J. Phys. Chem. 1993,97, 5499-5505
5499
Nanosecond Circular Dichroism Spectral Measurements: Extension to the Far-Ultraviolet Region C.-F. Zhang,? J. W.Lewis,t R. Cerpa,* I. D.Kuntz,*ts and D. S. Kliger'J Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, and Graduate Group in Biophysics and School of Pharmacy, University of California, San Francisco, California 94143 Received: November 16, 1992; In Final Form: February 12, 1993
The first measurements of time-resolved circular dichroism in the far-UV spectral region (far-UV-TRCD) with time resolution on the order of lO-'s are presented. The capability of making such measurements is demonstrated with ground- and excited-state CD spectra of (A)-Ru(bpy)3*+ between 190 and 290 nm as well as with C D spectra of a short peptide measured at different pHs. The properties of the far-UV-TRCD technique are discussed in detail, and comparisons between the static CD spectra obtained by this technique and those obtained by a conventional CD spectrometer are presented. Possible improvements to the current technique and application of the technique to resolve biophysical problems such as the kinetics of peptide helix+oil transition are also discussed.
Introduction Circular dichroism (CD), while difficult to observe, is important because it offers a direct spectroscopic indicator of the secondary structures of proteins. Determinations of secondary structures are best performed from CD measurements in the far-ultraviolet spectral region since strong peptide absorption bands can be found in the 190-230-nmregion. As a result, themost reliable structural determinations require CD measurements as far into the ultraviolet as possible.' Several years ago this laboratory developed a time-resolved circular dichroism (TRCD) method which increased temporal resolution by more than 4 orders of magnitude over previous CD measurements in thevisible and near-ultraviolet spectral regions.23 Since circular dichroism at these wavelengths is an indicator of protein sidechain and prosthetic chromophoreconformation, these techniques have enabled us to study with nanosecond time resolution the conformationalchanges of proteins such as carbon monoxymyoglobin,4 carbon mono~yhemoglobin,~and phytochrome6upon light excitation. To achieve nanosecond time resolution, we adopted an altemativeto the previous, highly evolved CD technology. Instead of using alternating left- and right-circularly polarized light as the probe light source, we use highly eccentric left- or rightelliptically polarized (LEP and REP, respectively) light produced by a linear polarizer followed by a birefringent element which introducesa small retardation (6 1O ) oriented at f45O relative to the polarization axis. A circularly dichroic sample, by virtue of its different absorptioncoefficients for left- and right-circularly polarized light, changes the eccentricity of the elliptical polarization and thus changes the relative intensities observed along the short axes of REP and LEP light. A second linear polarizer crossed relative to the first polarizer is placed after the sample to detect the change in polarization ellipticity and thus the CD or the change of the CD upon actinic light excitation. Because the size of the CD signal is proportional to Ae/6, using highly eccentric elliptically polarized light (and therefore small 6) to probe CD signals significantly improves the sensitivity of CD measurements and makes it possible to measure small CD ~ h a n g e s . ~At , ~the - ~ same time, however, since the intensity along the short axis of REP or LEP light is proportional to 6*, this method dramatically reduces the probe light intensity and hence increases the level of shot noise. These two factors cancel so that
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University of California, Santa Cruz. Graduate Group in Biophysics, University of California, San Francisco. School of Pharmacy, University of California, San Francisco.
0022-365419312097-5499$04.00/0
even with the increased noise from reduced probe intensity the CD signal-to-noise ratio is not affected. Enhanced time resolhion is achieved by increasing the detected intensity in several ways. First, a high-intensity flash lamp is used as a probe source to increase detected intensity during rapid measurements. Further advantage is gained by using multichannel detectors which avoid waste of the limited amount of far-UV light which comes from a UV optimized flash lamps3 Conventional circular dichroism instruments scan the spectrum sequentially, making relatively inefficient use of the already weak far-UV light present in their much dimmer probe beams, rendering that method unsuitable for high time resolution measurements. Because the measured signals vary as 6-l,it is important to use small retardations, and one must precisely control the probe light ellipticity. To achieve such accuracy requires the use of highquality linear polarizers. Glan-Taylor polarizers provide very pure linear polarization (extinction on the order of 10-6 with the flash lamp light source) and have performed well for the TRCD measurementsin the visible and near-UV region. Unfortunately, Glan-Taylor prisms are made of calcite, and absorption by this material below 250 nm makes them inappropriate for CD measurements in the far-UV region. It is just in this region, however, that peptide and protein secondary structure is most effectively st~died.~-s The amide bands of proteins and peptides, for example, lie between 190 and 230 nm and are the most reliable indicatorsof the kinetic transition between helix and random coil conformations. For thesereasons,wedecided toextend theTRCD technique to the far-UV region by employing the same principle described above but using Rochon-type, MgFz polarizers. Doubts existed about the suitability of these polarizers because, unlike calcite which has a relatively large difference of index of refraction (An = In, - ne[)allowing construction of reflection polarizers, birefringent materials such as quartz, MgF2, and ADP have relatively small An. They are thus ordinarily used for construction of less ideal, deflectionpolarizers. Rochon polarizers deflect the unwanted polarization by only a few degrees so, among other problems, special caution in alignment is needed in order to use them to achieve even moderately good extinction, particularly with incoherent light sources. Spectra from two different systems are presented here which demonstrate that TRCD measurements can be made in the farUV region (far-UV-TRCD) with nanosecond time resolution. First, we obtain CD spectra of ground and excited states of a resolved ruthenium tri's(bipyrid3) compound, (A)-Ru(bpy)jz+. This compound conveniently demonstrates the time resolution of the present technique because it is a photostable material with 0 1993 American Chemical Society
Zhang et al.
5500 The Journal of Physical Chemistry, Vol. 97, No. 21, 1993 L1
P1
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ROCHON
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P2
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L2
ROCHON
FL Figure 1. Experimental diagram for TRCD measurements in the far-UV spectral region. The principal components in the diagram are explained in Experimental Methods. The flash lamp probe beam must enter the side of the Rochon prisms with the optic axis in the direction of beam propagation. Abbreviations: L1, lens that collimates the diverging flash lamp light source; L2, lens that focuses the collimated probe beam onto the entrance slit of the spectrograph; P1, MgF2 polarizer that separates the unpolarized flash lamp light into vertically polarized, undeflected, ordinary ( 0 ) beam and horizontally polarized, deflected, extraordinary (e) beam, with the deflection angle approximately 2O; P2, MgF2 polarizer in the crossed position (90’) relative to P1; SP, strain plate with approximately 2O retardation and the compression (fast) strain axis at + 4 5 O or - 4 5 O relative to the polarization axis of P1, converting vertically polarized into elliptically polarized beam with highly elongated shape. The figure shows sectional patterns appropriate for left-elliptically polarized light incident on a negatively circularly dichroic sample.
largeand well-characterizedground- and excited-stateCD spectra in the visible and near-UV spectral region.12J4 In a second application we use the far-UV-TRCD technique to obtain CD spectra of a 30-residue polypeptide, GALA (see Experimental Methods for details), at different pHs. These spectra clearly demonstrate the viability of this technique for biophysical studies of polypeptide and protein systems. Experimental Methods The principle of the TRCD technique has been described in detail el~ewhere.~l~ The apparatus used here is shown in Figure 1. The probe light source is a xenon flash lamp (FX-249U) implemented with its associatedLitepac trigger transformer (FY7 12, EG&G Electro-Optics, Salem, MA). The sapphire window of this lamp provides optimal far-UV transmission. The light pulse is produced by discharging a 4-pF capacitor normally charged to 1200 V. The unpolarized beam is collimated by a fused-silica lens (Ll; SlUV, ESCO Products Inc., Oak Ridge, NJ) with the size of the beam reduced by an iris. The beam passes through the first MgF2 polarizer (Pl; PVR-10-2, Optics for Research, Caldwell, NJ), which separates vertically and horizontally polarized beams. The verticallypolarized beam then passes through a strain plate (SP) which produces approximately 2’ of retardation. The horizontally polarized beam, deflected by an angle of 2 O in the vertical plane by P1, is blocked by another iris. The strain plate is oriented with its fast (compression) axis at +45O or - 4 5 O measured counterclockwise relative to the polarization axis as viewed from the detector. It convertslinearly polarized light into right- or left-elliptically polarized light, REP (+45O) and LEP ( - 4 5 O ) , respectively, with the horizontally polarized component roughly 5000 times weaker than the vertically polarized component. The elliptically polarized light then enters a sample flow cell which consists of two fused silica windows glued to a metal cell body. (See ref 14 for details of cell construction.) The flow cell is connected to a peristaltic pump with the sample recycled through a flask used as a reservoir. Circularly dichroic samples change the circularly polarized components of REP and LEP and therefore change the horizontally polarized components of the elliptically polarized light as it passes through the flow cell. The collimated probe light then passes through the second MgF2 polarizer (P2) oriented in a crossed position relative to P1. The horizontally polarized light is then focused onto a 500-pm slit placed at the entrance of a Monospec-27 spectrograph (Therm0 Jarrel Ash, Franklin, MA). The vertically polarized light deflected by P2 in the horizontal plane by an angle of -2’ is blocked by a third iris. After entering the spectrograph, the probe light is dispersed by a 600 groove/mm grating blazed at 200 nm (Milton Roy, Rochester, NY) and is finally detected by a gated, UV-sensitive optical
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multichannel analyzer (OMA; Model 14204, EG&G, PARC, Princeton, NJ). The spectral resolution is approximately 3 nm. The time resolution is ultimately limited to 50 ns by the detector gating restrictions. In the far-UV-TRCD experiments the gate was normally set to 300 ns or longer to obtain enough photons for good signal/noise during signal averaging lasting a few minutes. It is important for these experiments to ensure that all optical components are carefully chosen and adjusted to optimize farUV transmissionand detection. This means not only the selection of the best available polarizers, optical windows, lenses, grating and detector but also that there is optimal alignment for the far-UV light. Polarizer manufacturers commonly will allow customers to test polarizers from their inventory so that they can purchase the ones most suitable for their needs. Besides varying in extinction, some Rochon prisms deviate both beams substantially, making alignment difficult. Cell windows must be tested before use, and 75% of those from typical manufacturers must be rejected. Lenses are strongly chromatic in the far-UV region, so care must be taken to correct the differencesin the focal lengths of the visible and far-UV light due to the wavelength dependence of the index of refraction. For simple quartz lenses a focal length of 6 in. in the visible region is equivalent to approximately 5 in. at 200 nm, and focal lengths of other values vary proportionally. In the previous TRCD experiments in the visible and near-UV regions, the collimated, unpolarized probe beam was weakly focused through Glan-Taylor polarizers and then collimated before being focused onto the spectrograph. With Rochon polarizers, however, a small and well-collimated probe beam must be used in order to achieve high extinction (on the order of 10-4). Otherwise, the beam with the desired polarization could not be separated effectively from the deflected beam. The two Rochon polarizers also must be slightly tilted to avoid the detection of multiple reflections from the surfaces of the polarizers. The aperture diameters of the irises and MgF2 polarizers were approximately 5 and 10 mm, respectively, resulting in a limiting aperture of --f/36. The calculations of extinction (E), retardation of the strain plate (a), and CD have been described previously.2 Here, the polarizer extinction is calculatedby comparing light levels obtained with the two polarizers in the crossed position (10) and after rotation from the crossed position by 2O (12):
E(X) = 2.8 X 10410/(12(X) -Io@))
(1)
The retardation (in degrees) can be calculated with or without “extinction correction”, i.e., the correction for the background light intensity obtained with P1 and P2 in the crossed position:
Nanosecond Circular Dichroism Spectral Measurements
The Journal of Physical Chemistry, Vol. 97, NO. 21, 1993 5501
w = 2t(Z,(X) - IO(W/(ZdN Io(X))]
6x1000 I
with correction (2A)
or = 2(Zs(A)/Zz(A))1’2
without correction
(2B)
where &(A) represents the light intensity with the strain plate between the two polarizers in the crossed position. The CD signal is calculated as
CD(V = ( I R ( N - IL(N)/(IR(X) + I L ( W - 2ZO(X)) (3) where ZR and IL are the intensities of the horizontally polarized components of the transmitted right- andleft-elliptically polarized light, respectively, and Zo(X) is the background intensity obtained with identical conditions except that the strain plate is removed. The CD signal can be converted into Ae withZ At(A) = (6(X)/2.3 Cr)CD(X) (4) in which 6(X) is the retardation of the strain plate in radians, C the sample concentration (in molarity), and 1 the cell path length (in centimeters). Note that in our experiments errors in the absolute value of Ae can arise from the usual sources such as uncertainties in the cell path length and also because the window strain may affect the CD signal in a complex way.I5 Figure 2A shows the flash lamp intensity profile between 190 and 280 nm for the far-UV-TRCD optical system described above with doubly distilled H20 flowing through the sample cell. The light below 185 nm is cut off mainly by atmospheric oxygen absorption. Apparently, the polarizers have imperfectextinction since the light level obtained with the two polarizers in the crossed position is small but nonnegligible compared to that obtained with thepolarizers rotated from thecrossedpositionby 2’. Hence, the extinction corrections for CD or strain calculations are important for the far-UV-TRCD measurements. This is illustrated in Figure 2C, in which the calculations of the strain plate retardation with and without extinction correction exhibit dramatic differences. Figure 2B shows, first, the degradation of the extinction of the MgFz polarizers in the far-UV region and, second, that such degradation is aggravated by the strain of the cell windows. The flow control of the peristaltic pump was set to be approximately 1 mL/min. Flowing at higher speed or with a viscous sample may generate significant pressure on the cell windows and therefore aggravate the strain, further degrading the extinction. The extinction measured in this system critically depends on the collimation of the measuring beam, optical alignment, and strain of the cell windows. The effect of window strain is much more pronounced in the far-UV region than the visible region, so optical windows must be tested in that region and glued carefully to the cell. The extinction also improves by an order of magnitude when using highly collimatedlight sources. The extinction of the same MgFz polarizers was found to reach 5 X lo” when tested with a He-Ne laser and roughly 2 X les with a low-power, 532-nm beam from the second harmonic of a Nd:YAG laser in the absence of a sample cell. Even under these conditions, however, we expect that the performance of the polarizers will be limited by the window strain when any currently available sample cell is present. For the far-UV-TRCD measurements using our flash lamp probe light source, collimation of the beam and aperture stopping through the use of small irises before the polarizers and lenses were critically important (see Figure 1). For excited-state CD measurements described below the actinic source was a 7-11s laser pulse at 532 or 266 nm produced by harmonicgenerationfrom the fundamental output from a Quanta Ray DCR- 1 Nd:YAG laser (Spectra-Physics, Mountain View, CA). The pulse energies used in the experiments were approximately 25 mJ/pulse (532 nm) and 10 mJ/pulse (266 nm). The actinic beam was focused slightly using a quartz lens (beam
7. 654.
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Wavelength (MI) Figure 2. Optical properties of the far-UV-TRCD instrument described in Figure 1. (A)Flash light intensityprofdesdetected by theUV-sensitive OMA: (-) probe lamp intensity with P1 and P2 in the crossed position and with the sample cell and the strain plate (oriented according to that described in Figure 1) between them; (--)and (- -) probelight intensities with PI and P2 in the crossed position and that turned off by Z0, respectively, with a sample cell but without the strain plate between them. (B) Extinction of the two MgFl polarizers observed by the farUV-TRCD apparatus. (-) and (- - -) extinction measured with or without the sample cell, respectively. (C) Wavelength dependence of the SP retardation (in degrees) measured with the sample cell between PI and P2. (-) and (.-) retardation calculated without (eq 2B)or with (eq 2A) extinction correction, respectively.
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diameter approximately 5 mm on the sample cell) and entered the sample cell at roughly 30° with respect to the probe beam (see Figure 1). CD spectra measured at specific times following laser excitation were collected by the gated OMA using a fourchannel digital delay/pulse generator (Model DG535, Stanford Research Systems, Inc., Stanford, CA) to control the delay time
5502 The Journal of Physical Chemistry, Vol. 97, No. 21, 1993 between the laser pulse and the opening of the OMA gate. The pulse width of the OMA gate used in the TRCD experimentswas 300 ns and the delay time 20 ns. Better time resolution with shorter gate width could be achieved at the expense of fewer photon counts and thus lower signal/noise per average. For static CD measurements, longer gate widths (e.g., 1 ps) were often used to obtain clean signals with a smaller number of averages. The far-UV transient absorption measurements shown here were performed under identical conditions to the far-UV-TRCD experiments to be able to make appropriate comparisons. Normally, far-UV transient absorption experiments measurements would be made without inclusion of crossed polarizers in the optical path so the light level could be increased by several orders of magnitude. In that case the limit of time resolution for this apparatus would be due to the 50-ns gating restrictions of the UV-sensitive OMA rather than considerations of signal/ noise. For those experiments, the probe light intensity between 190and 220 nm relative to that above 220 nm can be significantly improved using 200-W or 200-B band-pass filters (Acton Research Corporation, Acton, MA) which preferentially absorb near-UV and transmit far-UV light. The ( A ) - R ~ ( b p y ) ~used ~ + in the experiments was resolved according to the available standard procedures.I6 The GALA peptide containing 30 residues has the sequence WEAALA(EALA)2EHLAEALA(EAL)2AA.It was synthesized by the UCSF Biomolecular Resource Center on HMP resin using Fmoc-based chemistry on an Applied Biosystems Model 431A peptide synthesizer.17J8 The peptide was cleaved off the resin using 95%trifluoroacetic acid, 2.5% ethanedithiol, and 2.5%water. The crude peptide was purified by HPLC with a Hewlett-Packard liquid chromatograph using a reverse-phase C18 column (Rainin Dynamax)anda water/acetonitrilegradient. Peptide purity was greater than 90% as indicated by analytical C18 reverse-phase FPLC (Pharmacia). Peptide identity was confirmed by electrospray mass spectrometry at the UCSF Mass Spectrometry Facility with the calculated averaged mass 303 1.39 and observed mass 3032.33 f 1.75. The peptide was titrated to different pHs in solution using dilute HCl. All samples weredissolved in doubly distilled H2O. GALA concentrations in the 10 MMrange were chosen for measurements in the 2-mm-path length CD cell so that the optical density at 200 nm would be approximately 1. No birefringence artifacts caused by the polarized excitation of the samples were found in the far-UV-TRCD measurements because all molecules used in this study had small sizes and randomized their orientations in less than 10 ns. Spectra were calibrated by selecting two known absorptionpeaks in the spectral region of interest and calculating the other wavelengths by linear interpolation and extrapolation. For convenience in the far-UV experiments, the spectra were calibrated by the 266-nm laser scatter and the 191-nm absorption peak of low-purity grade methanol. This calibration agreed with the nominal dispersion of the grating used and was checked by noting the positions of peaks in the flash lamp output at 205.3, 229.4, 242.1, 247.8, 260.5, and 273.4 nm. Conventional static CD measurements were taken on an Aviv circular dichroism spectrometer (Model 60DS, Aviv Associates, Inc., Lakewood, NJ). All experiments were performed at room temperature. Results
Figure 3A shows the static CD of the ground state and the difference TRCD between the excited state and the ground state of (A)-Ru(bpy)32+in the near-UV region. Figure 3B shows the ground-state CD spectrum of the molecule measured by the Aviv CD spectrometerin the same spectral region. All the conversions from CD signal to A€were computed using eq4 with the extinction coefficient (3 of the ground state at 452 nm taken to be 15 700 M-1 cm-1.I2 The good agreement between the ground-state CD measured by the conventional CD spectrometer and by our
Zhang et al.
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Figure 3. Static and transient CD spectra of (A)-Ru(bpy)32+in the nearUV region. (A) Static and time-resolved CD spectra measured by the far-UV-TRCD instrument described in Figure 1. The sample was contained ina flow cell with 2-mm path length. (-) Static CD spectrum; (- - -) difference TRCD spectrum measured with 300-ns OMA gate starting at 20-ns delay after 25-mJ, 532-nm laser excitation. (B) Static CD spectrum measured on Aviv CD spectrometer. All CD signals have been converted into units of Ac.
instrument shows that the extended far-UV-TRCD apparatus works properly in this portion of the UV spectral region. The transient difference CD measured with 3004s gate starting 20 ns following laser excitation agrees well with the results on the excited stateof this molecule previously obtained using the TRCD technique in the visible and near-UV regions.13J4 In spite of the fact that fewer photons are detected, good agreement continues in the far-UV spectral region between the static CD spectra of (A)-R~(bpy)3~+ measured by our technique and measured by the conventional CD spectrometer, as shown in Figure 4, A and C, respectively. These results lend confidence to the capability of the current far-UV-TRCD technique for measuring transient CD changes in the far-UV region with nanosecond time resolution. Figure 4B presents the difference far-UV-TRCD signal of (A)R ~ ( b p y ) between ~ ~ + theexcitedand ground states. The transient differenceCDsignalwascollectedwith 300-nsOMAgatestarting 20 ns after excitation. The result shown was obtained by averaging eight data sets, each containing 256 measurements, and smoothing using a 15-pointSavitzky-Golaya1g0rithm.l~The REPand LEP measurements were taken in an alternating order to eliminate systematicartifacts such as sample degradationor drifts in window birefringence. No such artifacts were found, however, since the separate difference far-UV-TRCD results from the alternate measurements appeared identical within the noise level. Timeresolved absorptionmeasurements for (A)-Ru(bpy)++ in the farUV region were also performed with the same apparatus and conditions as the far-UV-TRCD experiments. The transient
Nanosecond Circular Dichroism Spectral Measurements
The Journal of Physical Chemistry, Vol. 97, No. 21, I993 5503
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Figure 4. Static and time-resolved CD and time-resolved absorption measurements of (A)-Ru(bpy)j*+ in the far-UV spectral region. The sample was contained in a flow cell with approximately 0.5-mm path length. (A) Static CD spectrum measured by the far-UV-TRCD instrument shown in Figure 1, (B) Duference far-UV-TRCD spectrum between the excited and ground states of (A)-Ru(bpy)3*+measured with 300-11s OMA gate starting a t 20-ns delay after 10-mJ laser excitation a t 266 nm. (C) Static CD spectrum obtained from Aviv CD spectrometer. (D) Difference time-resolved absorption spectrum measured under the same conditions as (B).
from the CD signals and offsetting under the assumption that the CD signals near 280 nm were zero.
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Disewion
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(A)-Ru(bpy)s2+ Results. Ru(bpy)P has been used as a photosensitizer for solar energy conversion,2°.21and the nature of its excited states has been studied e x t e n s i ~ e l y . ~The ~ - ~ ~bpy residues have absorption and CD bands in both the near-UV (between 250 and 350 nm) and far-UV (between 190 and 250 nm) regions and undergo electronic transitions to excited states upon excitationby UV or visiblelight. Wechose (A)-R~(bpy)3~+ as a system with which to demonstrate the feasibility of farUV-TRCD measurements because it exhibits large A€ in the near- and far-UV spectral regions and relatively large TRCD signals upon laser excitation (see Figures 3 and 4), is easy to resolve from the racemic mixture, and is stable during photolysis experiments. It is not our primary purpose in this paper to investigate the nature of the excited states; such discussion has been presented elsewhere in more detail.13J4 Excitation of R~(bpy)3~+ results in a metal-to-ligand chargetrahsfer (MLCT) lowest excited state. The intensity of the CD spectrum depends on the degree of coupling between equivalent bpy ligands. Thus, the CD intensity will differ dramatically depending on whether the transferred charge is delocalized on all three bpy ligands or is primarily localized on one bpy- ligand. Our previous study presented evidence of weak excited-state CD which must arise from coupling between one bpy- and two neutral bpy ligands.” The transient differenceabsorption signal between the MLCT and ground states shows a bleaching of the 280-nm band. At
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Wavelength (MI) Figure 5. Static CD spectra of GALA at different pHs. All data were -, collected by the far-UV-TRCD instrument shown in Figure 1. -, -,and -represent CD spectra a t pH 5.6,6.0,6.2, and 6.5, respectively.
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duference absorption spectrum obtained from 64 averages is shown in Figure 4D. In order to be comparable with the far-UV-TRCD results, the data were also collected with a 300-ns OMA gate starting at 20 ns delay after laser excitation. Finally, Figure 5 presents the static CD measurements in the far-UV region for GALA at pH 5.6,6.0,6.2, and 6.5. The CD signal at each pH was obtained by 256 averages with 1-fl OMA gate and was smoothed using a 15-point Savitzky4olay algorithm. The CD base lines were collected by the same method with doubly distilled H20 flowing through the sample cell. The results shown were computed by subtracting the CD base line
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The Journal of Physical Chemistry, Vol. 97, No. 21, 1993
room temperature in water, the MLCT state of (A)-R~(bpy),~+ decays with a lifetime of approximately 640 ns. The TRCD studies on the TU* transition of (P)-Ru(bpy),Z+ in the near-UV region suggested that the charge coupling between the b p y and bpy residues are lower in the MLCT state than that in the ground state but higher compared to an uncoupled complex.lj Since we have observed similar phenomena in the far-UV studies, we tentatively attribute the nature of the electronic transition in the far-UV region to be the same as that in the near-UV region. The bleaching of the absorption band near 280 nm (see the red edge of the transient difference absorption spectrum in Figure 4D) is attributed to the formation of an MLCT state of (A)R ~ ( b p y ) , ~which + , is described as Ru111(bpy)2(bpy)2+ with less coupling between the bpy and b p p than in the ground state of (A)-Ru(bpy),2+.13 The reduced coupling among the ligands is shown clearly by the reversal of the bimodal CD signal near 280 nm upon laser excitation. (See the difference far-UV-TRCD signal in Figure 3A.) Further study is needed to understand more precisely the nature of the CD bands in the far-UV region, as shown in Figure 4A,C. Here, we propose the same mechanism to explain the difference far-UV-TRCD signalof (A)-Ru(bpy)32+ shown in Figure 4B. We tentatively attribute the negative band near 200 nm and the two positive bands near 220 and 240 nm to the chiral absorption of the coupled bpy residues. Laser excitation creates an MLCT state, reduces the coupling among the bpy residues, and produces the transient CD changes with opposite signs to the ground-state CD spectrum. Our initial measurements of far-UV-TRCD signals were made on (A)-R~(bpy)~z+ since this is a stable system which exhibits large static as well as transient CD signals. However, our primary reason for developing the far-UV-TRCD technique was so that it could be applied to biologically important systems where less ideal and intense CD signals might be expected. We thus used the TRCD method to measure the peptide band CD spectrum of a model polypeptide. While static CD spectra were measured, these measurements were made with high time resolution to test the feasibility of future kinetic studies of helix changes by this method. Peptide Results. Figure 5 shows the ability of the current far-UV-TRCD technique to measure the CD changes of GALA caused by small pH changes in the helix-coil transition region of the peptide.Is The data at pH 6.0 and possibly 6.2 exhibit a minor base line sloping problem which may be caused by window strain effects. It should be possible to correct for this by more careful measurements of strain, extinction, and CD base lines. These spectra demonstrate, however, the feasibility of measuring the kinetic process of helix-coil transitions of peptides induced by instantaneous external perturbations such as light-induced pH changes. Previous studies on peptide helix-il kinetics have used indirect observationalmethods such as ultrasonic relaxation measurements,22fluorescence measurements following a temperature-jump perturbation,Z3 and conductance measurements following an electric field-jump perturbation.24 These observational methods are subject to various limitations; for example, dissociation reactions induced by the ultrasonic or electric field perturbations can complicate measurements.ZZJ4 Our approach avoids many of the problems associated with these methods. We expect that the far-UV-TRCD technique described in this paper now can be used to monitor such processes when coupled to a viable photochemical system that can efficiently produce a fast perturbation (such as a pH jump) to peptide structures. Taken together, the (A)-R~(bpy)3~+ and GALA results demonstrate for the first time the feasibility of measuring CD spectral changes in the far-UV region with submicrosecond time resolution. Despite the deteriorating extinction observed below 220 nm,chieflydue to theincreasein thestrainof thecell windows (see Figure 2B,C), the CD measurements between 190 and 220 nm are accurate, as shown by Figure 4A,C. The presence of low
Zhang et al. but nonnegligible background light intensity (detected with the two polarizers in the crossed position and without the strain plate between them) compared to the intensity of REP or LEP light (see Figure 2A) reduces the size of CD signals. The distorting effects of window strain on CD signals can be partly compensated by strain and extinction correctionsusing eqs 1 and 2. The effects of strain on CD signals can be complex,15 however, and care should be taken in data processing to avoid the “overcorrectionn of strain effects, as CD signals in the far-UV region seem to have reasonable profiles even without a strain correction. We have observed that cells with substantial intrinsic strain (on the order of 6) can result in base line signals which cause larger errors when they are subtracted from the data than when the data are left uncorrected. Clearly, the use of cells with lower strain levels is the solution to this problem. The TRCD technique as described here can be improved by a number of approaches. First, since cell window strain is a chief limiting factor on the extinction, as shown by Figure lB, the sensitivity of the far-UV-TRCD technique can be improved by developing more perfect sample cells with less window strain. This will help the method because smaller window strain allows CD measurements to be performed with smaller retardation (6) produced by the strain plate. Larger signals would then be observed because the CD signal is inversely proportional to 6 for a given A€, Second, time resolution better than 50 ns can be achieved with faster detectors. We have also used a UV-sensitive monochromatorand photomultipliertube (PMT) with a Tektronix digitizing system to perform the far-UV-TRCD measurements (data not shown). The time resolution can then reach approximately 10 ns, but the performance of this system is limited by the flash lamp light source in two ways. First, the light pulse from our high-voltage flash lamp has a duration of only a few microseconds and is therefore inappropriate for measurements over a longer period. While longer pulse flash lamps can be used, there is significant loss in UV output. Second, since flash lamps radiate in all directions and only a small cone of the light is used, most of the flash lamp light output is lost by converting the uncollimated light source to a collimated beam (see Figure 1). Up to this point flash lamps have provided the light intensities needed to overcome shot noise limits in the TRCD technique. We now believe, however, that the general quality of the far-UVTRCD technique could be improved by using an appropriate far-UV laser source. In addition to increasingprobelight intensity, which would reduce shot noise, a laser light source could also improve the extinction and make it possible to perform far-UVTRCD measurements with smaller 6 value and more sensitivity. As mentioned in Experimental Methods, the extinction of the MgF2 polarizers can be improved by an order of magnitude by replacing the diverging, incoherent flash lamp probe light with well-collimated, coherent light sources. The ideal probe sources are lasers which provide far-UV output between 190 and 220 nm. Work is in progress to find an appropriate far-UV laser in hopes of improving the detection sensitivity and time resolution of the current far-UV-TRCD technique. To make this far-UV-TRCD technique an important tool for studying helix-coil transitions, there must be, in addition to the ability to measure fast CD changes, available photochemical systems capable of inducing appropriate perturbations to peptide conformation. Conventional temperature jump and solution mixing are usually limited to millisecond time resolution and are thus not fast enough to resolve individual helix-coil transitions. It seems the most promising approach is to induce peptide conformational changes by using short light pulses (e.g., on the nanosecond scale) to generate pH, temperature, charge, or conformational changes of photochemical groups attached to the peptides. The last approach has been explored e x t e n s i ~ e l y , ~ ~ - ~ ~ but for most molecules the quantum yield of direct photoinduced
Nanosecond Circular Dichroism Spectral Measurements conformational change is too low to produce observable peptide structural change using a single laser pulse. One possibility is to perturb the peptide h e l i x e i l transformation by exciting a pH-jump compound mixed with the peptides in solution to produce a transient change of proton concentration by light pulses. An example of such a compound is 2-naphthol3,6-disulfonate, which releases protons efficiently upon UV excitation.** The limitations of pH-jump compounds are absorption in the far-UV region which reduces the probe light intensity and often creates absorption changes which interfere with the observation of the peptide spectral properties in this region. Second, the proton concentration change in the vicinity of peptides has a rise time of roughly 100 ns, due to proton diffusion, and decays within hundreds of microseconds in our system (data not shown). This puts a limit on the time scale of opticalmeasurements and makes it uncertain whether this method is suited for the studies of h e l i x e i l transition processes. Yet another approach is to link an individual peptide with a photochemical group which changes its charge upon light excitation. Photochemical molecules such as tetramethyl-pphenylenediamine (TMPD)29and p-aminobenzoic acid (PABA) are known to create solvated electrons and therefore change their charges upon laser excitation at 266 nm (data not shown). It has been observed that the charge change on the head group of a-helices affects the dipole moments of the helices and therefore the peptide conformation^.^^ At present, we are studying the feasibility of this approach and searching for the most efficient photochemical systems for the purposes discussed above. Whatever specific approach is used to produce rapid perturbations to samples of interest, the extension of the nanosecond TRCD technique to the far-UV should greatly expand the usefulness of this technique for biophysical studies. The region from 190 to 230 nm is the most important for determining the conformational states of biopolymers, but it has also been the most difficult for time-resolved measurements. This work clearly demonstrates that this longstanding barrier to direct observation of dynamic structural changes in biologically important molecules has been breached.
Acknowledgment. We thank Dr. S.Maller for experimental assistance and Professor F. Szoka at UCSF for kindly providing us the peptide sample. We also thank Drs. S.C. Bjcrling, R. Goldbeck, S. Milder, and S. Paquette for helpful discussions. This work is supported by the National Institutes of Health through Grant GM-35158 to D.S.K. and GM-19267 to I.D.K. The UCSF Mass Spectrometry Facility is supported by NIH National Center for Research Resources Grant RR01614 and
The Journal of Physical Chemistry, Vol. 97, No. 21, 1993 5505
NSF Biological Instrumentation Program Grant DIR-8700766. R. Cerpa is a Howard Hughes Medical Institute Predoctoral Fellow.
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