J. Phys. Chem. 1996, 100, 3273-3277
3273
Picosecond Structural Dynamics of Myoglobin following Photolysis of Carbon Monoxide Timothy P. Causgrove* and R. Brian Dyer CST-4, M.S.J586, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 ReceiVed: August 24, 1995; In Final Form: NoVember 3, 1995X
We have used picosecond infrared (IR) transient absorption spectroscopy in the amide I band to probe the dynamics of protein motion of myoglobin (Mb) following the photolysis of carbon monoxide. The rise time of the deoxy protein conformation is shown to be about 8 ps. The spectrum of amide I changes was also measured at 50 ps after photolysis and found to be similar to static IR difference spectra and to time-resolved IR spectra taken at times longer than 100 ns. By comparing the results obtained here with other picosecond results on photolysis of CO from Mb, we conclude that the majority of changes seen in the amide I spectra are due to global motion on the proximal side of the heme. The time scale for amide I changes are compared to the results of molecular dynamics calculations.
Introduction Protein motion is the link between the three-dimensional structure of a protein in solution and its physiological function. For most proteins, a change in structure is essential to allow the protein to fulfill its given function. Due to technological advancements in recent years in X-ray diffraction, molecular modeling and multidimensional NMR, a tremendous amount of progress has been made on the determination of crystal structures and solution structures of proteins. However, the relation of structure to function has advanced at a slower pace due in part to the lack of a method for observing protein motion. The model system of choice for the relation of protein structure to function has been myoglobin (Mb). The loss of a ligand such as O2, CO, or CN- from the binding site in Mb results in a conformational shift which alters the ligand binding properties of the protein. This conformational transition is related to the allosteric effect in hemoglobin.1 Ligands such as CO can be released from the protein photonically, which provides a convenient, very fast, repeatable, and reproducible trigger for the conformational shift from liganded to unliganded form. The three-dimensional structures of carbonmonoxy Mb (MbCO) and of deoxy Mb (Mb), as well as several mutant proteins,2,3 have been determined by X-ray4,5 and neutron6 diffraction. The protein is approximately 80% R-helix, with the eight helices customarily labeled A-H, and contains a single heme. The heme iron atom is 6-coordinate in MbCO; four of the ligands are the heme nitrogens, the fifth is the proximal histidine, and the sixth is CO. In the deoxy form, the iron is 5-coordinate, with the sixth position vacant. The area vacated by the sixth ligand is largely filled by rearrangement of nearby side chains, primarily His64, the distal histidine.4 In sperm whale Mb, Arg45 also shows a relatively large difference in the structures but is replaced by lysine in horse Mb. Differences in crystal structures between the liganded carbonmonoxy and unliganded deoxy forms of myoglobin may be described as being separated into changes on the distal side of the heme as opposed to changes on the proximal side of the heme. The changes on the distal side are primarily the rearrangement of the side chains to fill the heme pocket. These differences are * To whom correspondence should be addressed at Division of Science & Mathematics, Box W-100, Mississippi University for Women, Columbus, MS 39701. X Abstract published in AdVance ACS Abstracts, January 15, 1996.
0022-3654/96/20100-3273$12.00/0
local in nature; there is a relatively large displacement of a small number of atoms. The changes on the proximal side are caused by the motion of the heme iron, which results in a global conformational change, or the small displacement of a large number of atoms. This global motion modulates the ligand binding affinity and provides a relation between structure and function in myoglobin. The dynamics of this process are less well understood. It has been proposed that the conformational transition following photolysis of the heme-CO bond may be viewed as being triggered by the out-of-plane motion of the heme iron. The heme iron becomes high spin, moves away from the ligand binding site, and pushes on the proximal histidine (His93). This results in a concerted motion of the F helix toward the EF corner and an accompanying rearrangement of the remaining helixes. In hemoglobin, this motion results in the transition between R and T conformations, altering the binding affinity of the other three ligand binding sites. The kinetics of the photolysis process at room temperature requires at least two steps, i.e. hν
MbCO 98 Mb*CO f Mb* + CO where Mb* denotes the deoxy conformation, and Mb the liganded conformation. The geminate pair state Mb*CO is a significantly populated intermediate in which the CO ligand remains in the heme pocket, although not bound to the heme. The final state Mb* + CO represents the CO ligand free in the solvent and the protein in its equilibrium deoxy conformation. It should be noted that the intermediate state in the scheme above may exist as a set of substates with the ligand located various distances from the heme,7 but such substates are apparently not spectrally or temporally resolved at room temperature. The appropriate model for the transition from liganded to deoxy conformation is largely dependent on the amount of initial energy transferred to the protein compared to the barrier between conformational states. A small amount of initial energy would correspond to the conformational substates model,8,9 which arose from low-temperature experiments on rebinding of CO. This model describes the dynamics of protein motion as a random walk through many different conformations before settling in the lowest energy state. At the opposite extreme is a collective motion model,10-12 in which a large amount of initial energy overcomes the potential barriers and the conformation switches from liganded to deoxy by the most direct route possible, with © 1996 American Chemical Society
3274 J. Phys. Chem., Vol. 100, No. 8, 1996 no sampling of conformations which do not lie along the reaction coordinate. A very large amount of data has been collected on motion of the ligand and heme immediately following photolysis of MbCO due largely to their convenient absorbances in the infrared and visible regions, respectively. However, very little data exists on the protein response to photolysis. Other groups have measured resonance Raman,13,14 band III absorption,15 solution refractive index,10-12 and circular dichroism16 on a picosecond time scale, but none of these are a direct measurement of protein conformation. Extensive work has been done on characterization of ligand motion at cryogenic temperatures, where there is little or no formation of the final state, Mb*+ CO. However, very little is known about the geminate pair state, Mb*CO, or the conformational change at higher (near physiological) temperatures, primarily because current methods of measurement on the relevant time scales probe the strongly absorbing heme rather than the protein itself. One method of observing protein motion is by time-resolved infrared (TRIR) spectroscopy. Unlike methods based on visible or near-UV light, the use of an IR probe bypasses the heme absorption and reports directly on changes in protein structure. One region of the infrared which is commonly used due to its sensitivity to secondary structure is the amide I region centered about 1650 cm-1. The exact frequency of amide I absorption is dependent on the details of the hydrogen-bonding environment and on coupling, which is sensitive to protein backbone geometry. Frequency assignments have been made for common secondary structure elements.17-19 Although there are a large number of overlapping absorbances in the amide I band of even a small protein, spectral congestion is avoided by the fact that TRIR is inherently a difference technique. The time-resolved spectrum depends only on the components of amide I which shift following the photolytic perturbation, isolating the functional changes from the static majority. In a previous paper,20 we measured the time-resolved differences in the amide I region on the submicrosecond to millisecond time scale. The difference spectrum (Mb*-MbCO) was determined by both time-resolved measurements and static FTIR difference spectroscopy. The time scale of the protein conformational transition following photolysis was determined to be faster than about 300 ns, the response time of the instrument. For the reverse process, the transition from deoxy conformation to MbCO conformation occurred concurrently with the recombination (was limited by the recombination rate). Here we have probed in the amide I region on faster time scales in order to resolve the kinetics of the protein conformational transition. The set of experiments discussed above indicate a process occurring on a tens of picoseconds time scale. We have made measurements on absorption of protein ligands near 2000 cm-1 on a 1-2 ps time scale21 and now extend those experiments to the amide I spectral range.
Causgrove and Dyer
Figure 1. TRIR transient at 1943 cm-1 following photolysis of MbCO (solid line) and fit to the experimentally determined instrument response (dashed line).
a CARY spectrophotometer, and the sample was continuously moved to avoid a buildup of long-lived states. The picosecond infrared absorption spectrometer has been described previously22 and was used with minor modifications. Briefly, a Coherent Antares 76s Nd:YAG laser was used to synchronously pump a dye laser. The 1064 nm output of the Antares was regeneratively amplified at 30 Hz; the second harmonic of the amplified pulse was used to pump three dye stages which amplified the dye laser output. A part of the amplified dye pulse was difference mixed with excess 1064 nm light from the regenerative amplifier, generating a pulse at about 1.3 µm. This pulse was then differenced again with 1064 nm, producing a picosecond probe pulse which was tuned in the range 1600-2000 cm-1 by changing the dye laser wavelength. The probe pulses were split into two beams, one of which passed through the sample cell and the other through a reference cell. The sample and probe reference pulses were detected directly with two matched HgCdTe detectors. The amplified dye pulse also served as the excitation pulse and was in the range 581594 nm. The power of pump pulse was typically about 2.5 µJ focused to an approximately 120 µm spot. The dye laser was usually run with a three-plate birefringent filter in order to narrow the spectral distribution of the infrared probe to about 1.5 cm-1 at the expense of temporal pulse width. Time-resolved spectra were measured by setting the time delay at a fixed value and determining the change in absorbance at discrete dye laser wavelengths. The time delay for time-resolved spectra was chosen much longer than the instrument response in order to eliminate effects due to dispersion and variations in laser pulse width across the spectrum. Each point in the spectrum is the result of about 40 sets of 100 laser shots; error bars for ∆A in the picosecond spectrum were taken as the standard deviation of the individual measurements. Results
Experimental Section Samples were prepared by dissolving lyophilized Mb from horse skeletal muscle (Sigma) into 50 mM sodium phosphate buffer, pD 7.4 (in D2O). The heme was reduced with ∼8 mM sodium dithionite and liganded with CO by several cycles of degassing and back-filling with CO. No attempt was made to completely exchange the protein hydrogen for deuterium, although based on the number of exchangeable hydrogen atoms in lyophilized Mb, the exchange should be >98%. Final samples were about 3 mM (5 mM when probing at 1943 cm-1) in 100 µm cells with CaF2 windows. Sample integrity was checked by obtaining UV-vis spectra of the R-band region on
Previous TRIR experiments on Mb, particularly on the picosecond time scale, have concentrated on the bleaching of absorption at 1943 cm-1 and the rise in absorption at 2100 cm-1 due to unbound CO.23-25 Although these kinetics are too fast to be observed with current TRIR instruments, they are useful as an internal measure of the instrument cross-correlation. Figure 1 shows the response of MbCO at 1943 cm-1 to photolysis, the bleach of absorption due to bound CO. Also shown is a fit to the experimentally determined (using a Si wafer) instrument response function.26 On the time scale of the present experiment, the traces show the same rise time, indicating that the breaking of the Fe-CO bond occurs faster
Picosecond Structural Dynamics of Myoglobin
Figure 2. TRIR transients taken at (a) 1666 and (b) 1656 cm-1 following photolysis of MbCO. Thin solid lines are experimental data, dashed lines are fits to experimentally determined instrument response functions, and heavy solid lines are best fit to convolution of instrument response and exponential.
than the resolution of the instrument. This is consistent with subpicosecond UV-vis measurements which indicate the appearance of a deoxy-like UV-vis absorption spectrum on the subpicosecond time scale.27 The disappearance of infrared absorption following photolysis of the Fe-CO bond in hemoglobin has already been observed by Anfinrud et al.23 and in Mb by Jedju et al.24,28 Our earlier paper20 established the use of amide I bands as a marker for protein motion. In Figure 2, we show a picosecond transient in the amide I band probed at 1666 cm-1 upon photolysis of MbCO, with excitation at 583 nm. This probe wavelength corresponds to the largest (negative) change in the static and 1 µs difference spectra. Also shown (dashed line) is the instrument response function, which clearly does not match the rise of the data. A good fit to the data is obtained by convoluting the instrument response with a 8.2 ps exponential rise (shown by the heavy solid line). Also shown in Figure 2 is a transient taken with the probe tuned to 1656 cm-1, which corresponds to a positive change in absorbance in the static and 1 µs spectra. This transient is best fit by a convolution of the instrument response with a 6.1 ps exponential rise (heavy solid line). TRIR traces similar to those in Figure 2 have been obtained at 1682 and 1620 cm-1 although with a lower signalto-noise ratio. To compare the state formed on the 6-8 ps time scale with the final deoxy conformation, the TRIR spectrum was measured in the amide I region at a fixed 50 ps delay between pump and probe pulses. This spectrum is shown in the bottom portion of Figure 3; the top portion shows the static FTIR difference spectrum and in the middle the TRIR spectrum determined previously at 1 µs delay. The shape of the spectra are generally the same, although some differences can be expected because CO is present in the heme pocket at 50 ps but not in the static FTIR or at 1 µs.25,29 Discussion We have shown previously20 that TRIR in the amide I region is an excellent probe of protein motion in the range of 300 ns to a few milliseconds. In that work, the kinetics of changes in
J. Phys. Chem., Vol. 100, No. 8, 1996 3275
Figure 3. Top: static FTIR difference spectrum Mb-MbCO in the amide I region. Middle: difference spectrum at 1 µs determined from amplitudes of TRIR transients. Bottom: difference spectrum at 50 ps from measurements of δ absorbance at fixed delay between pump and probe pulses.
the amide I region of the infrared spectrum of Mb were indistinguishable from the recombination kinetics (measured at 1943 cm-1), consistent with a conformational transition occurring upon recombination. No rising kinetics were observed subsequent to 300 ns, indicating that (without glycerol) the final state is complete within 300 ns. Here, we have extended the TRIR measurements to the picosecond time scale in order to follow the kinetics of structural changes which occur following photolysis of CO. The previous paper determined the difference spectrum of the (Mb* + CO)MbCO couple in the amide I region. With the current time resolution, we are able to resolve the kinetics associated with the protein conformational transition (Mb*CO-MbCO). Previous results based on time-resolved resonance Raman,13,30 molecular dynamics simulations,31,32 band III absorption,15,33 and transient grating spectroscopy12 have all indicated structural changes taking place on a picosecond time scale, largely probing the heme and its immediate surroundings. By probing in the amide I region, we are able to observe conformational dynamics which are inaccessible to other experimental methods. Although the overwhelming majority of absorption in the 1600-1700 cm-1 region is due to the protein amide I band, it is possible that heme vibrations also contribute to the small changes observed in the difference spectra of Figure 3. The highest frequency ring vibration observed in Raman spectra is at about 1630 cm-1,30 lower than the major features observed in our TRIR spectra. The only other vibrations which should contribute in this region are the heme propionates, which appear at about 1700 cm-1. Therefore, the spectra observed in Figure 3 may be assigned largely to protein amide I vibrations with possible contributions from amino acid side chains. In our previous microsecond TRIR study of photodissociation of MbCO, we showed that heating of the sample by the pump pulse did not affect the results by using a deoxy Mb sample in place of MbCO and found that the signal was negligible. In a picosecond experiment, the situation is complicated by the fact
3276 J. Phys. Chem., Vol. 100, No. 8, 1996 that excess vibrational energy is diffusing away from the heme on the time scale of the experiment. Diffusion of vibrational energy in Mb has been studied using TRIR in the 1800-1900 cm-1 region,34 molecular dynamics simulations,35 and timeresolved resonance Raman.36 The molecular dynamics (MD) simulations35 showed that following excitation of deoxy Mb with a 530 nm photon, the heme temperature rose by 300-500 K, sufficient for large changes in the optical absorption of the heme. The average temperature of the protein atoms, however, rose by less than 20 K. This temperature increase may be regarded as an absolute upper limit for the temperature change of the protein, as the simulations did not include solvation. In terms of the present experiment, the maximum increase in protein temperature would be much smaller for several reasons: our 583 nm photon adds less energy to the system than a 530 nm photon; breaking of the Fe-CO bond requires about 16 kcal/mol of the 49 kcal/ mol of photon energy;37 and finally, raising the protein from its liganded to unliganded conformation (Mb to Mb*) requires about 2.4 kcal/mol.38,39 Therefore, on the picosecond time scale, 62% of our photon energy is added as excess thermal energy upon photolysis of MbCO. Taking into account these corrections, the maximum increase in temperature of the protein in our experiment (neglecting heat transfer to the solvent) would be about 10 K. In actuality, the rise in temperature of the protein would be much smaller based on the experiments of Lian et al.,34 who determined that cooling of the heme is the bottleneck process in thermal diffusion from the heme to the solvent. FTIR spectra of MbCO taken at 5 °C intervals show that changes in amide I absorption due to temperature are small and are spectrally very distinct from the difference spectra in Figure 3. This indicates that the changes seen are not due to simple heating of the protein. In addition to heating of the protein, transients could arise from heating of the D2O solvent. This effect has been studied extensively34 by probing in the 1800-1900 cm-1 region following 580 nm excitation of deoxy Mb. Using 15 mM Mb in 100 µm path length cells, they found that at 1843 cm-1 a bleach (due to solvent heating) of 0.003 absorbance units appeared with nonexponential kinetics on a time scale of about 10 ps. This result may be applied directly to the D2O heating expected in our experiments by correcting for the Mb concentration (a factor of 5 smaller for our samples) and the temperature dependence of D2O absorbance at the probe wavelengths used. The effect of heating on D2O absorption in the amide I region is a small bleach, the amplitude of which is nearly independent of probe wavelength. This bleach is also linearly dependent on temperature; therefore, even though at 15 mM concentration the sample is heated nonuniformly, only the average temperature is observed. These two corrections, in addition to the 62% correction discussed in the previous paragraph, gives an expected change in absorbance of 9 × 10-4 absorbance units. This calculation may be corroborated by using the known pulse energy (3 µJ), absorption of the sample at 583 nm (0.30), and pump spot size (120 µm). This calculation gives an expected change in absorbance of D2O of about 6 × 10-4. By either calculation, the change in absorbance due to water is considerably smaller than the observed transients, indicating that the observed signals cannot be due to simple heating of the solvent. The calculations above, as well as the spectra in Figure 3, indicate that the signals observed are due to protein motion, which occurs in about 6-8 ps. As mentioned above, several other picosecond experiments have been performed on photodissociation of CO from Mb. Recent measurements include
Causgrove and Dyer the time-dependent changes in absorption of band III, a weak Fe-to-porphyrin charge transfer band near 760 nm.15,33 The position of band III is generally taken to be proportional to the Fe out-of-plane distance. Results in buffer (without glycerol) show a highly nonexponential relaxation of band III to its equilibrium position with time constants of 3.5, 83, and 3300 ps.15 These kinetics were found to match the final phase of Fe out-of-plane displacement in MD simulations.32 The final phase of motion was from 0.45 to 0.615 Å out of the heme plane, the final 27% of iron motion. The nonexponential final phase was attributed to protein motion which acts to resist the out-of-plane motion. One interpretation of the origin of this longer phase is that it represents the global protein motion on the proximal side of the heme, as the heme iron moves in concert with protein over the final 27% of its total travel. Complex motion such as this would explain the origin of nonexponential geminate recombination of NO.31 Such behavior may be connected with a conformational substates model, in which the deviation from exponentiality corresponds to motion from or through conformational substates of the protein with a distribution of strain energies. Although these experiments indicate that the ultrafast heme iron motion is followed by a slower nonexponential phase, other experiments indicate that the motion is complete on the ultrafast time scale. Competing MD simulations40,41 have shown that the heme iron motion is complete within 1 ps; the results of the simulations are apparently quite sensitive to the particular force field used. Iron out-of-plane distance has also been related to the intensity of νFe-His, the iron-proximal histidine stretching mode. Recent picosecond resonance Raman experiments30,42 indicate that νFe-His has reached 90% ((10%) of its full intensity within 1 ps. In addition, transient grating experiments have been performed with time resolution of 10 ps.10-12 These experiments are sensitive to solution refractive index, and by carefully eliminating the thermal contribution to the signal, changes in density due to global protein rearrangement are detected within the time resolution available. These fast processes are consistent with a collective motion mechanism, in which a protein vibration corresponding to the reaction coordinate is essentially directly excited by photodissociation of CO. The time scale and mechanism of protein motion following photolysis of MbCO is still in question largely because previous probes were not uniquely sensitive to protein conformational changes. The transients in Figure 2 indicate that the majority of protein motion occurs on a 6-8 ps time scale; the difference in the lifetimes at the two probe wavelengths is probably not significant given the signal-to-noise ratio of the data. While most of the change occurs within 6-8 ps, it is clear that further relaxation occurs on a slower time scale, with kinetics that we are unable to resolve with the sensitivity of our experiment. It is possible that this slower process exhibits highly nonexponential kinetics, consistent with the kinetics observed for the relaxation of the band III spectrum.15 However, differences between the band III and amide I kinetics could reasonably be expected. One reason is that the two techniques measure different effects and are not necessarily sensitive to the same molecular parameters. Also, the initial amplitude of the kinetics (band position at zero time) is not known in the band III experiment. If a significant portion of the band shift occurs in the first 2 ps, then the proportion of the decay extending to longer times would tend to be overestimated. The similarity of the spectra in Figure 3 indicates that the final deoxy protein conformation has been substantially formed by 50 ps, probably with a 6-8 ps time constant. These results are
Picosecond Structural Dynamics of Myoglobin consistent with the transient grating experiments10-12 and tend to support a collective motion mechanism for the transition from liganded to deoxy conformation. It should be noted that the reverse process (deoxy to liganded conformational transition) is fundamentally different in terms of initial energy imparted to the protein, and the conformational substates model is most likely an accurate description of protein motion on rebinding. Although the 50 ps spectrum in Figure 3 is similar to the longer time and static spectra, some differences exist which are likely related to the local differences between the deoxy protein and the intermediate geminate pair state, primarily on the distal side of the heme. The geminate pair state was the subject of a recent X-ray diffraction study43 at 12 K, where rebinding is extremely slow. It was found that the proximal histidine undergoes a large scale motion, and the F helix is displaced by about 80% of its full motion. Although done at cryogenic temperatures, this structure is useful in interpreting the differences between the 1 µs and 50 ps spectra (Figure 3b,c). Differences between these two spectra cannot be due to heme absorption, because the heme does not change appreciably between 50 ps and 1 µs. The major deviation between the spectra is in the region of 1645-1655 cm-1; the only known side-chain absorption in this region is by asparagine,44 which is located at positions 12, 122, and 140 in horse Mb. The side chain of each of these residues is exterior to the protein, and they are thus unlikely to contribute to difference spectra. Differences between the 50 ps and 1 µs spectra in this region must therefore be due to backbone conformational changes, most likely on the distal side of the heme due to the presence of CO at 50 ps. The region near 1650 cm-1 has been assigned to R-helix,19 so these differences may be due to strain induced in the E helix by the large-scale motion of the distal histidine.43 The experiments described here have the potential to give a detailed picture of the protein response to photolysis of MbCO. Further developments will depend on increasing the signal-tonoise ratio of the kinetic scans (which is available using high repetition rate Ti:sapphire lasers) as well as on assignment of the vibrational bands observed in Figure 3 to separate regions of the protein. These assignments are aided by TRIR experiments on mutants such as H93G(Im), in which the proximal histidine is replaced by glycine and exogenous imidazole is incorporated as the proximal ligand.45 This should allow amide I changes to be tentatively assigned to proximal side and distal side contributions; such studies are underway. Acknowledgment. We wish to thank Dr. Mark Hoffbauer for the use of his laser equipment and Dr. Stefan Franzen for helpful discussions. This work was supported by NIH Grant GM45807 to R.B.D. References and Notes (1) Perutz, M. F.; Fermi, G.; Luisi, B.; Shaanan, B.; Liddington, R. C. Acc. Chem. Res. 1987, 20, 309. (2) Hubbard, S. R.; Hendrickson, W. A.; Lambright, D. G.; Boxer, S. G. J. Mol. Biol. 1990, 213, 215. (3) Maurus, R.; Bogumil, R.; Luo, Y.; Tang, H.-L.; Smith, M.; Mauk, A. G.; Brayer, G. D. J. Biol. Chem. 1994, 269, 12606. (4) Takano, T. J. Mol. Biol. 1977, 110, 569. (5) Kuriyan, J.; Wilz, S.; Karplus, M.; Petsko, G. A. J. Mol. Biol. 1986, 192, 133. (6) Hanson, J. C.; Schoenborn, B. P. J. Mol. Biol. 1981, 153, 117. (7) Steinbach, P. J.; et al. Biochemistry 1991, 30, 3988.
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