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Annu. Rev. Phys. Chem. 1997. 48:453–79 c 1997 by Annual Reviews Inc. All rights reserved Copyright
FAST NATURAL AND MAGNETIC CIRCULAR DICHROISM SPECTROSCOPY Robert A. Goldbeck, Daniel B. Kim-Shapiro,1 and David S. Kliger Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064-1077; e-mail:
[email protected] KEY WORDS:
time-resolved spectroscopy, nanosecond, picosecond, optical rotatory dispersion, linear dichroism
ABSTRACT The addition of circular or, more generally, elliptical polarization state detection to fast optical absorption spectroscopy can increase the amount of electronic and nuclear conformational information obtained about transient molecular species. To accomplish this, fast circular dichroism methods have emerged over the past decade that overcome the millisecond limit on time resolution associated with conventional modulation techniques and enable structural studies of excited states and kinetic intermediates. This article reviews techniques for time-resolved natural and magnetic circular dichroism spectroscopy covering the picosecond to millisecond time regimes and their applications, with particular emphasis on quasi-null ellipsometric techniques for nanosecond multichannel measurements of circular dichroism. Closely related quasi-null polarimetric techniques for nanosecond optical rotatory dispersion and linear dichroism measurements are also discussed.
INTRODUCTION As the time resolution of optical absorption methods, now at the femtosecond level, approaches the uncertainty principle limit for chemical energies, 1 This author has published previously as Daniel B. Shapiro. Present address: Department of Physics, Wake Forest University, Winston-Salem, North Carolina 27109-7507.
453 0066-426X/97/1001-0453$08.00
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control of the photon polarization provides one avenue to further increase the information-resolving power of fast spectral methods. With the development in the 1960s of the electro-optic (1) and photoelastic (2–4) modulator techniques underlying most modern circular dichroism (CD) instrumentation, polarized light became more widely used to illuminate the equilibrium structures of chiral molecules, an approach born of the work of Biot, Pasteur, and Cotton in the last century. The study of absolute stereochemistry in chiral centers of organic molecules and helicity in the secondary structure of biopolymers such as proteins and DNA are prominent applications of natural CD spectroscopy. Magnetic circular dichroism (MCD) spectroscopy, closely related to the eponymous optical rotation effect discovered by Faraday (4a), uses similar instrumentation to study molecular structure as it is reflected in the presence of spin and orbital degeneracies, or near degeneracies, in the electronic states. Nonequilibrium structures are now studied with commercial CD and MCD instruments on time scales as short as milliseconds, but the extension of rapid modulation and phaselocked detection methods to the study of molecular structures evolving on faster time scales is not straightforward. One solution to the problem of extending time-resolved CD (TRCD) and MCD (TRMCD) measurements to the nanosecond and microsecond time scales is to avoid rapid modulation techniques altogether and use a modified version of an ellipsometric method introduced before the modern era of CD instrumentation (5). Historically, a quasi-null half-shade ellipsometric apparatus was the method of choice for CD measurements from 1930 until the development of photoelectric detection methods in the 1950s (6). A similar quasi-null ellipsometric principle is now used in nanosecond multichannel CD measurements, with modifications that permit multichannel measurements without the circular birefringence (CB) artifacts associated with earlier methods (7). The extension of TRCD and TRMCD measurements into the picosecond regime has been accomplished with modulator and phase-locked detection techniques (8, 9), albeit considerably modified from the conventional rapid modulation approach, as described below. The use of phase-locked techniques for the detection of chirality in emissive molecules, through techniques such as lifetime-resolved fluorescence-detected CD (10) and circularly polarized luminescence (11), are considered below. We also briefly describe a crosspolarized transient grating technique for CD detection that may potentially be used for TRCD measurements with nanosecond or picosecond resolution (12, 13). In addition to discussing time-resolved CD techniques, both ellipsometric and phase-locked detection based, their applications to the natural CD and MCD of transient molecular species, and artifacts and orientation effects associated with photoselection, we examine quasi-null polarimetric methods for fast optical
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rotatory dispersion (ORD) (14) and linear dichroism (LD) (15) measurements that are closely related to CD ellipsometry.
MILLISECOND CD We first briefly review methods using rapid modulation and phase-locked detection for moderate time-resolution CD. Millisecond time-resolved CD detection techniques, introduced in 1974, have been coupled to flash photolysis (16), temperature jump (17, 18), pressure jump (19), and rapid mixing (stopped flow) (20, 21) techniques for the initiation of chemical kinetics. Stopped-flow CD, which was preceded briefly by the development of stopped-flow optical rotation measurements (22), has been applied widely to the study of conformational changes in macromolecules, especially in the area of protein folding (23). The instrumentation for stopped-flow CD has not changed significantly since the early incorporation of light sources extending measurements into the near and far UV (24, 25). The conventional stopped-flow CD spectrophotometer is composed essentially of a photoelastic modulator (PEM)–based static CD instrument coupled with a stopped-flow mixing device. A static CD spectrum is obtained on a conventional instrument by scanning wavelength while adjusting the modulation amplitude of the PEM. However, wavelength scanning is generally too slow for millisecond measurements, so conventional stopped-flow instruments necessarily measure CD at a single wavelength as a function of time. Although stopped-flow CD has been successful in providing valuable information concerning the dynamics of conformational changes of macromolecules, the time resolution of these instruments is insufficient to monitor many important reactions. A notable example arises in the study of protein folding. Early steps in the formation of secondary structure during the folding process can occur much more rapidly than milliseconds (26–28), whereas the factor limiting time resolution in stopped-flow CD is usually the mixing time, about 1 ms. Even if this dead time is avoided by initiating the reaction in a manner more rapid than stopped-flow mixing, e.g. photolysis or laser temperature-jump, the modulation frequency of the PEM nevertheless remains a barrier to resolving events below about 100 µs. Although reactions occurring in less than a few milliseconds are not currently monitored with stopped-flow CD for these reasons, gated detectors, such as those described below in CPL measurements, may enable researchers to extend the time resolution of a PEM-based CD measurement below the microsecond range. Stopped-flow CD measurements are collected a single wavelength at a time, which can limit the amount of structural information obtained. One improvement to this technique is to monitor absorbance and fluorescence simultaneously with CD (29, 30), providing more information on which to base the
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interpretation of conformational changes. The availability of a multichannel or rapid-scanning stopped-flow CD spectrophotometer capable of obtaining CD spectra with millisecond resolution could significantly quicken the pace of discovery in macromolecular structure and function studies, a possibility that has led more than one commercial instrument company to undertake development efforts along these lines.
LIFETIME-RESOLVED FLUORESCENCE-DETECTED CD A CD method that exploits the emission properties of excited states to monitor the steady state CD of ground state species is fluorescence-detected CD (FDCD), which measures the difference in emission intensity from a fluorophore excited with a left versus a right circularly polarized excitation beam. The information provided by this measurement is identical to that provided by an absorptive CD measurement, except that more specificity can be obtained in FDCD through localization of the detected optical activity to a particular fluorophore. In measurements on multichromophore systems such as proteins, for instance, FDCD can be used to probe the protein environment local to only those chromophores that fluoresce. FDCD is detected with a PEM-based apparatus similar to that of absorptive CD except that the detector is moved from a position where transmitted light is detected to one where emitted light is detected. FDCD can be more selective than absorptive CD, but localization to a single fluorophore may be complicated by the presence of several identical or similar fluorophores in a macromolecule. Separation of the fluorophore CD contributions in such cases may still be possible by careful analysis of the excitation and emission spectra, but such an analysis can be difficult. A more promising method for determining the individual contributions of multiple fluorophores to FDCD relies on phase-shift measurements of the fluorescence lifetime (10, 31, 32). Lifetime-resolved FDCD can identify individual fluorophore contributions in this way because of the strong influence of fluorophore environment on fluorescence lifetime. Lifetimes are measured in this method by examining the phase shift and demodulation of an intensity-modulated excitation beam. The intensity modulation is achieved by passing the excitation beam through a linear polarizer followed by a half-wave Pockels cell (an electro-optic modulator similar in action to a PEM, with square wave modulation frequencies from 1 to 240 Mhz) and a final polarizer orthogonal to the first. Passage through the Pockels cell modulates the linearly polarized light between horizontal and vertical orientations, and the light exiting the polarizer placed after the Pockels cell is modulated in intensity. The lifetime and amplitude of different components of fluorescence are measured for both right and left circularly
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polarized exciting beams. Circular polarization is achieved with a BabinetSoliel compensator, a variable retarder that can be adjusted in this application to produce the equivalent of ±λ/4 retardance at the excitation wavelength. Different fluorophores will have different fluorescence lifetimes, whereas a particular fluorophore will have the same fluorescence lifetime for incident right and left circularly polarized light. The difference in amplitudes for right and left circularly polarized excitation for a given component, separated from others according to fluorescence lifetime, yields the lifetime-resolved FDCD. The lifetime-resolved technique is capable of measuring FDCD with nanosecond fluorescence lifetime resolution (32). We point out, however, that the lifetime information for these measurements is typically obtained from separate, unpolarized emission measurements. Moreover, as mentioned above, although this method monitors the emission of short-lived species (the excited states), no independent dynamical information is obtained from the polarization measurements, which reflect the steady state CD properties of ground state species.
FAST CIRCULARLY POLARIZED LUMINESCENCE An emission-based method that offers the possibility of true time-resolved chiral spectroscopy is circularly polarized luminescence (CPL), defined as the difference in intensity of left minus right circularly polarized emission. CPL measures optical activity in the excited states of luminophores (fluorescent or phosphorescent) and is typically measured after excitation with unpolarized light, although additional information may be obtained from oriented CPL measured after excitation with linearly polarized light (33). The degree of circular polarization in the emitted light can be measured with a PEM and a linear polarizer functioning as a modulated circular polarization analyzer. A similar arrangement is used to conduct fluorescence anisotropy (FA) measurements in which CPL and FA are convoluted in a manner similar to that shown below in Equation 8 for CD and LD. In situations where one quantity does not completely dominate the other, CPL and FA can be separated by their dependencies on the modulation frequency. A review of CPL, with some reference to time-resolved measurements, has been published previously in Reference 11. Time-resolved CPL offers the same advantage over steady-state CPL that lifetime-resolved FDCD offers over steady-state FDCD; different contributions to the CPL can be resolved by examining the emission lifetime components. In addition, time-resolved CPL can (in principle) provide information concerning the chirality of excited states of intermediates of the luminophores or their surroundings. To this end, use of a gated photon-counting device with a PEM-based circular polarizer analyzer enables CPL measurements with 20-µs
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resolution (34–37), a time resolution that is limited by the modulation frequency of the PEM. The use of more complicated counting procedures and a completely random relation between the excitation pulses and modulation frequency can extend this time resolution (38). Several applications of these techniques have been made in the areas of chemical racemization and enantioselective quenching of lanthanide complexes (34–36, 39, 40). Additionally, application of a gated photon-counting and PEM-based system developed to measure the timeresolved CPL of protein phosphorescence has demonstrated the uniqueness of individual decay times responsible for the CPL in these proteins (41). The adaptation of a high-speed time-to-digital converter and histogramming memory to this system enables CPL measurements with nanosecond resolution, which is a capability that has been applied to measurements on the NADH chromophore in horse liver alcohol dehydrogenase and that has promise for applications in other protein systems and in the protein-folding problem (42).
NANOSECOND CD Neither ground states nor nonemissive chromophores can be studied with CPL. A more general method for nanosecond time-resolved CD that has been applied to a variety of transient molecular species, both excited states and (ground state) kinetic intermediates, takes advantage of the ability of chiral chromophores to alter the ellipticity of polarized light.
Strain-Plate Ellipsometric Methods for Multichannel Time-Resolved CD Ellipsometric CD methods may be classified as null or quasi-null methods. The most sensitive type of CD ellipsometer would use a crossed pair of linear polarizers to completely block light transmission from a source to a detector. Placing a circularly dichroic sample between the polarizers spoils the extinction and causes light to be transmitted with an intensity proportional to CD2 [we always assume that CD, defined as 1.1511εcz (where 1ε = the difference in extinction coefficients between left and right circularly polarized light, c = molar concentration, and z = pathlength), is much smaller than unity and that the small angle approximation is valid]. This null type of ellipsometric measurement is not a very accurate measure of CD, however, because circular birefringence (CB) also contributes to transmission, as does any linear dichroism (LD) or linear birefringence (LB) present. A quasi-null ellipsometer, in which a weakly linearly birefringent element is also placed between the polarizers, is more accurate. By adding a known component of phase retardance off-axis from the polarizers, this element introduces a known amount of ellipticity into the probe beam that serves as a comparison against which the ellipticity of the
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sample may be gauged. The key to accuracy in this method is the introduction of a retardance that is large compared to the CD of the sample. Sensitivity and precision, on the other hand, are maximized when the retardance is much smaller than a quarter wave. A compromise between these considerations is usually struck with a diagonal component of retardance in the 1–10◦ range, a retardance that is provided in multichannel TRCD measurements by orienting the fast axis of a weakly birefringent plate along a diagonal to the polarizer axes. The difference between transmitted intensities for the two possible diagonal orientations of the plate (IR − IL, where IR and IL are the intensities for right and left elliptical polarizations, respectively), normalized to the sum, defines a signal that is proportional to CD/δ, where δ is the retardance (43). The signal-to-noise advantage of this approach over conventional CD measurements lies in a reduced sensitivity to instrumental noise sources, such as arc wander, that are proportional in magnitude to the average source intensity (44). (The signal-to-noise merits of ellipsometric and conventional measurements are identical with respect to photon shot noise, which is proportional to the root of average intensity.) The signal-to-noise ratio in the presence of noisy light sources, such as xenon flash lamps, is effectively amplified by a factor of 1/δ, or about 100 in an ellipsometric measurement with δ = 1◦ , allowing for greater source brightness in the typical trade-off between brightness and stability. The nanosecond ellipsometric CD apparatus shown in Figure 1 has been applied to a variety of chemical systems (44) and has undergone several technical improvements, such as the addition of multichannel detection (45), since its introduction in 1985 (43, 46). Calcite Glan-Taylor polarizers, originally introduced for their high extinction (10−6) in near-UV and visible band TRCD studies, are replaced by MgF2 Rochon polarizers for TRCD studies of far-UV bands (47), such as the peptide bands in proteins. [Polarizers with high light extinction are a critical requirement for ellipsometric CD measurements, as are strain free optics (48).] The potential importance of fast secondary structure probes for use in peptide- and protein-folding studies has driven the development of improved far-UV CD detection methods. In this regard, the xenon flash lamps used as probe light sources in multichannel TRCD studies provide relatively little output in the far-UV, and correspondingly increased signal averaging is required for adequate signal-to-noise ratios at high time resolutions. Wen et al (49) introduced an upconverted Ti:sapphire laser as a probe source for single-wavelength far-UV TRCD studies. Two advantages of this apparatus, shown in Figure 2, are (a) high light intensity, which decreases the amount of signal averaging required to achieve the desired measurement precision in the presence of photon shot noise; and (b) high beam collimation, which improves the level of light extinction achieved under the narrow acceptance
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Figure 1 Nanosecond multichannel ellipsometric TRCD apparatus using a strain plate, crossed polarizers, and laser photolysis apparatus (described in 68). The strain plate (SP) is a fused silica plate slightly compressed along an axis diagonal to the polarizer axes by an anvil mechanism mounted on a motorized rotation stage. Broad band light from a microsecond flash is linearly polarized by the first polarizer (P1) and converted to highly eccentric, elliptically polarized light by the plate. A circularly dichroic sample adds to or subtracts from this ellipticity, as detected by the analyzing polarizer (P2) and gated optical multichannel analyzer (OMA). Magnet and Faraday rotation compensators (RC) are added for TRMCD measurements.
angle constraints imposed by Rochon polarizers and improves the accuracy of TRCD measurements. The excited states of inorganic complexes (50–53) and conformational changes in photolyzed hemeprotein complexes (54–56) and the plant pigment phytochrome (57–59) have been particular areas of focus for multichannel nanosecond TRCD studies. TRCD spectra were used to assign electronic states and examine ligand-ligand electronic interactions in ruthenium (50, 52), chromium (53), and iron trisbipyridyl (51) complexes, the last having an 800-ps lifetime.
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Figure 2 Ti:sapphire laser-based system for nanosecond TRCD in the far UV. A stream of 150-fs IR pulses, separated by 13 ns, from the Ti:sapphire laser, upconverted to the far UV with a harmonic generator, provides a quasi-continuous wave probe source for fast, single-wavelength TRCD measurements using crossed Rochon polarizers (P1, P2) and a strain plate (SP1). Signal detection with a photomultiplier tube and digitizing oscilloscope is synchronized to the pump laser pulse (e.g. Nd:YAG doubled to 532 nm) with a photodiode (PIN).
Nanosecond TRCD spectroscopy was also useful in determining a triplet mechanism for photo cross-linking of 4-thiouridine in tRNA (60). The first TRCD studies of the carbonmonoxy complex of the oxygen storage protein myoglobin (MbCO) (43, 54) established that the bulk of the protein structural relaxation triggered by photodissociation of the CO ligand occurred within the nanosecond resolution of these measurements, at least as these changes are reflected in the heme band CD, which arises mainly from induced dipole-dipole interactions with aromatic amino acid residues. Because of the compactness of the X-ray structures for MbCO and unliganded Mb, the role of dynamic conformational fluctuations of amino acid side chains in facilitating ligand migration through the protein has elicited much interest. In this regard, although dynamic fluctuations are too ephemeral for direct observation in a nanosecond measurement, a nanosecond TRCD study of the near-UV spectral region found a 110-µs process that suggests the possible involvement of tyrosine residue motions in a net relaxation of conformation corresponding to an early stage of ligand rebinding (55). Much more dramatic structural relaxations are encountered after the photodissociation of carbonmonoxyhemoglobin (HbCO) as the initial, unliganded photoproduct undergoes tertiary and quaternary structural relaxations that move it from the R quaternary state of HbCO to the T state characteristic of equilibrium deoxyHb. The R-to-T transition, corresponding ˚ displacement of dimeric subunits to a 15◦ relative rotation and an almost 1 A
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within the Hb tetramer, has been generally identified in room temperature timeresolved absorption studies with an intraprotein relaxation observed to have a time constant near 20 µs (61). Recent evidence from time-resolved resonance Raman (62) and TRCD (56) studies of the near-UV aromatic bands, however, indicates that some, if not most, of the R-to-T conformational change occurs with a time constant closer to 500 ns, suggesting that the R → T transition in hemoglobin is actually a compound process that proceeds through at least one distinct intermediate (63). Low-temperature trapping is used with low time-resolution spectral methods to study kinetic intermediates. However, the structures of frozen intermediates may differ from their high-temperature analogs (particularly in large, complex molecules such as proteins), either because entropically disfavored conformations that are inaccessible at high temperature become accessible at a lower temperature or because structural transformations encounter temperaturedependent kinetic bottlenecks, i.e. entropically disfavored transition states. Such a situation apparently arises in the phototransformation cycle of the plant pigment phytochrome, in which the visible band TRCD of metastable intermediates was measured at room temperature and found to differ significantly from the static CD spectra of the corresponding species trapped at low temperature (57). Further, far-UV TRCD studies of phytochrome photoactivation show that secondary structure change—specifically, an increase in α-helical folding near the protein N terminus—is associated with decay of the long-lived (40 ms) meta-Ra2 intermediate (58). Interestingly, loss of helicity in the photoreversion reaction proceeds with a 300-µs time constant, about two orders of magnitude more quickly than the formation step, but the folding and unfolding time constants may not be closely related; the photoactivation and photoreversion reactions seem to proceed through different kinetic intermediates (59).
Quarter-Wave–Plate Ellipsometric Methods for Time-Resolved CD An alternative to the strain-plate method is available for situations where fast CD is to be measured at a single wavelength or in a very small wavelength region. This alternative entails placing a quarter-wave–plate retarder (for the desired wavelength) and the sample between initially crossed polarizers, the wave plate axes oriented with the transmission axes of the crossed polarizers. A measurement is performed by rotating the polarizer that is before the sample by a small angle, β. Another measurement is then performed with this polarizer rotated by the opposite angle, −β. The difference divided by the sum of the measured intensities in these measurements yields CD/β. The CD of (+)589tris(ethylenediamine) cobalt(III) 3 − [Co(en)3]3+ taken with the quarterwave–plate and the strain-plate ellipsometric methods are shown in Figure 3.
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Figure 3 The circular dichroism of (+)589tris(ethylenediamine) cobalt(III) (3 − [Co(en)3]3+) measured with two quasi-null ellipsometric techniques at comparable detected light intensities: (a) (solid line) the strain plate method used in the multichannel nanosecond TRCD apparatus in Figure 1 (δ = 1◦ ), and (b) (dashed line) the quarter-wave plate (at 633 nm), polarizer rotation method used in premodern CD instruments (β = 2◦ ). The strain-plate method is free of the ORD artifact that slightly distorts the spectrum obtained with the quarter-wave plate method, shifting the trough at 480 nm to shorter wavelengths.
The advantage of the strain-plate method over the quarter-wave–plate method is that the former can be used for spectral measurements without the need for achromatic retarders. Consider an example in which CD is to be measured over the spectral region from 300 to 600 nm. If a simple, chromatic retarder is a quarter wave at 600 nm, then the wave plate retards a half wave at 300 nm. Thus at 300 nm, the quarter-wave plate would be measuring CB with this method and not CD (14). On the other hand, if a strain plate is used to retard 1◦ at 600 nm, it will retard 2◦ at 300 nm. Thus the CD signal at 600 nm will be magnified two times more than at 300 nm, a factor that is easily corrected with the known dispersion of the retarder. The advantages of conducting spectral measurements have been discussed above and underlie the current use of the strain-plate method. Although nearly achromatic quarter-wave retarding devices such as Fresnel rhombs are available, they typically have too much residual strain for use in these measurements. However, a strain-free water-filled rhomb was used successfully in the early half-shade quasi-null ellipsometric CD instrument mentioned above (6). In general, this quarterwave–plate method should be regarded as most useful for measurements at
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single wavelengths or over small spectral regions. Moreover, although strain plates are simple to make, the commercial availability of quarter-wave plates (at many wavelengths) may make quarter-wave–plate methods more attractive to many laboratories. Quarter-wave–plate methods also offer a practical advantage in that β is freely and reversibly varied with a rotation mount, whereas hysteresis due to frictional forces is frequently encountered in the anvil mechanism used to vary δ in the strain-plate method [although new strain-plate designs using circular quartz plates address such problems (64)]. A second quarter-wave–plate method for quasi-null ellipsometric CD measurements uses exactly crossed polarizers and skews the wave-plate axes by a small angle, β, from the polarizer axes, an approach that shows more promise for multichannel TRCD than does the previous quarter-wave–plate method employing a polarizer rotation. The difference between the two possible waveplate orientations (fast or slow axis rotated by β from the first polarizer axis), normalized to the sum, defines a signal that is proportional to CD/β. The advantage of this approach over the previous quarter-wave–plate method is that the wave plate need not be achromatic in order to make artifact-free multichannel measurements. Deviations from quarter-wave retardance do not introduce CB artifacts (to first order) and affect only the magnitude of the measured signal by changing the effective value of β in this method (64a).
NANOSECOND MCD In principle, applying a magnetic field to any chromophore induces a CD; the resulting MCD strongly reflects electronic structural features such as spin and orbital degeneracies from which information about the spatial structure and coordination of the chromophore may be inferred. Accordingly, applications have principally focused on chromophores with unpaired spins (metal complexes), rotational symmetries (aromatics, particularly the porphyrins), or both (hemes). The effect of the applied field is linear and comprises three terms—A, B, and C—that phenomenologically characterize MCD spectra (65). If either the initial or final electronic state is degenerate at zero field, the splitting of degenerate components in the applied field produces a derivative-shaped A term (inversely proportional in magnitude to the transition bandwidth) that may be considered the generalization of atomic Zeeman spectroscopy to the broad band shapes of condensed-phase molecules. Degeneracy in the initial state also gives rise to an MCD contribution, a C term, as a result of the difference in the Boltzmann populations of degenerate components as they become split in the applied field (inversely proportional in magnitude to the average thermal energy, kB T ). Finally, perturbation of the wavefunctions of the initial and final electronic states by the applied field produces a B term that is present, although often weakly, even in the absence of electronic degeneracies (proportional in second-order
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perturbation theory to a sum over intermediate states containing individual terms inversely proportional to energy differences between intermediate and initial or final states). These terms contribute to the MCD as · µ ¶ ¸ C A ∂g(ν) −2k N H + B+ g(ν) , 1. α(ν) = 3 h ∂ν kB T where H is the field strength, ν is frequency, k = 8π 3ν/hc, h is the Planck constant, kB is the Boltzmann constant, c is the speed of light, T is absolute temperature, N = molecules/cm3, and g(ν) is the band shape function. Quantum mechanical expressions for A, B, and C are given below for isotropic and photoselection-oriented cases. The extension of the TRCD apparatus in Figure 1 to time-resolved MCD measurements is not as straightforward as simply adding a field to magnetize the sample, because measures must be taken to counter the concomitant Faraday rotation imposed on the probe beam’s elliptical polarization as it travels through the cell windows and solvent (66). As the magnitude of δ sets the scale for the amount of polarization rotation that may be tolerated, rotation cancellation schemes must reduce the net rotation to much less than δ in order to avoid signal distortion and must do so achromatically for multichannel measurements. The most precisely achromatic compensation is provided by using a matching solvent blank in an equal and opposite applied field (67). An inexpensive alternative to placing a solvent blank in a second magnet is to use optically active solutions, e.g. aqueous solutions of resolved sucrose or fructose, a Faraday rotation compensation method that works quite well in the visible spectral region where the optical rotary dispersion (ORD) and magnetic ORD (MORD) of many transparent substances have similar wavelength dependencies. A further advantage of using a second field as a compensator is the opportunity to match any inhomogeneity in the applied field, which enables more complete Faraday compensation (68). As in conventional MCD measurements, any natural CD exhibited by the sample adds to the TRMCD signal and may be canceled by taking the difference of opposed field TRMCD measurements. Nanosecond TRMCD may be applied to the excited states (66) or kinetic intermediates (44) of magneto-optically active chromophores, and applications have focused mainly on the photodissociation intermediates of hemeproteinligand complexes, as hemeproteins generally have intense, room temperature MCD signals throughout the heme absorption bands. In studies of mammalian cytochrome c oxidase (cytochrome aa3) (69, 70), the terminal enzyme in the respiratory chain, as well as its bacterial counterpart cytochrome ba3 (71), a bacterial oxidase, and cytochrome c3, a bacterial electron transport protein (72), TRMCD was used to follow the ligation state of heme iron after photodissociation of the CO complex. Loss of the axial ligand allows d-shell electrons
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on the heme iron of the diamagnetic complex to become unpaired in the photodissociation product, the intersystem crossing taking place in femtoseconds. The resulting paramagnetism introduces a C term that dramatically enhances the Soret band MCD signal and serves as a marker for pentacoordinate ligation (an imidazole nitrogen of a protein residue, the proximal histidine, axially coordinates the iron atom as it sits nearly in-plane with the four nitrogens of the porphyrin ring) in these enzymes. Researchers want to uncover the mechanism by which cytochrome oxidase couples the energy released in the reduction of oxygen to the pumping of protons across the inner mitochondrial membrane and thereby creates a gradient that is used to produce ATP as an energy source for cellular processes. TRMCD studies show that a pentacoordinate MCD signal arises in cytochrome oxidase promptly after nanosecond photolysis of the cytochrome a3–CO complex, as would be expected for cleavage of the Fe-CO bond (69–71), but, interestingly, low-intensity time-resolved resonance Raman (TR3) spectra taken at the same time interval after photolysis are missing the Fe-N stretch signal that is a Raman marker for high-spin, pentacoordinate cytochrome a3 (69). The apparent discrepancy in coordinations implied for cytochrome a3 by the TRMCD and TR3 markers is resolved in a ligand shuttle model that postulates the loss of Fe-Nimidazole coordination, which accounts for the absent Raman band, and the binding of an unspecified ligand endogenous to the protein at the distal site vacated by CO, which is consistent with the pentacoordinate TRMCD signal. Although this model must be considered speculative (although features such as the displacement of the proximal histidine by binding of a distal ligand are not without precedent), it does suggest one mechanism by which redox events at cytochrome a3 could serve as a gate for proton pumping (69). The binding of an endogenous ligand, in this case a histidine residue located in the distal region of the heme pocket, has been observed after the photodissociation of cytochrome c3–CO. Although ligand binding probably plays no role in the physiological function of this electron transport enzyme, CO displaces the distal histidine to form a stable complex that may be photodissociated. In their TRMCD spectral study, O’Connor et al (72) noted a kinetic competition between photodissociated CO and histidine for rebinding to iron by the transient appearance of an A term in the visible band region, characteristic of the (highly symmetric) bis-histidine complex.
NANOSECOND ORD AND LD In 1953 Keston & Lospalluto (73) described a quasi-null method for ORD measurements, which was closely related to the ellipsometric CD method, and incorporated it into a polarimetric attachment for the Beckman DU spectrophotometer. The same approach was incorporated into a flash apparatus for
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Figure 4 Probe beam optics for nanosecond quasi-null polarimetric TRORD (TRMORD) apparatus. Polarizers are crossed except for a small rotation angle ± β applied with a motorized rotation stage. A circularly birefringent sample adds or subtracts from this rotation, as detected by the analyzing polarizer and optical multichannel analyzer.
microsecond time-resolved ORD (TRORD) measurements of triplet states in aromatics (74), and a similar apparatus for single-wavelength OR measurements using a CW He-Ne laser was applied to phototransformation intermediates of rhodopsin (75). Most recently, the technique was applied to a nanosecond TRORD study of HbCO photolysis intermediates using a multichannel laser photolysis apparatus similar to the nanosecond TRCD apparatus described above (14). In the nanosecond TRORD apparatus shown schematically in Figure 4, the front polarizer is rotated to ± β from horizontal, where β is a small angle, typically 1◦ . The optical rotation of the sample adds or subtracts to the net rotation of the beam from horizontal, as detected by the analyzing polarizer. The difference in transmitted intensities for the two polarizer orientations, normalized to the sum, defines a signal that is proportional to CB/β. In anisotropic samples possessing a linear dichroism, the apparatus in Figure 4 more generally measures a linear combination of CB and LD0 , the latter being the component of LD diagonal to the horizontal and vertical axes. The sensitivity of this technique to both CB and LD0 is evident when one recalls that both optical effects rotate the probe beam’s plane of polarization. The measurement of either CB or LD0 can be maximized through appropriate alignment of the crossed polarizers, as shown in Figure 5. With a vertically polarized pump beam, orienting the probe beam polarizers vertically and horizontally
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Figure 5 Both the ORD and the photoselection-induced LD spectrum of photolyzed HbCO are obtained separately on the apparatus in Figure 4 by appropriate orientation of the laser polarization vector relative to the probe beam polarization axes. ORD (solid line) is obtained with the pump polarization aligned along the axis of the fixed probe polarizer (signal of 0.1 equals an optical rotation of 0.006◦ ), and LD (dashed line) is obtained with the pump polarization bisecting the probe polarization axes (signal of 0.3 equals an absorption difference of 0.009).
will eliminate LD0 effects and produce a pure CB measurement, whereas probe polarizers oriented at +45◦ and −45◦ will maximize the LD0 measurement and yield, in most cases, a pure LD signal. The apparatus in Figure 4 has been applied to ultrasensitive TRLD measurements in bacteriorhodopsin purple membrane suspension, measurements that placed an upper limit of 8◦ on the extent of chromophore reorientation during the K-to-L intermediate transformation (76, 77) (see Figure 6). In similar measurements on sickle hemoglobin polymers, the small amount of heme-bound CO that photodissociates from the polymer was found to recombine several orders of magnitude more slowly than ligand recombination in monomers (78, 79). The nanosecond optical rotatory dispersion method just described has also been extended to time-resolved measurements of magnetically induced ORD (TRMORD), and applications to HbCO and MbCO photolysis are currently in progress (79a). In this case, taking the difference of opposed field measurements cancels any natural ORD or any LD contributing to the TRMORD signal at a particular field configuration.
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Figure 6 TRLD spectra of bacteriorhodopsin, observed 500 ns after photolysis with the 532-nm pulse of a frequency-doubled Nd:YAG laser, showing the signal-to-noise improvement of the quasinull polarimetric method over a conventional measurement. The noisy signal was obtained by the standard method with 64 signal averages; the smooth signal was obtained after 6 averages with the quasi-null polarimetric technique.
PICOSECOND CD AND MCD The ellipsometric technique used in nanosecond TRCD may in principle be extended to picosecond or femtosecond measurements. However, the high repetition rates available in picosecond lasers make absorptive CD measurement using circularly polarized light in this time regime practical, as demonstrated by Xie & Simon in 1989 (8). Their apparatus uses two electro-optically cavity dumped dye lasers operated at 1 kHz to produce tunable 50-ps pulse-width pump and probe pulses. To reduce LB and LD artifacts, the pump pulses are passed through a depolarizer and spinning half-wave plate before exciting the sample. The laser pulse timing is synchronized to a photoelastic modulator placed after a linear polarizer in the probe beam to produce alternating circular polarizations in the beam by arranging for probe pulses to arrive when alternate +λ/4 and −λ/4 voltages are applied to the modulator. The polarized probe beam is sent through the region of the sample excited by the pump, the relative timing of pump and probe pulses being adjusted with a variable delay line placed in the pump beam. A lock-in amplifier detects the CD signal in the output of the photomultiplier (PMT) monitoring the transmitted probe beam. Xie & Simon (80, 81) used picosecond TRCD measurements to follow the dynamics of protein relaxation after photodissociation of CO from myoglobin and found a several-hundred-picosecond conformational relaxation that is not
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apparent in the unpolarized transient absorption data. Xie & Simon (82) studied the primary intermediates in the photocycle of bacterial photosynthetic reaction centers with picosecond TRCD as well, and their CD spectra do not appear to support earlier assignments of exciton coupling as the source of optical activity in these intermediates. These authors extended the picosecond TRCD technique to magnetic CD measurements, and in an application to the MbCO system with picosecond TRMCD after ligand photodissociation, they showed that the low to high spin state change of the heme iron occurs within the 20 ps risetime of the instrument (9).
TRANSIENT-GRATING DETECTED CD This nonlinear, null background method offers high sensitivity and the potential for nanosecond or picosecond time resolution in CD detection (12, 13). Two light beams of equal amplitude and linearly polarized in orthogonal directions intersect at an acute angle to produce an interference pattern that is spatially modulated between left and right circular polarizations. A circularly dichroic sample differentially absorbs light in the spatial regions irradiated with different circular polarizations, resulting in an index of refraction grating that Bragg diffracts a third probe beam into a detector with an intensity proportional to CD2. An advantage of this technique, in addition to sensitivity, is the ability to use an unpolarized probe beam, which avoids polarization artifacts at the detector. Disadvantages are sensitivity to LD effects and the need to accommodate dependence of the Bragg angle on wavelength when scanning a spectrum. The thermal grating created by circularly dichroic absorption decays with the thermal diffusion time constant, which is several microseconds in aqueous solution. Nanosecond and picosecond time resolutions have already been demonstrated in two-step excitation transient grating experiments without circular polarization (83), and comparable time resolutions should also be possible in transient grating CD measurements (12).
PHOTOSELECTION IN TRCD AND TRMCD Photoexcitation partially orients a molecular sample through the process of photoselection (84); the extent of orientation depends on the symmetry properties of the chromophoric transition; the intensity, polarization, and propagation properties of the light; and the orientation and excitation relaxation properties of the sample. Photoselection-induced orientation can influence TRCD and TRMCD measurements directly, through the effect of molecular anisotropy on the orientation-averaged CD and MCD, and indirectly, through measurement artifacts associated with photoselection-induced LD and LB (discussed below). In fluid samples, both these direct and (first order) indirect effects decay
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with the rotational diffusion lifetime, so (neglecting population decay to the prephotolysis state) the return to the isotropic TRCD or TRMCD signal is given by 1α(t) − 1αt=∞ = (1αt=0+ − 1αt=∞ ) exp[−6DR t],
2.
where 1α = αL − αR is the apparent difference in absorption coefficients for left and right circularly polarized probe light, respectively; 1α t = 0+ is the rotationally unrelaxed TRCD/MCD immediately after photolysis; and DR is the rotational diffusion constant. For isotropic molecules, the diffusion constant is given by DR = kBT/(6ηV ), where η is the viscosity and V is the hydrodynamic volume of the solute.
CD of Samples Oriented by Photoselection The orientation-averaged CD of a photoselected sample with nondegenerate transitions at the probe frequency ν and the pump frequency ν 0 is given (in the low conversion limit) by α L − αR =
2 0 kk N φ I g(ν)g(ν 0 ) 15 × {3|µ0 | Im(µ · m) + (µ · µ0 )Im(µ0 · m) 2
+ (πν/c)(µ0 × µ)Qµ0 }
3.
for a collinear excitation geometry, where φ = photoconversion quantum efficiency; I = photons/cm2, µ, m, and Q are the electric dipole, magnetic dipole, and electric quadrupole moments for the probe transition, respectively; and the primes indicate quantities for the pump transition (85). The quadrupole term, which does not appear in the CD of isotropic samples, vanishes from the analogous expression for the degenerate transition case. General expressions for the CD of photoselected samples and their application to special symmetry cases can be found in Reference 85.
MCD of Samples Oriented by Photoselection The orientation-averaged MCD of a photoselected sample excited by a linearly polarized pump beam propagating perpendicularly to the probe beam (crossed beams) is given (again, in the low conversion limit) by · µ ¶ ¸ −2Hkk 0 N I φ 0 a ∂g(ν) c X−beam 1α = g (ν) + b+ g(ν) , 4. 15 h ∂ν kB T where a = A1 + A2 sin2 θ + A3 cos2 θ, ª © b = −Im B 1 + B 2 sin2 θ + B 3 cos2 θ ,
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and c = C 1 + C 2 sin2 θ + C 3 cos2 θ.
5.
θ is the azimuthal angle of the pump polarization relative to the pump-probe propagation plane, and the subscripted A, B, and C MCD terms, generalized for photoselection, are given by 0
A1 = D A ¡ ¡ ¢ ¢ i X ∗ A2 = µ ˜ (i a 00 f b00 ) · µ ˜ ∗ i a0 0 f b00 µ(i ˜ i a0 0 f b00 ˜ a fb ) × µ 0 dd 0 00 aa a bb0 b00
¤ £ ˜ a 00 i a )δbb00 ˜ f b f b00 )δaa 00 − m(i · m( ¡ ¢ ∗¡ 0 0 ¢ i X A3 = µ(i ˜ a fb ) × µ ˜ ∗ (i a 00 f b00 ) · µ ˜ i a0 0 f b00 µ ˜ i a 0 f b0 0 dd 0 00 aa a bb0 b00
¤ £ ˜ a 00 i a )δbb00 ˜ f b f b00 )δaa 00 − m(i · m( 0 D X µ(i ˜ a fb ) d ab # " Xµ ˜ ∗( j fb ) X µ ˜ ∗ (i a j) ˜ ∗ (i a j) × m ˜ ∗( j fb ) × m + · Ej − Ef E j − Ei j6= f j6=i # " ˜ ∗( j fb ) X µ ˜ ∗ (i a j) 1 X ∗ ¡ 0 0 ¢ X µ ˜ ∗ (i a j)m ˜ ∗ ( j f b )m B2 = µ ˜ i a 0 f b0 · + dd 0 Ej − Ef E j − Ei aa 0 j6= f j6=i
B1 =
bb0
¡ ¢ X ¡ 0 0¢ ˜ i a0 0 f b00 − µ ˜ i a 0 f b0 · µ(i ˜ a fb ) × µ aa 0 bb0
" ·
X µ(i ˜ j fb ) ˜ a j) × m( j6= f
Ej − Ef
¡ ˜ ∗ i a0 0 f b00 ×µ ˜ ∗ (i a f b ) · µ
+
¢
X µ( ˜ a j) ˜ j f b ) × m(i j6=i
E j − Ei
#
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B3 =
473
2 X ¡ 0 0¢ µ ˜ i a 0 f b0 × µ(i ˜ a fb ) dd 0 0 " ·
aa bb0
Xµ ˜ ∗( j fb ) ˜ ∗ (i a j)m j6= f
Ej − Ef
+
Xµ ˜ ∗ (i a j) ˜ ∗ ( j f b )m j6=i
#
E j − Ei
¡ ¢ ·µ ˜ ∗ i a0 0 f b00
0
C1 = D C ¡ ¡ ¢ ¢ i X ∗ ˜ a 00 i a ) C2 = µ ˜ (i a 00 f b ) · µ ˜ ∗ i a0 0 f b00 µ(i ˜ i a0 0 f b00 · m(i ˜ a fb ) × µ 0 dd 0 00 aa a bb0
C3 =
¡ ¢ ∗¡ 0 0 ¢ i X ˜ a 00 i a ). µ(i ˜ a fb ) × µ ˜ ∗ (i a 00 f b ) · µ ˜ i a0 0 f b00 µ ˜ i a 0 f b0 · m(i 0 dd 0 00
6.
aa a bb0
Ei , Ef , and Ej denote the energies of initial (i), final ( f ), and intermediate ( j) states; d and d 0 indicate the (ground state) degeneracies of probe and pump transitions, respectively; and the subscripts a and b label degenerate levels within initial and final states (86). More general expressions for the oriented MCD as a function of excitation geometry and polarization are given in Reference 86. The isotropic A, B, and C terms, first derived by Buckingham & Stephens (86a), and D (dipole strength) term appearing above are given by £ ¤ i X ˜ f b f b0 )δaa 0 − m(i ˜ a 0 i a )δbb0 A= µ(i ˜ a fb ) × µ ˜ ∗ (i a 0 f b0 ) · m( 2d 0 aa bb0
( " Xm ˜ ∗( j fb ) · µ 1X ˜ ∗ (i a j) B= Im µ(i ˜ a fb ) × d ab Ej − Ef j6= f +
Xm ˜ ∗ (i a j) · µ ˜ ∗( j fb ) j6=i
#)
E j − Ei
C=
i X ˜ a0 ia ) µ(i ˜ a fb ) × µ ˜ ∗ (i a 0 f b ) · m(i 2d aa 0 b
D=
1X |µ(i ˜ a f b )|2 . d ab
Expressions for the effects of photoselection-induced orientation on CD and MCD such as those in Equations 3–7, combined with the time evolution in
7.
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Equation 2 for fluid samples, may be used to describe alterations in CD and MCD imposed by the orienting influence of photoexcitation in rigid samples and resulting dynamic changes in the TRCD and TRMCD of fluid samples undergoing rotational relaxation (85, 86).
ARTIFACTS Before examining potential artifacts that can arise in TRCD and TRMCD methods, we first consider how conventional phase-locked detection CD measurements are performed. We then discuss photoselection-induced LB and LD artifacts in ellipsometric TRCD and TRMCD measurements on phototransformed samples.
Phase-Locked Detection Methods Phase-locked detection methods use rapid polarization modulation, accomplished with either an electro-optic or photoelastic modulator, to provide the signal-to-noise advantage needed to detect a circular dichroism that is typically less than one part in 103 of total absorption. The PEM consists of a piezoelastic crystal coupled to another crystal such as calcite or MgF2. A sinusoidally varying voltage is applied across the piezoelectric crystal so that a time-dependent retardance is achieved in the crystal. A common configuration is one in which light is passed through a vertical polarizer before traversing the PEM oriented at 45◦ with respect to the polarizer. The light is then passed through the sample, resulting in a transmitted intensity (for optically thin samples) of e−A {1 + LD cos[δ(t)] + CD sin[δ(t)]},
8.
where A is the absorbance. The retardance, δ(t), is given by A sin(ωt),with A = (2π ds)/λ, where d is the width of the crystal, ω is the frequency of modulation of the applied driving voltage that is set equal to the resonant frequency of the crystal, and s is a strain factor that depends on the properties of the crystal and the voltage across it. The amplitude of the retardance is usually kept constant during a spectral circular dichroism measurement by varying the voltage amplitude as the wavelengths are scanned. The cos (δ) and sin (δ) terms can be expanded in terms of Bessel functions as follows: cos(δ) = J0 (A) + 2J2 (A) cos(2ωt) + 2J4 (A) cos(4ωt) + · · · and sin(δ) = 2J1 (A) sin(ωt) + 2J3 (A) cos(3ωt) + · · ·
9.
A lock-in amplifier discriminates between the different frequency components to measure either CD (most applications) or LD.
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In most experiments, the voltage is set so that J1(A) is a maximum. Alternatives are possible such as setting J0(A) equal to zero. Residual strain in the PEM can result in some mixing of the cos(δ) and sin(δ) components so that if LD is nonzero, LD can be mixed into a CD measurement or CD can appear in a LD measurement. LB of the front window of the sample cell will convolute and distort the LD and CD signals; thus low strain cells are necessary. Finally, the purity of CD, LD, or any polarization measurement depends on all polarization effects being relatively small. If a sample has a large linear birefringence, then an experiment designed to measure CD will yield a signal that is distorted by birefringence. A discussion of artifacts as they arise in phase-locked detection methods for picosecond TRCD is presented in Reference 87.
Ellipsometric CD The ellipsometric approach, while having the time-resolution advantages discussed above over the phase-locked approach, is also more sensitive to artifacts. LB effects, in particular, enter into the TRCD signal to first order, as shown in Equation 10 (46, 48, 88). This is in contrast to the situation in phase-lock detection, discussed above, where LB and LD artifacts enter only as higher-order couplings between optical effects. Photoselection-induced LB is particularly pernicious in nanosecond TRCD experiments because its spectral variation and time evolution tend to mimic a bona fide TRCD transient. In this regard, knowledge of the rotational diffusion time constant helps to distinguish transients that may correspond to photoselection effects. 2CD − 2LB0 − CB · LB + CD · LD IR − IL = 10. IR + IL δ The second-order artifacts in the numerator of Equation 10 can usually be neglected, with the exception of the CB · LB coupling in the context of TRMCD. The Faraday rotation of cell and solvent can contribute a very substantial CB that renders the product of CB and LB nonnegligible compared with the MCD of the sample (this coupling is not mitigated by Faraday rotation compensation schemes, as these are not applied within the sample where coupling takes place). On the other hand, taking the difference of opposed-field measurements cancels the first order contribution of LB0 to the TRMCD signal, an advantage not available to natural ellipsometric TRCD (48). For detailed discussions of artifacts in TRCD and TRMCD measurements, and strategies to minimize these, see References 46, 48, and 88. TRCD/MCD signal =
FUTURE PROSPECTS There is tremendous interest in unraveling the early (submillisecond) events in protein and peptide folding, and far-UV TRCD promises to be a powerful
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tool for monitoring fast secondary structure formation in such studies. Further progress in this area depends on developing peptide and protein systems in which folding or unfolding can be initiated by a rapid perturbation such as the laser-initiated phototransformation of an appropriate internal chromophore or a laser-induced temperature jump, to name two possibilities. Laser temperature jump has already been used as an ultrafast trigger in time-resolved IR studies of helix melting and formation in peptides (89), but shock wave–induced birefringence artifacts must be overcome in coupling this method to nanosecond TRCD spectroscopy. Current efforts in this direction are focused on the nanosecond TRORD approach, which is much less sensitive to birefringence transients (89a). Ligand photodissociation or photoinitiated redox reactions at the heme chromophore offers another possible basis for triggering folding and unfolding processes in hemeproteins (90, 91). Fast protein-folding switches and applications are reviewed in Reference 92. Extension of TRMCD studies into the near-UV spectral region would open up aromatic molecules smaller than porphyrins for study, which could be used to monitor processes in the wide variety of proteins that contain MCD-active aromatic residues such as tryptophan and tyrosine. Study of the allosteric transition in hemoglobin through the TRMCD of tryptophan and tyrosine residues at the dimer-dimer interface is one example, although the possibilities are not limited to hemeproteins. The sensitivity of MCD to heteroatom protonation, illustrated by the elegant work of Michl (93), suggests possible applications of TRMCD spectroscopy in the study of intramolecular proton transfer dynamics in excited states of heteroatomic aromatic molecules. Ellipsometric TRCD techniques should be compatible with femtosecond laser technologies, and the kilohertz repetition rates typically available in these systems also make phase-locked modulation-detection methods appropriate. [The large CD of circularly polarized excitons in solid state semiconductors enabled a femtosecond TRCD study of GaAs quantum wells at 4 K to make separate absorption measurements for left and right circularly polarized probe beams (94)]. Looking ahead, femtosecond TRMCD would be particularly suited to observing ultrafast electronic processes, such as spin state conversions, in porphyrins and heme systems. Whereas it is clear that the features of electronic structure to which TRMCD spectroscopy is most sensitive can change on ultrafast time scales, because of the small mass of the electron, how rapidly the secondary, tertiary, and quaternary structures of biopolymers can respond to ultrafast perturbations ultimately remains an open question for future TRCD studies. ACKNOWLEDGMENTS This work was prepared with support from the National Institutes of Health— grants GM-35158, GM-38549 (DSK), and NRSA HL08969 (DBKS)—and
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from the US Department of Energy, through funds provided by the University of California for discretionary research by Los Alamos National Laboratory (RAG). Visit the Annual Reviews home page at http://www.annurev.org.
Literature Cited 1. Grosjean M, Legrand M. 1960. C. R. Acad. Sci. 251:2150–52 2. Kemp JC. 1969. J. Opt. Soc. Am. 59:950– 54 3. Billardon M, Badoz J. 1966. C. R. Acad. Sci. Ser. B 263:139–42 4. Jasperson SN, Schnatterly SE. 1969. Rev. Sci. Instrum. 40:761–67 4a. Faraday M. 1846. Philos. Trans. R. Soc. London 136:1–20 5. Kliger DS, Lewis JW. 1987. Rev. Chem. Intermed. 8:367–98 6. Kuhn W, Braun E. 1930. Z. Phys. Chem. B 8:445–54 7. Abu-Shumays A, Duffield JJ. 1966. Anal. Chem. 38(7):A29–A58 8. Xie XL, Simon JD. 1989. Rev. Sci. Instrum. 60:2614–27 9. Xie XL, Simon JD. 1990. J. Phys. Chem. 94:8014–16 10. Wu K, McGown LB. 1991. Appl. Spectrosc. 45:1–3 11. Riehl JP, Richardson FS. 1993. Methods Enzymol. 226:539–53 12. Terazima M. 1995. J. Phys. Chem. 99: 1834–36 13. Terazima M. 1996. Mol. Phys. 88:1223– 36 14. Shapiro DB, Goldbeck RA, Che D, Esquerra RM, Paquette SJ, Kliger DS. 1995. Biophys. J. 68:326–34 15. Che D, Shapiro DB, Esquerra RM, Kliger DS. 1994. Chem. Phys. Lett. 224:145– 54 16. Ferrone FA, Hopfield JJ, Schnatterly SE. 1974. Rev. Sci. Instrum. 45:1392–96 17. Bayley P, Martin S, Anson M. 1975. Biophys. Res. Commun. 66:303–8 18. Anson M, Martin SR, Bayley P. 1977. Rev. Sci. Instrum. 48:953–62 19. Gruenewald B, Knoche W. 1978. Rev. Sci. Instrum. 49:797–801 20. Bayley PM, Anson M. 1974. Biopolymers 13:401–5 21. Anson M, Bayley PM. 1974. J. Phys. E 7:481–86 22. Hiromi K, Sˆozaburo O, Itoh S, Nagamura T. 1968. J. Biochem. 64:897–900
23. Pflumm M, Luchins J, Beychok S. 1986. Methods Enzymol. 130:519–34 24. Nitta K, Segawa T, Kuwajima K, Sugai S. 1977. Biopolymers 16:703–6 25. Luchins J, Beychok S. 1978. Science 199:425–26 26. Gruenewald B, Nicola CU, Lustig A, Schwarz G, Klump H. 1979. Biophys. Chem. 9:137–47 27. Ikeguchi M, Kuwajima K, Mitani M, Sugai S. 1986. Biochemistry 25:6965–72 28. Kuwajima K, Yamaya H, Miwa S, Sugai S, Nagamura T. 1987. FEBS Lett. 221:115–18 29. Ramsay G, Ionescu R, Eftink MR. 1995. Biophys. J. 69:701–7 30. Wada A, Tachibana H, Hayashi H, Saito Y. 1980. J. Biochem. Biophys. Methods 2:257–69 31. Geng L, McGown LB. 1992. Anal. Chem. 64:68–74 32. Wu K, Geng L, Joseph MJ, McGown LB. 1993. Anal. Chem. 65:2339–45 33. Tinoco I Jr, Ehrenberg B, Steinberg IZ. 1977. J. Chem. Phys. 66:916–20 34. Metcalf DH, Snyder SW, Wu S, Hilmes GL, Riehl JP, et al. 1989. J. Am. Chem. Soc. 111:3082–83 35. Metcalf DH, Snyder SW, Demas JN, Richardson FS. 1990. J. Am. Chem. Soc. 112:469–79 36. Metcalf DH, Snyder SW, Demas JN, Richardson FS. 1990. J. Am. Chem. Soc. 112:5681–95 37. Blok PML, Schakel P, Dekkers HPJM. 1990. Meas. Sci. Technol. 1:126–30 38. Rexwinkel RB, Schakel P, Meskers SCJ, Dekkers HPJM. 1993. Appl. Spectrosc. 47:731–40 39. Metcalf DH, Snyder SW, Demas JN, Richardson FS. 1990. J. Phys. Chem. 94:7143–53 40. Metcalf DH, Stewart JMM, Snyder SW, Grisham CM, Richardson FS. 1992. Inorg. Chem. 31:2445–55 41. Schauerte JA, Steel DG, Gafni A. 1992. Proc. Natl. Acad. Sci. USA 89:10154–58 42. Schauerte JA, Schlyer BD, Steel DG,
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43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 64a. 65.
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GOLDBECK, KIM-SHAPIRO & KLIGER Gafni A. 1995. Proc. Natl. Acad. Sci. USA 92:569–73 Lewis JW, Tilton RF, Einterz CM, Milder SJ, Kuntz ID, Kliger DS. 1985. J. Phys. Chem. 89:289–94 Lewis JW, Goldbeck RA, Kliger DS, Xie XL, Dunn RC, Simon JD. 1992. J. Phys. Chem. 96:5243–54 Lewis JW, Yee GG, Kliger DS. 1987. Rev. Sci. Instrum. 58:939–44 Einterz CM, Lewis JW, Milder SJ, Kliger DS. 1985. J. Phys. Chem. 89:3845– 53 Zhang CF, Lewis JW, Cerpa R, Kuntz ID, Kliger DS. 1993. J. Phys. Chem. 97:5499– 505 Che D, Goldbeck RA, McCauley SW, Kliger DS. 1994. J. Phys. Chem. 98: 3601–11 Wen YX, Chen E, Lewis JW, Kliger DS. 1996. Rev. Sci. Instrum. 67:3010–16 Gold JS, Milder SJ, Lewis JW, Kliger DS. 1985. J. Am. Chem. Soc. 107:8285– 86 Milder SJ, Gold JS, Kliger DS. 1986. J. Am. Chem. Soc. 108:8295–96 Milder SJ, Gold JS, Kliger DS. 1988. Chem. Phys. Lett. 144:269–72 Milder SJ, Gold JS, Kliger DS. 1990. Inorg. Chem. 29:2506–11 Milder SJ, Bj¨orling SC, Kuntz ID, Kliger DS. 1988. Biophys. J. 53:659–64 Chen EF, Kliger DS. 1996. Inorg. Chim. Acta. 242:149–58 Bj¨orling SC, Goldbeck RA, Paquette SJ, Milder SJ, Kliger DS. 1996. Biochemistry 35:8619–27 Bj¨orling SC, Zhang CF, Farrens DL, Song PS, Kliger DS. 1992. J. Am. Chem. Soc. 114:4581–88 Chen EF, Parker W, Lewis JW, Song PS, Kliger DS. 1993. J. Am. Chem. Soc. 115:9854–55 Chen EF, Lapko V, Song PS, Kliger DS. 1997. Biochemistry. In press Milder SJ, Weiss PS, Kliger DS. 1989. Biochemistry 28:2258–64 Hofrichter J, Sommer JH, Henry ER, Eaton WA. 1983. Proc. Natl. Acad. Sci. USA 80:2235–39 Jayaraman V, Rodgers KR, Mukerji I, Spiro TG. 1995. Science 269:1843–48 Goldbeck RA, Bj¨orling SC, Paquette SJ, Kliger DS. 1996. Biochemistry 35:8628– 39 Esquerra RM, Lewis JW, Kliger DS. 1997. Rev. Sci. Instrum. 68:1372–76 Goldbeck RA, Esquerra RM, Kliger DS. Unpublished results Piepho SB, Schatz PN. 1983. Group Theory in Spectroscopy with Applications to
66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 79a. 80. 81. 82. 83. 84. 85. 86. 86a. 87. 88.
Magnetic Circular Dichroism. New York: Wiley Goldbeck RA, Dawes TD, Milder SJ, Lewis JW, Kliger DS. 1989. Chem. Phys. Lett. 156:545–49 Shashoua VE. 1973. Methods Enzymol. 27:796–810 Goldbeck RA, Kliger DS. 1993. Methods Enzymol. 226:147–77 ´ Dyer RB, Woodruff WH, Einarsd´ottir O, Bagley KA, Palmer G, et al. 1991. Proc. Natl. Acad. Sci. USA 88:2588–92 ´ Goldbeck RA, Dawes TD, Einarsd´ottir O, Woodruff WH, Kliger DS. 1991. Biophys. J. 60:125–34 ´ Dawes TD, Goldbeck RA, Einarsd´ottir O, O’Connor DB, Surerus KK, et al. 1992. Biochemistry 31:9376–87 O’Connor DB, Goldbeck RA, Hazzard JH, Kliger DS, Cusanovich MA. 1993. Biophys. J. 65:1718–26 Keston A, Lospalluto J. 1953. Fed. Proc. 12:229 Carapellucci PA, Richtol HH, Strong RL. 1967. J. Am. Chem. Soc. 89:1742–43 Tsuda M. 1979. Photochem. Photobiol. 29:175–77 Che DP, Shapiro DB, Esquerra RM, Kliger DS. 1994. Chem. Phys. Lett. 224:145–54 Esquerra RM, Che D, Shapiro DB, Lewis JW, Bogolmoni RA, et al. 1996. Biophys. J. 70:962–70 Shapiro DB, Esquerra RM, Goldbeck RA, Ballas SK, Mohandas N, Kliger DS. 1995. J. Biol. Chem. 270:26078–98 Shapiro DB, Esquerra RM, Goldbeck RA, Ballas SK, Mohandas N, Kliger DS. 1996. J. Mol. Biol. 259:947–56 Esquerra RM, Goldbeck RA, Shapiro DB, Kliger DS. Unpublished results Xie XL, Simon JD. 1990. J. Am. Chem. Soc. 112:7802–3 Xie XL, Simon JD. 1991. Biochemistry 30:3682–92 Xie XL, Simon JD. 1991. Biochim. Biophys. Acta 1057:131–39 Miller RJD, Pierre M, Rose TS, Fayer MD. 1984. J. Phys. Chem. 88:3021–25 Albrecht AC. 1961. J. Mol. Spectrosc. 6:84–108 Che D, Goldbeck RA, Kliger DS. 1994. J. Chem. Phys. 100:8602–13 Goldbeck RA, Che D, Kliger DS. 1996. J. Chem. Phys. 104:6930–37 Buckingham AD, Stephens PJ. 1966. Annu. Rev. Phys. Chem. 17:399–432 Xie XL, Simon JD. 1990. Opt. Soc. Am. B 7:1673–84 Bj¨orling SC, Goldbeck RA, Milder SJ, Randall CE, Lewis JW, Kliger DS. 1991.
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FAST CD AND MCD J. Phys. Chem. 95:4685–94 89. Williams S, Causgrove TP, Gilmanshin R, Fang KS, Callender RH, et al. 1996. Biochemistry 35:691–97 89a. Wen Y-X, Strauss CEM, Goldbeck RA, Kliger DS. Unpublished results 90. Pascher T, Chesick JP, Winkler R, Gray HB. 1996. Science 271:1558–60 91. Jones CM, Henry ER, Hu Y, Chan CK,
479
Luck SD, et al. 1993. Proc. Natl. Acad. Sci. USA 90:11860–64 92. Chen E, Goldbeck RA, Kliger DS. 1997. Annu. Rev. Biophys. Biomol. Struct. 26:327–55 93. Michl J. 1980. Pure Appl. Chem. 52: 1549–63 94. Stark JB, Knox WH, Chemla DS. 1992. Phys. Rev. Lett. 68:3080–83
