(-1)) are presented and used in the data analysis. In addition, the recent accurate determination of the OD vibrational lifetime (1.45 ps) and the time scale for the production of vibrational relaxation induced broken hydrogen bond "photoproducts" ( approximately 2 ps) aid in the data analysis. The data are analyzed using time dependent diagrammatic perturbation theory to obtain the frequency time correlation function (FTCF). The results are an improved FTCF compared to that obtained previously with vibrational echo correlation spectroscopy. The experimental data and the experimentally determined FTCF are compared to calculations that employ a polarizable water model (SPC-FQ) to calculate the FTCF. The SPC-FQ derived FTCF is much closer to the experimental results than previously tested nonpolarizable water models which are also presented for comparison.     

Dynamics of nanoscopic water: vibrational echo and infrared pump-probe studies of reverse micelles

The dynamics of water in nanoscopic pools 1.7-4.0 nm in diameter in AOT reverse micelles were studied with ultrafast infrared spectrally resolved stimulated vibrational echo and pump-probe spectroscopies. The experiments were conducted on the OD hydroxyl stretch of low-concentration HOD in the H2O, providing a direct examination of the hydrogen-bond network dynamics. Pump-probe experiments show that the vibrational lifetime of the OD stretch mode increases as the size of the reverse micelle decreases. These experiments are also sensitive to hydrogen-bond dissociation and reformation dynamics, which are observed to change with reverse micelle size. Spectrally resolved vibrational echo data were obtained at several frequencies. The vibrational echo data are compared to data taken on bulk water and on a 6 M NaCl solution, which is used to examine the role of ionic strength on the water dynamics in reverse micelles. Two types of vibrational echo measurements are presented: the vibrational echo decays and the vibrational echo peak shifts. As the water nanopool size decreases, the vibrational echo decays become slower. Even the largest nanopool (4 nm, approximately 1000 water molecules) has dynamics that are substantially slower than bulk water. It is demonstrated that the slow dynamics in the reverse micelle water nanopools are a result of confinement rather than ionic strength. The data are fit using time-dependent diagrammatic perturbation theory to obtain the frequency-frequency correlation function (FFCF) for each reverse micelle. The results are compared to the FFCF of water and show that the largest differences are in the slowest time scale dynamics. In bulk water, the slowest time scale dynamics are caused by hydrogen-bond network equilibration, i.e., the making and breaking of hydrogen bonds. For the smallest nanopools, the longest time scale component of the water dynamics is approximately 10 times longer than the dynamics in bulk water. The vibrational echo data for the smallest reverse micelle displays a dependence on the detection wavelength, which may indicate that multiple ensembles of water molecules are being observed.     

Conformational dynamics and stability of HP35 studied with 2D IR vibrational echoes

Two-dimensional infrared (2D IR) vibrational echo spectroscopy was used to measure the fast dynamics of two variants of chicken villin headpiece 35 (HP35). The CN of cyanophenylalanine residues inserted in the hydrophobic core were used as a vibrational probe. Experiments were performed on both singly (HP35-P) and doubly CN-labeled peptide (HP35-P(2)) within the wild-type sequence, as well as on HP-35 containing a singly labeled cyanophenylalanine and two norleucine mutations (HP35-P NleNle). There is a remarkable similarity between the dynamics measured in singly and doubly CN-labeled HP35, demonstrating that the presence of an additional CN vibrational probe does not significantly alter the dynamics of the small peptide. The substitution of two lysine residues by norleucines markedly improves the stability of HP35 by replacing charged with nonpolar residues, stabilizing the hydrophobic core. The results of the 2D IR experiments reveal that the dynamics of HP35-P are significantly faster than those of HP35-P NleNle. These observations suggest that the slower structural fluctuations in the hydrophobic core, indicating a more tightly structured core, may be an important contributing factor to HP35-P NleNle's increased stability.     

Anharmonic vibrational modes of nucleic acid bases revealed by 2D IR spectroscopy

Polarization-dependent two-dimensional infrared (2D IR) spectra of the purine and pyrimadine base vibrations of five nucleotide monophosphates (NMPs) were acquired in D(2)O at neutral pH in the frequency range 1500-1700 cm(-1). The distinctive cross-peaks between the ring deformations and carbonyl stretches of NMPs indicate that these vibrational modes are highly coupled, in contrast with the traditional peak assignment, which is based on a simple local mode picture such as C═O, C═N, and C═C double bond stretches. A model of multiple anharmonically coupled oscillators was employed to characterize the transition energies, vibrational anharmonicities and couplings, and transition dipole strengths and orientations. No simple or intuitive structural correlations are found to readily assign the spectral features, except in the case of guanine and cytosine, which contain a single local CO stretching mode. To help interpret the nature of these vibrational modes, we performed density functional theory (DFT) calculations and found that multiple ring vibrations are coupled and delocalized over the purine and pyrimidine rings. Generally, there is close correspondence between the experimental and computational results, provided that the DFT calculations include explicit waters solvating hydrogen-bonding sites. These results provide direct experimental evidence of the delocalized nature of the nucleotide base vibrations via a nonperturbative fashion and will serve as building blocks for constructing a structure-based model of DNA and RNA vibrational spectroscopy.     

Detection of electronic and vibrational coherences in molecular systems by 2D electronic photon echo spectroscopy

Two-dimensional optical photon echo spectra are simulated for model systems which exhibit vibrational, electronic and a combination of electronic and vibrational coherent dynamics. The coherent motion manifests itself as periodic beatings of the spectrum cross-peak intensity with the population time. The intensity modulations are compared to evolution of the excited-state population and coordinate expectation value. The advantageous capabilities of the technique as well as possible difficulties in spectra interpretations are outlined. Possibilities for distinguishing electronic and vibrational coherences are discussed.     

Fe vibrational spectroscopy of myoglobin and cytochrome f

The Fe vibrational density of states (VDOS) has been determined for the heme proteins deoxymyoglobin, metmyoglobin, and cytochrome f in the oxidized and reduced states, using nuclear resonance vibrational spectroscopy (NRVS). For cytochrome f in particular, the NRVS spectrum is compared with multiwavelength resonance Raman spectra to identify those Raman modes with significant Fe displacement. Modes not seen by Raman due to optical selection rules appear in the NRVS spectrum. The mean Fe force constant extracted from the VDOS illustrates how Fe dynamics varies among these four monoheme proteins, and is correlated with oxidation and spin state trends seen in model heme compounds. The protein's contribution to Fe motion is dominant at low frequencies, where coupling to the backbone tightly constrains Fe displacements in cytochrome f, in contrast to enhanced heme flexibility in myoglobin.     

Differentiation of beta-sheet-forming structures: ab initio-based simulations of IR absorption and vibrational CD for model peptide and protein beta-sheets.

Ab initio quantum mechanical computations of force fields (FF) and atomic polar and axial tensors (APT and AAT) were carried out for triamide strands Ac-A-A-NH-CH(3) clustered into single-, double-, and triple-strand beta-sheet-like conformations. Models with phi, psi, and omega angles constrained to values appropriate for planar antiparallel and parallel as well as coiled antiparallel (two-stranded) and twisted antiparallel and parallel sheets were computed. The FF, APT, and AAT values were transferred to corresponding larger oligopeptide beta-sheet structures of up to five strands of eight residues each, and their respective IR and vibrational circular dichroism (VCD) spectra were simulated. The antiparallel planar models in a multiple-stranded assembly give a unique IR amide I spectrum with a high-intensity, low-frequency component, but they have very weak negative amide I VCD, both reflecting experimental patterns seen in aggregated structures. Parallel and twisted beta-sheet structures do not develop a highly split amide I, their IR spectra all being similar. A twist in the antiparallel beta-sheet structure leads to a significant increase in VCD intensity, while the parallel structure was not as dramatically affected by the twist. The overall predicted VCD intensity is quite weak but predominantly negative (amide I) for all conformations. This intrinsically weak VCD can explain the high variation seen experimentally in beta-forming peptides and proteins. An even larger variation was predicted in the amide II VCD, which had added complications due to non-hydrogen-bonded residues on the edges of the model sheets.     

Dynamics of the C(1D)+D2 reaction: a comparison of crossed molecular-beam experiments with quasiclassical trajectory and accurate statistical calculations

In this paper we report a combined experimental and theoretical study on the dynamics of the insertion reaction C((1)D)+D(2) at 15.5 kJ mol(-1) collision energy. Product angular and velocity distributions have been obtained in crossed beam experiments and quasiclassical trajectory (QCT) and rigorous statistical calculations have been performed on the recent and accurate ab initio potential energy surface of Bussery-Honvault, Honvault, and Launay at the energy of the experiment. The molecular-beam results have been simulated using the theoretical calculations. Good agreement between experiment and both QCT and statistical predictions is found.     

Native and unfolded cytochrome c--comparison of dynamics using 2D-IR vibrational echo spectroscopy

Unfolded vs native CO-coordinated horse heart cytochrome c (h-cyt c) and a heme axial methionine mutant cyt c552 from Hydrogenobacter thermophilus ( Ht-M61A) are studied by IR absorption spectroscopy and ultrafast 2D-IR vibrational echo spectroscopy of the CO stretching mode. The unfolding is induced by guanidinium hydrochloride (GuHCl). The CO IR absorption spectra for both h-cyt c and Ht-M61A shift to the red as the GuHCl concentration is increased through the concentration region over which unfolding occurs. The spectra for the unfolded state are substantially broader than the spectra for the native proteins. A plot of the CO peak position vs GuHCl concentration produces a sigmoidal curve that overlays the concentration-dependent circular dichroism (CD) data of the CO-coordinated forms of both Ht-M61A and h-cyt c within experimental error. The coincidence of the CO peak shift curve with the CD curves demonstrates that the CO vibrational frequency is sensitive to the structural changes induced by the denaturant. 2D-IR vibrational echo experiments are performed on native Ht-M61A and on the protein in low- and high-concentration GuHCl solutions. The 2D-IR vibrational echo is sensitive to the global protein structural dynamics on time scales from subpicosecond to greater than 100 ps through the change in the shape of the 2D spectrum with time (spectral diffusion). At the high GuHCl concentration (5.1 M), at which Ht-M61A is essentially fully denatured as judged by CD, a very large reduction in dynamics is observed compared to the native protein within the approximately 100 ps time window of the experiment. The results suggest the denatured protein may be in a glassy-like state involving hydrophobic collapse around the heme.