Peak Shift Spectroscopy Research Articles

Direct in situ observation of the percolation transition in VO2 thin film by peak-shift spectroscopy

When a thin film undergoes a percolative insulator-to-metal phase transition, the real part of the dielectric constant dramatically changes; it diverges close to the percolation threshold and becomes negative in the metallic phase. To date, the percolative nature of this phase transition has been monitored by near-field imaging or conventional spectroscopy. Although such methods are successful, they require laborious raster scanning or complicated theoretical fitting processes. In this study, using the strong coupling between slot antennas and a phase-transition material, a direct step-by-step investigation of the percolation transition can be demonstrated without any scanning or fitting processes. The percolation threshold can be identified in real time by monitoring the peak position, because the resonance transmission through the slot antenna array is highly affected by the dielectric environment. The work presented in this study could potentially contribute to the characterization of phase-transition materials to be used in memory devices or active metamaterials.

Structure and Dynamics of Stacking Interactions in an Antibody Binding Site.

For years, antibodies (Abs) have been used as a paradigm for understanding how protein structure contributes to molecular recognition. However, with the ability to evolve Abs that recognize specific chromophores, they also have great potential as models for how protein dynamics contribute to molecular recognition. We previously raised murine Abs to different chromophores and, with the use of three-pulse photon echo peak shift spectroscopy, demonstrated that the immune system is capable of producing Abs with widely varying flexibility. We now report the characterization of the complexes formed between two Abs, 5D11 and 10A6, and the chromophoric ligand that they were evolved to recognize, 8-methoxypyrene-1,3,6-trisulfonic acid (MPTS). The sequences of the Ab genes indicate that they evolved from a common precursor. We also used a variety of spectroscopic methods to probe the photophysics and dynamics of the Ab-MPTS complexes and found that they are similar to each other but distinct from previously characterized anti-MPTS Abs. Structural studies revealed that this difference likely results from a unique mode of binding in which MPTS is sandwiched between the side chain of PheH98, which interacts with the chromophore via T-stacking, and the side chain of TrpL91, which interacts with the chromophore via parallel stacking. The T-stacking interaction appears to mediate relaxation on the picosecond time scale, while the parallel stacking appears to mediate relaxation on an ultrafast, femtosecond time scale, which dominates the response. The anti-MPTS Abs thus not only demonstrate the simultaneous use of the two limiting modes of stacking for molecular recognition, but also provide a unique opportunity to characterize how dynamics might contribute to molecular recognition. Both types of stacking are common in proteins and protein complexes where they may similarly contribute to dynamics and molecular recognition.

Adaptive mutations alter antibody structure and dynamics during affinity maturation.

While adaptive mutations can bestow new functions on proteins via the introduction or optimization of reactive centers, or other structural changes, a role for the optimization of protein dynamics also seems likely but has been more difficult to evaluate. Antibody (Ab) affinity maturation is an example of adaptive evolution wherein the adaptive mutations may be identified and Abs may be raised to specific targets that facilitate the characterization of protein dynamics. Here, we report the characterization of three affinity matured Abs that evolved from a common germline precursor to bind the chromophoric antigen (Ag), 8-methoxypyrene-1,3,6-trisulfonate (MPTS). In addition to characterizing the sequence, molecular recognition, and structure of each Ab, we characterized the dynamics of each complex by determining their mechanical response to an applied force via three-pulse photon echo peak shift (3PEPS) spectroscopy and deconvoluting the response into elastic, anelastic, and plastic components. We find that for one Ab, affinity maturation was accomplished via the introduction of a single functional group that mediates a direct contact with MPTS and results in a complex with little anelasticity or plasticity. In the other two cases, more mutations were introduced but none directly contact MPTS, and while their effects on structure are subtle, their effects on anelasticity and plasticity are significant, with the level of plasticity correlated with specificity, suggesting that the optimization of protein dynamics may have contributed to affinity maturation. A similar optimization of structure and dynamics may contribute to the evolution of other proteins.

Protein Dynamics and the Diversity of an Antibody Response

The immune system is remarkable in its ability to produce antibodies (Abs) with virtually any specificity from a limited repertoire of germ line precursors. Although the contribution of sequence diversity to this molecular recognition has been studied for decades, recent models suggest that protein dynamics may also broaden the range of targets recognized. To characterize the contribution of protein dynamics to immunological molecular recognition, we report the sequence, thermodynamic, and time-resolved spectroscopic characterization of a panel of eight Abs elicited to the chromophoric antigen 8-methoxypyrene-1,3,6-trisulfonate (MPTS). Based on the sequence data, three of the Abs arose from unique germ line Abs, whereas the remaining five comprise two sets of siblings that arose by somatic mutation of a common precursor. The thermodynamic data indicate that the Abs recognize MPTS via a variety of mechanisms. Although the spectroscopic data reveal small differences in protein dynamics, the anti-MPTS Abs generally show similar levels of flexibility and conformational heterogeneity, possibly representing the convergent evolution of the dynamics necessary for function. However, one Ab is significantly more rigid and conformationally homogeneous than the others, including a sibling Ab from which it differs by only five somatic mutations. This example of divergent evolution demonstrates that point mutations are capable of fixing significant differences in protein dynamics. The results provide unique insight into how high affinity Abs may be produced that bind virtually any target and possibly, from a more general perspective, how new protein functions are evolved.

Bimodal dynamics of mechanically constrained hydrogen bonds revealed by vibrational photon echoes

We have investigated the dynamics of the hydrogen bonds that connect the components of a [2]rotaxane in solution. In this rotaxane, the amide groups in the benzylic-amide macrocycle and the succinamide thread are connected by four equivalent N-H⋅⋅⋅O=C hydrogen bonds. The fluctuations of these hydrogen bonds are mirrored by the frequency fluctuations of the NH-stretch modes, which are probed by means of three-pulse photon-echo peak shift spectroscopy. The hydrogen-bond fluctuations occur on three different time scales, with time constants of 0.1, 0.6, and ≥200 ps. Comparing these three time scales to the ones found in liquid formamide, which contains the same hydrogen-bonded amide motif but without mechanical constraints, we find that the faster two components, which are associated with small-amplitude fluctuations in the strength of the N-H⋅⋅⋅O=C hydrogen bonds, are very similar in the liquid and the rotaxane. However, the third component, which is associated with the breaking and subsequent reformation of hydrogen bonds, is found to be much slower in the rotaxane than in the liquid. It can be concluded that the mechanical bonding in a rotaxane does not influence the amplitude and time scale of the small-amplitude fluctuations of the hydrogen bonds, but strongly slows down the complete dissociation of these hydrogen bonds. This is probably because in a rotaxane breaking of the macrocycle-axle contacts is severely hindered by the mechanical constraints. The hydrogen-bond dynamics in rotaxane-based molecular machines can therefore be regarded as liquidlike on a time scale 1 ps and less, but structurally frozen on longer (up to at least 200 ps) time scales.

Three‐Pulse Photon‐Echo Peak Shift Spectroscopy and Its Application for the Study of Solvation and Nanoscale Excitons

An understanding of chemical reactivity begins with an understanding of the dynamics involved regarding system-bath interactions. Spectroscopic studies of these interactions in condensed-phase multichromophoric systems are intricate and contain much information. Photon-echo spectroscopy has proven to be a useful tool for probing these interactions. A description of three-pulse photon-echo peak shift spectroscopy (3PEPS)--theory, experiment, and application--for the study of solvation and dynamics of nanoscale excitons is presented. Also, we discuss how two-dimensional photon-echo spectroscopy (2DPE) relates to 3PEPS and show how 3PEPS data can be extracted from 2D photon-echo data.

Photon echo studies of photosynthetic light harvesting

The broad linewidths in absorption spectra of photosynthetic complexes obscure information related to their structure and function. Photon echo techniques represent a powerful class of time-resolved electronic spectroscopy that allow researchers to probe the interactions normally hidden under broad linewidths with sufficient time resolution to follow the fastest energy transfer events in light harvesting. Here, we outline the technical approach and applications of two types of photon echo experiments: the photon echo peak shift and two-dimensional (2D) Fourier transform photon echo spectroscopy. We review several extensions of these techniques to photosynthetic complexes. Photon echo peak shift spectroscopy can be used to determine the strength of coupling between a pigment and its surrounding environment including neighboring pigments and to quantify timescales of energy transfer. Two-dimensional spectroscopy yields a frequency-resolved map of absorption and emission processes, allowing coupling interactions and energy transfer pathways to be viewed directly. Furthermore, 2D spectroscopy reveals structural information such as the relative orientations of coupled transitions. Both classes of experiments can be used to probe the quantum mechanical nature of photosynthetic light-harvesting: peak shift experiments allow quantification of correlated energetic fluctuations between pigments, while 2D techniques measure quantum beating directly, both of which indicate the extent of quantum coherence over multiple pigment sites in the protein complex. The mechanistic and structural information obtained by these techniques reveals valuable insights into the design principles of photosynthetic light-harvesting complexes, and a multitude of variations on the methods outlined here.

Three-pulse photon echo peak shift spectroscopy as a probe of flexibility and conformational heterogeneity in protein folding

We investigate the equilibrium unfolding of Zn-cytochrome c in guanidine hydrochloride by three-pulse photon echo peak shift (3PEPS) spectroscopy. Unexpectedly, the measurements reveal that inhomogeneous broadening of the sample at the midpoint of the denaturation is larger than that of either native or unfolded states. To interpret this finding, we present simulations of the peak shift for both two-state and three-state unfolding models. Both the denaturant concentration dependence of the asymptotic peak shift (APS) and the wavelength dependence of the APS at the midpoint of the denaturation are different for the two models. Our data are consistent with two-state unfolding.

Probe of flexibility and conformational heterogeneity in Zn-cytochrome c (Zn-cyt c) folding by Three-Pulse Photon Echo Peak Shift (3PEPS) Spectroscopy

We investigated the dynamics and conformational heterogeneity of Zn-cyt c with 3PEPS, a type of femtosecond nonlinear spectroscopy that is used to quantify the spectrum of sub-nanosecond protein motions and the inhomogeneous broadening of the system. Wavelength-dependent measurements were performed on the Soret transitions of the folded state, the guanidine hydrochloride unfolded state (4.5 M), and the state at the midpoint (2.5 M) of the denaturant titration. The measurements resolve a substantial increase in the inhomogeneous broadening of the Soret band upon unfolding, as expected due to an increase in conformational diversity upon unfolding.

Exploring the Energy Landscape of Antibody−Antigen Complexes: Protein Dynamics, Flexibility, and Molecular Recognition

The production of antibodies that selectively bind virtually any foreign compound is the hallmark of the immune system. While much is understood about how sequence diversity contributes to this remarkable feat of molecular recognition, little is known about how sequence diversity impacts antibody dynamics, which is also expected to contribute to molecular recognition. Toward this goal, we examined a panel of antibodies elicited to the chromophoric antigen fluorescein. On the basis of isothermal titration calorimetry, we selected six antibodies that bind fluorescein with diverse binding entropies, suggestive of varying contributions of dynamics to molecular recognition. Sequencing revealed that two pairs of antibodies employ homologous heavy chains that were derived from common germline genes, while the other two heavy chains and all six of the light chains were derived from different germline genes and are not homologous. Interestingly, more than half of all the somatic mutations acquired during affinity maturation among the six antibodies are located in positions unlikely to contact fluorescein directly. To quantify and compare the dynamics of the antibody-fluorescein complexes, three-pulse photon echo peak shift and transient grating spectroscopy were employed. All of the antibodies exhibited motions on three distinct time scales, ultrafast motions on the <100 fs time scale, diffusive motions on the picosecond time scale, and motions that occur on time scales longer than nanoseconds and thus appear static. However, the exact frequency of the picosecond time scale motion and the relative contribution of the different motions vary significantly among the antibody-chromophore complexes, revealing a high level of dynamic diversity. Using a hierarchical model, we relate the data to features of the antibodies' energy landscapes as well as their flexibility in terms of elasticity and plasticity. In all, the data provide a consistent picture of antibody flexibility, which interestingly appears to be correlated with binding entropy as well as with germline gene use and the mutations introduced during affinity maturation. The data also provide a gauge of the dynamic diversity of the antibody repertoire and suggest that this diversity might contribute to molecular recognition by facilitating the recognition of the broadest range of foreign molecules.

Measuring Electronic Coupling in the Reaction Center of Purple Photosynthetic Bacteria by Two-Color, Three-Pulse Photon Echo Peak Shift Spectroscopy

One- and two-color, three-pulse photon echo peak shift spectroscopy (1C and 2C3PEPS) was used to estimate the electronic coupling between the accessory bacteriochlorophyll (B) and the bacteriopheophytin (H) in the reaction center of the purple photosynthetic bacterium Rhodobacter sphaeroides as approximately 170 +/- 30 cm-1. This is the first direct experimental determination of this parameter; it is within the range of values found in previously published calculations. The 1C3PEPS signal of the Qy band of the bacteriochlorophyll B shows that it is weakly coupled to nuclear motions of the bath, whereas the 1C3PEPS signal of the Qy band of the bacteriopheophytin, H, shows that it is more strongly coupled to the bath, but has minimal inhomogeneous broadening. Our simulations capture the major features of the data with the theoretical framework developed in our group to separately calculate the response functions and population dynamics.

One- and Two-Color Photon Echo Peak Shift Studies of Photosystem I

Wavelength-dependent one- and two-color photon echo peak shift spectroscopy was performed on the chlorophyll Qy band of trimeric photosystem I from Thermosynechococcus elongatus. Sub-100 fs energy transfer steps were observed in addition to longer time scales previously measured by others. In the main PSI absorption peak (675-700 nm), the peak shift decays more slowly with increasing wavelength, implying that energy transfer between pigments of similar excitation energy is slower for pigments with lower site energies. In the far-red region (715 nm), the decay of the peak shift is more rapid and is complete by 1 ps, a consequence of the strong electron-phonon coupling present in this spectral region. Two-color photon echo peak shift data show strong excitonic coupling between pigments absorbing at 675 nm and those absorbing at 700 nm. The one- and two-color peak shifts were simulated using the previously developed energy transfer model (J. Phys. Chem. B 2002, 106, 10251; Biophysical Journal 2003, 85, 140). The simulations agree well with the experimental data. Two-color photon echo peak shift is shown to be far more sensitive to variations in the molecular Hamiltonian than one-color photon echo peak shift spectroscopy.

Photon echo spectroscopy of porphyrins and heme proteins: Effects of quasidegenerate electronic structure on the peak shift decay

Three pulse photon echo peak shift spectroscopy and transient grating measurements on Zn-substituted cytochrome c, Zn-tetraphenylporphyrin, and Zn-protoporphyrin IX are reported. The effects of protein conformation, axial ligation, and solvent are investigated. Numerical simulations of the peak shift and transient grating experiments are presented. The simulations employed recently derived optical response functions for square-symmetric molecules with doubly degenerate excited states. Simulations exploring the effects of excited-state energy splitting, symmetric and asymmetric fluctuations, and excited-state lifetime show that the time scales of the peak shift decay in the three-level system largely reflect the same dynamics as in the two-level system. However, the asymptotic peak shift, which is a clear indicator of inhomogeneous broadening in a two-level system, must be interpreted more carefully for three-level systems, as it is also influenced by the magnitude of the excited-state splitting. The calculated signals qualitatively reproduce the data.

Two Dimensional Electronic Spectroscopy

Two different electronically resonant two-dimensional spectroscopies are described. The first, two-color photon echo peak shift spectroscopy, is sensitive to correlations in transition frequency between the initial and probed (final) states. It provides new insight into the mechanism of ultrafast solvation and should prove useful for characterizing dynamics in inhomogeneous systems in general. The second technique, fifth order threepulse scattering, contains two coherence periods whose durations are controlled. The entire two-dimensional surface was recorded for a dye molecule in dilute solution and a photosynthetic light-harvesting complex. The data provide insight into the short-time dynamics of solvation and exciton relaxation, respectively.

Two-color three pulse photon echo peak shift spectroscopy

Two-color three pulse photon echo peak shift spectroscopy (2C3PEPS) has been used to probe correlation in electronic transitions in two different regions of the electronic spectrum of a chromophore in the condensed phase. The 2C3PEPS can be done in an “uphill” or a “downhill” sense, where the first two interactions are of lower (higher) frequency than the final interaction with the radiation field. In both cases no correlation between the two spectral regions is observed at very short times. Different parts of the spectrum gain correlation owing to ultrafast solvent motion and the accompanying Stokes shift. We propose a model for the spectral shift that incorporates a conditional probability distribution for transition frequencies in the two spectral regions. The model qualitatively reproduces all the features of complete numerical simulations. Our results show that memory is partially conserved during the spectral diffusion process from the pump to the probe region. The downhill difference peak shift is very sensitive to the time scale of the inertial component of solvation and seems very promising for an accurate determination of this time scale.

Flexibility of an Antibody Binding Site Measured with Photon Echo Spectroscopy

The lock-and-key and induced fit models of antigen binding by antibodies are topics of considerable importance to immunology and protein biochemistry in general. These models ascribe different degrees of flexibility to the antibody combining site and may therefore be distinguished by measuring the response of the protein to the force exerted by charge redistribution and structural reorganization of an optically excited antigen. Spectroscopic characterization of the magnitude and time scale of this protein reorganization is therefore a basis for comparing the flexibility of different antibodies. This approach is used to study a panel of antibodies elicited to the chromophoric antigen 8-methoxypyrene-1,3,6,trisulfonic acid. The reorganization energies of the chromophore-antibody complexes are determined and are found to be distributed over a wide range. Three pulse photon echo peak shift spectroscopy is used to measure the time scales of the reorganization process in one complex. The antibody motions occur on time scales ranging from 75 femtoseconds to 67 picoseconds. Structural origins of the observed protein motions are discussed.

The interaction of solutes with their environments

The origins of electronic spectral broadening in solutions, glasses and proteins are discussed via three pulse stimulated echo peak–shift measurements. Spectral densities for dye molecules in polar solutions are presented and discussed in terms of molecular models for the dynamics. The breakdown of the harmonic bath picture at low frequencies is presented. Finally, echo measurements on light harvesting complexes of purple bacteria are presented and it is shown that the energy transfer timescale can be obtained from peak–shift measurements.