Observing Vibrational Energy Flow in a Protein with the Spatial

Sep 5, 2014 - Adolfo Bastida , José Zúñiga , Alberto Requena , Beatriz Miguel , María Emilia Candela , and Miguel Angel Soler. The Journal of Phys...
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Observing Vibrational Energy Flow in a Protein with the Spatial Resolution of a Single Amino Acid Residue Naoki Fujii, Misao Mizuno, Haruto Ishikawa, and Yasuhisa Mizutani* Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan S Supporting Information *

ABSTRACT: One of the challenges in physical chemistry has been understanding how energy flows in a condensed phase from the microscopic viewpoint. To address this, spaceresolved information at the molecular scale is required but has been lacking due to experimental difficulties. We succeeded in the real-time mapping of the vibrational energy flow in a protein with the spatial resolution of a single amino acid residue by combining time-resolved resonance Raman spectroscopy and site-directed single-Trp mutagenesis. Anti-Stokes Raman intensities of the Trp residues at different sites exhibited different temporal evolutions, reflecting propagation of the energy released by the heme group. A classical heat transport model was not able to reproduce the entire experimental data set, showing that we need a molecular-level description to explain the energy flow in a protein. The systematic application of our general methodology to proteins with different structural motifs may provide a greatly increased understanding of the energy flow in proteins. SECTION: Spectroscopy, Photochemistry, and Excited States the heme migrates inside of the protein and finally dissipates to solvent water. The energy dissipation from the protein to the water solvent has been studied in femtosecond time-resolved infrared17 and transient phase grating studies 18,19 that monitored the heating of water caused by the photoexcitation of protein. These studies showed that vibrational energy was transferred to the water interface through the protein matrix in less than 20 ps. Thus, the time scales of energy relaxation in heme and energy release to water have been well-characterized. In contrast, much less is understood about energy flow within a protein. Elucidation of the pathways by which energy is released by the heme to the protein matrix is fundamental to understanding the mechanisms underlying energy transfer in proteins. To address this issue, we used time-resolved antiStokes ultraviolet resonance Raman (UVRR) spectroscopy to observe the vibrationally excited population in an amino acid residue following photoexcitation of the heme group. Timeresolved anti-Stokes Raman spectroscopy is selective for vibrationally excited populations and therefore is a powerful approach for measuring vibrational energy transfer.15,16,20 In addition, the resonance effect by using UV light allows us to selectively measure the Raman spectra of aromatic amino acid residues.21,22 Accordingly, information on the time evolution of the vibrational energy transferred to these residues in proteins can be provided by time-resolved anti-Stokes UVRR spectra of aromatic amino acids. We recently applied this technique to cytochrome c and demonstrated its usefulness in studying

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nergy is the origin of many fascinating dynamics in nature. Thus, the energy flow in a condensed phase is fundamentally important for chemical dynamics.1−5 Although heat transfer through a material is macroscopically welldescribed by Fourier’s law,6 microscopic descriptions of energy flow in a condensed phase remain very poorly defined. Temporally resolved mapping of energy flow at the molecular scale is a prerequisite for better understanding the microscopic mechanism of energy flow. One approach to studying energy flow is to excite a specific group and monitor energy redistribution in the surroundings. The energy flow can be studied by observing both the energy decay of the initially excited group and the energy rise in the accepting groups.7−9 Using this approach, it is possible to elucidate the energy flow pathways in a condensed phase. Hemeproteins are ideal systems for studying energy flow in proteins. Because the nonradiative lifetime of electrically excited heme in proteins is extremely short (within 100 fs),10 it is possible to optically deposit about 25000 cm−1 of excess vibrational energy selectively at the heme group fast enough to resolve the subsequent energy redistribution processes. The heme group is relatively isolated from the rest of the protein and thus can approximate a solute in solution. Energy flow in hemeproteins has been studied both experimentally and theoretically. Pioneering work on the energy flow was reported by Hochstrasser and his co-workers, who performed molecular dynamics simulations for cytochrome c and myoglobin (Mb) with initial energy deposits corresponding to photoexcitation.11 Time-resolved absorption12,13 and resonance Raman studies14−16 have shown that the energy of the vibrationally excited heme was transferred to the surrounding protein moiety with time constants of a few picoseconds. The energy released from © 2014 American Chemical Society

Received: July 11, 2014 Accepted: September 5, 2014 Published: September 5, 2014 3269

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vibrational energy flow in proteins.23 This technique is even more powerful and applicable to the time- and space-resolved observation of energy migration in proteins when combined with site-directed mutagenesis. The position of a Trp residue can be changed in a protein by amino acid substitution. Thus, by comparing data from mutants in which the distances of the Trp residue from the heme are different, it is possible to map the energy flow in a protein by moving the position of the probe residue. The protein moiety of a hemeprotein is a “quasisolvent” in which the three-dimensional structure is wellcharacterized and can be modified by site-directed mutagenesis. Hemeproteins therefore provide significant advantages for studying energy flow in the condensed phase rather than the solution phase because the distance and relative orientation of the heater (heme) and probe group (Trp residue) can be fixed in proteins but not in solution (where the two molecules would diffuse freely). Here, we applied this approach to determining the distance dependence of vibrational energy migration in Mb. Figure 1a

Figure 2 shows the time-resolved anti-Stokes UVRR spectra measured for the Mb mutants. The top trace in each panel is

Figure 2. Time-resolved anti-Stokes UVRR spectra of the Mb mutants for time delays from −5 to 50 ps. (a) Trp68 mutant, (b) Trp28 mutant, and (c) Trp14 mutant. Wavelengths of pump and probe pulses are 405 and 230 nm, respectively. The top traces (blue) are the probe-only spectra corresponding to the anti-Stokes UVRR spectra of the Mb mutants. The asterisks represent the sulfate band at 983 cm−1, used as an intensity standard. The other traces (red) are time-resolved difference spectra, which were generated by subtracting the probe-only spectrum from the pump−probe spectrum at each delay time. The accumulation time for obtaining each spectrum is 72 and 120 min for the Trp68 and the other two mutants, respectively. (d) Atomic displacement vectors of the W16 and W18 modes of 3-methylindole.21 Atomic displacements are enlarged three times.

Figure 1. Crystallographic structure of sperm whale Mb. (a) Wild-type Mb. The two original Trp residues are shown as space-filling spheres in yellow, and the protein is shown in a green ribbon representation with a superimposed gray space-filling representation. (b) Mutant Mb. The heme and probe Trp residues are modeled as space-filling spheres in red and cyan, respectively. Figures of the Trp68 mutant (PDB ID: 2OH9) and the Trp28 mutant (PDB ID: 2OH8) were produced using structural data from the protein data bank; the figure of the Trp14 mutant is based on the structural data of wild-type Mb (PDB ID: 1BZ6). The figures were produced with PyMOL (http://www.pymol. org/). The center-to-center distance between the heme group and the Trp residue for each mutant is shown.

the spectrum obtained with the probe pulse alone; this represents the anti-Stokes UVRR spectrum for each mutant at thermal equilibrium at room temperature. The band intensities in the probe-only spectra reflect the population in the vibrationally excited state at room temperature. Raman bands due to the Trp residues in the UVRR spectra were identified by comparison with UVRR spectra of aqueous amino acid solutions. We adopted the mode assignments made by Harada and co-workers.22 The UVRR bands at 760, 876, and 1012 cm−1 in the probe-only spectrum of the Trp68 mutant are due to the W18, W17, and W16 modes, respectively, of the Trp residue. Atomic displacements of the W16 and W18 modes are shown in Figure 2d. The band at 983 cm−1 marked by the asterisk arose from the sulfate ion added to the sample solutions as an internal standard of Raman intensity to correct for self-absorption. A pump pulse of 405 nm excites the heme into the electronically excited state and generates vibrationally excited states. To eliminate the spectral contribution of unphotoexcited molecules in the pump−probe spectra, difference spectra attributed to vibrationally excited populations generated by the pump pulse were calculated for the Mb mutants, as shown in Figure 2a−c. The Raman bands due to the sulfate ion were used as an intensity standard to calculate the difference spectra. At a −5 ps delay, no difference features were observed in the time-resolved spectra; this result corroborates the measured cross correlation width of 3.7 ps. In contrast, the spectra at positive delay times showed the positive anti-Stokes Raman bands of the Trp residue. The positive bands vanished within 50 ps in the difference spectra. Similar intensity changes were observed for the Trp28 mutant (Figure 2b). For the Trp14

shows the three-dimensional structure of sperm whale Mb (PDB ID: 1BZ6). Mb is a relatively small protein composed of 153 amino acids and a heme prosthetic group, shown as spacefilling spheres. Wild-type sperm whale Mb has two Trp residues, Trp7 and Trp14. First, we prepared mutants devoid of Trp residues by replacing Trp7 and Trp14 with Tyr and Phe, respectively.24 Then, we prepared three mutants with a single Trp at a specific position. These Mb mutants are shown in Figure 1b. One mutant has a Trp residue at position 68 and is thus located in the vicinity of the heme. The center-to-center distance between the heme and Trp68 is 6.8 Å (PDB ID: 2OH9). Another mutant has a Trp residue at position 28. The distance between the heme and Trp28 is 12.4 Å (PDB ID: 2OH8). In the third mutant, Trp7 was replaced with Tyr while Trp14 remained unaltered; the distance from the heme to Trp14 is 15.0 Å. The Trp residues in all three mutants are located in a similar direction from the heme, but their distances from the heme differ significantly. By comparing the data obtained using these mutants, we explored the distance dependence of energy flow from the heme to the Trps. Crystallographic data of these mutants25 showed that the threedimensional structures of the mutants are very close to that of wild-type Mb. 3270

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mutant (Figure 2c), no anti-Stokes UVRR band was evident in the difference spectra. The anti-Stokes band intensity changes due to change in the population of the vibrational excited state and/or change in the UVRR cross section of the mode. The UVRR cross section is affected by structure of Trp residues. For example, the environment around the residue and/or conformation of the side chain affect the cross section. If the changes in the antiStokes intensity are due to the change in the cross section, intensity changes of the Stokes UVRR bands in the timeresolved difference spectra relative to those of the probe-only spectra must be comparable to those of the anti-Stokes UVRR bands. We examined this possibility by measuring the timeresolved Stokes UVRR spectra of Mb (Supporting Information, Figure S1) and found that the intensity changes of the Stokes W16 and W18 bands were much smaller than those of the antiStokes bands. The contribution of the change in the cross section of all of the mutants can be neglected, consistent with the photochemical inertness of ferric Mb.18 Thus, the changes in the anti-Stokes intensity are attributed to changes in the vibrationally excited population. Figure 2a−c compares the time-resolved anti-Stokes UVRR spectra of the three mutants. The band intensities in the three panels were normalized based on the probe-only spectra of the Mb mutants, allowing the anti-Stokes intensities in the timeresolved difference spectra of the Mb mutants to be compared. The anti-Stokes intensities of the mutants reflect the amount of energy delivered to the Trp residues at different positions. The time-resolved spectra in Figure 2 show that the anti-Stokes intensities decreased as the heme-Trp distance increased. This can be explained by the classical thermal diffusion model showing that the excess energy becomes spatially less dense as the energy diffuses in the protein. Figure 3 depicts the temporal evolution of the anti-Stokes intensity of the W18 and W16 bands in the time-resolved

difference spectra relative to that in the probe-only spectrum. The changes in the anti-Stokes intensity bands were fitted by a convolution of the instrument response with an exponential rise and an exponential decay for the Trp68 and Trp29 mutants. The time constants of the rise of the W18 band were 3.0 ± 0.4 and 4.0 ± 0.6 ps for the Trp68 and Trp28 mutant, respectively. For the W16 band, time constants of 3.0 ± 0.7 and 4.9 ± 1.1 ps for the rise were obtained for the Trp68 and Trp28 mutant, respectively. Thus, the intensity rise for Trp28 is consistently slower than that for Trp68. This indicates that it takes longer for the excess energy to arrive at position 28 (Trp28, 12.4 Å) than at position 68 (Trp68, 6.8 Å). Accordingly, the observed distance dependence is qualitatively consistent with classical thermal diffusion. These data provide important insights into the mechanism of energy migration in proteins. We revealed that the energy transferred to the Trp residue is smaller and the buildup time of the excited population is longer as the heme-Trp distance increases. This is consistent with the prediction from the classical thermal transport model. Next, we compared these data and with qualitative data calculated based on the model. Li and Champion presented a classical two-boundary thermal transport model to simulate the thermal dynamics of transient cooling in chromophoric biomolecules.26 We calculated the temperature distribution in each protein based on the twoboundary heat transport model, as illustrated in the inset of Figure 4a (details of the calculations are given in the

Figure 4. (a) Temperature as a function of position and time calculated from the model using the parameters shown in Supporting Information. The inset represents the two-boundary classical heat transport model for a solvated hemeprotein with a radius of 20 Å containing an embedded heme with a radius of 4 Å. (b) Changes in the Boltzmann factor of the W18 mode at 8 ps for the three mutants calculated based on the model (solid line) as a function of the heme− Trp distance and the intensity of the anti-Stokes W18 band at 8 ps (closed circles). Figure 3. Temporal changes of the anti-Stokes W18 (a) and W16 (b) band intensities in the range of −5 to 50 ps upon excitation at 405 nm. Closed triangles, circles, and squares indicate the band intensity of the Trp68, Trp28, and Trp14 mutants, respectively, measured at each delay time relative to that in the probe-only spectrum. Solid lines show the best fits to a double-exponential function of the form I1[exp(−t/ τdecay) − exp(−t/τrise)] convoluted with the instrument response function. Broken lines are the Boltzmann factor based on the temperature calculated in the two-boundary classical heat transport model. (a) The time constants of the rise and decay of the Trp68 mutant were 3.0 ± 0.4 and 9.6 ± 1.0 ps, respectively. For the Trp28 mutant, time constants of 4.0 ± 0.6 and 19.2 ± 2.7 ps were obtained for the rise and decay, respectively. (b) The time constants of the rise and decay of the Trp68 mutant were 3.0 ± 0.7 and 9.2 ± 1.8 ps, respectively. For the Trp28 mutant, the time constants of 4.9 ± 1.1 and 14.9 ± 3.4 ps were obtained for the rise and decay, respectively.

Supporting Information). Figure 4a shows the calculated temperature as a function of time and position of the Trp residue. The temperature dependence of the anti-Stokes Raman intensity is expressed by the Boltzmann factor. On the basis of the calculated temperature, we calculated the Boltzmann factor for the W18 and W16 modes to determine the temporal evolution of the anti-Stokes intensities. The broken lines in Figure 3 represent temporal profiles of the Boltzmann factor based on the model. The model reproduced well the temporal behavior of the anti-Stokes intensities of the W16 and W18 bands for both the Trp68 and Trp28 mutants, whereas it reproduced poorly the temporal behavior for the Trp14 mutant. 3271

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Notes

Figure 4b compares the anti-Stokes Raman intensities at 8 ps and the Boltzmann factors calculated based on the twoboundary heat transport model. The model reproduced the observed anti-Stokes intensities for the Trp68 and Trp28 mutants. However, the observed intensity for the Trp14 mutant distinctly deviated from the calculated value. Accordingly, the classical heat transport model is not able to reproduce the entire present data set, suggesting that a more realistic model at the molecular level is necessary for describing energy flow in proteins. In fact, Leitner discussed that energy flow is intrinsically anisotropic due to the geometry of proteins.27 The present data experimentally corroborate this picture. Vibrational cooling of the heme in Mb was reported to occur in 1−6 ps.12,14−16,28 The time constants of the intensity rise of the Trp68 mutant are close to the cooling time constant, indicating that energy transfer from the heme to the residue at position 68 in Mb occurs very efficiently. A study by molecular dynamics simulations on Mb predicted that the residue 68 is seen to exhibit a large local energy diffusivity to the heme, that is, energy transport between the heme and this nearby residue,29 which is consistent with the present results. There is no covalent bond between the heme and the E helix where Trp68 is located. However, X-ray crystallographic data show that Trp68 in the V68W mutant has a nonbonded contact with the heme group.25 Recently, a molecular dynamics simulation study suggested that the contact mechanism is important in energy migration in proteins.30 Ye et al. suggested that the Fe− His93 covalent bond is not an efficient channel of the energy release from the locally hot heme.13 In contrast, an experiment of heme substitution demonstrated that van der Waals contacts of the heme with the protein/solvent matrix is important in cooling the hot heme.13 The energy transfer between the heme and Trp68 was fast, despite the lack of a direct covalent linkage between them. This experimental finding strongly suggests that the energy is transmitted not through the main chain of the protein but through atomic contacts. The present technique of energy mapping based on the antiStokes UVRR intensity of Trp residues fully utilizes two unique characteristics of Raman spectroscopy, site-specific observation by the resonance Raman effect and selective observation of the vibrationally excited population by anti-Stokes scattering. In this study, we further developed this technique by combining it with site-directed mutagenesis. This advancement allows the long-anticipated ability to map the energy flow in a protein with the spatial resolution of a single amino acid residue. Systematic application of our general methodology to proteins with different structural motifs may provide a greatly increased understanding of the energy flow in proteins.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Professor Paul M. Champion (Northeastern University) for his valuable suggestion of using the twoboundary heat transport model. We thank Professor Akinori Kidera (Yokohama City University) for stimulating discussions on the energy transfer mechanism. We are grateful to Professors Koichiro Ishimori and Takeshi Uchida (Hokkaido University) for kindly supplying the Mb plasmid. This work was supported by a Grant-in-Aid for Scientific Research in the Priority Area “Molecular Science for Supra Functional Systems” (No. 19056013) to Y.M. from The Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-inAid for Scientific Research on Innovative Areas “Soft Molecular Systems” (No. 25104006) to Y.M. from The Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Grant-in-Aid for Scientific Research (B) (No. 20350007) to Y.M. from the Japan Society for the Promotion of Science.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details (sample preparation, UVRR measurements, and time-resolved Stokes UVRR spectra) and details of calculations based on the two-boundary thermal transport model. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

(1) Owrutsky, J. C.; Raftery, D.; Hochstrasser, R. M. Vibrational Relaxation Dynamics in Solutions. Annu. Rev. Phys. Chem. 1994, 45, 519−555. (2) Elsaesser, T.; Kaiser, W. Vibrational and Vibronic Relaxation of Large Polyatomic Molecules in Liquids. Annu. Rev. Phys. Chem. 1991, 42, 83−107. (3) Seilmeier, A.; Kaiser, W. In Ultrashort Laser Pulses; Kaiser, W., Ed.; Springer: New York, 1993; p 279. (4) Zinth, W.; Kaiser, W. In Ultrashort Laser Pulses; Kaiser, W., Ed.; Springer: New York, 1993; p 235. (5) Proteins: Energy, Heat and Signal Flow; Leitner, D. M., Straub, J. E., Eds.; CRC Press: Boca Raton, FL, 2009. (6) Carslaw, H. S.; Jaeger, J. C. Conduction of Heat in Solids; Oxford University Press: Oxford, U.K., 1959. (7) Muller-Werkmeister, H. M.; Bredenbeck, J. A Donor−Acceptor Pair for the Real Time Study of Vibrational Energy Transfer in Proteins. Phys. Chem. Chem. Phys. 2014, 16, 3261−3266. (8) Helbing, J.; Devereux, M.; Nienhaus, K.; Nienhaus, G. U.; Hamm, P.; Meuwly, M. Temperature Dependence of the Heat Diffusivity of Proteins. J. Phys. Chem. A 2011, 116, 2620−2628. (9) Li, G.; Magana, D.; Dyer, R. B. Anisotropic Energy Flow and Allosteric Ligand Binding in Albumin. Nat. Commun. 2014, 5, 3100. (10) Champion, P. M.; Lange, R. On the Quantitation of Light Emission from Cytochrome c in the Low Quantum Yield Limit. J. Chem. Phys. 1980, 73, 5947−5957. (11) Henry, E. R.; Eaton, W. A.; Hochstrasser, R. M. Molecular Dynamics Simulations of Cooling in Laser-Excited Heme Proteins. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8982−8986. (12) Lim, M.; Jackson, T. A.; Anfinrud, P. A. Femtosecond Near-IR Absorbance Study of Photoexcited Myoglobin: Dynamics of Electronic and Thermal Relaxation. J. Phys. Chem. 1996, 100, 12043−12051. (13) Ye, X.; Demidov, A.; Rosca, F.; Wang, W.; Kumar, A.; Ionascu, D.; Zhu, L.; Barrick, D.; Wharton, D.; Champion, P. M. Investigations of Heme Protein Absorption Line Shapes, Vibrational Relaxation, and Resonance Raman Scattering on Ultrafast Time Scales. J. Phys. Chem. A 2003, 107, 8156−8165. (14) Franzen, S.; Bohn, B.; Poyart, C.; Martin, J. L. Evidence for SubPicosecond Heme Doming in Hemoglobin and Myoglobin: A TimeResolved Resonance Raman Comparison of Carbonmonoxy and Deoxy Species. Biochemistry 1995, 34, 1224−1237. (15) Mizutani, Y.; Kitagawa, T. Direct Observation of Cooling of Heme Upon Photodissociation of Carbonmonoxy Myoglobin. Science 1997, 278, 443−446.

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Corresponding Author

*E-mail: [email protected]. Phone: +81-6-68505776. Fax: +81-6-6850-5776. 3272

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(16) Mizutani, Y.; Kitagawa, T. Ultrafast Dynamics of Myoglobin Probed by Time-Resolved Resonance Raman Spectroscopy. Chem. Rec. 2001, 1, 258−275. (17) Lian, T.; Locke, B.; Kholodenko, Y.; Hochstrasser, R. M. Energy Flow from Solute to Solvent Probed by Femtosecond IR Spectroscopy: Malachite Green and Heme Protein Solutions. J. Phys. Chem. 1994, 98, 11648−11656. (18) Genberg, L.; Bao, Q.; Gracewski, S.; Miller, R. J. D. Picosecond Transient Thermal Phase Grating Spectroscopy: A New Approach to the Study of Vibrational Energy Relaxation Processes in Proteins. Chem. Phys. 1989, 131, 81−97. (19) Genberg, L.; Heisel, F.; McLendon, G.; Miller, R. J. D. Vibrational Energy Relaxation Processes in Heme Proteins: Model Systems of Vibrational Energy Dispersion in Disordered Systems. J. Phys. Chem. 1987, 91, 5521−5524. (20) Okamoto, H.; Nakabayashi, T.; Tasumi, M. Analysis of AntiStokes Resonance Raman Excitation Profiles as a Method for Studying Vibrationally Excited Molecules. J. Phys. Chem. A 1997, 101, 3488− 3493. (21) Asher, S. A. Uv Resonance Raman Studies of Molecular Structure and Dynamics: Applications in Physical and Biophysical Chemistry. Annu. Rev. Phys. Chem. 1988, 39, 537−588. (22) Harada, I.; Takeuchi, H. Raman and Ultraviolet Resonance Raman Spectra of Proteins and Related Compounds. In Spectroscopy of Biological Systems; Clark, R. J. H., Hester, R. E., Eds.; John Wiley & Sons: Chichester, U.K., 1986; pp 113−175. (23) Fujii, N.; Mizuno, M.; Mizutani, Y. Direct Observation of Vibrational Energy Flow in Cytochrome c. J. Phys. Chem. B 2011, 115, 13057−13064. (24) Sirangelo, I.; Tavassi, S.; Martelli, P. L.; Casadio, R.; Irace, G. The Effect of Tryptophanyl Substitution on Folding and Structure of Myoglobin. Eur. J. Biochem. 2000, 267, 3937−3945. (25) Olson, J. S.; Soman, J.; Phillips, G. N. Ligand Pathways in Myoglobin: A Review of Trp Cavity Mutations. IUBMB Life 2007, 59, 552−562. (26) Li, P.; Champion, P. M. Investigations of the Thermal Response of Laser-Excited Biomolecules. Biophys. J. 1994, 66, 430−436. (27) Leitner, D. M. Energy Flow in Proteins. Annu. Rev. Phys. Chem. 2008, 59, 233−259. (28) Li, P.; Sage, J. T.; Champion, P. M. Probing Picosecond Processes with Nanosecond Lasers: Electronic and Vibrational Relaxation Dynamics of Heme Proteins. J. Chem. Phys. 1992, 97, 3214−3227. (29) Leitner, D. M. Frequency-Resolved Communication Maps for Proteins and Other Nanoscale Materials. J. Chem. Phys. 2009, 130, 195101. (30) Martínez, L.; Figueira, A. C. M.; Webb, P.; Polikarpov, I.; Skaf, M. S. Mapping the Intramolecular Vibrational Energy Flow in Proteins Reveals Functionally Important Residues. J. Phys. Chem. Lett. 2011, 2, 2073−2078.

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