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2009, 113, 7991–7994 Published on Web 05/14/2009
Efforts toward Developing Probes of Protein Dynamics: Vibrational Dephasing and Relaxation of Carbon–Deuterium Stretching Modes in Deuterated Leucine Jo¨rg Zimmermann,† Kenan Gundogdu,‡ Matthew E. Cremeens,† Jigar N. Bandaria,‡ Gil Tae Hwang,† Megan C. Thielges,† Christopher M. Cheatum,*,‡ and Floyd E. Romesberg*,† Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla California 92037, and Department of Chemistry, UniVersity of Iowa, Iowa City, Iowa 52242 ReceiVed: January 18, 2009; ReVised Manuscript ReceiVed: March 31, 2009
The spectral position of C-D stretching absorptions in the so-called “transparent window” of protein absorption (1800-2300 cm-1) makes them well suited as probes of protein dynamics with high temporal and structural resolution. We have previously incorporated single deuterated amino acids into proteins to site-selectively follow protein folding and ligand binding by steady-state FT IR spectroscopy. Ultimately, our goal is to use C-D bonds as probes in time-resolved IR spectroscopy to study dynamics and intramolecular vibrational energy redistribution (IVR) in proteins. As a step toward this goal, we now present the first time-resolved experiments characterizing the population and dephasing dynamics of selectively excited C-D bonds in a deuterated amino acid. Three differently deuterated, Boc-protected leucines were selected to systematically alter the number of additional C-D bonds that may mediate IVR out of the initially populated bright C-D stretching mode. Three-pulse photon echo experiments show that the steady-state C-D absorption linewidths are broadened by both homogeneous and inhomogeneous effects, and transient grating experiments reveal that IVR occurs on a subpicosecond time scale and is nonstatistical. The results have important implications for the interpretation of steady-state C-D spectra and demonstrate the potential utility of C-D bonds as probes of dynamics and IVR within a protein. Proteins and other biological molecules are unique in that they have been evolved for function. Thus, their properties, including their dynamics, might be fundamentally different from those of small molecules. Unfortunately, direct experimental characterization of specific protein vibrations is limited by the spectral congestion inherent to their IR spectra as well as by extensive coupling. Isotopic substitution has been used to overcome these limitations,1 and carbon-deuterium (C-D) bonds are particularly promising in this regard.2,3 The unique absorption frequency of C-D bonds (2100-2300 cm-1) not only facilitates their observation but also simplifies their analysis as they are adiabatically decoupled from all other protein vibrations. In addition, unlike non-native vibrational probes, isotopic substitution is nonperturbative and it does not introduce artificial interactions.4 However, the nonpolar nature of C-D bonds makes them challenging to characterize due to rather small molar extinction coefficients of 5 to 30 M-1cm-1, and thus their characterization requires signal-to-noise ratios in excess of 105 and careful data analysis. Nevertheless, we have previously used site-selectively deuterated proteins and steadystate Fourier transform infrared (FT IR) spectroscopy to characterize protein folding, ligand binding, and conformational heterogeneity with residue specific detail.2,4,5 Ultimately, our goal is to use C-D bonds as probes in time-resolved IR spectroscopy to study dynamics and intramolecular vibrational * To whom correspondence should be addressed. E-mail: (C.M.C.)
[email protected]; (F.E.R.)
[email protected]. † The Scripps Research Institute. ‡ University of Iowa.
10.1021/jp900516c CCC: $40.75
Figure 1. Structures of deuterated leucines characterized. (d3)Leu is a 1:1 mixture of the two isotopologues where each Cδ methyl group is deuterated.
energy redistribution (IVR) within proteins. As a step toward this goal, we present here the first time-resolved experiments characterizing the dephasing and population dynamics of selectively excited C-D bonds within a deuterated amino acid. We first characterized the steady-state FT IR absorption spectra of the three deuterated, Boc-protected leucine isotopologues in chloroform (Figures 1 and 2) and performed ab initio quantum chemical calculations to aid in band assignment (Supporting Information). Each deuterated Cδ methyl group gives rise to a symmetric stretch around 2060 cm-1 and two nearly degenerate asymmetric stretches around 2220 cm-1. In the case of (d3)Leu, only one of the two Cδ methyl groups is deuterated, and the spectrum shows three bands that are assigned as one symmetric (2060 cm-1) and two asymmetric stretches (2210 and 2225 cm-1). With (d7)- and (d10)Leu, both Cδ methyl groups are deuterated, and two symmetric stretches are observed at ∼2060 and ∼2080 cm-1 in (d7)Leu and at ∼2060 and ∼2100 cm-1 in (d10)Leu. The asymmetric Cδ stretches of both (d7)and (d10)Leu are overlapped and give rise to a single intense absorption band at ∼2220 cm-1. The weak absorption at 2160 2009 American Chemical Society
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Letters TABLE 1: Exponential Fits of Transient Grating and Three-Pulse Photon Echo Dataa transient gratingb τ1/fs
a1/%
τ2/ps
three-pulse photon echo a2/%
(d3)Leu 170 ( 70 95 ( 90 3.1 ( 0.7 5 ( 14 (d7)Leu 220 ( 90 80 ( 80 2.4 ( 0.9 16 ( 17 (d10)Leuc 420 ( 60 66 ( 11 2.8 ( 0.2 34 ( 5
τ/fs 280 ( 70 300 ( 50 320 ( 50
At the 99.73% confidence level for delay times >250 fs (TG) or 300 fs (3PE), see text and Supporting Information for details. b Data fit to {a1 · exp(-t/τ1) + a2 · exp(-t/τ2)}2. c An additional damped sinus function, a3 · exp(t/τD) · sin(ωt + φ), was included to account for the oscillatory feature in the TG and 3PE data; fits yielded τD ) (290 ( 40) fs, ω ) (40 ( 3) cm-1 for the TG decay, and τD ) (120 ( 50) fs, ω ) (37 ( 33) cm-1 for the 3PE decay of (d10)Leu, respectively. a
Figure 2. Absorption spectra of each deuterated leucine in CHCl3.
Figure 3. Normalized transient grating (A) and three-pulse photon echo (B) decays.
cm-1 in (d7)- and (d10)Leu is attributed to the deuterated Cγ methine. In addition, mixing of the CR and antisymmetric Cβ stretching modes of (d10)Leu gives rise to the feature on the red side of the asymmetric Cδ stretches between 2180 and 2200 cm-1, while the symmetric Cβ stretch is nearly degenerate with the Cδ symmetric stretch at ∼2100 cm-1. The weak absorption around 2125 cm-1 observed in all three deuterated leucines is likely a combination band or overtone involving C-D bending. Conveniently, the molar extinction of the absorption at ∼2220 cm-1 is larger in (d10)Leu than in (d7)Leu due to overlap of the asymmetric Cδ and Cβ stretches, which makes (d10)Leu a comparatively strong IR chromophore. To begin to explore their dynamics, we characterized the lifetime of selectively excited C-D bonds using transient grating (TG) spectroscopy. A laser wavelength of 2210 cm-1 was used to excite primarily the asymmetric Cδ stretching modes, but the initially prepared C-D bright state also includes minor contributions from other modes within the bandwidth of the excitation pulse (150 cm-1). The normalized TG decays for deuterium labeled and unlabeled leucine species show a large signal around time zero due to a nonresonant solvent response, which decays completely within ∼250 fs (Figure 3A). The shape of this nonresonant peak is the same for the labeled and unlabeled samples, thus the nonresonant response prevents observation of C-D specific dynamics on time scales less than 250 fs. For times greater than 250 fs, the TG signal originates exclusively from C-D vibrational dynamics, as no signal was observed with the unlabeled sample. For the deuterated leucines, the TG signal decays nonexponentially but is well fit by the squared sum of two exponentials (Table 1). The slower ∼3 ps component of the decay varies only by about 20% between the isotopologues, and the changes appear to be uncorrelated to the degree of deuteration (τ2,(d3)Leu < τ2,(d10)Leu < τ2,(d7)Leu). Conversely, the subpicosecond decay component is ∼3-fold shorter for (d3)Leu and ∼2-fold shorter for (d7)Leu compared to (d10)Leu. An oscillatory feature is also apparent in the TG decay of (d10)Leu with a frequency of ∼40 cm-1 (see Supporting Information). These quantum beats are likely associated with the coherent
excitation of the multiple C-D transitions that fall within the laser bandwidth, which also explains their increased amplitude in (d10)Leu. It is not a priori clear whether the observed subpicosecond component in the TG decay is due to population dynamics or to vibrational dephasing occurring on a similar time scale.6 Thus, to deconvolute the contributions of population and dephasing dynamics, we performed three-pulse photon echo (3PE) experiments with a zero wait time between second and third pulse, which are equivalent to a two-pulse photon echo experiment, to directly measure the dephasing rates of the initially prepared C-D excited states (Figure 3B). The unlabeled leucine exhibits a large nonresonant solvent response around time zero that completely decays within ∼250 fs. The decays for the deuterated samples are well fit by single exponential functions with time constants of ∼300 fs for all three isotopologues (Table 1). Thus, the dephasing rate of the C-D bright state is essentially independent of the density of nearly resonant dark states. The nonresonant signal peak also broadens toward positive delay times, most noticeably for (d7)Leu. This observation suggests that there is a fast dephasing process convoluted with the solvent response on a ∼100 fs time scale. For (d7)Leu, the maximum of the 3PE signal also shifts to positive delay times. Such photon-echo peak shifts are indicative of inhomogeneous broadening of the absorption band.6 No peak shift was observed for (d3)Leu or (d10)Leu, but a shift may have been obscured by the relatively strong superimposed nonresonant solvent response around time zero. Guided by the experimentally observed single-exponential 3PE decays, we used a Kubo model7 to describe the line broadening of the C-D absorptions in which the two-point time-correlation function of transition frequency fluctuations is given by C(t) ) ∆2 exp(-t/τ), where ∆2 is the mean-squared amplitude of the frequency fluctuations, and τ is their correlation time. Nonlinear response function theory8 was used to determine ∆ and τ from simultaneous fits to the absorption line shape of the asymmetric stretches at 2220 cm-1 and the 3PE decays (Supporting Information). Neither of these measurements alone is sufficient to uniquely specify ∆ and τ, but taken together they provide adequate constraints on the Kubo parameters. For (d3)Leu, the asymmetric absorption contains two transitions of nearly equal intensity; hence, we calculated the absorption spectrum and 3PE decay for a pair of uncoupled oscillators with center frequencies of 2209 and 2222 cm-1, assuming that the oscillators have the same frequency correlation function. For (d7)Leu and (d10)Leu the asymmetric absorption appears to be a single Gaussian line, although it must be a superposition of the four asymmetric stretches of the two Cδ deuterated methyl groups. This suggests that the four stretching
Letters vibrations are (nearly) degenerate, most likely because the two Cδ methyl groups are chemically equivalent in the isotropic solvent environment. It is thus reasonable to assume that the four transitions have identical line shapes and transition frequencies, and thus they can be modeled as a single oscillator. The approximations used are somewhat primitive, but there are insufficient constraints to support a more sophisticated model. Nevertheless, both the measured absorption line shapes and 3PE decays are well fit using this model (Supporting Information, Figure S2). For all of the different deuterated leucine species, τ is ∼0.3 to ∼0.35 ps and ∆ is ∼1.9 ps-1. The characteristic product, ∆ · τ ≈ 0.6, indicates that the C-D stretching absorptions are not fully in the homogeneous limit (∆ · τ , 1), but significantly broadened by inhomogeneous effects,8,9 in contrast to other vibrational probes of protein dynamics, for example, amide, carbonyl, nitrile, or azide vibrations, whose absorptions appear to be predominantly homogenously broadened (∆ · τ < 0.1) when incorporated into small molecules.10 This result suggests the presence of structural inhomogeneity in the leucine side chain, which may for instance be due to librational motions expected to occur on the >100 ps time scale.11 This in turn suggests that the C-D stretch vibrations are sensitive to these conformational changes, which is significant for the development of C-D chromophores as probes of protein dynamics, as it supports the interpretation of the line width changes in terms of inhomogeneous changes in the protein environment, that is, protein heterogeneity.3a,12 However, since the lines are clearly not in the purely inhomogeneously broadened limit, lifetime broadening may also contribute to line width in some cases, as has been suggested previously.13 Importantly, because the 3PE decays are approximately independent of the extent of amino acid deuteration, we can conclude that the observed deuteration-dependent biphasic TG decays are a result of population dynamics. Interestingly, the dependence of the lifetimes on the extent of deuteration, τ1,(d10)Leu > τ1,(d7)Leu > τ1,(d3)Leu, is the opposite of that expected for statistical IVR. With statistical IVR, increasing the extent of deuteration would reduce the population lifetime of the excited C-D stretch, because it increases the density of isoenergetic states.14 The data suggest that the local density of isoenergetic states does not control IVR within the amino acid. The dynamics of C-D vibrations are expected to be similar to those of C-H vibrations, which have been studied extensively in small molecules using anti-Stokes Raman,15,16 transient electronic absorption,17,18 and IR pump-probe19 spectroscopy. In general, these studies also observed biphasic IVR after excitation of C-H stretches. This behavior has typically been interpreted in terms of fast energy redistribution between the excited C-H state and a few other modes that are strongly coupled, followed by slower dissipation of the energy into the bath of states provided by the remainder of the molecule’s degrees of freedom.16,17 Strong and selective coupling between stretching and bending motions also results in the well-known and widely observed phenomenon of Fermi resonance.20 A similar biphasic population relaxation has also been observed in recent measurements of the relaxation of the C-D stretching vibration of deuterated haloforms.21 In the current measurements, IVR from the initially excited C-D state into associated bending modes also appears to be selective, despite the relatively large size of the molecule (37 atoms). This must result from an adiabatic decoupling between the C-D bonds and the vast majority of other molecular vibrations. Thus, the initial IVR likely proceeds through pathways involving energy transfer to a subset of other C-D stretches and/or bending motions that are strongly coupled to the initially prepared excited state and not statistically through the bath of states at the energy of the
J. Phys. Chem. B, Vol. 113, No. 23, 2009 7993 C-D vibration. Moreover, the observed increase in lifetime with deuteration suggests that additional C-D bonds can actually detune key resonances, thereby increasing the time required for initial equilibration. While the details of IVR in (dn)Leu require further study, our experiments demonstrate the feasibility of time-resolved IR experiments on C-D stretch vibrations in deuterated amino acids and are encouraging with respect to ultimately using C-D labels to study protein dynamics with time-resolved IR spectroscopy. Such experiments would allow for the characterization of protein dynamics with the same bond-specific detail previously possible only in small molecules. For example, it would be fascinating to determine whether the observed IVR pathways remain important in a protein, where despite the astronomically high density of states, only a few are likely to remain strongly coupled to any given C-D vibration. It is also interesting to speculate that specific couplings might facilitate energy flow among other bonds within a protein, possibly facilitating conformation-dependent IVR with potentially profound implications for biological function. Acknowledgment. This research was supported by the National Science Foundation under Grant MCB 034697 (to F.E.R.) and by the Roy J. Carver Charitable Trust and NSF CHE-0644410 (to C.M.C.). Supporting Information Available: Information detailing experimental procedures, the ab initio quantum chemical calculations, and modeling of the 3PE data. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Tadesse, L.; Nazarbaghi, R.; Walters, L. J. Am. Chem. Soc. 1991, 113, 7036. (b) Barth, A. Biopolymers 2002, 67, 237. (c) Decatur, S. M. Acc. Chem. Res. 2006, 39, 169. (2) Chin, J. K.; Jimenez, R.; Romesberg, F. E. J. Am. Chem. Soc. 2001, 123, 2426. (3) (a) Fujisaki, H.; Straub, J. E. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6726. (b) Becker, J.; Becher, F.; Hucke, O.; Labahn, A.; Koslowski, T. J. Phys. Chem. B 2003, 107, 12878. (c) Mirkin, N. G.; Krimm, S. J. Phys. Chem. A 2007, 111, 5300. (d) Naraharisetty, S. R. G.; Kurochkin, D. V.; Rubtsov, I. V. Chem. Phys. Lett. 2007, 437, 262. (e) Romesberg, F. E. Chembiochem 2003, 4, 563. (4) Thielges, M.; Case, D. A.; Romesberg, F. E. J. Am. Chem. Soc. 2008, 130, 6597. (5) (a) Sagle, L.; Zimmermann, J.; Dawson, P.; Romesberg, F. J. Am. Chem. Soc. 2006, 128, 14232. (b) Sagle, L.; Zimmermann, J.; Matsuda, S.; Dawson, P.; Romesberg, F. J. Am. Chem. Soc. 2006, 128, 7909. (c) Weinkam, P.; Zimmermann, J.; Sagle, L.; Matsuda, S.; Dawson, P.; Wolynes, P.; Romesberg, F. E. Biochemistry 2008, 47, 13470. (6) Joo, T.; Jia, Y.; Yu, J.; Lang, M.; Fleming, G. J. Chem. Phys. 1996, 104, 6089. (7) Kubo, R. J. Phys. Soc. Jpn. 1962, 17, 1100. (8) Mukamel, S. Principles of nonlinear optical spectrsocopy; Oxford University Press, New York, 1995. (9) Berg, M. A.; Rector, K. D.; Fayer, M. D. J. Chem. Phys. 2000, 113, 3233. (10) (a) Zanni, M. T.; Asplund, M. C.; Hochstrasser, R. M. J. Chem. Phys. 2001, 114, 4579. (b) Mukherjee, P.; Krummel, A. T.; Fulmer, E. C.; Kass, I.; Arkin, I. T.; Zanni, M. T. J. Chem. Phys. 2004, 120, 10215. (c) Inaba, R.; Tominaga, K.; Tasumi, M.; Nelson, K. A.; Yoshihara, K. Chem. Phys. Lett. 1993, 211, 183. (d) Hamm, P.; Lim, M.; Hochstrasser, R. M. Phys. ReV. Lett. 1998, 81, 5326. (11) Compton, D. A. C.; Montero, S.; Murphy, W. F. J. Phys. Chem. 1980, 84, 3587. (12) (a) Sagle, L.; Zimmermann, J.; Dawson, P.; Romesberg, F. J. Am. Chem. Soc. 2004, 126, 3384. (b) Chin, J.; Jimenez, R.; Romesberg, F. J. Am. Chem. Soc. 2002, 124, 1846. (13) Cremeens, M.; Fujisaki, H.; Zhang, Y.; Zimmermann, J.; Sagle, L.; Matsuda, S.; Dawson, P.; Straub, J.; Romesberg, F. J. Am. Chem. Soc. 2006, 128, 6028. (14) Cheatum, C. M.; Heckscher, M. M.; Bingemann, D.; Crim, F. F. J. Chem. Phys. 2001, 115, 7086.
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(15) (a) Alfano, R.; Shapiro, S. Phys. ReV. Lett. 1972, 29, 1655. (b) Lauberau, A.; von der Linde, D.; Kaiser, W. Phys. ReV. Lett. 1972, 28, 1162. (c) Hartl, I.; Zinth, W. J. Phys. Chem. A 2000, 104, 4218. (d) Fischer, S.; Lauberau, A. Chem. Phys. Lett. 1978, 55, 189. (e) Hoffmann, M.; Graener, H. Chem. Phys. 1996, 206, 129. (f) Iwaki, L.; Dlott, D. Chem. Phys. Lett. 2000, 321, 419. (16) Fendt, A.; Fischer, S.; Kaiser, W. Chem. Phys. 1981, 57, 55. (17) Heckscher, M.; Sheps, L.; Bingemann, D.; Fleming Crim, F. J. Chem. Phys. 2002, 117, 8917. (18) Charvat, A.; Assmann, J.; Abel, B.; Schwarzer, D. J. Phys. Chem. A 2001, 105, 5071.
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JP900516C
