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J. Phys. Chem. B 2006, 110, 1056-1062
Femtosecond Study of Partially Folded States of Cytochrome C by Solvation Dynamics Kalyanasis Sahu, Sudip Kumar Mondal, Subhadip Ghosh, Durba Roy, Pratik Sen, and Kankan Bhattacharyya* Physical Chemistry Department, Indian Association for the CultiVation of Science, JadaVpur, Kolkata 700 032, India ReceiVed: July 15, 2005; In Final Form: October 15, 2005
Using femtosecond time-resolved fluorescence spectroscopy, it is shown that the solvation dynamics in the two partially folded states (IS′ and IS′′) of a protein, cytochrome C, are very different. In the case of IS′ (formed by the addition of 2 mM sodium dodecyl sulfate, SDS) almost the entire dynamic solvent shift of coumarin 153 (C153) is captured in a picosecond setup and the contribution of the ultrafast component (0.5 ps) is very small (5%). Solvation dynamics of IS′′ (formed by 2 mM SDS and 5 M urea) displays a major component (47%) of 1.3 ps. This indicates that the structure of IS′′ is much more open and exposed compared to that of IS′. The difference in the dynamics of IS′ and IS′′ is attributed to differences in their structure, particularly near the heme region, and the presence of urea in IS′′.
1. Introduction The unique biologically active (native or folded) 3-dimensional structure of a protein represents a delicate balance of electrostatic and hydrophobic interactions.1 A slight imbalance caused by certain additives (e.g., urea or surfactants) results in partial or complete unfolding of the protein and impairs its biological activity. Study of an unfolded protein is important to understand protein folding pathways. For many proteins, the native and the unfolded (denatured) states are separated by distinct protein folding intermediates. Different folding intermediates differ in the degree of exposure and penetration of water and also in the dynamics of side chain of the protein and associated water molecules. With a view to understand the relation between structure and function of a protein there have been many studies on protein folding intermediates using stopped flow,2 circular dichroism (CD),2,3 small-angle X-ray scattering,4 NMR,5 fluorescence,6 calorimetry,7 and vibrational spectroscopy.8 Very recently, solvation dynamics,9-13 large scale computer simulation,14 quasi elastic neutron scattering (QENS),15 and NMR5 have been introduced as a tool to study dynamics in a protein. Many of these studies reveal an ultraslow component of solvation dynamics which is slower by 2-3 orders of magnitude compared to bulk water.16 It is proposed that the ultraslow dynamics originates primarily from the presence of motionally restricted quasi-bound water molecules in the immediate vicinity of the protein.17 Most recently, protein folding intermediates have been studied by solvation dynamics. Solvation dynamics in the molten globule state of a protein, glutaminyl t-RNA synthetase (GlnRS), is found to be slower than that in bulk water and appreciably different from that in the native state.11 The marked slow solvation dynamics in the molten globule state compared to bulk water indicates the presence of considerable residual structure in the molten globule state. A recent NMRD study also reported slow dynamics of water molecules in the molten globule state.5a * To whom correspondence should be addressed. E-mail: pckb@ mahendra.iacs.res.in. Fax: (91)-33-2473-2805.
For many proteins, there are more than 1 folding intermediates. The structural differences in different folding intermediates should give rise to different dielectric response (solvation dynamics). In the present work, we demonstrate that solvation dynamics of 2 folding intermediates of a protein cytochrome C differ in the ultrafast component, and, thus, femtosecond spectroscopy may be used to distinguish between different folding intermediates. The mitochondrial respiratory protein cytochrome C is cationic in nature and carries a net positive charge (+8) at a neutral pH (∼7).2-3,4c It resides in the inner mitochondrial membrane and transfers electron to cytochrome oxidase. The cationic protein cytochrome C forms partially folded or molten globule-like states on binding with anionic surfactants,2 lipids,3a polyvinyl sulfate,3b sugars,3c and polyols,3d and in acidic pH.3e Surfactant induced unfolding of cytochrome C plays an important role in apoptosis of eukaryotic cells.2a-b Similar unfolding is often encountered for other proteins on binding to membranes.1d-e According to a flow-cytometry study,2a apoptosis in mouse involves conformational change in cytochrome C on binding to the membrane.2b In this work, we report on partial unfolding of cytochrome C on binding to an anionic surfactant (sodium dodecyl sulfate, SDS) which is analogous to unfolding of cytochrome C by lipid membranes. It is reported2c-d that unfolding of cytochrome C by urea and SDS from the native state (N) to the fully unfolded state (U) involves two intermediates, IS′ and IS′′
N f IS′ f IS′′ f U IS′ is formed in 2 mM SDS at pH 7, whereas IS′′ is formed in the presence of 2 mM SDS and 4-6 M urea. The structure of the folding intermediates of cytochrome C has been studied by far-UV, near-UV, and visible (around 400 nm) circular dichroism (CD) spectroscopy (see Discussion, later).2c The formation of the intermediate states (IS′ and IS′′) of cytochrome C is accompanied by a marked change of the emission intensity of the lone tryptophan residue (Trp 59). In the native state, fluorescence of the tryptophan residue of cytochrome C is very weak because of efficient Forster energy
10.1021/jp0538924 CCC: $33.50 © 2006 American Chemical Society Published on Web 12/16/2005
Partially Folded States of Cytochrome C SCHEME 1: (A) Necklace Model. (B) Structure of Coumarin 153 (C153)
transfer to heme.2c Addition of 2 mM SDS causes a dramatic increase in the fluorescence intensity of tryptophan. This is attributed to partial unfolding of cytochrome C which results in an increase in the distance between tryptophan and heme and consequent suppression of the energy transfer process.2c In a recent work, we have studied solvation dynamics of coumarin 153 (C153, Scheme 1B) in the two partially unfolded states, IS′ and IS′′, of cytochrome C using picosecond fluorescence spectroscopy.12 According to this study, solvation dynamics in IS′ is described by two components 90 and 400 ps. For IS′′, the components of solvation dynamics are 60 and 170 ps. While the components of the picosecond solvation dynamics are not too different for the 2 folding intermediates (IS′ and IS′′), the amounts of solvation missed in our picosecond study are very different. Using the Fee-Maroncelli procedure18b we estimated that in IS′ almost the entire (100%) solvation dynamics is captured in a picosecond setup while only ∼22% of the solvation dynamics of IS′′ is detected in a picosecond setup.12 This suggests that the ultrafast components of solvation dynamics are very different in the two intermediate states. In this work, using a femtosecond upconversion setup, we show that this is indeed the case. In this study, we have used coumarin 153 (C153) as a probe. C153 is a very well-known solvation probe18c,19 and has been used to study solvation dynamics in many proteins.13 2. Experimental Section Coumarin 153 (C153, Scheme 1B, Exciton), cytochrome C (from horse heart, Sigma), urea (Sigma), and sodium dodecyl sulfate (SDS, Aldrich) were used as received. The steady-state absorption and emission spectra were recorded in a Shimadzu UV-2401 spectrophotometer and a Perkin-Elmer 44B spectrofluorimeter, respectively. All solutions were made in a 50 mM sodium phosphate buffer of pH 7. In our femtosecond upconversion setup (FOG 100, CDP) the sample was excited at 405 nm using the second harmonic of a
J. Phys. Chem. B, Vol. 110, No. 2, 2006 1057 mode-locked Ti-sapphire laser (Tsunami, Spectra Physics) pumped by 5-W Millennia (Spectra Physics). The Tsunami produces 810 nm laser pulses having pulse duration of 50 fs, repetition rate 80 MHz, and pulse energy ∼8.5 nJ. The fundamental 810 nm beam was frequency doubled in a nonlinear crystal (1 mm BBO, θ ) 25°, φ ) 90°). The polarization of the second harmonic excitation beam was rotated by a Berek compensator so as to collect the emission decay at magic angle polarization. To avoid possible photodegradation, the laser power was reduced to ∼4 mW by placing neutral density filters before the sample and the sample was placed in a rotating cell of path length 1 mm. The fluorescence emitted from the sample was upconverted in a nonlinear crystal (0.5 mm BBO, θ ) 38°, φ ) 90°) using a gate beam of 810 nm. The upconverted light was dispersed in a monochromator and detected using photon counting electronics. A cross-correlation function obtained using the Raman scattering from ethanol displayed a full width at halfmaximum (fwhm) of 350 fs. The femtosecond fluorescence decays were fitted using a Gaussian shape for the exciting pulse. To accurately determine the long components of the fluorescence decays we measured the slow components (in >100 ps time scale) in a picosecond set up and used them to fit the femtosecond decays. For picosecond measurements, the samples were excited at 405 nm using a picosecond diode laser (IBH Nanoled-07) in an IBH FluoroCube apparatus. The emission was collected at a magic angle polarization using a Hamamatsu MCP photomultiplier (5000U-09). The time-correlated single photon counting (TCSPC) setup consists of an Ortec 9327 CFD and a Tennelec TC 863 TAC. The data were collected with a PCA3 card (Oxford) as a multichannel analyzer. The typical fwhm of the system response using a liquid scatterer is about 80 ps. The fluorescence decays were deconvoluted using IBH DAS6 software. The time-resolved emission spectra (TRES) were constructed following the procedure prescribed by Fleming and Maroncelli,18a and using the parameters of best fit to the fluorescence decays and the steady-state emission spectrum. The emission maxima of the TRES at various times were obtained by fitting the data to a log-normal spectral function.18a The solvation dynamics is described by the decay of the solvent correlation function C(t), defined as
C(t) )
ν(t) - ν(∞) ν(0) - ν(∞)
(1)
where ν(0), ν(t), and ν(∞) are the peak frequencies at time 0, t, and ∞, respectively. ν(∞) was determined by fitting ν(t) as ν(t) ) ν(∞) + [ν(0) - ν(∞)]Σai exp(-t/τi). According to Fee and Maroncelli,18b the true emission frequency at time zero, νpem(0), may be calculated using the absorption frequency (νpabs) in a polar medium (i.e., cytochrome C and SDS solution), using the following relation: np νpem(0) ) νpabs - [νnp abs - νem]
(2)
np where νnp em and νabs, respectively, denote the steady state frequencies of emission and absorption of C153 in a nonpolar solvent. As a nonpolar solvent, we used cyclohexane in which absorption and emission maxima of C153 are, respectively, at 393 and 455 nm. To study fluorescence anisotropy decay, the analyzer was rotated at regular intervals to get fluorescence decay at perpendicular (I⊥) and parallel (I|) polarization. Then the anisotropy function, r(t), was calculated using the formula
1058 J. Phys. Chem. B, Vol. 110, No. 2, 2006
r(t) )
I|(t) - GI⊥(t) I|(t) + 2GI⊥(t)
Sahu et al.
(3)
The G value of the setup was determined to be 1.8. 3. Results 3.1. Steady-State Emission Spectra of C153 Bound to IS′ and IS′′ of Cytochrome C. Steady-state emission properties of C153 are very sensitive to solvent polarity. With decrease in polarity, emission quantum yield (φf) of C153 increases 7.5 times from 0.12 in water (or phosphate buffer) to 0.9 in cyclohexane while its emission maximum (λmax em ) shifts from 550 nm in water to 455 nm in cyclohexane.19 λmax em and φf of C153 in an aqueous phosphate buffer solution remain unchanged on addition of 5 µM cytochrome C. This suggests that C153 does not bind to cytochrome C in the native state. On addition of 2 mM SDS, when cytochrome C forms a partially unfolded intermediate (IS′), λmax em of C153 exhibits a marked blue shift to 538 nm in IS′. The blue shift of λmax em clearly indicates that C153 binds to the folding intermediate, IS′, and the microenvironment of C153 in IS′ is less polar than that in phosphate buffer. When 5 M urea is added to a solution containing 5 µM cytochrome C and 2 mM SDS the protein forms another partially unfolded state (IS′′). In this state, λmax em of C153 is at 546 nm. This indicates that in IS′′ the microenvironment is more polar compared to IS′. Figure 1 describes emission spectra of 5 µM C153 in IS′ and IS′′ of 5 µM cytochrome C at excitation wavelengths (λex) 295, 405, and 430 nm. At λex ) 295 nm, both C153 and tryptophan of cytochrome C are excited, while at λex ) 405 nm, along with C153, heme group of cytochrome C absorbs strongly. At 430 nm C153 absorbs predominantly with a very small contribution from the heme group and, hence, is relatively free from inner filter effect (due to absorption by heme). It is readily seen that for excitation at 430 nm the emission intensity of C153 in IS′ is higher than that in IS′′ and both are higher than that of C153 in phosphate buffer (Figure 1). From the emission intensities and the position of emission maximum it is inferred that the polarity of IS′ is lower than that of IS′′ while both are less polar than bulk water. We now examine the possibility of energy transfer from the lone tryptophan residue (Trp 59) of cytochrome C to C153. In both IS′ and IS′′ the fluorescence intensity of tryptophan (at ∼350 nm) decreases only slightly on addition of C153 (Figure 1). The slight decrease in the emission intensity of Trp 59 may be attributed to the inner filter effect caused by partial absorption of the exciting light at 295 nm by C153. Thus it seems that energy transfer from tryptophan to C153 is negligible in the IS′ and IS′′ states of cytochrome C and the probe C153 resides far from Trp 59 in these two states. For excitation at 405 nm (heme absorption region), the emission intensity of C153 in the presence of cytochrome C is found to be lower compared to C153 in buffer. This rules out the possibility of fluorescence energy transfer from heme to C153. The decrease in emission intensity of C153 (for λex ) 405 nm) in cytochrome C may be due to very strong (80%) absorption of the light by the heme unit. Addition of SDS to cytochrome C causes a distinct blue shift of the sharp Soret absorption band of heme and disappearance of the 695 nm band.2e The disappearance of the 695 nm band is ascribed to the disruption of the heme-methionine bond on addition of SDS.2e This indicates SDS binds to cytochrome C at a location near the heme unit. Since C153 does not bind to
Figure 1. (A) Emission spectra of (i) C153 in phosphate buffer (-‚‚); (ii) C153 in 5 µM cytochrome C, 2 mM SDS and 5 M urea (IS′′) (- -); (iii) C153 in 5 µM cytochrome C and 2 mM SDS (IS′) (‚‚‚); (iv) 5 µM cytochrome C and 2 mM SDS solution (s); (v) 5 µM cytochrome C, 2 mM SDS, and 5 M urea (-‚-), λex ) 295 nm. The inset shows the emission around the tryptophan region (∼350 nm). (B) Emission spectra of C153 containing (i) 5 µM cytochrome C; (ii) 5 µM cytochrome C, 2 mM SDS, and 5 M urea (IS′′); (iii) 5 µM cytochrome C and 2 mM SDS (IS′); (iv) 2 mM SDS and 5 M urea; and (v) phosphate buffer, λex ) 405 nm. (C) Emission spectra of C153 in (i) phosphate buffer; (ii) 5 µM cytochrome C, 2 mM SDS, and 5 M urea (IS′′); (iii) 5 µM cytochrome C and 2 mM SDS (IS′); and (iv) 2 mM SDS and 5 M urea; λex ) 430 nm.
cytochrome C in the absence of SDS, it seems that C153 binds to a region close to where SDS binds i.e., near the heme unit. Note, the heme is located far from Trp 59 in IS′ and IS′′. This
Partially Folded States of Cytochrome C
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Figure 2. Femtosecond fluorescence transients of C153 in 50 mM phosphate buffer (pH 7) containing 5 µM cytochrome C, 2 mM SDS, and 5 M urea (IS′′) at 500, 545, and 620 nm.
Figure 3. Time-resolved emission spectra of C153 in 50 mM phosphate buffer (pH 7) containing 5 µM cytochrome C, 2 mM SDS, and 5 M urea (IS′′) at 0 ps (9), 1 ps (O), 4 ps (2), and 1000 ps (3).
suggests that in IS′ and IS′′ C153 and heme are both far from Trp 59 and hence, may be close to each other. In summary, in the partially unfolded states of cytochrome C, C153 resides near the heme unit. 3.2. Time-Resolved Studies. 3.2.1. Femtosecond SolVation Dynamics of C153 in IS′′: Cytochrome C, 2 mM SDS, and 5 M Urea. In IS′′ (i.e., 5 µM cytochrome C with 2 mM SDS and 5 M urea) the fluorescence decays of C153 vary quite significantly with the emission wavelength. The femtosecond studies reveal that at the blue end (500 nm) the fluorescence decay is triexponential with three decay components of 0.7 ps (50%), 130 ps (17%), and 2000 ps (33%), while at the red end (620 nm) the decay of time constants 620 and 2000 ps is preceded by a distinct rise with a time constant of 1.4 ps (Figure 2). TRES of C153 in cytochrome C, 2 mM SDS, and 5 M urea are shown in Figure 3. The decay of C(t) is found to be triexponential (Figure 4, Table 1) with a very fast component of 1.3 ps (67%) and two slow components of 60 ps (18%) and 170 ps (15%) (Table 1). The total Stokes shift for C153 in 5 µM cytochrome C, 2 mM SDS, and 5 M urea (in IS′′) is found to be 600 ( 50 cm-1. This is higher than the Stokes shift detected earlier in a picosecond setup (190 ( 50 cm-1) for IS′′.12 Following the Fee-Maroncelli procedure,18b we calculated about 30% of total Stokes’ shift is missed in IS′′ state of cytochrome C even in a femtosecond setup. Thus, the overall contribution of the 1.3, 70, and 170 ps components are, respectively, 47%, 12.5%, and 10.5%.
Figure 4. (A) Initial parts of the decay of response function, C(t) of C153 in 50 mM phosphate buffer (pH 7) containing 5 µM cytochrome C and 2 mM SDS (IS′) (0); 5 µM cytochrome C, 2 mM SDS, and 5 M urea (IS′′) (b); 2 mM SDS and 5 M urea (2); and 55 mM SDS (3). The points denote the actual values of C(t) and the solid line denotes the best fit to the decay. (B) The full range decay of response function, C(t) of C153 in 50 mM phosphate buffer (pH 7) containing 5 µM cytochrome C and 2 mM SDS (IS′) (0); and5 µM cytochrome C, 2 mM SDS, and 5 M urea (IS′′) (b). The points denote the actual values of C(t) and the solid line denotes the best fit to the decay.
3.2.2. Femtosecond SolVation Dynamics in IS′: Cytochrome C and 2 mM SDS. We have reported earlier that in the case of C153 in IS′ (5 µM cytochrome C and 2 mM SDS) almost 100% of the total solvent shift is detected in a picosecond setup. Our femtosecond study is consistent with this. In this case, the contribution of the ultrafast component (0.5 ps) is found to be very small (5%). In this state, the slow components of 90 and 400 ps contribute, respectively, 85% and 10%. The decay of C(t) for IS′ is shown in Figure 4 and the decay parameters are tabulated in Table 1. 3.2.3. SolVation Dynamics of C153: Effect of Buffer, SDS, and Urea. As a control, we studied femtosecond solvation dynamics in the absence of the protein to confirm that the femtosecond dynamics is indeed associated with the protein. Note, CMC of SDS decreases from 8 mM in bulk water to 2 mM in the presence of buffer.16 In phosphate buffer containing 5 M urea CMC of SDS is about 3.5 mM. Thus in buffer 2 mM SDS corresponds to the micellar region in the absence of urea and to the pre-micellar region in the presence of urea. We studied solvation dynamics in 2 mM SDS and 5 M urea which is in the pre-micellar region and is free from complication due to the probe molecules in the SDS micelles.
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TABLE 1: Decay Parameters of C(t) of C153 in 50 mM Phosphate Buffer of pH 7 in Different Systems system
∆νa (cm-1)
a1b
τ1b (ps)
a2b
τ2b (ps)
a3b
τ3b (ps)
fraction observed (fobs)
5 µM Cytochrome C + 2 mM SDS (IS′) 5 µM Cytochrome C + 2 mM SDS + 5 M urea (IS′′) 2 mM SDS + 5 M urea 55 mM SDS
615 570 560 450
0.05 0.68 0.90 0.40
0.5 1.3 0.9 1.2
0.85 0.18 0.10 0.02
90 60 5 45
0.10 0.15
400 170
0.58
200
1.00 0.70 0.46 0.37
a
(50 cm . (10%. -1 b
component of 800 ps (15%). In the absence of the protein, the anisotropy decay in 2 mM SDS and 5 M urea (τR )150 ps) is much faster than that in the 2 folding intermediates. The time constants for anisotropy decay in SDS micelle (2 mM SDS in 50 mM sodium phosphate buffer) are 180 ps (65%) and 900 ps (35%). This is much longer than those in 2 mM SDS and 5 M Urea (i.e., pre-micellar region). Also, the initial anisotropy (r0) in 2 mM SDS and 5 M urea is lower than that in IS′, IS′′, and SDS micelle (Table 2, Figure 5). Thus, the anisotropy decays clearly show that the microenvironments of C153 in IS′, IS′′, and in SDS micelle are very different from that in the pre-micellar SDS solution. 4. Discussion Figure 5. Fluorescence anisotropy decays of C153 in 50 mM phosphate buffer (pH 7) containing (i) 5 µM cytochrome C, 2 mM SDS (IS′) (3); (ii) 5 µM cytochrome C, 2 mM SDS, and 5 M urea (IS′′) (b); (iii) 2 mM SDS (9); and (iv) 2 mM SDS and 5 M urea (4). The solid lines are the fitted curves. The inset shows the initial portion of the fitted curves.
TABLE 2: Decay Parameters of Rotational Anisotropy Decay, r(t), of C153 in Different Systems in 50 mM Phosphate Buffer of pH 7 system 5 µM Cytochrome C + 2 mM SDS (IS′) 5 µM Cytochrome C + 2 mM SDS + 5 M urea (IS′′) 2 mM SDS 2 mM SDS+ 5 M urea a
τ1a (ps)
a2a
τ2a (ps)
0.25 0.5
180
0.5
850
0.35 0.85
120
0.15
800
0.20 0.65 0.12 1.00
180 150
0.35
900
r0
a1a
(10%.
In 2 mM SDS and 5 M urea the fluorescence decays of C153 do not show any wavelength dependence in a picosecond setup. Since in this case there is no SDS micelle, C153 stays in bulk water and exhibits ultrafast solvation dynamics. The femtosecond study shows that in 2 mM SDS and 5 M urea the solvation dynamics of C153 is indeed very fast and displays a major (90%) component of 0.9 ps and a small component of 5 ps (Figure 4, Table 1). This is much faster than the solvation dynamics in the two protein folding intermediates IS′ and IS′′. We also studied solvation dynamics of C153 in SDS micelles in the absence of urea (Table 1 and Figure 4). In this case only 37% of the total dynamic shift is detected even in a femtosecond setup. As shown in Figure 4 and Table 1 it is evident that the solvation dynamics of C153 in SDS is described by an ultrafast component of 1.2 ps and a slow component of 200 ps. The overall decay is faster than that in IS′. 3.3. Fluorescence Anisotropy Decay. The fluorescence anisotropy decays of C153 in IS′ (cytochrome C with 2 mM SDS) and IS′′ (cytochrome C with 2 mM SDS and 5 M urea) are shown in Figure 5 and the parameters of anisotropy decay are listed in Table 2. In IS′, the decay of the fluorescence anisotropy is described by two components: 180 ps (50%) and 850 ps (50%). For IS′′, the fluorescence anisotropy decay displays a major (85%) component of 120 ps and a long
The main aim of this work is to understand solvation dynamics of two partially unfolded states of cytochrome C formed in the presence of SDS and urea. As noted earlier, the SDS-induced unfolding of cytochrome C is analogous to the lipid-induced unfolding involved in the apoptosis in eukaryotic cells.2a The structure of the folding intermediates (IS′ and IS′′) of cytochrome C has been studied in detail using CD spectroscopy.2c The far-UV CD spectrum of IS′ (i.e., cytochrome C in 2 mM SDS) is not very different from that of the native state. Detailed analysis of the far-UV CD spectrum suggests that in IS′ the protein retains more than 80% of its secondary structure.2c The near-UV CD spectrum of cytochrome C changes quite significantly on addition of SDS. This is attributed to the formation of a partially unfolded or “molten globule” state, IS′ with complete loss of tertiary structure.2c The far-UV CD of the intermediate IS′′ (cytochrome C in 2 mM SDS and ∼5 M urea or IS′ plus 5 M urea) suggests that ∼50% of the native secondary structure is retained in IS′′.2c The near-UV CD of IS′′ resembles that of IS′ and this indicates that in IS′′ the tertiary structure is completely lost as in the case of IS′.2c The visible CD spectrum of IS′′ around 400 nm (absorption maximum of heme) is very different from that of IS′ and also from the completely unfolded (U) protein.2c (In the presence of SDS, cytochrome C does not unfold completely even in 10 M urea).2c The structural differences between IS′ and IS′′ may be summarized as follows. In both IS′ and IS′′, the tertiary structure is almost completely lost. IS′ retains almost 80% of the native secondary structure while IS′′ retains about 50%. The structure around the heme unit is very different for IS′ and IS′′. We now consider the role of vibrational cooling of the partially unfolded states following excitation at 400 nm. For excitation at ∼400 nm, the heme unit of cytochrome C is excited along with the fluorescent probe (C153).20 Excitation of the heme unit is followed by transfer of excitation energy to vibrational modes of the protein and surrounding water molecules. This results in ultrafast heating.20 The subsequent cooling has been detected using transient IR,20a-b transient grating,20c and transient Raman spectroscopy.20d Using transient IR20a-b and computer simulations20e Hochstrasser and co-workers showed that in several heme proteins (myoglobin, deoxyhemo-
Partially Folded States of Cytochrome C globin, and cytochrome C) vibrational cooling occurs in ∼10 ps time scale. The rate of vibrational cooling depends on the density of vibrational states. Obviously, the folding intermediates (IS′ and IS′′) of cytochrome C should not differ much in density of states, and, hence, would display similar cooling rates. Thus, if the observed time-dependent fluorescence Stokes shift were due to vibrational cooling, both the folding intermediates should exhibit similar ultrafast components of decay. However, the magnitude and relative contributions of the relaxation components of the two intermediates are markedly different. For IS′, contribution of the ultrafast component (0.5 ps) is very small (5%). For IS′′, the contribution of the ultrafast component of solvation dynamics is very large. In IS′′, about 30% of the total Stokes shift is missed even in a femtosecond setup and there is a major contribution (47%) of an ultrafast component (1.3 ps) followed by two other slow components of 70 ps (12.5%) and 170 ps (10.5%). The drastic difference between the ultrafast decay components of the 2 folding intermediates suggests that the observed time-dependent Stokes shift is not due to vibrational cooling. Thus, we interpret the time-dependent Stokes shift as solvation dynamics in the two protein folding intermediates IS′ and IS′′. While solvation dynamics represents motion of solvent dipoles, the anisotropy decay describes motion of the solute (fluorescence probe). For a protein bound fluorophore, motion of the macromolecular chains is superimposed on the motion of the probe. Thus, the anisotropy decay in IS′ and IS′′ may be ascribed to chain dynamics. In IS′′, the value of initial anisotropy is quite high. This suggests absence of any ultrafast component (
