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The Molecular Origin of Ultrafast Water-Protein Coupled Interactions Yangzhong Qin, Menghui Jia, Jin Yang, Dihao Wang, Lijuan Wang, Jianhua Xu, and Dongping Zhong J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.6b01954 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 4, 2016

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The Journal of Physical Chemistry Letters

J. Phys. Chem. Letters ---Article (2016)

The Molecular Origin of Ultrafast Water-Protein Coupled Interactions

Yangzhong Qin,1,2,¶ Menghui Jia,1,¶ Jin Yang,2 Dihao Wang,2 Lijuan Wang,2 Jianhua Xu1,* and Dongping Zhong2,*

1State

Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai

200062, China.

2

Department of Physics, Department of Chemistry and Biochemistry, and

Programs of Biophysics, Chemical Physics and Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA

*Corresponding

author. E-mail: [email protected] or [email protected].

ACS Paragon Plus Environment


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Abstract: The fluctuations of hydration water and the protein are coupled together at the protein surface and often such water-protein dynamic interactions are controlled presumably by hydration water motions. However, direct evidence is scarce and it requires measuring the dynamics of hydration water and protein sidechain simultaneously. Here, we use a unique protein with a single tryptophan to directly probe interfacial water and related sidechain relaxations with temperature dependence. With systematic mutations to change local chemical identity and structural flexibility, we found that the sidechain relaxations are always slower than hydration water motions and the two dynamic processes are linearly correlated with the same energy barriers, indicating the same origin of both relaxations. The charge mutations change the rates of hydration water relaxations but not the relaxation barriers. These results convincingly show that the water-protein relaxations are strongly coupled and the hydration water molecules govern such fluctuations on the picosecond timescales.

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Protein surface hydration is essential to its structural stability for a unique 3D architecture and to its dynamical fluctuations for the biological function.1-3 Hydration dynamics and protein fluctuations have been extensively studied by a series of powerful experimental techniques of NMR,4,5 neutron scattering,6,7 and optical spectroscopy3,8-11 and by computational molecular dynamics (MD) simulations.12-14 Most work separately studied either hydration water dynamics or local protein fluctuations. Direct measurement of both water and protein motions is difficult and challenging, especially due to the ultrafast nature of hydration water motions15-17 under physiological temperature. Significantly, a series of recent studies indicated ultrafast collective relaxations of coupled water-protein interactions at their interface on the picosecond timescales.18-22 Frauenfelder et al.23,24 proposed a slaving model of water-driven protein fluctuations, based on extensive lowtemperature studies of ligand-myoglobin interactions, to explain the observed various kinetics. If one of the interacting partners, water or protein, fluctuates with a significantly larger amplitude or relaxes on a faster time scale, that one could be the determinant in the subsequent interactions. Thus, the simultaneous examination of both hydration water and local protein fluctuations is critical to unraveling their ultimately coupled relationship. We have recently studied the hyperthermal protein of Dpo4 polymerase and simultaneously measured the hydration dynamics and related protein sidechain motions with systematic temperature changes using a tryptophan scan around the protein surfaces. Strong correlations of the relaxations between hydration water and local proteins with temperature changes were observed, strongly suggesting the same origin of both fluctuations.25 This observation is significant and the results may resolve the long-standing issue of hydration water-protein coupled motions. However, many urgent questions still

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remain: whether such correlations exclusively exist only for a hyperthermal protein or are robust enough to all proteins; what determines the relaxation barriers and how the barrier changes for different proteins; and what will happen to such correlations with modification of local environments from highly charged to completely hydrophobic. These questions are critical and urgent to directly addressing generality of such coupled fluctuations and revealing the nature of the driving force behind the coupled motions. In this work, we use Staphylococcal nuclease (SNase) as a model system (Figure 1).26 SNase is an ideal protein with a single tryptophan (W140) residue that is partially buried in the protein with a fluorescence emission peak at 332 nm. Extensive studies have shown that such a partially buried tryptophan reports only two relaxation time scales of a few to tens of picoseconds,19,22 probing only the inner-layer interfacial water molecules in the hydration shell, and cannot detect the outer-layer mobile water, making data analyses much easier. With systematic mutations of the three neighboring charged residues around W140 using an alanine scan, we have previously shown the dominant relaxation on the picosecond timescales from interfacial hydration water molecules.19,22 Here, we will use four proteins of wild type, single mutation of K110A, double mutation of K110AE129A and triple mutation of K110AE129AK133A (Fig. 1) by an alanine scan for the fixed probe (W140) site and simultaneously measure the relaxations of hydration water and local tryptophan with systematic temperature changes. Unlike our previous studies on Dpo4 to scan the probe around the protein surface, here we fix the probe position and systematically change its neighboring environment. With mutations around one specific probe site, we may alter the flexibility of local protein structures and hydration water networks. By careful examination of those changes of the dynamics with temperature, we

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will evaluate the various correlations of their relaxations at the same site with modification of local chemical and structural properties to reveal their ultimate relationship. Figure 2A shows five typical femtosecond fluorescence transients among more than ten gated ones from the blue to red side emission in the range of 310 to 370 nm for WT at 1 °C and 40 °C within a time window of 3.0 ns. All transients exhibit clear solvation signature with ultrafast decays at the blue side and rises at the red side. Two distinct decays in a few (3.7-6.1 ps at 1 °C and 1.9-3.0 ps at 40 °C) and tens-to-a hundred (104-204 ps at 1 °C and 53-120 ps at 40 °C) of picoseconds were observed for the WT and the similar relaxation patterns were also observed for the other three mutant proteins. From 1 °C to 40 °C, the dynamics become faster by about a factor of 2 (Fig. 2A) for four proteins. As expected for a partially buried probe, we did not observe any ultrafast decay component in hundreds of femtoseconds from the outer-layer mobile water molecules. Here, we detected only the inner-layer interfacial water relaxations near the water-protein interface. Using the methodology we developed for tryptophan (also see Supporting Information),9,27 we constructed the solvation correlation functions of WT and three mutants at various temperatures. Since the solvation only contains two relaxation dynamics on the picosecond timescales, the data analyses are straightforward. We obtained in total 18 correlation functions (Table S1). Fig. 2B shows the derived results for the WT at five different temperatures and Fig. 2C gives the results for the four proteins at 1 °C. Clearly, these data exhibit the solvation dynamics on two distinct solvation timescales of τ2S and τ3S (we usually refer τ1S to ultrafast relaxation on the femtosecond time scale). For all four proteins, the hydration dynamics obviously slow down with temperature decrease, for example, from 2.8 to 5.1 ps and from 68 to 151 ps for the wild type from 40 °C to 1 °C,

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respectively (Fig. 2B). The similar trends were observed for all other three mutants (Table S1). Consistent to the previous studies,22 the hydration dynamics become faster with more alanine mutations that form a more hydrophobic local environment, from 5.1 to 3.7 ps and 151 to 82 ps from WT to KEK at 1 °C, respectively (Fig. 2C). The faster relaxation around a hydrophobic environment than that in a densely charged location is also consistent with our previous observation in apomyoglobin.18 Similarly, we measured the anisotropy dynamics of the tryptophan sidechain of the four proteins for different temperatures. Fig. 3A shows a typical result for WT at 1 °C and Fig. 3B gives the derived anisotropy dynamics r(t) at 1 °C and 40 °C. Fig. 3C also shows the results of r(t) for two mutants of K and KEK at 1 °C. All anisotropy dynamics have to be fitted with four decay components. The initial ultrafast component (τIC) in less than 100 fs (after deconvolution from the instrument response) is due to the internal conversion from 1Lb

to 1La of tryptophan through conical intersection.28 Two intermediate times around ten

picoseconds (τ2W) and hundreds of picoseconds (τ3W) have to be fit in the anisotropy dynamics (see Fig. S1) and their corresponding amplitudes are relatively small, representing the two local motions.29 The last one is the entire protein tumbling motion in nanoseconds (τT). With temperature decrease, the sidechain relaxation clearly becomes slower (Fig. 3B). At the same temperature, the four proteins show noticeable different relaxations (Fig. 3C). More significantly, the anisotropy dynamics also show two distinct timescales of local relaxations on the picosecond timescales, a similar pattern to the observed hydration dynamics above (Table S1). Similar to all proteins we studied before,9,15,22,25 the sidechain relaxations (τiW, i=2,3) are always slower than the corresponding hydration water motions (τiS),25 i.e., τiW≥τiS; also see Table S1. The

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fundamental question is whether the two relaxations from hydration water and the local protein sidechain are intrinsically correlated and if any, what is their ultimate relationship. Fig. 4 shows the plots of the pairs of two relaxations (τ2W, τ2S) in Fig. 4B and (τ3W, τ3S) in Fig. 4C with temperature dependence. On the top (Fig. 4A), we also show local snapshots of four proteins at 2 ns from a 20-ns MD simulation with two inner layers of about 60-70 water molecules within a distance of 10 Å to the indole-ring center of tryptophan. More than 90% of those water molecules are within 7 Å to the protein surface. Astonishingly, all eight plots in Figs. 4b and 4c are a straight line going through the original point (0,0) and follow a relationship of τiW=mτiS (m≥1). Thus, these relaxations must be from one origin. This observation is significant and the results clearly show that the relaxations of the local sidechain are intrinsically related to hydration water motions. Since all the sidechain motions are slower than hydration water relaxations and also the corresponding wobbling angles are very small, assuming a wobbling motion, it reasonably concludes that hydration water relaxations drive sidechain motions. Furthermore, because the sidechain is partially buried inside the protein, the hydration water layers must collectively modulate the protein surface fluctuations, resulting in the partially buried sidechain to follow the interfacial water relaxations. Assuming a simple Arrhenius rate process, k=Aexp(-ΔE/RT), we plotted all relaxation rates relative to temperature and Fig. 5 shows all 16 linear fittings for four mutants of both hydration water dynamics in Fig. 5A and tryptophan sidechain relaxations in Fig. 5B (Table 1). Note that these 16 sets of data for the local motions do not follow the linear relationship with the ratio of the dynamic viscosity over temperature (η/T) (Figs. S2 and S3) while the tumbling time perfectly follows a straight line (Fig. S4). Overall, each set

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of four lines for four proteins in Fig. 5 are nearly parallel, indicating similar barriers for the four proteins for each dynamical process. Due to the linear correlations between hydration water and sidechain relaxation in Fig. 4, the energy barriers for each correlated pair of each mutant should be the same. Fig. 6A shows the obtained 16 energy barriers (ΔE) and 16 corresponding prefactors (A) (Table 1). For each mutant, the energy barriers of both hydration water and sidechain relaxations are nearly similar within a standard deviation of 0.25 kJ/mol for ΔE2 and of 0.31 kJ/mol for ΔE3. The average energy barrier of ΔE2 is 11.0±0.4 kJ/mol and that of ΔE3 is 14.6±0.4 kJ/mol (Fig. 6A). These barriers are small and equivalent to 1 to 2 hydrogen-bond energies. The water-network relaxation in bulk water is in hundreds of femtoseconds with a barrier of about 5-6 kJ/mol,30,31 even less than one hydrogen-bond energy due to the hydrogen-bond network cooperativity. Here, the initial hydration water network relaxes in a few picoseconds, significantly slower with a double of the energy barrier 11 kJ/mol, only about breaking at most 2 hydrogen bonds. Such a low energy barrier reflects the collective relaxation of interfacial hydration water networks. Even for the second slow relaxation in tens-to-a hundred of picoseconds, the barrier is also small, only about 14.6 kJ/mol, slightly larger than the previous one. These water network arrangements, coupled with the local protein fluctuations, must be a cooperative relaxation on a relatively longtime scale, representing a concerted motion of breaking and making hydrogen bonds simultaneously.32-34 The prefactors are different for the hydration water motions and sidechain relaxations for each mutant, reflecting the local different flexibility relating to chemical and structural properties. For τ2S, the prefactors correspond to a time of 31-38 fs (A2S-1) for the four proteins and the WT is the slowest (38 fs), similar to the fastest water relaxation of

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inertial or liberation motion.31 The collective motions with a barrier of 11 kJ/mol slow down the initial water relaxations. For the related τ2W, the slaved sidechain relaxation (A2W1)

is in 60-140 fs and the mutant K is the slowest (140 fs) while KE is the fastest (60 fs). For

the second slow relaxation of water-network rearrangements, the triple mutant KEK has both the fastest motions of 140 fs (A3S-1) for hydration water molecules and 300 fs (A3W-1) for the tryptophan sidechain while both the wild type and mutant K have the slowest relaxations of 250 fs for hydration water and 380 fs for the sidechain. These results are consistent with the previous observations that in a hydrophobic environment water networks rearrange faster while in a heavy charged local position hydration water moves slowly.18 These different prefactors should directly reflect the mobility of the hydration water networks and local protein sidechains.35 We have used unique SNase with a partially buried single tryptophan residue to examine the coupled hydration water-protein interactions at the protein surface. By simultaneously probing both hydration water motions and local sidechain (tryptophan) relaxations, we can elucidate the nature of such coupling and determine which, either hydration water or the protein, is the determinant in such fluctuations. SNase is an ideal system and the single tryptophan reports only two relaxation timescales of interfacial water molecules in a few picoseconds and tens-to-a hundred of picoseconds, directly probing water-protein interactions. By systematic mutations of the neighboring charged residues and change of the local chemical identities and structural flexibility, we observed the relaxation times of both hydration water and the tryptophan sidechain. By variation of temperature, we derived the relaxation barriers for such dynamical interactions.

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Regardless of any change of the local environment, the barriers are nearly the same, 11.0 kJ/mol for the fast motion and 14.6 kJ/mol for the slow relaxation, even though the dynamics change significantly. Such small energy barriers must be from the cooperative relaxations by collectively breaking and making hydrogen bonds. Significantly, we observed that the hydration water motions are always faster than the buried sidechain motions but they exhibit linear correlations with the same energy barriers for relaxations. Thus, hydration-water relaxations drive protein surface fluctuations with two distinct timescales within sub-nanosecond, similar to the model proposed by Frauenfelder et al. for a βh-relaxation, i.e., hydration-layer fluctuations slaving protein surface motions. Such a dynamic process is illustrated in Fig. 6B and the coupled water-protein relaxations are limited in a restricted region due to the nature of slaved motions in water-protein interactions. Hydration water plays a significant, active role on ultrafast protein fluctuations, as shown here, at least around water-protein interfaces.

EXPERIMENTAL METHODS Mutant Proteins and Sample Preparation. The wild-type (WT) SNase and three mutants of K110A (K), K110AE129A (KE) and K110AE129AK133A (KEK), were prepared by the method of Kunkel as described in ref. 36. Protein expression and purification were performed by following the procedure given in ref. 37. The obtained proteins were finally dissolved in a buffer of 25 mM Tris and 50 mM NaCl at pH 8. The protein concentration used in femtosecond-resolved studies was 400–600 μM. The steady-state fluorescence emission was measured using a SPEX FluoroMax-3 spectrometer. The wide-type and three mutants have their emission peaks from 332-337 nm.19,22 For temperature studies, due to

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less stability of mutants KE and KEK, we only measured the dynamics with four different temperatures (1 °C, 10 °C, 20 °C and 30 °C) while for WT and mutant K, we examined the dynamics up to 40 °C with five different temperatures. The precise measurement of each protein sample temperature is detailed in Supporting Information. Femtosecond Methods and Tryptophan Lifetimes of Proteins. We used the femtosecond-resolved fluorescence up-conversion method for all ultrafast measurements and the detailed methodology has been described previously (also see Supporting Information).38 Briefly, a pump laser at 290 nm was used with the energy of about 100 nJ per pulse before focusing into the motor-controlled moving sample cell. The fluorescence emission was collected by a pair of parabolic mirrors and mixed with a gating pulse at 800 nm in a 0.2-mm β-barium borate (BBO) crystal through a nonlinear configuration. The instrumental response time under the current nonlinear configuration is about 450 fs as determined from the up-conversion signal of Raman scattering by water at ~320 nm. With the excellent signal-to-noise ratio, we can readily resolve the dynamics in ~200 fs. For solvation dynamics measurements, the magic-angle (54.70) condition was used. For fluorescence anisotropy measurements, the pump-beam polarization was rotated to be either parallel or perpendicular to the BBO acceptance axis to obtain the parallel (I||) and perpendicular (I⊥) signals, respectively. The resulting time-resolved anisotropy can be calculated: r(t)=(I||-I⊥)/(I||+2I⊥). In construction of solvation correlation functions,9,27 the tryptophan fluorescence lifetimes of all four proteins at each wavelength were measured by the time-correlated single photon counting method (TCSPC).

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ASSOCIATED CONTENT Supporting Information ACS Publications website at DOI: xxxx Materials and methods, data analyses, MD simulations, supporting figures and table, additional references, and complete reference 6 (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] or [email protected]. Author Contributions Y.Q. and M.J. contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We thank Prof. Bertrand Garcia-Moreno (Johns Hopkins University) for generously providing us the SNase plasmid. Mr. Menghui Jia was supported by the Fund of ECNU for Overseas and Domestic Academic Visits for five months working in the Zhong group in US. This work was supported in part by the National Science Foundation of China (61178085) to JX, the National Institute of Health (Grant GM118332) to DZ, and the Program of Introducing Talents of Discipline to Universities (B12024) for support of a short visit of DZ in ECNU. The MD simulations were supported in part by an allocation of computing time by the Ohio Supercomputer Center.

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REFERENCES (1)

Ball, P. Water as an Active Constituent in Cell Biology. Chem. Rev. 2008, 108, 74-108.

(2)

Levy, Y.; Onuchic, J. N. Water Mediation in Protein Folding and Molecular

Recognition. Annu. Rev. Biophys. Biomol. Struct. 2006, 35, 389-415. (3)

Pal, S. K.; Zewail, A. H. Dynamics of Water in Biological Recognition. Chem. Rev.

2004, 104, 2099-2123. (4)

Lewandowski, J. R.; Halse, M. E.; Blackledge, M.; Emsley, L. Direct Observation of

Hierarchical Protein Dynamics. Science 2015, 348, 578-581. (5)

Nucci, N. V.; Pometun, M. S.; Wand, A. J. Site-resolved Measurement of Water-Protein

Interactions by Solution NMR. Nat. Struct. Mol. Biol. 2011, 18, 245-249. (6)

Schiro, G.; Fichou, Y.; Gallat, F. X.; Wood, K.; Gabel, F.; Moulin, M.; Hartlein, M.;

Heyden, M.; Colletier, J. P.; Orecchini, A.; et al. Translational Diffusion of Hydration Water Correlates with Functional Motions in Folded and Intrinsically Disordered Proteins. Nat. Commun. 2015, 6, 6490. (7)

Doster, W.; Settles, M. Protein-Water Displacement Distributions. Biochim. Biophy.

Acta 2005, 1749, 173-186. (8)

Cohen, B. E.; McAnaney, T. B.; Park, E. S.; Jan, Y. N.; Boxer, S. G.; Jan, L. Y. Probing

Protein Electrostatics with a Synthetic Fluorescent Amino Acid. Science 2002, 296, 17001703. (9)

Zhong, D. Hydration Dynamics and Coupled Water-Protein Fluctuations Probed by

Intrinsic Tryptophan. Adv. Chem. Phys. 2009, 143, 83-149.

13



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

(10)

Page 14 of 28

Waegele, M. M.; Culik, R. M.; Gai, F. Site-specific Spectroscopic Reporters of the Local

Electric Field, Hydration, Structure, and Dynamics of Biomolecules. J. Phys. Chem. Lett. 2011, 2, 2598-2609. (11)

Nibali, V. C.; Havenith, M. New Insights into the Role of Water in Biological Function:

Studying Solvated Biomolecules Using Terahertz Absorption Spectroscopy in Conjunction with Molecular Dynamics Simulations. J. Am. Chem. Soc. 2014, 136, 12800-12807. (12)

Makarov, V.; Pettitt, B. M.; Feig, M. Solvation and Hydration of Proteins and Nucleic

Acids: A Theoretical View of Simulation and Experiment. Acc. Chem. Res. 2002, 35, 376-384. (13)

Bagchi, B. Water Dynamics in the Hydration Layer around Proteins and Micelles.

Chem. Rev. 2005, 105, 3197-3219. (14)

Li, T.; Hassanali, A. A.; Kao, Y. T.; Zhong, D.; Singer, S. J. Hydration Dynamics and

Time Scales of Coupled Water-Protein Fluctuations. J. Am. Chem. Soc. 2007, 129, 33763382. (15)

Zhong, D.; Pal, S. K.; Zewail, A. H. Biological Water: A Critique. Chem. Phys. Lett. 2011,

503, 1-11. (16)

Zhang, L.; Wang, L.; Kao, Y. T.; Qiu, W.; Yang, Y.; Okobiah, O.; Zhong, D. Mapping

Hydration Dynamics around a Protein Surface. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 18461-18466. (17)

King, J. T.; Kubarych, K. J. Site-specific Coupling of Hydration Water and Protein

Flexibility Studied in Solution with Ultrafast 2D-IR Spectroscopy. J. Am. Chem. Soc. 2012, 134, 18705-18712.

14


Page 15 of 28

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


(18)

Zhang, L.; Yang, Y.; Kao, Y.; Wang, L.; Zhong, D. Protein Hydration Dynamics and

Molecular Mechanism of Coupled Water-Protein Fluctuations. J. Am. Chem. Soc. 2009, 131, 10677-10691. (19)

Qiu, W.; Kao, Y. T.; Zhang, L.; Yang, Y.; Wang, L.; Stites, W. E.; Zhong, D.; Zewail, A. H.

Protein Surface Hydration Mapped by Site-specific Mutations. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 13979-13984. (20)

Abbyad, P.; Shi, X. H.; Childs, W.; McAnaney, T. B.; Cohen, B. E.; Boxer, S. G.

Measurement of Solvation Responses at Multiple Sites in a Globular Protein. J. Phys. Chem. B 2007, 111, 8269-8276. (21)

Biesso, A.; Xu, J. H.; Muino, P. L.; Callis, P. R.; Knutson, J. R. Charge Invariant Protein-

Water Relaxation in GB1 via Ultrafast Tryptophan Fluorescence. J. Am. Chem. Soc. 2014, 136, 2739-2747. (22)

Jia, M.; Yang, J.; Qin, Y.; Wang, D.; Pan, H.; Wang, L.; Xu, J.; Zhong, D. Determination of

Protein Surface Hydration by Systematic Charge Mutations. J. Phys. Chem. Lett. 2015, 6, 5100-5105. (23)

Fenimore, P. W.; Frauenfelder, H.; McMahon, B. H.; Parak, F. G. Slaving: Solvent

Fluctuations Dominate Protein Dynamics and Functions. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16047-16051. (24)

Frauenfelder, H.; Chen, G.; Berendzen, J.; Fenimore, P. W.; Jansson, H.; McMahon, B.

H.; Stroe, I. R.; Swenson, J.; Young, R. D. A Unified Model of Protein Dynamics. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5129-5134. (25)

Qin, Y.; Wang, L.; Zhong, D. Dynamics and Mechanism of Ultrafast Water-Protein

Interactions. Proc. Natl. Acad. Sci. U.S.A. 2016, 113, 8424-8429.

15



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

(26)

Page 16 of 28

Truckses, D. M.; Somoza, J. R.; Prehoda, K. E.; Miller, S. C.; Markley, J. L. Coupling

between trans/cis Proline Isomerization and Protein Stability in Staphylococcal Nuclease. Protein Sci. 1996, 5, 1907-1916. (27)

Qiu, W.; Zhang, L.; Kao, Y.; Lu, W.; Li, T.; Kim, J.; Sollenberger, G. M.; Wang, L.; Zhong,

D. Ultrafast Hydration Dynamics in Melittin Folding and Aggregation: Helix Formation and Tetramer Self-assembly. J. Phys. Chem. B 2005, 109, 16901-16910. (28)

Yang, J.; Zhang, L.; Wang, L.; Zhong, D. Femtosecond Conical Intersection Dynamics

of Tryptophan in Proteins and Validation of Slowdown of Hydration Layer Dynamics. J. Am. Chem. Soc. 2012, 134, 16460-16463. (29)

Qiu, W.; Zhang, L.; Okobiah, O.; Yang, Y.; Wang, L.; Zhong, D.; Zewail A. H. Ultrafast

Solvation Dynamics of Human Serum Albumin: Correlations with Conformational Transitions and Site-selected Recognition. J. Phys. Chem. B 2006, 110, 10540-10549. (30)

Jimenez, R.; Fleming, G. R.; Kumar, P. V.; Maroncelli, M. Femtosecond Solvation

Dynamics of Water. Nature 1994, 369, 471-473. (31)

Laage, D.; Hynes, J. T. A Molecular Jump Mechanism of Water Reorientation. Science

2006, 311, 832-835. (32)

Ghosh, R.; Banerjee, S.; Hazra, M.; Roy, S.; Bagchi, B. Sensitivity of Polarization

Fluctuations to the Nature of Protein-Water Interactions: Study of Biological Water in Four Different Protein-Water Systems. J. Chem. Phys. 2014, 141, 22D531. (33)

Matyushov, D. V. Nanosecond Stokes Shift Dynamics, Dynamical Transition, and

Gigantic Reorganization Energy of Hydrated Heme Proteins. J. Phys. Chem. B 2011, 115, 10715-10724.

16


Page 17 of 28

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(34)

Martin, D. R.; Matyushov, D. V., Dipolar Nanodomains in Protein Hydration Shells. J.

Phys. Chem. Lett. 2015, 6, 407-412. (35)

Fonseca, T.; Kim, H. J.; Hynes, J. T. Dynamics of Twisted Intramolecular Charge-

transfer Complexes in Polar-solvents. J. Mol. Liq. 1994, 60, 161-200. (36)

Kunkel, T. A. Rapid and Efficient Site-specific Mutagenesis without Phenotypic

Selection. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 488-492. (37)

Karp, D. A.; Gittis, A. G.; Stahley, M. R.; Fitch, C. A.; Stites, W. E.; Garcia-Moreno, B.

High Apparent Dielectric Constant inside a Protein Reflects Structural Reorganization Coupled to the Ionization of an Internal Asp. Biophys. J. 2007, 92, 2041-2053. (38)

Zhang, L.; Kao, Y.; Qiu, W.; Wang, L.; Zhong, D., Femtosecond Studies of Tryptophan

Fluorescence Dynamics in Proteins: Local Solvation and Electronic Quenching. J. Phys. Chem. B 2006, 110, 18097-18103.

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Figure Captions

Figure 1. Protein structure with the local probe site. Protein structure of WT SNase (PDB ID: 1SNO) is shown in ribbon (A) and surface (B) representations. The local hydration site around W140 (yellow) is closely surrounded by three charged residues K110, E129 and K133 within 5 Å. W140 is sandwiched by K110 and K133, and E129 and K133 forms a salt bridge. All charged residues were colored in red (negative) and in blue (positive), respectively.

Figure 2.

Ultrafast hydration dynamics probed by time-resolved fluorescence

emission. (A) Normalized femtosecond-resolved fluorescence transients of WT SNase gated at different wavelengths with temperatures of 1 °C (upper panel) and 40 °C (lower panel). (B) Solvation correlation functions of WT from 1 °C to 40 °C and the dynamics gradually becomes faster with temperature increase. (C) Solvation correlation functions of WT and three mutants at 1 °C. All proteins show only two distinct timescales in a few and tens-to-hundred of picoseconds.

Figure 3.

Protein sidechain dynamics probed by time-resolved fluorescence

anisotropy. (A) Relative fluorescence transients probed at two polarizations (parallel and perpendicular) for WT SNase at 1 °C. The inset shows a close-up view of the transients in a short time range. (B) Fluorescence anisotropy dynamics of WT at 1 °C and 40 °C. The 18


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dynamics becomes faster with temperature increase. (C) Fluorescence anisotropy dynamics of K and KEK at 1 °C for comparison. All proteins show a total of four relaxations including ultrafast internal conversion τIC

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