Carbon−Deuterium Bonds as Site-Specific and Nonperturbative

LETTER pubs.acs.org/JPCL

Carbon-Deuterium Bonds as Site-Specific and Nonperturbative Probes for Time-Resolved Studies of Protein Dynamics and Folding J€org Zimmermann, Megan C. Thielges, Wayne Yu, Philip E. Dawson, and Floyd E. Romesberg* Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States

bS Supporting Information ABSTRACT: Carbon-deuterium (C-D) bonds are nonperturbative spectroscopic probes that absorb in a region of the IR spectrum that is free of other protein absorptions. We explore the use of these probes under time-resolved conditions to follow the unfolding of cytochrome c from a photostationary state that accumulates after CO is photodissociated from the protein’s heme prosthetic group. Our results clearly show that C-D bonds are well-suited to characterize protein folding and dynamics. SECTION: Biophysical Chemistry

T

he experimental characterization of dynamics on all biologically relevant time scales is essential to understanding protein folding and function but has proven extraordinarily challenging due to the absence of probes with suitably high structural and temporal resolution. Because vibrational spectroscopy directly characterizes the vibrations that give rise to dynamics and because it has inherently high time resolution (sufficient to resolve even rapidly interconverting species), it provides an ideal approach to this problem.1,2 However, its application to proteins is typically precluded by spectral congestion. Interestingly, this problem may be circumvented with the use of carbon-deuterium (C-D) bonds, which absorb in an otherwise transparent region of the spectrum (between ∼2100 and 2200 cm-1). Moreover, C-D bonds are nonperturbative, ensuring that the observed dynamics may be interpreted in terms of the native protein. Previously, we site-specifically incorporated C-D bonds throughout cytochrome c (cyt c),3-5 an SH3 domain,6 and dihydrofolate reductase,7 and experimental characterization of these proteins, along with other computational8-12 and model13-16 studies, have clearly demonstrated the potential utility of the approach. While these studies all characterized the C-D absorptions under steady-state conditions, the most exciting application of the approach is in the characterization of protein motions under time-resolved conditions. However, given their small extinction coefficient (∼10 M-1 cm-1), it is not clear whether C-D bonds will be observable under time-resolved conditions where signal-tonoise ratios are inherently lower. Here, we report the development of an approach to phototrigger the unfolding of cyt c, and using it, we demonstrate that the absorptions of a single CD3 group within the protein may be time-resolved via rapid-scan FTIR spectroscopy. r 2011 American Chemical Society

Figure 1. Structure of cyt c (PDB 1GIW) showing the heme iron (orange), native ligands (blue), and the deuteration site (red).

Cyt c shuttles electrons through the respiratory chain via the Fe(II) and Fe(III) states of its heme prosthetic group and has served as a paradigm for the study of protein folding and, in particular, the contribution of ligand exchange to the folding and unfolding processes.17,18 In the folded state, along with His18, Met80 ligates the iron center of the heme (Figure 1), but it dissociates early during the unfolding process, during which it may be replaced by other protein ligands, for example, His or Lys residues,19-21 or by exogenously added ligands such as CO.22 In fact, under mildly denaturing conditions (e.g., 4 M guanidine hydrochloride (GdnHCl)), displacement of Met80 by CO causes the reduced protein to unfold. Consequently, photodissociation of Received: January 4, 2011 Accepted: January 28, 2011 Published: February 08, 2011 412

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Table 1. Spectral Fit Parameters for Steady-State Spectra and LIDS ν (cm-1)

A (mOD)

fwhm (cm-1)

Steady State cyt c-COa

15.7 ( 0.6

1955.6 ( 0.2

25 ( 1

1.6 ( 0.4

1974 ( 1 2125.6 ( 0.3

11.5 ( 0.6 6.5 ( 0.4

0.21 ( 0.01

2131.1 ( 0.3

11.2 ( 0.9

(d3)Met80b,c (d3)Met80-COb

Bleach a

cyt c-CO

b

(d3)Met80-CO

-5.7 ( 0.5

1955.6d

25d

-0.7 ( 0.1

d

11.5d

d

11.2d

-0.10 ( 0.01

1974 2131.1

Figure 2. FTIR spectra (top) and LIDS (bottom) in the CO stretch region (left) and CD3 symmetric stretch region (right) for (d3)Met80 (dotted lines), (d3)Met80-CO (solid lines), and cyt c-CO (dasheddotted lines). See Supporting Information for experimental details.

Induced Absorption cyt c-COa

0.61 ( 0.07

1966 ( 1

17 ( 1

(d3)Met80-COb

0.15 ( 0.03 0.18 ( 0.04

2136 ( 1 2125.8 ( 0.3

25 ( 1 7.2 ( 0.1

CO stretch. b Symmetric CD3 stretch. c From ref 5. d Fixed during fit, see Supporting Information. a

signal at around 1955 cm-1 (Table 1 and Figure 2), which is ascribed to depopulation of the CO-bound unfolded state. The amplitude of the bleach signal is about 40% of the steady-state CO absorption amplitude, suggesting that a substantial amount of the CO is photodissociated. Both LIDS show two induced absorptions, one at 2136 cm-1, which was assigned to the stretch vibration of unbound CO, and one at 1966 cm-1, which must correspond to a photostationary state with CO bound to the heme center. The frequency of the induced absorption at 1966 cm-1 is similar to that observed for CO bound to folded cyt c,24,27 and thus, it may originate from a transient species in which CO has rebound to a folded or nearly folded protein, and the reduced amplitude would result from the short lifetime of the species. Another possibility is that the induced absorption corresponds to the population of a small amount of a species where CO rebinding has trapped the protein in a partially folded state. Interestingly, the LIDS of (d3)Met80-CO also revealed a derivative-like absorption superimposed on the induced absorption of free CO, which was well fit by two Gaussian functions (Figure 2 and Table 1). One Gaussian with a negative amplitude is assigned to the bleach of the CD3 absorption in the CObound unfolded state and the other to an induced absorption in the photostationary state. The frequency and amplitude of the induced absorption is indistinguishable from the symmetric CD3 stretch absorption of (d3)Met80 in the folded state, suggesting that most of the protein in the photostationary state has an intact Fe(II)-Met80 bond. Because Met80 association is thought to be the last step in folding,17,18 the data suggest that after preirradiation, a substantial amount of the protein does indeed fold and assume a native-like structure. This conclusion is further supported by changes observed in the LIDS of the UV/vis absorption spectra of cyt c-CO in 4 M GdnHCl, which shows bleach signals corresponding to the unfolded protein and induced absorptions that are indistinguishable from the folded protein (see Supporting Information). On the basis of the time scales for CO binding to the folded protein,26 rapid-scan FTIR spectroscopy would appear to be well-suited for the characterization of the unfolding dynamics of the photostationary state. Before evaluating the use of a C-D probe, we first characterized the relatively strong CO bleach signal. As expected, identical results were obtained with both cyt c-CO and (d3)Met80-CO. The buildup of the CO bleach

the bound CO was thought to initiate folding,22 and submillisecond time scale changes in the UV/vis and circular dichroism spectra following photodissociation were initially interpreted in terms of early folding events, but it was shown later that these signals are associated mainly with CO rebinding.22-25 However, in their initial report, Jones et al. noted that a small fraction of CO-photodissociated cyt c populates a long-lived state whose UV/vis spectral signature is virtually identical to that of the native state,22 suggesting that a small amount of protein escapes CO rebinding and instead undergoes folding. In addition, Kumar et al. reported that CO rebinding to the folded protein takes place on a rather slow (seconds or greater) time scale.26 We thus reasoned that folded protein might accumulate with sustained irradiation and that halting irradiation could then serve as a trigger to initiate unfolding that would be amenable to experimental characterization by rapid-scan FTIR spectroscopy. Previously, we synthesized cyt c with (methyl-d3) methionine incorporated at position 80 ((d3)Met80) and showed that for oxidized (d3)Met80, both the line width and frequency of the CD3 symmetric stretch vibration at ∼2130 cm-1 are different in the folded and unfolded states.4 To examine the CD3 absorptions under conditions suitable for CO photodissociation-induced (un)folding, we examined the IR spectra of reduced (d3)Met80 in 4 M GdnHCl (Table 1 and Figure 2). In the absence of CO, the FTIR spectrum of (d3)Met80 in 4 M GdnHCl consists of a single major absorption at 2125.6 cm-1, which was previously assigned to the symmetric CD3 stretch vibration of Fe(II)-bound Met80.5 In the presence of CO, we found a single major absorption at 2131.1 cm-1, which was assigned to the symmetric CD3 stretch vibration of unligated, solvent-exposed Met80.5 In the latter case, a strong absorption at 1956 cm-1 and a minor absorption at 1974 cm-1 were also observed and assigned to the stretch vibration of CO bound to the heme of the unfolded protein.24,27 Next, to assess the overall spectral changes in the FTIR spectrum associated with preirradiation, we measured the lightinduced difference spectra (LIDS) of both (d3)Met80-CO and cyt c-CO in 4 M GdnHCl after 90 s of preirradiation with the second harmonic of a Nd:YAG laser (532 nm, ∼10 ns pulse width, 20 Hz repetition rate). Both LIDS show a strong bleach 413

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Met80 is already displaced by another ligand, such as another side chain of the protein (likely either Lys or His20,22) or a water molecule.28 Additional experiments employing C-D bonds incorporated at other positions within the protein will help differentiate these possibilities. The LIDS and time-resolved studies clearly indicate that repeated photodissociation of CO from cyt c-CO during preirradiation in subdenaturing concentrations of GdnHCl allows a substantial amount of the protein to fold, despite the low quantum yield of the process and an environment that thermodynamically favors the unfolded state.22 Thus, after preirradiation, the kinetics of unfolding may be conveniently monitored. In this study, we used rapid-scan FTIR spectroscopy to follow CO rebinding, and the dynamics associated with native ligand displacement were directly monitored via the C-D absorptions of (d3)Met80. In all, the observed CO and C-D kinetics suggests that two photostationary states accumulate, but only in the dominant state does CO binding displace Met80 directly. The less-populated photostationary state, in which Met80 displacement is not required for CO binding, likely represents a partially folded intermediate, perhaps similar to those observed using other techniques.20,29 The characterization of cyt c with C-D bonds site-selectively incorporated at other positions should allow for an unprecedented characterization of the cyt c unfolding process, including the characterization of any protein contribution to the minor process observed with the CO probe. The incorporation of C-D bonds throughout other proteins, perhaps in conjunction with other initiation techniques, should allow for an unprecedented characterization of their folding or unfolding as well. Most importantly, it is clear that C-D bonds are well-suited for the characterization of protein dynamics in real time. While C-D bonds are weaker IR chromophores than cyano or azide groups, which absorb in the same spectral region and have been incorporated into proteins30-33 or model systems,34-37 they are less likely to be perturbative. Compared to 13Cd18O chromophores, which have been used to characterize individual amide bonds,38-40 C-D bonds are again the weaker chromophores, but they are more general in that they maybe used to characterize side chain and backbone motions, and they also have significantly less background absorption, which in practice, complicates analysis for 13Cd18O chromophores. Moreover, while strong coupling renders 13Cd18O chromophores excellent probes of secondary structure, their absorptions are more difficult to interpret in terms of local factors, such as the electrostatic environment, than are those of C-D bonds, which have greater local-mode character.41 Finally, in addition to their use as probes, the nonperturbative nature and unique absorption frequency of C-D bonds should allow for site-specific deposition of energy within a protein. Thus, C-D bonds should find unique applications in the effort to site-specifically characterize a variety of biological processes, such as folding, ligand binding, energy flow, and perhaps even catalysis, although the latter applications will require the resolution of the C-D signals on a time scale much faster than that reported here. Efforts toward these goals are currently in progress.

Figure 3. (Left) Time dependence of CO bleach (closed circles) and C-D bleach (open circles). (Right) Exponential fits of the postirradiation decays of CO bleach (closed circles) and C-D bleach (open circles). See Supporting Information for experimental details.

signal during laser irradiation is well fit by two exponential functions with time constants (relative amplitudes) of 3 ( 1 (34 ( 8%) and 11 ( 3 s (66 ( 8%). The postirradiation decay of the CO bleach signal is also well fit by two exponential functions with time constants (relative amplitudes) of 2.1 ( 0.9 (5 ( 2%) and 20 ( 4 s (95 ( 2%). Therefore, the biphasic buildup simply reflects the biphasic decay as relaxation between laser pulses during preirradiation results in a biphasic repopulation of the ground state. The observed biexponential kinetics most likely reflect the accumulation of two distinct photostationary states to which CO rebinds with distinct time constants, although further experiments are required to unambiguously demonstrate this. Finally, to directly explore the role of Met80 in the accumulation and/or depopulation of the photostationary states and to evaluate the utility of C-D probes in characterizing the process, we examined whether it is possible to collect transient C-D absorption spectra of (d3)Met80. Indeed, we found that the transient IR absorptions of (d3)Met80 are observable (Figure 3), with a maximum signal of 0.5 mOD, which is at least 10-fold greater than the background signal. The buildup was well fit with biexponential kinetics with time constants (relative amplitudes) of 2 ( 1 (30 ( 10%) and 19 ( 4 s (70 ( 10%), again likely reflecting depletion of the ground state via two distinct photostationary states. The approximate coincidence of the time constants as probed by CO or C-D absorptions suggests that CO is directly displaced by Met80 without the intervention of any long-lived intermediates. In contrast to the CO transients, the postirradiation decay of the C-D absorption was well fit by a single exponential with a time constant of 20 ( 4 s. Further evidence that the faster, loweramplitude process observed in the CO transients is not present in the C-D transients is provided by a fit of the C-D data to two exponential functions with time constants and relative amplitudes fixed to those of the CO data. The resulting fits were of significantly lower quality (Figure S4, Supporting Information), confirming that Met80 does not participate in the faster process. The time constant of the dominant, slower process observed with CO is identical to that observed with (d3)Met80, and when corrected for protein concentration, it is consistent with that measured previously for CO binding to the folded protein.26 This again suggests that the majority of the protein folds during the preirradiation period and identifies the observed process as the direct displacement of Met80 from the Fe(II) center by CO. The minor, faster process observed with the CO probe may be associated with CO rebinding to a small amount of protein where

’ ASSOCIATED CONTENT

bS

Supporting Information. Detailed methods and spectroscopic data. This material is available free of charge via the Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION

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Corresponding Author

*E-mail: fl[email protected].

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