Femtosecond infrared (IR) pump probe and dynamic hole burning experiments were used to examine the ultrafast response of the modes in the 1600-1700 cm -1 region (the so-called amide I modes) of N-methylacetamide (NMA) and three small globular peptides, apamin, scyllatoxin, and bovine pancreatic trypsin inhibitor (BPTI). A value of 16 cm -1 was found for the anharmonicity of the amide I vibration. Vibrational relaxation of the amide I modes of all investigated peptides occurs in ca. 1.2 ps. An even faster value of 450 fs is obtained for NMA, a model for the peptide unit. The vibrational relaxation is dominated by intramolecular energy redistribution (IVR) and reflects an intrinsic property of the peptide group in any environment. Dynamic hole burning experiments with a narrow band pump pulse which selectively excites only a subset of the amide I eigenstates reveal that energy migration between different amide I states is slow compared with vibrational relaxation. Two-dimensional pump-probe (2D-IR) spectra that display the spectral response of the amide I band as a function of the frequency of the narrow band pump pulse show that the amide I states are nevertheless delocalized along the peptide backbone. A simple excitonic coupling model describes the nonlinear pump-probe spectrum, and it reproduces the experimental 2D-IR spectra. It is estimated that the accessible peptide excitons are delocalized over a length of ca. 8 Å.
A method is introduced for subdiffraction imaging that accumulates points by collisional flux. It is based on targeting the surface of objects by fluorescent probes diffusing in the solution. Because the flux of probes at the object is essentially constant over long time periods, the examination of an almost unlimited number of individual probe molecules becomes possible. Each probe that hits the object and that becomes immobilized is located with high precision by replacing its point-spread function by a point at its centroid. Images of lipid bilayers, contours of these bilayers, and large unilamellar vesicles are shown. A spatial resolution of Ϸ25 nm is readily achieved. The ability of the method to effect rapid nanoscale imaging and spatial resolution below Rayleigh criterion and without the necessity for labeling with fluorescent probes is proven.diffusion controlled ͉ fluorescence ͉ single molecule F or the purpose of visualizing biological processes and structures, it is essential to develop new methods that can determine the spatial locations of molecules with the highest possible precision. The submolecular methods of spatial resolution such as x-ray and electron topography, atomic force microscopy, and near-field optical microscopy have contributed enormously toward this goal. However, new approaches that are not invasive to biological objects and are accessible to bulk, as well as surface structures, could have a significant impact and could address many new questions in the life sciences (1). Optical methods based on fluorescence detection do have single-molecule sensitivity and can be arranged to be nondestructive even when performed in natural environments, incorporating such complex and sensitive structures as living cells. In principle, optical responses can probe the entire 3D space. Although diffraction limits the spatial resolution of optical methods, many approaches have been developed recently that allow distance measurements on scales that are much shorter than the wavelength of the light.It is possible to specify the location of a single molecule with very high precision from measurements of its fluorescence by fitting the emitted intensity distribution to the 2D spatial parameters of the point-spread function (PSF). This approach has been shown to locate emitters with Ϸ1-nm precision (2). The distance between two optically different molecules can be estimated by FRET methods that can serve as molecular rulers at nanometer accuracy (3). Two molecules emitting light at different frequencies have also been resolved by means of PSF measurements. Even for molecules having the same spectra, the process of photobleaching causes the fluorescence source to be switched between molecules. When combined with a PSF measurement, this approach has been able to distinguish pairs (4) and quartets (5) of molecules with nanometer precision. Another approach has been to analyze the blinking trajectory of the emission of quantum dots to distinguish between a single dot and groups of them clustered on the nanom...
We describe a new four-wave rectification method for the generation of intense, ultrafast terahertz (THz) pulses from gases. The fundamental and second-harmonic output of an amplified Ti:sapphire laser is focused to a peak intensity of ~5x10(14)W/cm (2) . Under these conditions, peak THz fields estimated at 2 kV/cm have been observed; the measured power spectrum peaks near 2 THz. Phase-dependent measurements show that this is a coherent process and is sensitive to the relative phases of the fundamental and second-harmonic pulses. Comparable THz signals have been observed from nitrogen and argon as well as from air.
A form of two-dimensional (2D) vibrational spectroscopy, which uses two ultrafast IR laser pulses, is used to examine the structure of a cyclic penta-peptide in solution. Spectrally resolved cross peaks occur in the off-diagonal region of the 2D IR spectrum of the amide I region, analogous to those in 2D NMR spectroscopy. These cross peaks measure the coupling between the different amide groups in the structure. Their intensities and polarizations relate directly to the three-dimensional structure of the peptide. With the help of a model coupling Hamiltonian, supplemented by density functional calculations, the spectra of this penta-peptide can be regenerated from the known solution phase structure. This 2D-IR measurement, with an intrinsic time resolution of less than 1 ps, could be used in all time regimes of interest in biology.The three-dimensional (3D) structure of peptides and proteins and their fluctuations are essential properties responsible for the extremely high specificity of biological reactions. Progress in understanding biological processes such as enzyme reactions originates from the detailed knowledge of the secondary, tertiary, and quarternary structures of the participating biomolecules. Two major spectroscopic techniques are responsible for this development: x-ray scattering, which maps out the electron density of the molecule, and two-dimensional (2D)-NMR spectroscopy (1-3), which can measure the distances between pairs of protons. The next step must be the determination of structures in motion over a wide range of time scales. We believe that multidimensional IR spectroscopy can address this new challenge.The IR spectra of the amide transitions provide information about secondary structural motifs of proteins and peptides. The so-called amide I band, which consists of mostly the stretching motion of the peptide backbone CϭO groups, is a strong IR absorber, which is spectrally isolated from other vibrational modes such as those from amino acid side groups. The amide I states can be viewed as vibrational excitons (4, 5) with the individual peptide groups considered as separated but coupled units. The coupling could be either through-space, such as multipole or even dipole-dipole interaction as proposed by Krimm and Bandekar (4), or through-bond, involving charge shifts and kinematic coupling via the C ␣ atoms of the backbone. The states, which have one excitation present in the whole assembly, can be interrogated by conventional (linear) IR absorption spectroscopy, but the information obtained is insufficient to determine the coupling Hamiltonian, from which a structure might be deduced. More information is available from nonlinear third-order spectroscopic techniques (6) such as the 2D experiment presented here. In a separated system picture both the one-exciton and two-exciton states contribute to the nonlinear IR signal in such a way that all couplings between the separate amide units are available, and in principle a 3D structure of the peptide can be deduced.The success of NMR sp...
The involvement of chemical exchange in 2D IR heterodyne echo spectroscopy is characterized through the hydrogen-bond exchange between CH3OH and the CN of CH3CN. The exchange dynamics on the hydrogen-bond potential surfaces associated with different quantum states of the high-frequency CN stretching mode contributes to strong cross peaks between CN groups in two different chemical configurations and provides firm evidence of the hydrogen exchange between them. In analogy with NMR, the chemical exchange is seen in both slow and dynamic regimes. The relative magnitudes of the cross peaks at various population periods measure the picosecond regime time constants for H-bond transfer, whereas the temperature dependences indicate that the activation energy for the exchange from the H-bonded state to the free state is Ϸ6.2 kJ⅐mol ؊1 . The results suggest that the hydrogenbond dynamics is very similar in both vibrational quantum states of CN, suggesting that this stretching mode is not strongly coupled to the H-bond breaking reaction coordinate. The likely manifestations of chemical exchange in 2D IR experiments are discussed.photon echo ͉ nonlinear spectroscopy ͉ dynamic exchange ͉ liquid V ibrational spectroscopy has provided important experimental access to the microscopic aspects of ultrafast hydrogenbond processes in complex systems. The dynamics of OOH or OOD stretching vibrational modes in water (1-10) or alcohol oligomers (11), the vibrations of molecular (12, 13) and atomic (14) aqueous ions, and the N-H and CAO stretching modes, including those in peptides or proteins in water (15-23) and alcohols (24), are very sensitive to and correlated with the structural and dynamical properties of hydrogen bonds. In principle, the shape of the conventional IR absorption spectrum provides information on the equilibrium dynamics of a hydrogen-bonded system. However, in many cases, the line shapes are determined by population lifetimes and spectral diffusion processes that often cannot be reduced to the unique set of parameters needed to describe the frequencies and amplititudes of coupled solvent nuclear motions. With the help of multidimensional nonlinear spectroscopic techniques in the IR spectral region, it has become possible to probe these hydrogen-bond dynamics and extract more details on the structures and dynamics with high time resolution (25). Dynamical information on the O-H, O-D, and N-H stretching modes of intermolecular hydrogen-bonded systems such as alcohols, water, and amides has been obtained in the form of vibrational lifetimes, energy transfer, hydrogen-bond breaking and reforming rates, and the time dependence of spectral diffusion. In addition, the motions of hydrogen bonds in peptides and liquids are of importance in many chemical and biological processes. In protein secondary structures, the amide carbonyl group is very often involved in hydrogen bonding to water, N-H groups, or both. Much remains to be learned from experiments on the vibrational populations and coherences about the dynamics of thes...
Two-dimensional infrared spectra of peptides are introduced that are the direct analogues of two-and three-pulse multiple quantum NMR. Phase matching and heterodyning are used to isolate the phase and amplitudes of the electric fields of vibrational photon echoes as a function of multiple pulse delays. Structural information is made available on the time scale of a few picoseconds. Line narrowed spectra of acyl-proline-NH2 and cross peaks implying the coupling between its amide-I modes are obtained, as are the phases of the various contributions to the signals. Solvent-sensitive structural differences are seen for the dipeptide. The methods show great promise to measure structure changes in biology on a wide range of time scales.
Articles you may be interested inSite-specific vibrational dynamics of the CD3ζ membrane peptide using heterodyned two-dimensional infrared photon echo spectroscopy Coupling and orientation between anharmonic vibrations characterized with two-dimensional infrared vibrational echo spectroscopyThe stimulated infrared photon echo of N-methylacetamide-D ͓NMAD; CH 3 ͑CO͒ND͑CH 3 )] was measured and used to determine the vibrational frequency correlation function. The correlation function was modeled as a single exponential plus a constant, and it was found that most of the NMAD vibrational frequency distribution is motionally narrowed with a pure dephasing time of 1.12 ps. The two-dimensional infrared ͑2D IR͒ spectrum of NMAD was also obtained by heterodyning the echo field with a weak local oscillator pulse. The real and imaginary portions of the 2D IR spectrum exhibit multiple peaks due to ϭ0-1 and 1-2 coherences that are excited, which are not resolved in the absolute magnitude of the 2D IR spectrum. Using the correlation function determined from the stimulated photon echo, the 2D IR spectrum was accurately simulated. Resolution enhancement of the 2D IR spectrum was performed by manipulating the photon echo field with window functions. The enhanced experimental and simulated 2D IR spectra are dramatically narrowed.
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