Correlations of structure and rates of energy transfer for through-bond energy-transfer cassettes.

Fluorescent DNA-labeling cassettes are designed to have a common absorbing chromophore matched to a single exciting laser wavelength, but up to four different emitters. Experiments reported here have examined the energy-transfer rates and fluorescence polarization characteristics for two different types of cassette, involving three distinct relative orientations of the donor and acceptor transition moments and the axis of the rigid linker. Energy-transfer times range from <200 fs to approximately 20 ps, the fastest transfer times occurring when the transition moments of the donor and acceptor species are aligned parallel to the linker axis. Experimental evidence is presented that supports a through-bond energy-transfer mechanism, in contrast with a commercial DNA-labeling agent, which exhibits much slower transfer times controlled by FRET. These rigid cassettes also exhibit polarized fluorescence from the acceptor species, so that this particular type of DNA-labeling probe has some of the advantages of single-molecule probes such as rhodamine and coumarin dyes.

Conformational preferences and vibrational frequency distributions of short peptides in relation to multidimensional infrared spectroscopy.

Molecular dynamics simulations of the structural distributions and the associated amide-I vibrational modes are carried out for dialanine peptide in water and carbon tetrachloride. The various manifestations in nonlinear-infrared spectroscopic experiments of the distributions of conformations of solvated dialanine are examined. The two-dimensional infrared (2D-IR) spectrum of dialanine exhibits the coupling between the amide oscillators and the correlations of the frequency fluctuations. An internally hydrogen-bonded conformation exists in CCl(4) but not in H(2)O where two externally hydrogen-bonded forms are preferred. Simulations of solvated dialanine show how the 2D-IR spectra expose the underlying structural distributions and dynamics that are not deducible from linear-infrared spectra. In H(2)O the 2D-IR shows cross-peaks from large coupling in the alpha-helical conformer and an elongated higher frequency diagonal peak, reflecting the broader distribution of structures for the more flexible acetyl end. In CCl(4), the computed cross-peak portion of the 2D-IR shows evidence of two amide-I transitions in the high-frequency region which are not apparent from the diagonal peak profile. The vibrational frequency inhomogeneity of the amide-I band arises from fluctuations of the instantaneous normal modes of these conformers rather than the shifts induced by hydrogen bonding. The simulation shows that there are correlations between fluctuations of the acetyl and amino end frequencies in H(2)O that arise from mechanical coupling and not from hydrogen bonding at the two ends of the molecule. The angular relationships between the two amide units which also show up in 2D-IR were computed, and spectral manifestations of them are discussed. The simulations also permit a calculation of the rate of energy transfer from one side of the molecule to the other. From these calculations, 2D-IR spectroscopy in conjunction with simulations is seen to be a promising tool for determining dynamics of structure changes in dipeptides.

Two-dimensional IR spectroscopy can be designed to eliminate the diagonal peaks and expose only the crosspeaks needed for structure determination.

The power of two-dimensional (2D) IR spectroscopy as a structural method with unprecedented time resolution is greatly improved by the introduction of IR polarization conditions that completely eliminate diagonal peaks from the spectra and leave only the crosspeaks needed for structure determination. This approach represents a key step forward in the applications of 2D IR to proteins, peptides, and other complex molecules where crosspeaks are often obscured by diagonal peaks. The technique is verified on the model compound 1,3-cyclohexanedione and subsequently used to clarify the distribution of structures that the acetylproline-NH(2) dipeptide adopts in chloroform. In both cases, crosspeaks are revealed that were not observed before, which, in the case of the dipeptide, has led to additional information about the structure of the amino group end of the peptide.

Two-dimensional infrared spectroscopy: a promising new method for the time resolution of structures.

Recently, new methods for determining time-evolving structures using infrared analogs of NMR spectroscopy have been introduced that have outstanding potential in structural biology. Already, within the past two years, structures of dipeptides, tripeptides and pentapeptides have been determined on much faster timescales than the conformational dynamics. Also, two-dimensional infrared correlation spectra of some proteins and isotopically edited alanine-rich helices have been examined.

Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy.

We report single-molecule measurements on the folding and unfolding conformational equilibrium distributions and dynamics of a disulfide crosslinked version of the two-stranded coiled coil from GCN4. The peptide has a fluorescent donor and acceptor at the N termini of its two chains and a Cys disulfide near its C terminus. Thus, folding brings the two N termini of the two chains close together, resulting in an enhancement of fluorescent resonant energy transfer. End-to-end distance distributions have thus been characterized under conditions where the peptide is nearly fully folded (0 M urea), unfolded (7.4 M urea), and in dynamic exchange between folded and unfolded states (3.0 M urea). The distributions have been compared for the peptide freely diffusing in solution and deposited onto aminopropyl silanized glass. As the urea concentration is increased, the mean end-to-end distance shifts to longer distances both in free solution and on the modified surface. The widths of these distributions indicate that the molecules are undergoing millisecond conformational fluctuations. Under all three conditions, these fluctuations gave nonexponential correlations on 1- to 100-ms time scale. A component of the correlation decay that was sensitive to the concentration of urea corresponded to that measured by bulk relaxation kinetics. The trajectories provided effective intramolecular diffusion coefficients as a function of the end-to-end distances for the folded and unfolded states. Single-molecule folding studies provide information concerning the distributions of conformational states in the folded, unfolded, and dynamically interconverting states.

Two-dimensional infrared spectroscopy of peptides by phase-controlled femtosecond vibrational photon echoes.

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-NH(2) 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.

Intense terahertz pulses by four-wave rectification in air.

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.

The dynamics of structural deformations of immobilized single light-harvesting complexes.

Single assemblies of the intact light-harvesting complex LH2 from Rhodopseudomonas acidophila were bound to mica surfaces at 300 K and examined by observing their fluorescence after polarized light excitation. The complexes are generally not cylindrically symmetric. They act like elliptic absorbers, indicating that the high symmetry found in crystals of LH2 is not present when the molecules are immobilized on mica. The ellipticity and the principal axes of the ellipses fluctuate on the time scale of seconds, indicating that there is a mobile structural deformation. The B850 ring of cofactors shows significantly less asymmetry than B800. The photobleaching strongly depends on the presence of oxygen.

Heme protein dynamics revealed by geminate nitric oxide recombination in mutants of iron and cobalt myoglobin.

Nitric oxide myoglobin (MbNO) at 300 K was photodissociated with 405 nm pulses. The NO recombination in several mutants of iron and cobalt myoglobins was investigated at a time resolution of ca. 70 fs. The geminate recombination of NO was nonexponential on sub-nanosecond time scales. For both metals, the change of the detailed structure of the heme pocket (position 68 mutations) caused significant changes in the rates of recombination; however, the metal substitution influenced the recombination much less than did amino acid substitution. The results indicate a primary role of the heme pocket structure in the dynamics, and they suggest that proximal protein relaxation is not the limiting factor in the geminate recombination process. Recombination in cobalt derivatives is somewhat more efficient on the sub-nanosecond time scales than in corresponding iron myoglobins, consistent with other results that show a greater intrinsic reactivity toward the NO of cobalt compared with the iron heme. A comparison of results using Soret band excitation with previous Q-state excitation studies demonstrates that the ligand dissociates with a similar kinetic energy in both cases, suggesting fast intramolecular energy redistribution before dissociation.

The two-dimensional IR nonlinear spectroscopy of a cyclic penta-peptide in relation to its three-dimensional structure.

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.

Peptidyl transferase center activity observed in single ribosomes.

We demonstrate the functional activity of single ribosomal complexes, opening the way for detailed studies of the trajectories of protein synthesis. Our approach employs a single-molecule detection system, capable of picoseconds to minutes resolution, to observe a growing peptide labeled at its N terminus with the fluorophore tetramethylrhodamine (TMR). Single complexes of mRNA-programmed ribosomes with TMR-Met-tRNAMetf or TMR-Met-Phe-tRNAPhe are immobilized on mica and observed by fluorescence. Immobilized ribosome.mRNA.TMR-Met-tRNAMetf complexes form peptide bonds with puromycin. Single-molecule detection reveals dynamics on the scale of seconds at the ribosomal peptidyl transferase center.

Protein fluctuations are sensed by stimulated infrared echoes of the vibrations of carbon monoxide and azide probes.

The correlation functions of the fluctuations of vibrational frequencies of azide ions and carbon monoxide in proteins are determined directly from stimulated photon echoes generated with femtosecond infrared pulses. The asymmetric stretching vibration of azide bound to carbonic anhydrase II exhibits a pronounced evolution of its vibrational frequency distribution on the time scale of a few picoseconds, which is attributed to modifications of the ligand structure through interactions with the nearby Thr-199. When azide is bound in hemoglobin, a more complex evolution of the protein structure is required to interchange the different ligand configurations, as evidenced by the much slower relaxation of the frequency distribution in this case. The time evolution of the distribution of frequencies of carbon monoxide bound in hemoglobin occurs on the approximately 10-ps time scale and is very nonexponential. The correlation functions of the frequency fluctuations determine the evolution of the protein structure local to the probe and the extent to which the probe can navigate those parts of the energy landscape where the structural configurations are able to modify the local potential energy function of the probe.

Fluorescence and photobleaching dynamics of single light-harvesting complexes.

Single light-harvesting complexes LH-2 from Rhodopseudomonas acidophila were immobilized on various charged surfaces under physiological conditions. Polarized light experiments showed that the complexes were situated on the surface as nearly upright cylinders. Their fluorescence lifetimes and photobleaching properties were obtained by using a confocal fluorescence microscope with picosecond time resolution. Initially all molecules fluoresced with a lifetime of 1 +/- 0.2 ns, similar to the bulk value. The photobleaching of one bacteriochlorophyll molecule from the 18-member assembly caused the fluorescence to switch off completely, because of trapping of the mobile excitations by energy transfer. This process was linear in light intensity. On continued irradiation the fluorescence often reappeared, but all molecules did not show the same behavior. Some LH-2 complexes displayed a variation of their quantum yields that was attributed to photoinduced confinement of the excited states and thereby a diminution of the superradiance. Others showed much shorter lifetimes caused by excitation energy traps that are only approximately 3% efficient. On repeated excitation some molecules entered a noisy state where the fluorescence switched on and off with a correlation time of approximately 0.1 s. About 490 molecules were examined.

Nonexponential kinetics of a single tRNAPhe molecule under physiological conditions.

The fluorescence decay functions of individual, specifically labeled tRNAPhe molecules exhibit nonexponential character as a result of conformational dynamics occurring during the measurement on a single molecule. tRNAPhe conformational states that interchange much more slowly are evidenced by the distribution of lifetimes observed for many individual molecules. A structural model for the nonexponential decay indicates that the tRNAPhe-probe adduct fluctuates between two states, one of which provides conditions that quench the probe fluorescence.

Ultrafast thermally induced unfolding of RNase A.

A temperature jump (T-jump) method capable of initiating thermally induced processes on the picosecond time scale in aqueous solutions is introduced. Protein solutions are heated by energy from a laser pulse that is absorbed by homogeneously dispersed molecules of the dye crystal violet. These act as transducers by releasing the energy as heat to cause a T-jump of up to 10 K with a time resolution of 70 ps. The method was applied to the unfolding of RNase A. At pH 5.7 and 59 degrees C, a T-jump of 3-6 K induced unfolding which was detected by picosecond transient infrared spectroscopy of the amide I region between 1600 and 1700 cm-1. The difference spectral profile at 3.5 ns closely resembled that found for the equilibrium (native-unfolded) states. The signal at 1633 cm-1, corresponding to the beta-sheet structure, achieved 15 +/- 2% of the decrease found at equilibrium, within 5.5 ns. However, no decrease in absorbance was detected until 1 ns after the T-ump. The disruption of beta-sheet therefore appears to be subject to a delay of approximately 1 ns. Prior to 1 ns after the T-jump, water might be accessing the intact hydrophobic regions.

Femtosecond coherent transient infrared spectroscopy of reaction centers from Rhodobacter sphaeroides.

Protein and cofactor vibrational dynamics associated with photoexcitation and charge separation in the photosynthetic reaction center were investigated with femto-second (300-400 fs) time-resolved infrared (1560-1960 cm-1) spectroscopy. The experiments are in the coherent transient limit where the quantum uncertainty principle governs the evolution of the protein vibrational changes. No significant protein relaxation accompanies charge separation, although the electric field resulting from charge separation modifies the polypeptide carbonyl spectra. The potential energy surfaces of the "special pair" P and the photoexcited singlet state P* and environmental perturbations on them are similar as judged from coherence transfer measurements. The vibrational dephasing time of P* modes in this region is 600 fs. A subpicosecond transient at 1665 cm-1 was found to have the kinetics expected for a sequential electron transfer process. Kinetic signatures of all other transient intermediates, P, P*, and P+, participating in the primary steps of photosynthesis were identified in the difference infrared spectra.

Regenerative amplification of 30-fs pulses in Ti:sapphire at 5 kHz.

18-fs pulses from a self-mode-locked Ti:sapphire laser are amplified to 60 microJ of energy at 4.9 kHz with chirped-pulse amplification in a Ti:sapphire regenerative amplifier. After recompression, 30-35-fs, near-transform-limited pulses are obtained.

Near-field time-resolved fluorescence studies of poly(phenylmethyl silane) with subwavelength resolution.

We present an example of the first time-correlated single-photon counting (TCSPC) near-field optical measurement. The aperture size of our prepared aluminun-coated fiber-optic probe was approximately 50 nm, which represents a spatial resolution of λex/7 for our UV measurements. Near-field fluorescence decays of poly(phenylmethyl silane) in solid thin films excited in the range 325-360 nm were obtained and the steady-state excitation spectra compared with the excitation spectral information obtained in the far field. Fluorescence decays showed single exponential lifetimes ranging from 45 to 277 ps, which was dependent on the excitation wavelength and the selected near-field tip. The proximity of the metal-coated tip to the sample may be the reason for the modulation in fluorescence lifetime.

Picosecond infrared studies of the dynamics of the photosynthetic reaction center.

The changes in the vibrational transitions of the protein and redox cofactors of the photosynthetic reaction center were examined by picosecond infrared spectroscopy. The spectra in the vibrational mid-infrared region (1800-1550 cm-1) of hydrated and partially dehydrated reaction centers were investigated from 50 ps to 4 ns after photoinitiation of the electron transfer. Features in the infrared difference spectra were identified with both protein and redox cofactor vibrational modes and correlated with electron transfer events whose kinetics were measured in the infrared and visible regions. The observed protein response is confined to a few amide I transitions (1644 cm-1, 1661 cm-1, 1665 cm-1) and carboxylic residues (1727 cm-1). About 85% of the observed signal corresponded to alterations in the cofactor-associated ester and keto carbonyls. The amide I and carboxylic transitions appeared prior to 50 ps, suggesting that the primary electron transfer event is coupled with a specific piece of the protein backbone and to glutamic or aspartic residues nearby the special pair. Infrared absorption changes accompanying bacteriochlorophyll-dimer cation formation dominated the signal at all times investigated. Infrared spectral changes observed in hydrated and partially dehydrated reaction centers were distinctly different; a band at 1665 cm-1 with a spectral width of 6 cm-1 in the hydrated protein, corresponding to a protein amide I bleach, was not present in the dehydrated film. These differences are discussed in terms of the markedly different electron transfer kinetics observed in the presence of water.