Importance of Atomic Contacts in Vibrational Energy Flow in Proteins

Subscriber access provided by UNIV LAVAL

Letter

Importance of Atomic Contacts in Vibrational Energy Flow in Proteins Masato Kondoh, Misao Mizuno, and Yasuhisa Mizutani J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.6b00785 • Publication Date (Web): 10 May 2016 Downloaded from http://pubs.acs.org on May 10, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry Letters is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry Letters

Importance of Atomic Contacts in Vibrational Energy Flow in Proteins Masato Kondoh†, Misao Mizuno, and Yasuhisa Mizutani* Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan.

AUTHOR INFORMATION Corresponding Author *Yasuhisa Mizutani, Tel.: +81-6-6850-5776. Fax: +81-6-6850-5776. E-mail: [email protected]. Present Addresses †

Department of Chemistry, Graduate School of Pure and Applied Sciences, University of

Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan.

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 22

ABSTRACT Vibrational energy flow in proteins was studied by monitoring the time-resolved anti-Stokes ultraviolet resonance Raman scattering of three myoglobin mutants in which a Trp residue substitutes a different amino acid residue near heme. The anti-Stokes Raman intensities of the Trp residue in the three mutants increased with similar rates after depositing excess vibrational energy at heme, despite the difference in distance between heme and each substituted Trp residue along the main chain of the protein. This indicates that vibrational energy is not transferred through the main chain of the protein but rather through atomic contacts between heme and the Trp residue. Distinct differences were observed in the amplitude of the band intensity change between the Trp residues at different positions, and the amplitude of the band intensity change exhibits a correlation with the extent of exposure of the Trp residue to solvent water. This correlation indicates that atomic contacts between an amino acid residue and solvent water play an important role in vibrational energy flow in a protein.

TOC GRAPHICS

KEYWORDS Resonance Raman spectroscopy, anti-Stokes scattering, vibrational energy relaxation, myoglobin, thermal diffusion


Page 3 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Energy exchange between many degrees of vibrational freedom accompanies a wide range of chemical dynamics that closely affect the functions of proteins. Consequently, understanding the process of energy exchange in a protein is crucial for obtaining profound insights into protein dynamics and function1-2 and requires elucidating the microscopic mechanisms of energy flow in proteins. Many experimental3-6 and theoretical7-10 studies to date have revealed the energy flow in proteins, particularly in hemeproteins. Hemeproteins are an ideal system to study energy flow because the heme group exhibits ultrafast internal conversion (within 100 fs),11 and thus excess vibrational energy can be deposited locally at the heme site immediately following photoexcitation. Subsequent energy relaxation processes can be studied by time-resolved spectroscopy. The time scales of the energy relaxation of heme3-4 and energy dissipation into water5-6 have been characterized. It was shown that energy transfer from a vibrationally excited heme to the surrounding protein moiety occurs with time constants of a few picoseconds3-4 and that the energy is dissipated from the protein into the solvent in less than 20 ps.5-6 However, much less is understood about energy flow within the protein moiety, and characterization of this flow requires a technique for site-selective observation in a protein molecule. Such a technique has been lacking due to experimental difficulties. We have adopted anti-Stokes ultraviolet resonance Raman (UVRR) spectroscopy12-13 to address this need. Due to the resonance Raman effect, UVRR spectroscopy selectively probes Raman bands of aromatic amino acid residues14 and therefore allows site-selective detection of energy at the level of a single amino acid residue in a large protein molecule. In addition, time-resolved anti-Stokes Raman spectroscopy is selective for vibrationally excited populations and can be suitable for studying vibrational energy flow. In our first report, we succeeded in the direct observation of vibrational energy flow in cytochrome c, and



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 22

demonstrated that time-resolved anti-Stokes UVRR Raman spectroscopy is powerful for monitoring vibrational energy flow in a protein.12 We further developed this technique by combining it with site-directed mutagenesis. By changing the position of the probe aromatic amino acid residue relative to heme by amino acid substitution, we could examine the distance dependence of intraprotein energy flow in myoglobin (Mb).13 In this study, we investigated vibrational energy flow in the surroundings of the heme group in the ferric form of Mb using picosecond time-resolved anti-Stokes UVRR spectroscopy. Figure 1A shows the three-dimensional structure of sperm whale Mb. Mb is a relatively small globular protein containing a heme group, which is shown as space filling orange spheres in Figure 1. We prepared three Mb mutants, F43W, V68W, and L89W. The Trp residue at position 43, 68, or 89 in each mutant locates in the vicinity of heme, as represented by space-filling purple spheres in Figure 1B. The distance from heme to the Trp43, Trp68 or Trp89 residue in each mutant is 7.4, 6.4, and 6.4 Å, respectively. These values were obtained from the coordinates of the iron atom of the heme group and the center of mass of each Trp residue, including the main chain atoms. Wild-type sperm whale Mb has two Trp residues, Trp7 and Trp14, which are also present in the Mb mutants. The distance from heme to Trp7 and Trp14 is 22.0 and 16.6 Å, respectively. These distances are significantly longer than those from heme to the introduced Trp residues. Consistent with this, the previous study showed that the intensities of the time-resolved anti-Stokes UVRR band of Trp14 in Mb is negligibly weak as compared with that of Trp68.13 Accordingly, the observed anti-Stokes intensities for the F43W, V68W, and L89W mutants are attributed to the probe residues, namely, Trp43, Trp68, and Trp89, respectively.


Page 5 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. X-ray crystal structure of sperm whale Mb. (A) Wild-type Mb (PDB ID: 1BZ6). The protein is shown in gray ribbon representation with a superimposed pale gray surface. Heme is represented as orange space-filling spheres. The positions of the two intrinsic Trp residues are indicated by representing their α-carbons as purple space-filling spheres. The α-carbons of the residues at positions 43, 68, and 89 are shown as space-filling spheres colored blue, red, and green, respectively. (B) Structures around heme in the Mb mutants [PDB ID: 2EY2 (F43W), 2OH9 (V68W), and 1CH3 (L89W)]. Heme and probe Trp residues are represented as spacefilling spheres in orange and purple, respectively. The distance between heme and the Trp residue for each mutant is shown.

Figure 2 shows time-resolved anti-Stokes UVRR spectra of F43W, V68W, and L89W after photoexcitation of heme. The top trace in each panel is a probe-only spectrum representing a steady-state anti-Stokes UVRR spectrum for each mutant. The probe-only spectra for all the mutants contain anti-Stokes UVRR bands at 760, 876, and 1010 cm−1, which are assigned to the W18, W17, and W16 modes of the Trp residues, respectively, based on the mode assignments reported previously.14 The band at 983 cm−1, marked by an asterisk, is due to sulfate ion added to



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 22

Figure 2. Time-resolved anti-Stokes UVRR spectra of the Mb mutants probed at 230 nm after photoexcitation of heme at 405 nm. (A) F43W, (B) V68W, and (C) L89W. The top traces are the probe-only spectra representing the anti-Stokes UVRR spectra of the steady states for the Mb mutants. The asterisks represent the band due to sulfate ion at 983 cm−1. The other traces are time-resolved difference spectra, obtained by subtracting the probe-only spectrum from the pump−probe spectrum at each delay time. The accumulation time for obtaining each spectrum was 82 minutes.

the sample solutions as an internal standard of Raman intensity. The band intensities in the probeonly spectra arise from the thermal population in vibrationally excited states at room temperature. The other traces in each panel in Figure 2 are time-resolved difference spectra for each mutant and


Page 7 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were obtained by subtracting the probe-only spectrum from the pump-probe spectrum at each delay time after intensity correction for the self-absorption effect and drift in laser power. Positive antiStokes UVRR bands for the W18 and W16 modes appeared in the difference spectra at positive delay times. These positive bands were observed in the time-resolved difference spectra for all the Mb mutants, but were negligibly weak in spectra for wild-type Mb (Figure S1). The appearance of the positive bands indicates that the anti-Stokes UVRR band intensities of Trp43, Trp68, and Trp89 increased after photoexcitation of heme. Anti-Stokes UVRR band intensities can reflect populations of the vibrational excited state and/or UVRR cross sections of the corresponding modes. If the observed increase in the anti-Stokes Trp band intensities arises from changes in the cross sections upon photoexcitation, the corresponding UVRR band intensities on the Stokes side must exhibit comparable changes. To estimate the contribution from the changes in the cross sections, we measured time-resolved Stokes UVRR spectra of the three Mb mutants. For all the mutants, the band intensity changes relative to the intensity in the probe-only spectra on the Stokes side were much smaller than those on the antiStokes side (Figure S2). Therefore, the observed increase in the anti-Stokes UVRR intensity reflects the increase in the vibrationally excited populations of the Trp residue resulting from energy transfer from the photoexcited heme. The differences in the temporal intensity changes in the anti-Stokes UVRR bands between the three positions of the Trp residue provide space-resolved information on the vibrational energy flow in Mb. We compared the time-resolved anti-Stokes UVRR intensities of the Trp43, Trp 68, and Trp89 residues in the Mb mutants. In Figure 2, the band intensities in the three panels were normalized using the W18 band intensity of the corresponding probe-only spectrum. We plotted the anti-Stokes W18 band intensities of the Trp43, Trp 68, and Trp89 residues in the time-resolved



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 22

difference spectra of each mutant against the delay time, as shown in Figure 3, to unambiguously compare their temporal behaviors. The vertical axis represents the photoinduced change in the W18 band intensity relative to that in the probe-only spectrum. For all the mutants, the band intensity increased up to 6-8 ps, and then decayed to almost zero within 50 ps. The intensity rise and decay correspond to the increase and decrease, respectively, of the vibrationally excited populations of the probe Trp residue. The population increase is caused by the vibrational energy transfer from heme to the Trp residue, whereas the decrease arises from the energy transfer from the Trp residue to its surroundings. The temporal changes at the three Trp positions were similar, implying that the vibrational energy is transferred from heme to the three positions (positions 43, 68, and 89) with similar rates. Trp89 is located in the F-helix, where His93 forms a covalent linkage with heme. Trp68 is in the center of the E-helix, and Trp43 locates in the CD corner. Thus, the distances between heme and Trp43, Trp68, and Trp89 along the main chain of the protein are completely different. If the excess energy is transferred to the Trp residues through the main chain, the build-up times of the vibrationally excited population would be completely different between the three positions, but that is not the case. Non-bonded contacts of Trp43, Trp68, and Trp89 with heme are evident in Xray crystallographic data (see Figure 1B).15-16 The similar rates of energy transfer from heme to the Trp residues suggest that the energy is transferred not through the heme-His93 covalent linkage and the protein main chain, but through atomic contacts between the heme group and the residues. The anti-Stokes UVRR intensities of the Trp band in the time-resolved difference spectra reflect the amount of vibrational energy deposited transiently to the Trp residues. The amplitudes of the band intensity changes in Figure 3 show distinct difference among the three Trp positions: the


Page 9 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


amplitude is largest for Trp68, then Trp43, followed by Trp89. The observed difference implies that the energy flow at the three positions is different. There are two possibilities for the origin of this difference: (i) the rate of energy flow into the Trp residue is different, or (ii) the rate of energy flow out of the Trp residue is different. To consider the former possibility, we first estimated the extent of contacts between heme and each probe Trp residue. As the extent of contacts become larger, the rate of energy flow into the Trp residue likely increases. The larger inflow rate increases the amount of energy transferred to the Trp residue, resulting in an increase in the anti-Stokes Raman band intensity of the Trp residue. The extent of heme-Trp contacts was estimated as follows. We calculated the interatomic distances for all the atomic pairs between an atom in the heme group and an atom of the probe Trp residue, then counted the number of pairs whose interatomic distance is smaller than a certain threshold distance (Figure S3). We consider the number of pairs in the range from 3.4 to 4.4 Å as the threshold value, based on the van der Waals radius of a carbon atom (1.7 Å). Then, we compared the number of pairs between heme and each probe Trp residue. The number of contacts was estimated to be nearly the same for Trp43 and Trp89, but was smaller for Trp68. Distances between the closest atoms of heme and the Trp residue found in the PDB data are 3.28 Å (proximal carbon atom of the vinyl group−indole nitrogen atom), 3.66 Å (terminal carbon atom of the vinyl group−indole ξ3 carbon atom), and 3.11 Å (pyrrole α carbon atom−indole nitrogen atom) for Trp43, Trp68, and Trp89, respectively. All of them are nonpolar contacts. If the mechanism is primarily driven by possibility (i), we expected that the anti-Stokes Raman band intensity of Trp43 and Trp89 would be larger than that of Trp68, but this prediction is clearly not supported by the experimental data shown in Figure 3. This indicates that possibility (i) is not the dominant mechanism and cannot explain the observed positional dependence of the energy flow.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 22

Figure 3. Temporal profiles of the photoinduced anti-Stokes W18 band intensity of the Trp43, Trp 68, and Trp89 residues in the range from −5 to 50 ps following photoexcitation. Solid lines show the best fits to a double-exponential function, exp

/

exp

, convoluted using the instrument response function.

Next, we investigated possibility (ii) based on the molecular environment around each Trp residue. The X-ray crystallographic structures of the mutants show that Trp68 and Trp43 are buried inside the protein, whereas Trp89 locates near the protein surface and faces the solvent.15-16 Based on these structural data, we calculated the values of the solvent accessible surface area (SASA) of each probe Trp residue, including the main chain atoms. The SASA values were 2.9, 5.1, and 12.7 Å2 for Trp68, Trp43, and Trp89, respectively. The amplitudes of the anti-Stokes band intensity changes as shown in Figure 3 are inversely correlated with the SASA values: the larger the SASA value, the smaller the amplitude. This correlation can be reasonably understood by the assumption that solvent water accepts the energy of the Trp residues. It is known that solvent water is an


Page 11 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


efficient acceptor of vibrational energy.17-19 The microscopic mechanism of cooling was discussed for solvent water in detail.20 The rate of energy dissipation from the Trp residues is expected to increase as the SASA value increases, and the larger dissipation rate would reduce the amount of energy accumulated transiently in the Trp residue. Consequently, the amplitude of the anti-Stokes band intensity change of the Trp band decreases when the Trp residue has a larger SASA value. The observed intensity difference between the three positions shows that the mechanism associated with possibility (ii) is dominant. The difference in the temporal profiles of the anti-Stokes UVRR intensity changes among the Trp residues at the three positions was qualitatively explained by the following picture of vibrational energy flow. Vibrational energy flows from heme to the probe Trp residue through atomic contacts. The energy in the Trp residue subsequently flows to its surroundings at different rates, depending on the solvent accessibility of the Trp residue at that position. To examine this picture, we analyzed the temporal profiles of Trp43, Trp68, and Trp89 simultaneously using a biexponential function,

/

exp

exp

, convoluted with

instrument response. This function is obtained from scheme 1. Q0 is the amount of energy initially deposited into heme, kHd is the energy relaxation rate from heme, kTr is the energy flow rate into the probe Trp residue from heme, and kTd is the energy relaxation rate from the Trp residue.

Scheme 1: Model to analyze the temporal profile of anti-Stokes Raman intensities



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 22

In the fitting, only the parameter kTd was allowed to adopt different values among the three temporal profiles and the other parameters were forced to share common values. The temporal profiles were reproduced within the error range with the parameter kHd-1 = 4.69 ± 1.0 ps. Vibrational cooling of heme in Mb was reported to occur in 1-6 ps.3-4 The obtained time constant kHd-1 was similar to the reported time constant of the vibrational cooling of heme. The kTd-1 values were obtained to be 13.5 ± 3.0, 9.0 ± 1.8 and 4.70 ± 0.8 ps for the Trp68, Trp43, and Trp89 residues, respectively. We confirmed that the difference in the amplitude of the band intensity change observed for the three Trp residues around heme is explained by the difference in the energy dissipation rate from each Trp residue. The energy dissipation rate kTd showed a larger value as the SASA value at the corresponding position becomes larger (Figure S4). The correlation between the dissipation rate and SASA supports our proposal that the solvent water is an efficient acceptor of vibrational energy. Furthermore, temporal profiles for the anti-Stokes W16 band intensity changes of Trp43, Trp68, and Trp89 were similar to those observed in the W18 band (Figure S5). We therefore demonstrated that our minimalist model of vibrational energy flow can reasonably explain the temporal changes of vibrationally excited populations in proteins. In summary, we studied vibrational energy flow in Mb by monitoring the time-resolved antiStokes UVRR scattering of a specific Trp residue near heme and used the Trp as a probe. The present study established the importance of atomic contacts in vibrational energy flow in proteins. It was clearly shown that the dominant channel for energy flow from the heme group to the protein moiety is not through the covalent linkage of heme-His93 and the protein main chain, but rather through atomic contacts between heme and the residues. We propose that atomic contact between an amino acid residue and solvent water is an important channel for energy dissipation in proteins. Systematic application of our methodology to proteins with a probe residue at different positions


Page 13 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


will provide a more detailed map and, therefore, increase our understanding of vibrational energy flow in proteins.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 22

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:. Materials and methods (sample preparation, UVRR measurements, and SASA calculations) and supporting results (time-resolved anti-Stokes UVRR spectra of WT Mb, time-resolved Stokes UVRR spectra, evaluation of the heme-Trp contact, relationship between the energy dissipation rate and the SASA value, and temporal profiles of the anti-Stokes W16 band intensity change).

AUTHOR INFORMATION Corresponding Author *

[email protected]

Present Addresses †

Department of Chemistry, Graduate School of Pure and Applied Sciences, University of Tsukuba,

1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We would like to thank Professor Akio Kitao and Dr. Kazuhiro Takemura at the University of Tokyo for providing the SASA calculation data for the Mb mutants. We are grateful for financial support from the Ministry of Education, Science, Culture, and Sports (MEXT) of Japan through a Grant-in-Aid for Scientific Research on Innovative Areas “Soft Molecular Systems” (No.


Page 15 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25104006) to Y.M., and from the Japan Society for the Promotion of Science through a Grant-inAid for Scientific Research (B) (No. 26288008) to Y.M.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 22

References 1. Leitner, D. M. Heat Transport in Proteins. In Proteins: Energy, Heat and Signal Flow, Leitner, D. M.; Straub, J. E., Eds. CRC Press: Boca Raton, Florida, 2010; pp 247-269. 2. Xhang, Y.; Straub, J. E. Directed Energy Funneling in Proteins: From Structure to Function. In Proteins: Energy, Heat and Signal Flow, Leitner, D. M.; Straub, J. E., Eds. CRC Press: Boca Raton, Florida, 2010; pp 199-228. 3. Mizutani, Y.; Kitagawa, T. Direct Observation of Cooling of Heme Upon Photodissociation of Carbonmonoxy Myoglobin. Science 1997, 278, 443-446. 4. 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. 5. 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. 6. 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. 7. Henry, E. R.; Eaton, W. A.; Hochstrasser, R. M. Molecular Dynamics Simulations of Cooling in Laser-Excited Heme Proteins. Proc. Natl. Acad. Sci. USA 1986, 83, 8982-8986. 8. Moritsugu, K.; Miyashita, O.; Kidera, A. Vibrational Energy Transfer in a Protein Molecule. Phys. Rev. Lett. 2000, 85, 3970-3973. 9. Leitner, D. M. Frequency-Resolved Communication Maps for Proteins and Other Nanoscale Materials. J., Chem. Phys. 2009, 130, 195101. 10. Buchenberg, S.; Leitner, D. M.; Stock, G. Scaling Rules for Vibrational Energy Transport in Globular Proteins. Journal of Physical Chemistry Letters 2016, 7, 25-30. 11. 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. 12. Fujii, N.; Mizuno, M.; Mizutani, Y. Direct Observation of Vibrational Energy Flow in Cytochrome c. J. Phys. Chem. B 2011, 115, 13057-13064. 13. Fujii, N.; Mizuno, M.; Ishikawa, H.; Mizutani, Y. Observing Vibrational Energy Flow in a Protein with the Spatial Resolution of a Single Amino Acid Residue. J. Phys. Chem. Lett. 2014, 5, 3269-3273. 14. 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. 15. Olson, J. S.; Soman, J.; Phillips, G. N. Ligand Pathways in Myoglobin: A Review of Trp Cavity Mutations. IUBMB Life 2007, 59, 552-562. 16. Watanabe, Y.; Nakajima, H.; Ueno, T. Reactivities of Oxo and Peroxo Intermediates Studied by Hemoprotein Mutants. Acc. Chem. Res. 2007, 40, 554-562. 17. Gao, Y.; Koyama, M.; El-Mashtoly, S. F.; Hayashi, T.; Harada, K.; Mizutani, Y.; Kitagawa, T. Time-Resolved Raman Evidence for Energy ‘Funneling’ through Propionate Side Chains in Heme ‘Cooling’ Upon Photolysis of Carbonmonoxy Myoglobin. Chem. Phys. Lett. 2006, 429, 239-243. 18. Koyama, M.; Neya, S.; Mizutani, Y. Role of Heme Propionates of Myoglobin in Vibrational Energy Relaxation. Chem. Phys. Lett. 2006, 430, 404-408.


Page 17 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


19. Sagnella, D. E.; Straub, J. E. Directed Energy “Funneling" Mechanism for Heme Cooling Following Ligand Photolysis or Direct Excitation in Solvated Carbonmonoxy Myoglobin. J. Phys. Chem. B 2001, 105, 7057-7063. 20. Park, S.-M.; Nguyen, P. H.; Stock, G. Molecular Dynamics Simulation of Cooling: Heat Transfer from a Photoexcited Peptide to the Solvent. J., Chem. Phys. 2009, 131, 184503.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. X-ray crystal structure of sperm whale Mb. (A) Wild-type Mb (PDB ID: 1BZ6). The protein is shown in gray ribbon representation with a superimposed pale gray surface. Heme is represented as orange spacefilling spheres. The positions of the two intrinsic Trp residues are indicated by representing their α-carbons as purple space-filling spheres. The α-carbons of the residues at positions 43, 68, and 89 are shown as space-filling spheres colored blue, red, and green, respectively. (B) Structures around heme in the Mb mutants [PDB ID: 2EY2 (F43W), 2OH9 (V68W), and 1CH3 (L89W)]. Heme and probe Trp residues are represented as space-filling spheres in orange and purple, respectively. The distance between heme and the Trp residue for each mutant is shown. 170x68mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 18 of 22

Page 19 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Time-resolved anti-Stokes UVRR spectra of the Mb mutants probed at 230 nm after photoexcitation of heme at 405 nm. (A) F43W, (B) V68W, and (C) L89W. The top traces are the probe-only spectra representing the anti-Stokes UVRR spectra of the steady states for the Mb mutants. The asterisks represent the band due to sulfate ion at 983 cm−1. The other traces are time-resolved difference spectra, obtained by subtracting the probe-only spectrum from the pump−probe spectrum at each delay time. The accumulation time for obtaining each spectrum was 82 minutes. 150x135mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Temporal profiles of the photoinduced anti-Stokes W18 band intensity of the Trp43, Trp 68, and Trp89 residues in the range from −5 to 50 ps following photoexcitation. Solid lines show the best fits to a double-exponential function, {k_Tr Q_0/(k_Hd-k_Td)}{exp (-k_Td t)-exp(-k_Hd t)}, convoluted using the instrument response function. 97x65mm (300 x 300 DPI)


Page 20 of 22

Page 21 of 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1: Model to analyze the temporal profile of anti-Stokes Raman intensities 41x10mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TOC graphic 60x60mm (300 x 300 DPI)


Page 22 of 22

Importance of Atomic Contacts in Vibrational Energy Flow in Proteins

Recommend Documents