Determination of Protein Surface Hydration by Systematic Charge

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Letter

Determination of Protein Surface Hydration by Systematic Charge Mutations Dongping Zhong, Menghui Jia, Jin Yang, Yangzhong Qin, Dihao Wang, Haifeng Pan, Lijuan Wang, and Jianhua Xu J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.5b02530 • Publication Date (Web): 04 Dec 2015 Downloaded from http://pubs.acs.org on December 5, 2015

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The Journal of Physical Chemistry Letters

JPC Letters (2015)

Determination of Protein Surface Hydration by Systematic Charge Mutations

Menghui Jia,1,¶ Jin Yang,2,¶ Yangzhong Qin,2 Dihao Wang,2 Haifeng Pan,1 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] and Tel: (614)2923044.

ACS Paragon Plus Environment


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Abstract: Protein surface hydration is critical to its structural stability, flexibility, dynamics and function. Recent observations of surface solvation on picosecond time scales have evoked debate on the origin of such relatively slow motions, from hydration water or protein charged sidechains, especially with molecular dynamics simulations. Here, we used a unique nuclease with a single tryptophan as a local probe and systematically mutated three neighboring charged residues to differentiate the contributions from hydration water and charged sidechains. By various mutations of one, two and all three charged residues, we observed slight increases in the total tryptophan Stokes shifts with less neighboring charged residue(s) and found insensitivity of charged sidechains to the relaxation patterns. The dynamics is correlated with hydration water relaxation with the slowest time in a dense charged environment and the fastest time at a hydrophobic site. On such picosecond time scales, the protein surface motion is restricted. The total Stokes shifts are dominantly from hydration water relaxation and the slow dynamics is from water-driven relaxation, coupled with local protein fluctuations.

TOC

Key Words:

Hydration dynamics, site-directed mutation, collective water-network

relaxation, insensitivity of charge sidechain

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Protein surface hydration has been heavily studied in the recent decade by a series of methods such as NMR,1,2 neutron scattering,3 dielectric relaxation,4 optical spectroscopy including 2D-IR,5-7 THz absorption8,9 and fluorescence Stokes shift,10-13 and molecular dynamics simulations (MD).14,15 Besides the ultrafast water motions occurring within a few picoseconds,3,6,14,16,17 a longtime solvation process in tens of picoseconds was also observed.2,4,10-13,15 Extensive studies on different proteins indicated that the observed longtime relaxation mainly results from a collective water-network motion.1,2,8-13,18-21 Such a longtime hydration dynamics must induce a local protein motion, a similar process that has been called as a βh-relaxation, i.e., protein motions slaved by the hydration shell fluctuations.22,23

We have carried out extensive studies of protein surface hydration using a tryptophan scan to probe different locations through site-directed mutagenesis and by recording the dynamic fluorescence Stokes shifts of tryptophan, both relaxation energies and times, with femtosecond resolution.11,12,17,18,21 From numerous proteins studied, we found that the two or three relaxation processes were observed depending on the locations of tryptophan relative to the protein surface. If tryptophan is exposed to the protein surface (typically λmax≥338 nm), three relaxation processes were observed, two ultrafast motions arising from inner (a few picoseconds) and outer (hundreds of femtoseconds) water layers of the hydration shell and the other longtime relaxation (tens of picoseconds) representing inner water-network rearrangements.16-18,21 If tryptophan becomes partially buried inside a protein (λmax≤338 nm), only two relaxation dynamics were observed and both are from the inner-layer water motions.16-18,21 In our recent study of Staphylococcus nuclease

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(SNase),19,24 the protein is an ideal system with only a single tryptophan residue with the fluorescence emission peak at around 332-333 nm. We observed only two distinct relaxation time scales, one in a few picoseconds and the other in around one hundred of picoseconds,19 for the wild type and single mutations for each of three charges, K110, E129 and K133, surrounding the partially buried tryptophan in 3-5 Å (Fig. 1). From the observed relaxation energies and time scales that are insensitive to any single charge mutations, we concluded that the hydration water plays a dominant role and water seems to drive the entire relaxation within sub-nanoseconds. We also clearly stated the critical interactions of hydration water with the local protein in the longtime relaxation.19 Such “slaved” local protein fluctuations may conversely facilitate the longtime water relaxation due to the strong water-protein coupling at their interface.19

Several MD simulations have been tried to elucidate the origin of the longtime relaxation.14,15,25-27 Earlier studies claimed the local protein relaxation responsible for the longtime process14 but a recent MD simulation on SNase reported both contributions from the local protein and hydration water.27 These MD simulations mainly attributed neighboring charged residues to the longtime relaxation. In SNase, the three charged residues encircle the single tryptophan (W140) and the single charge mutations have already showed nearly no change of the total Stokes shifts and related dynamics.19 In this study, we use an alanine (A) scan to further mutate any of two charged residues each time and finally replace all three charged residues to have a hydrophobic environment around W140. Combined with the previous single charge mutations,19 such systematic studies by various combinations of mutating one, two and all three charged residues around the probe W140 will carefully examine the dependence of the longtime solvation dynamics on local 4


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charged residues and finally ascertain the contribution and dynamics of hydration water molecules.

Figure 2 shows the fluorescence emission spectra of WT and four mutants. Using an alanine scan to mutate any of two or the total three neighboring charged residues around W140, the emissions gradually shift to the red side, but not to the blue side, indicating more Stokes shifts with less charged residues. The wild type has an emission peak at 333 nm and with the complete mutation of all three charged residues (KEK) and leaving a hydrophobic environment around W140, the emission peak moves to 337 nm, a 4-nm more Stokes shift. The inset in Fig.2 shows the circular dichroism (CD) spectra of WT and the four mutants with similar profiles, indicating negligible changes in structure by mutations. The mutation of charged residues no doubt modifies the local water network and hydrogen-bond configurations. Thus, the more Stokes shifts by mutations could result from the contributions of water solvation, not the charged residues, because the KEK mutant has the largest Stokes shift without any neighboring charged residues in a hydrophobic environment. Also, the corresponding dynamics of water relaxation could also change through mutations (see below). Thus, the dominant contributions of the Stokes shifts from the three charged residues (K110, E129 and K133) obtained from the recent MD simulations27 seem unlikely as shown here by systematic mutations of charged residues.

Figure 3 shows six typical fluorescence transients out of more than 10 gated ones from the blue to red side in the range from 310 to 370 nm for the triple mutant of KEK. Clearly, we only observed two distinct solvation dynamics (1.8-3.7 ps and 26-77 ps) for all blue-side transients gated at wavelengths less than the emission peak (337 nm). At the red

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side, the transients show a rise component (~1.6 ps) and the lifetime decay. We did not observe any ultrafast decay component in hundreds of femtoseconds. For WT and all other three double mutants, the transients show the similar patterns with the two distinct solvation dynamics in a few and tens of picoseconds. As we observed before,18 for the emission peaks at less than 338 nm, it indicates that W140 is partially buried inside the protein, consistent with the X-ray structure.28 As observed here again, we detected only two solvation dynamics in a few and tens of picoseconds, representing the inner-layer water relaxations (see below). The ultrafast outer-layer water relaxations in hundreds of femtoseconds cannot be probed by W140 due to the far distances and weak perturbations. Using the methodology we developed for tryptophan,11,29 we can construct the solvation correlation functions of WT and the four mutants. Since the solvation only contains two relaxation dynamics on the picosecond timescale, the data analyses are straightforward. Figure 4A shows the final derived results, clearly exhibiting two distinct solvation timescales; also see Table 1. For the first component (τ2S), the dynamics for four mutants are nearly similar and in 2.1 to 2.4 ps. For WT, the time becomes longer and is 3.2 ps (we usually refer τ1S to ultrafast relaxation on the femtosecond time scale), consistent with the previous observation that water relaxes slowly in a dense charged environment.13,18 For the second long component (τ3S), the WT also has the longest relaxation timescale of 102 ps. For the three double mutants, the relaxations are also similar and take 82-92 ps. For the triple mutant with a hydrophobic environment, the time becomes noticeably shorter and is 56 ps, again consistent with our observation in apomyoglobin that the tryptophan probe located at the hydrophobic sites gives the faster solvation while the probe surrounded by dense charged residues yields the longer

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relaxation time.13,18 Nevertheless, all solvation times for WT and the four mutants are a few and tens of picoseconds and the observed difference is correlated with the hydration water relaxations in different environments. We also carefully measured the anisotropy dynamics of the tryptophan sidechain and the result for the double mutant EK is shown in inset of Figure 4B. The derived anisotropy r(t) is shown in Fig. 4B and the relaxation times are also listed in Table 1. The dynamics can be best 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 through conical intersection.30 The two resolved relaxation times are 6 (τ2W) and 120 (τ3W) ps, although their amplitudes are relatively small, representing the local wobbling motions. The last one is the entire protein tumbling motion in nanoseconds (τT). Very significantly, as observed in apomyoglobin,18 we found that the solvation dynamics (τiS, i=2, 3) are always faster than the sidechain relaxations (τiW). Figure 5 shows such a striking correlation with τiW≥τiS, indicating that the hydration relaxations may drive the sidechain motions. Combined with our previous single mutations of three charged residues (K110, E129 and K133),19 all solvation dynamics are biphasic and have two distinct timescales of a few and tens of picoseconds. By gradually removing charged residue(s) using an alanine scan (Fig. 6) and with intact structures, the Stokes shifts do not decrease but increase slightly. By completely removing all three neighboring charged residues and leading to a hydrophobic environment, the Stokes shift increases by 4 nm to the red side. Clearly, the Stokes shifts are not dominantly from the contributions of charged-residue relaxations. In a recent MD simulation of single charge mutations27 to interpret our previous experimental

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observations, it is interesting to find that for WT and E129A, the hydration water and local charged residues contributed 30% and 70% to the total Stokes shifts, respectively. For K110A and K110C, the results are nearly the same and the hydration water and local charged residues contributed 50% each. But, for K133A, the hydration water contributed 70% while the local charged residues caused 30%. It claimed that the main contributions are from two positive charged residues (K110 and K133) and various noticeable longrange protein contributions cancel each other, leading to insensitivity to the total Stokes shifts by the single charge mutations.27 Here, by mutating any two of the three charged residues, the dynamics are nearly similar and the Stokes shifts increase slightly. Interestingly, the Stokes shift remains nearly the same as WT by mutating two positive residues of K110AK133A. Thus, the Stokes shifts are not mainly from the charged-residue relaxation but must be from surrounding hydration water motions. For the triple mutant (K110AE129AK133A), we observed even the largest Stokes shift with all hydrophobic residues around the probe. If the charged residues made significant contributions to the total Stokes shifts, it is difficult to rationalize the largest shift for a hydrophobic site. On the other side, such a shift can result from the hydration water relaxation due to the change of water networks by charge mutations. Thus, the insensitivity of the total Stokes shifts and dynamic solvation patterns to the local charges clearly indicates that the charged residues do not make significant contributions to the observed total Stokes shifts and cannot be the origin of the observed solvation dynamics. The only possibility is that the hydration water is dominantly responsible for the total shifts and related dynamics. As we have already pointed out in our previous report,19 even for the single charge mutations, the hydration water dynamics can induce the local protein fluctuations on the

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longer time scale of tens of picoseconds and their interactions are coupled. Hydration dynamics is an integrated process of water-protein coupled relaxation because both the hydration water and the protein interact together at the protein surface. Any motion, either from the hydration water or local protein residues, will affect each other. However, on the picosecond time scale, hydration water motions are much easier and the water-network rearrangements, by breaking and making hydrogen bonds, are much feasible. The protein sidechains may fluctuate but are much restricted on such an ultrafast time scale. The observed wobbling motions, strongly correlated with the solvation dynamics, could be the consequence of the coupled water-protein relaxations. But, the amplitudes are very small and the wobbling cone semiangle is only in about 6°-12° (inset in Fig. 5).31 Thus, by sudden excitation of the probe tryptophan, the nonequilibrated hydration water network relaxes by rearranging various hydrogen bonds to a new configuration. This process couples with local protein (mostly sidechains) motions to reach the final, new equilibrated state. Seemingly, the hydration water drives such relaxation processes. We have performed MD simulations to examine how many water molecules fluctuate around the probe tryptophan. By considering dipole-dipole interactions, we mainly consider water molecules within 10 Å away from the probe, which mostly covers 34 layers of water molecules. The water molecules beyond 10 Å can also be perturbed as observed by THz measurements32 but may not contribute significantly to the Stokes shifts observed here. Fig. 6 shows the total water molecules within 10 Å to the center of the indole ring. The probe tryptophan is partially buried on the protein surface and around 70 water molecules (the right panels in Fig. 6) are found to be near the probe, regardless of any double or triple mutations. Only 5 water molecules are within 5 Å, consistent with the

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other MD report.27 However, among 70 water molecules, 45 ones (64%) are in the first hydration layer within 5 Å away from the protein surface and 63 ones (90%) are in the second hydration layer within 7 Å from the surface. Thus, the response upon excitation of the probe is mainly from the first and second layers of water molecules in the hydration shell. These two layers of water molecules consist of the inner layers of hydration water. These water molecules interact with the protein and their dynamics take longer times in a few and tens of picoseconds to relax to a new configuration. As we pointed out early,11,13,1821

the first component in a few picoseconds is from the local relaxation of the hydrogen-

bond network and the second one in tens of picoseconds attributes to the rearrangement of the water network. It is unlikely that only 5 water molecules near the probe make the dominant contributions to the Stokes shifts as reported recently.27 For tryptophan exposed to the protein surface, the responding water molecules within 10 Å from the probe may contain third and fourth layers of water molecules in the hydration shell. These water molecules consist of the outer-layer hydration water and have a relaxation dynamics on hundreds of femtoseconds as extensively observed by many systems.16,17,21 The dynamics observed here are from the inner-layer (~2 layers) water relaxations in the hydration shell and the hydration water motions are significantly lengthened by the hydration network and the coupling with the protein. In conclusion, we have used unique SNase with a partially buried single tryptophan residue to probe the local solvation dynamics and thus address the recent argument about the origin of our observed long hydration dynamics in tens of picoseconds. MD simulations often showed significant contributions from neighboring charged residues to the long relaxation, both in Stokes shifts and time, opposite to our experimental observations. With

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three charged residues surrounding the probe tryptophan in SNase, we have used an alanine scan and systematically mutated charged residue(s) by one, two and all three to examine the changes of the total Stoke shifts and relaxation dynamics. Significantly, we basically observed slight increases in the Stokes shifts with less surrounding charged residues and found insensitivity of the solvation patterns to the neighboring charged residues. With the complete mutation of three charged residues, we observed the largest Stokes shift with a hydrophobic local environment. It should be cautious to invoke MD results on such ultrafast time scales as we suggested before11,19 due to a series of sensitive treatments in simulations.

The observed solvation dynamics are correlated with hydration water relaxations. For a dense charge environment, the relaxation takes the longest time as observed here for WT. For a hydrophobic environment, the relaxation is the fastest as shown here for the triple mutant. Thus, it is unlikely that the charged residues make significant contributions to the Stokes shifts. On tens of picoseconds, the hydration water network in the inner layers near the protein surface relaxes and no doubt couples with the local protein. On such a timescale, the protein motions are limited and water relaxations are dominant. It seems that the inner-layer hydration water drives the local sidechain motions on the picosecond time scale.

EXPERIMENATL METHODS Mutant Proteins and Sample Preparation. The wild-type (WT) SNase and four mutants

of

K110AE129A

(KE),

E129AK133A

11

(EK),


K110AK133A

(KK)

and


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K110AE129AK133A (KEK), were prepared by the method of Kunkel as described in ref. 33. Protein expression and purification were performed by following the procedure given in ref. 34. 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 fluorescence emission was measured using a SPEX FluoroMax-3 spectrometer. Because the buffer condition and pH value are different from those used in the previous study,17 the wild-type protein shows a slightly different emission peak, 333 nm currently vs. 332 nm previously, and could also lead to slightly different relaxation dynamics. Femtosecond Methods and Mutant Lifetimes. All experimental measurements were carried out using the femtosecond-resolved fluorescence up-conversion method described previously.35 Briefly, a pump laser at 290 nm was used with the energy of about 140 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 between 400-500 fs as determined from the up-conversion signal of Raman scattering by water at ~320 nm. For solvation dynamics measurements, the magic-angle (54.70) condition was used and 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, the

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fluorescence lifetimes of mutants at each wavelength were measured by the time correlated single photon counting method (TCSPC).

AUTHOR INFORMATION

Corresponding Author

[email protected]

Author Contributions

¶These

authors contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

We thank Prof. Bertrand Garcia-Moreno (Johns Hopkins University) for generously providing us the SNase plasmid. Mr. Menghui Jia thanks the Fund of ECNU for Overseas and Domestic Academic Visits for support of five months working in the Zhong group. This work was supported in part by the National Institute of Health (Grant GM095997) to DZ, the National Science Foundation of China (61178085) to JX, and the Program of Introducing Talents of Discipline to Universities (B12024) for support of a short visit of DZ in ECNU.

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26. Toptygin, D.; Woolf, T. B.; Brand, L.; Picosecond Protein Dynamics: The Origin of the TimeDependent Spectral Shift in the Fluorescence of the Single Trp in the Protein GB1. J. Phys. Chem. B 2010, 114, 11323-11337. 27. Scott, J. N.; Callis, P. R. Insensitivity of Tryptophan Fluorescence to Local Charge Mutations. J. Phys. Chem. B 2013, 117, 9598-9605. 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. 29. Lu, W.; Kim, J.; Qiu, W.; Zhong, D. Femtosecond Studies of Tryptophan Solvation: Correlation Function and Water Dynamics at Lipid Surfaces. Chem. Phys. Lett. 2004, 388, 120-126. 30. 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. 31. 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. 32. Ebbinghaus, S.; Kim, S. J.; Heyden, M.; Yu, X.; Heugen, U.; Gruebele, M.; Leitner, D. M.; Havenith, M. An Extended Dynamical Hydration Shell around Proteins. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 20749-20752. 33. Kunkel, T. A. Rapid and Efficient Site-specific Mutagenesis without Phenotypic Selection. Proc. Natl. Acad. Sci. U. S. A. 1985, 82, 488-492. 34. Karp, D.A.; Gittis, A.G.; Stahley, M.R.; Fitch, C.A.; Stites, W.E.; Garcia-Moreno, B. High

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Apparent Dielectric Constant inside a Protein Reflects Structural Reorganization Coupled to the Ionization of an Internal Asp. Biophy. J. 2007, 92, 2041-2053. 35. Zhang, L.; Kao, Y.-T.; 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. X-ray ribbon structure of wild-type SNase (PDB ID code 1SNO) and a circle highlighting the hydration sites around single tryptophan W140 (yellow) with a surface map of the protein and a close-up view of three charged residues (K110, E129 and K133) around W140.

Figure 2. Fluorescence emission spectra of WT and four mutant proteins. All mutant emissions slightly shift to the red side and the triple mutant has the longest emission peak. Inset is the circular dichroism spectra for WT and all four mutants, showing the similar profiles and indicating no structural changes.

Figure 3. Normalized femtosecond-resolved fluorescence transients of W140 from the triple mutant (KEK) of SNase on short (A) and long (B) time scales with a series of gated fluorescence emissions. Note that no ultrafast decay components in less than one picosecond were observed.

Figure 4. (A) Correlation functions for WT and four mutants show two distinct relaxation times in a few and tens of picoseconds. (B) The anisotropy dynamics of the double mutant (EK), derived from parallel and perpendicular measurements (inset), clearly shows four

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decay components representing the ultrafast internal conversion, two wobbling motions corresponding to two hydration dynamics, and entire protein tumbling.

Figure 5. A correlation between the solvation dynamics and the sidechain (W140) relaxations. A relationship of τiW≥τiS was observed. Inset shows the cone semiangles of tryptophan wobbling motions for WT and all mutants. Note that the angles are very small in 8°-12°.

Figure 6. MD simulations on WT and all four mutants in a few nanoseconds. (Left panel) Total water molecules in a distance from 5 Å and 10 Å to the center of the probe indole and from 5 Å and 7 Å to the protein surface fluctuate with time. Note that nearly 90% of water molecules surrounding W140 are in the first two layers of surface hydration water (7 Å). (Right panel) Surface maps of local WT and four mutants with positive (blue) and negative (red) charged groups around W140 (yellow) with all water molecules (sticks) within 10 Å. Note a hydrophobic environment around W140 in the triple mutant (KEK).

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Table 1. Results obtained from the hydration dynamics c(t) and the anisotropy dynamics r(t) of WT SNase and four mutant proteins.a SNase WT KK EK KE KEK

τ2S (ps) 3.2 2.1 2.2 2.4 2.3

τ3S (ps) 102 86 92 82 56

E2 (cm-1) 569 548 558 602 860

E3 (cm-1) 231 248 329 233 288

τ2W (ps) 8.5 7.4 6.0 5.2 9.4

τ3W (ps) 108 91 120 148 99

θ2 (deg) 7.2 6.9 11.2 8.8 8.7

θ3 (deg) 7.7 10.3 9.8 9.2 12.2

aThe four mutants are: KK, K110AK133A; KE, K110AE129A; EK, E129AK133A; KEK, K110AE129AK133A.

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W140

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Determination of Protein Surface Hydration by Systematic Charge

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