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Dielectric Relaxations in Confined Hydrated Myoglobin Giorgio Schiro`,*,† Antonio Cupane,† Eugenio Vitrano,† and Fabio Bruni‡ CNISM and Dipartimento di Scienze Fisiche ed Astronomiche, UniVersita` di Palermo, Palermo, Italy, and CNISM and Dipartimento di Fisica “E. Amaldi”, UniVersita` di Roma Tre, Rome, Italy ReceiVed: February 16, 2009; ReVised Manuscript ReceiVed: April 23, 2009
In this work we report the results of a broadband dielectric spectroscopy study on the dynamics of a globular protein, myoglobin, in confined geometry, i.e. encapsulated in a porous silica matrix, at low hydration levels, where about only one or two water layers surround the proteins. In order to highlight the specific effect of confinement in the silica host, we compared this system with hydrated myoglobin powders at the same hydration levels. The comparison between the data relative to the two different systems indicates that geometrical confinement within the silica matrix plays a crucial role in protein-water dielectric relaxations, the effect of sol-gel encapsulation being essentially a suppression of cooperative relaxations that involve the coherence/ cooperativity of solvent motions and solvent-coupled protein dynamics. We also provide direct evidence that protein relaxations inside the gel depend on the hydration level and are “slaved” to the solvent β-relaxation. Introduction Biochemical studies performed in past years on protein activity in the intracellular environment1,2 made the biophysical community aware that molecular crowding and geometrical confinement could be extremely relevant factors in the comprehension of the relationship among dynamics, structure and function of proteins in their real biological environment. In this sense standard biophysical studies on protein solutions in a bulk phase and at low concentration may limit the understanding and neglect some key factors which determine structure, dynamics and also stability of biomolecules. An essential aspect of this complex scenario is therefore the role of confinement in restricted geometries and the influence of solid surfaces on the dynamics of globular proteins. In order to get a description of the effects of geometrical confinement on the dynamics of a protein-solvent system, we have chosen a suitable approach to obtain solid host matrices where we could confine inside nanometric cavities both protein and solvent through the sol-gel technique.3-7 Depending on sample treatment, it is possible to encapsulate proteins at different solvent composition (e.g., water-glycerol) and hydration level. In our previous works on the dynamical properties of sol-gel encapsulated heme-proteins,8-11 we showed that effects of confinement on protein dynamics, essentially a drastic reduction of molecular motions (either in time and space, in agreement with MD simulations on proteins inside glassy systems12), are mediated by the solvent: indeed relaxation times of solvent molecules are remarkably larger than in the corresponding bulk phase.8 In particular, elastic neutron scattering studies,10,11 performed on myoglobin (Mb) encapsulated in silica gel at various levels of average hydration, revealed that the confinement effect, i.e. the reduction of anharmonic protein motions observed in sol-gel encapsulated samples, depends nonmonotonically on the average protein hydration and presents * Corresponding author. E-mail:
[email protected]. Tel: +39091-6234-219/302/205. Fax: +39-091-6162-461. † CNISM and Dipartimento di Scienze Fisiche ed Astronomiche, Universita` di Palermo. ‡ CNISM and Dipartimento di Fisica “E. Amaldi”, Universita` di Roma Tre.
a maximum at an hydration level of about h ) 0.35 (hydration h defined as h ≡ g of D2O/g of dry protein). The confinement effect was attributed to a perturbation of the collective dynamics (R-relaxation-like) of the solvent in the hydration shell due to confinement, more than to direct protein-matrix interactions. However, as it is well-known, elastic neutron scattering on D2O-hydrated deuterated protein powders explores mainly motions of the nonexchangeable hydrogen atoms of the protein and only in the time scale of picoseconds to nanoseconds, depending on instrumental resolution. In view of the scenario presented above, the importance of studying molecular relaxations of both proteins and solvent in confinement on a wider time scale is evident. Broadband dielectric spectroscopy (BDS) seems to be an ideal technique to this purpose since it enables one to extend the time scale of investigated motions from about 100 ns to about 100 s; moreover, being sensitive to relaxations of polar and charged molecular groups, it investigates the dynamics of water molecules and of protein side chains. The temperature dependence of dielectric parameters (dielectric strengths ∆ε and relaxation times τ) allows one to obtain a dynamic description of the processes involved in dielectric molecular rearrangements. Dielectric spectroscopy studies on hydrated powders of proteins and other biological systems date back to about 30 years. In their pioneering studies Careri and Rupley studied the protonic conduction process in lysozyme powders at various hydrations, as well as in many other biological systems, and advanced a model based on the percolation theory.13-15 More recently, BDS has been used by several groups to investigate the dynamics of biological macromolecules16-22 (mainly protein hydrated powders) and the glass forming properties of polymer-water mixtures.23,24 In particular, the temperature dependence of dielectric relaxation times in a mixture waterglycerol-Mb measured by Swenson and co-workers20 has been recently discussed by Ngai and co-workers,25 and the behavior observed in water-protein systems was described in terms of a universal property shown by mixtures of water and glassforming polymers. It should be noted, however, that the dynamic properties of water in confined geometries and in the hydration shell of proteins (and in particular the possible presence of a
10.1021/jp901420r CCC: $40.75 2009 American Chemical Society Published on Web 06/19/2009
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fragile to strong crossover at a critical temperature) are still a highly debated issue in the current literature.21,26-29 In this paper we present a BDS study of myoglobin encapsulated in silica hydrogel at two different levels of hydration (i.e., the same samples used in our previous investigations with elastic and quasi-elastic neutron scattering10,11), and in the temperature interval of 120-300 K. To study the effect of different confinements, the data are compared with data on Mb powders at the same hydrations. The aim of the paper is to study the effect of confinement on dynamics on a wide time scale and to obtain a description of the coupling between solvent in the hydration shell and protein in the various environments. Experimental Methods Samples. Silica Gels. Lyophilized horse Mb (Sigma Aldrich) was dissolved, at room temperature, in 0.1 M K-phosphate buffer pH 7. Addition of a 4-fold molar excess of an oxidant agent (potassium ferricyanide, C6FeK3N6) guaranteed that protein was in the ferric (met) state; after equilibration, the excess potassium ferricyanide was removed by dialysis against a 0.1 M K-phosphate pH 7 buffer solution at 7 °C. Deuteration of protein solution was then obtained via several H2O-D2O (Euriso-Top, purity 99.97%) dilution/concentration steps. The final D2O/H2O molar ratio was greater than 99. The final protein concentration (w/w), as measured by optical absorption, was 26.5%. The experimental protocol to perform protein encapsulation in silica hydrogels was already described in previous works:10 a solution containing 60% v/v TMOS (Sigma Aldrich), 38% v/v D2O (Euriso-Top, purity 99.97%) and 2% v/v HCl 0.04 M was sonicated for 20 min in an ice bath; immediately after sonication it was mixed in a 1:1 proportion (in volume) and at 7 °C with the met-Mb/D2O solution. In these conditions formation of a gel about 1 mm thick occurred in about 1 min. The gel was then left aging in a controlled atmosphere of N2/ D2O and the hydration levels were determined from the observed mass change on drying (hydration h is here defined as h ≡ g of D2O/g of dry protein). Hydrated Powders. Met-Mb powder was prepared with the following procedure: lyophilized horse met-Mb was dissolved in D2O (Euriso-Top, purity 99.97%) at a concentration of 50 mg/mL and held at room temperature for approximately one day. The solution was then centrifuged for 20 min at 10 °C and lyophilized. The resulting protein powder was held for about 30 h under vacuum at 45 °C: the product of the above procedure was considered the dry (h ) 0) sample. However, note that the method used is unable to remove the strongly bound water molecules, which amount to approximately h ) 0.02: the reported powder hydration levels should therefore be considered as slightly underestimated. In order to reach the required hydration, met-Mb powders were held in a controlled atmosphere of D2O; hydration value h was determined by measuring the mass change. Measurements. Dielectric spectroscopy measurements were performed in the frequency range 10-2-107 Hz using a Novocontrol Alpha analyzer. Further details on the experimental setup and sample holder have been already reported.30 Accuracy of dielectric spectra is ∼2%. Dielectric measurements have been performed on samples of met-Mb in a silica gel and met-Mb powder at hydration h ) 0.3 and h ) 0.5, in the temperature range 120-300 K. Sample diameter and thickness were 28 and 1 mm, respectively. Differential scanning calorimetry measurements were performed with a Pyris Diamond (Perkin-Elmer) calorimeter.
Figure 1. Example of a typical fit on the dielectric spectra of met-Mb powder at the hydration h ) 0.5. Upper panel: real part (permittivity). Lower panel: imaginary part (dielectric loss). Low-frequency components of permittivity have been translated along the y axis for clarity.
Samples of Mb hydrated powders were sealed in steel pans of ∼60 µL. Samples were first cooled to 90 K with a cooling rate of 20 K/min; calorimetric upscans from 90 to 300 K were then performed with a heating rate of 20 K/min. Data Analysis. All spectra, namely, the complex permittivity ε* vs frequency ν, were fitted (real and imaginary part simultaneously, i.e. by minimizing a χ2 which contained both components) by a combination of Havriliak-Negami functions31 describing each relaxation process, a term for direct conductivity and an additional term to take into account effects of electrode and interface polarization30,32 due to the inhomogeneous nature of the samples:
ε* ) ε∞ +
∆ε
j ∑ [1 + (i2πντ )R ]β j
j
j
j
+i
σ + (a + ib)ν-λ 2πν (1)
where ε∞ is the high frequency limit of permittivity, ∆εj is the dielectric strength and τj is the relaxation time of the jth relaxation process. The empirical parameters 0 < R e 1 and 0 < β e 1 describe respectively the broadening and the asymmetry of each relaxation process. 0 < λ < 1 is a parameter describing the fractal character of the underlying interface polarization process. A typical fit is shown in Figure 1; we believe that only simultaneous fittings of the real and imaginary parts of the complex dielectric constant permit one to unambiguously identify the various relaxations present in complicated spectra and to follow accurately their temperature dependence. Results Figure 2 reports three-dimensional plots of dielectric losses measured in met-Mb powder as a function of frequency and temperature (panel a, h ) 0.3; panel b, h ) 0.5); analogous plots relative to encapsulated met-Mb are shown in Figure 3 (panel a, h ) 0.3; panel b, h ) 0.5). Figure 4 reports two typical dielectric loss spectra obtained in the hydrated powder samples, at selected temperatures and relative to the two different hydrations investigated (left panels, a, h ) 0.3; b. h ) 0.5). As
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Figure 2. Dielectric losses in met-Mb powder at two different hydrations: h ) 0.3 (panel a) and h ) 0.5 (panel b).
Figure 3. Dielectric losses in encapsulated met-Mb at two different hydrations: h ) 0.3 (panel a) and h ) 0.5 (panel b).
Figure 4. Dielectric relaxations in met-Mb powders at h ) 0.3 (upper panels) and h ) 0.5 (lower panels). Panels a and b: typical dielectric spectra (imaginary part), at the temperature of 236 K (a) and 223 K (b); continuous lines are overall fittings of experimental data while dotted lines represent the individual processes. Panels c and d: Arrhenius plots of relaxation times; continuous and dotted lines are fittings with VFT or Arrhenius law, respectively. The vertical thick line indicates the temperature of the VFT-Arrhenius crossover in the intermediate relaxation (process II). Panels e and f: temperature dependence of dielectric strength; dashed lines are guides to the eye.
evident from Figures 2 and 4 three main relaxations are observed for the hydrated powder samples, while a conductivity/interface polarization contribution becomes relevant only in the low frequency tail and at high temperatures. Relaxation processes are identified in the figures as processes I, II and III in order of decreasing peak frequency at a given temperature. Relaxation
times and dielectric strengths are reported in the center and right panels of Figure 4 respectively as a function of the inverse temperature (panels c and e, h ) 0.3; panels d and f, h ) 0.5). To highlight the hydration effects on the observed relaxations, the temperature dependence of relaxation times at the two hydration levels investigated is compared in Figure 5.
Dielectric Relaxations in Confined Hydrated Myoglobin
J. Phys. Chem. B, Vol. 113, No. 28, 2009 9609 tion contribution becomes relevant only in the low frequency tail and at high temperatures. Relaxation times and dielectric strengths are reported in panels b and c of Figure 7 respectively, as a function of the inverse temperature (circles, h ) 0.3; squares, h ) 0.5). In Figure 8 we compare the temperature dependence of relaxation times relative to the encapsulated metMb samples with that relative to process III observed in the hydrated powders and with the one observed for water encapsulated in silica gels (“dry” samples, see ref 6). Discussion
Figure 5. Hydration dependence of the various relaxation times seen in hydrated met-Mb powders. Circles: h ) 0.3. Squares: h ) 0.5. Processes I, II and III are reported in panels a, b and c, respectively. Crosses in panels b and c refer to met-Mb at h ) 0.8 (data from ref 19); stars in panel (b) refer to Hb at h ) 0.8 (data from ref 18).
Figure 6. Calorimetric heating scans (scan rate ) 20 K/min) on metMb powders in the temperature range 100-240 K.
In Figure 6 we report the calorimetric heating scans on metMb powders in the 100-240 K temperature range. It is evident that a glass transition occurs in the region 175-205 K.33 It clearly involves hydration water, as indicated by the comparison with the scan on a dry (h ≈ 0) sample where no sign of a glass transition is detected. Figure 7 reports typical dielectric loss spectra measured on silica gel samples, as a function of frequency at T ) 261 K (panel a; circles, h ) 0.3; squares, h ) 0.5). As evident from Figures 3 and 7 only one main relaxation is observed for encapsulated samples; again, a conductivity/interface polariza-
Relaxations in Mb Hydrated Powders: Process I. In both powder samples a main relaxation process is observed (process I in Figure 2; white circles and white squares in Figure 4c,d,e,f), which shows a temperature dependence described by a VogelFulcher-Tamman (VFT) law τ ) τ0exp[DT0/(T-T0)]. Values of the VFT parameters for process I are reported in Table 1. This is the fastest process, and its relaxation time depends on hydration level, as shown in Figure 5c: in fact, relaxation times for h ) 0.5 and h ) 0.3 differ by about 3 orders of magnitude, lower hydration corresponding to slower relaxation times. Both relaxations are described by a Havriliak-Negami function, with an evident asymmetry (parameter β ∼ 0.6, see eq 1). In Figure 5c the relaxation times relative to the fastest process observed for Mb in water at h ) 0.8 (data taken from ref 19) are also reported, and confirm the VFT behavior and the strong hydration dependence of this relaxation. Following Swenson and co-workers18,19 we assign process I to the dynamics of interfacial water, i.e. of water in the protein hydration shell. Evidence for this assignment comes from: (a) the observation that the relative amplitudes of process I increase with increasing hydration (see Figures 4a and 4b). (b) the strong dependence of relaxation times for process I on hydration. Indeed, it must be considered that h ∼ 0.3 corresponds to an almost complete hydration layer around the protein;34-36 at higher hydration levels a number of water molecules does not interact directly with protein surface (in particular with polar groups): they move more freely37,38 and make faster the dynamics of first hydration layer. (c) the calorimetric data reported in Figure 6. Indeed, as already discussed in the Results section, the glass transition measured by DSC is clearly attributable to hydration water; on the other hand, the “dielectric” glass transition temperatures, estimated as usual from dielectric data by calculating the temperatures T100 that correspond to a relaxation time of 102 s (see Figures 4c and 4d), are largely compatible with the “calorimetric” glass transition temperature region found by DSC (see arrows in Figure 6). [As it is well-known, there are different ways of defining Tg from a DSC heating scan (onset, midpoint); moreover calorimetric Tg depend upon cooling and heating rates.39 A direct comparison between calorimetric Tg and dielectric T100 seems therefore unreliable.40 For these reasons, here we limit ourselves to remark the agreement between T100 and the calorimetric glass transition temperature region. In any case, the nonperfect coincidence of calorimetric glass transition (where both samples show the same behavior, independently of Tg definition) and dielectric T100 (where the two T100 are different) can be attributed to the effect of different thermodynamic and dielectric sample heterogeneity.] Since the glass transition arises mainly from the R-re-
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Figure 7. Panel a: typical dielectric spectra (imaginary part) of encapsulated met-Mb at two different hydrations, at the temperature of 261 K; circles, h ) 0.3; squares, h ) 0.5. Panel b: Arrhenius plot of relaxation times; symbols as in panel a. Lines are fittings with Arrhenius law. Panel c: temperature dependence of dielectric strength; symbols as in panel a; dashed lines are guides to the eye.
Figure 8. Temperature dependence of relaxation times relative to process III. Circles and squares: encapsulated met-Mb at h ) 0.3 and h ) 0.5, respectively. Dotted circles and dotted squares: met-Mb powder at h ) 0.3 and h ) 0.5, respectively. Stars and triangles: water encapsulated in silica gels (data from ref 6). Lines are fittings with Arrhenius law.
laxation, the coincidence between “calorimetric” glass transition temperature and “dielectric” T100 suggests that the solvent relaxation responsible for process I mainly involves the R-relaxation of water in the hydration shell of the protein. (d) As shown in Figures 4e and 4f, the dielectric strength of process I is very slightly dependent on temperature, as expected for a structural relaxation of hydration water in the supercooled state. In our samples process I shows a cooperative VFT behavior in the whole temperature range investigated. Conversely, Swenson and co-workers observe in their Mb sample at h ) 0.8 a VFT-Arrhenius crossover occurring at about 170 K. We may speculate that, in the low hydration conditions of our samples, process I becomes so slow that the VFT-Arrhenius crossover falls outside the observable frequency and temperature range. We stress also that our data do not suggest the presence of a fragile-to-strong dynamical crossover (FSC) recently observed for protein hydration water around 220 K.26 Such a dynamical transition, identified by a cusp in the temperature dependence of the relaxation time, has been observed by quasielastic neutron scattering (i.e., in the nanosecond time scale) and attributed to the existence of a critical point in the onephase region of supercooled water.27 While the presence of a FSC and its implications are indeed interesting, recent data do not seem to support this claim.20,21 Relaxations in Mb Hydrated Powders: Process II. The second fastest relaxation process observed (process II in Figure 2, black circles and black squares in Figures 4c and 4d, respectively) displays an interesting VFT-Arrhenius crossover at about 235 K: at both hydration levels the relaxation times
appear to follow a VFT behavior in the high temperature region (T g 235 K, as indicated by vertical lines in Figures 4c and 4d), while they show an Arrhenius-like behavior at lower temperatures, thus indicating that the activation energy of the process becomes constant. In order to make the identification of a crossover temperature independent of visual inspection, in Figure 9 we report the χ2 obtained by fitting the τ vs T values with a VFT law from room temperature down to Tf, as a function of 1000/Tf. Here χ2 is defined as χ2 ) [∑(τiexp - τiVFT)2/σi2]/ [degrees of freedom], where σi is the experimental error associated with each τiexp. As indicated by the arrow in Figure 9, it is evident that relaxation times follow a VFT behavior only down to about 235 K, where the χ2 values start to increase steeply. Values of the high temperature VFT parameters and low temperature Arrhenius parameters for process II are reported in Table 1. Our data also show that, for this process, the hydration dependence is much weaker than for process I. Following Swenson and co-workers,18,19 we attribute process II to relaxations of polar/charged side groups on the surface of the protein, possibly with contributions from tightly bound water molecules. This is confirmed by the good agreement between the absolute values of the relaxation time of process II with that measured with hydrated lyzozyme powders and attributed to intramolecular motions of the protein likely enhanced by the presence of adsorbed water molecules (see Figure 3 of ref 41) even though no attempts were made to identify a VFT-Arrhenius crossover. A detailed comparison of our data for process II with analogous data from refs 18 and 19 shows, however, some discrepancies. In Figure 5b we report data on the “protein” process analogous to our process II taken from refs 18 and 19 and relative to hemoglobin (Hb) and Mb powders at h ) 0.8. While for all samples the presence of a VFT-Arrhenius crossover is confirmed, the Mb data at h ) 0.8 exhibit, surprisingly, a relevant hydration dependence of relaxation times and a crossover temperature of 180 K, markedly different from about 210-230 K observed for the other samples. Clearly, more data at various hydrations are necessary to clarify this point. Concerning the crossover observed for process II, two different positions are present in the recent literature to interpret its physical origin. Swenson and co-workers18,19 attribute the crossover to an R-relaxation above Tg that changes to a local beta β-relaxation below Tg. Conversely, Capaccioli et al.24,25 explain the crossover according to a well-reported phenomenon typical of β-relaxation in many glass-forming liquids, where, on crossing Tg, the structural arrest imposes a frozen structure and so a change in the temperature dependence of the intermolecular barrier; such a transition affects the β-relaxation dynamics, changing its temperature behavior from VFT to
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TABLE 1: Fit Parameters of Arrhenius Plots for met-Mb Powders and met-Mb in Silica Gel VFT parameters τ0 [s] process I powder, h ) 0.3 process I powder, h ) 0.5 process II powder, h ) 0.3 process II powder, h ) 0.5 process III powder, h ) 0.3 process III powder, h ) 0.5 process III silica, h ) 0.3 process III silica, h ) 0.5
-11
(1.5 ( 0.3) × 10 (2.9 ( 0.5) × 10-12 (1.0 ( 0.3) × 10-10 (2.1 ( 0.4) × 10-10
Arrhenius parameters
T0 [K]
D
(146 ( 12) (143 ( 10) (158 ( 10) (158 ( 12)
(9.6 ( 1.2) (7.4 ( 1.0) (8.4 ( 1.2) (7.5 ( 0.9)
τ0 [s]
∆H [kJ/mol]
(4.0 ( 0.8) × 10-20 (9.7 ( 1.8) × 10-17 (8.1 ( 1.4) × 10-16 (2.7 ( 0.6) × 10-18 (2.6 ( 0.5) × 10-14 (1.0 ( 0.3) × 10-14
(75 ( 7) (59 ( 6) (70 ( 6) (82 ( 10) (70 ( 5) (69 ( 5)
Arrhenius. To single out which of these two interpretations is correct is far outside the scope of this work, and, as a matter of fact, our data do not provide clear-cut evidence on this subject. However, the data in Figure 4 panels e and f seem to favor the interpretation of Capaccioli et al., since they indicate that the dielectric strength of process II increases with increasing temperature and therefore this process has a β-relaxation character. It is also interesting to note that the crossover occurs in the same temperature region (around 230 K) where elastic neutron scattering reveals, although in the different time scale of about 100 ps, the activation of large scale protein side chains motions10,11 (that in the framework of the two-well model42 are attributed to R-relaxations and/or to large amplitude β-relaxations), strongly coupled with solvent motions.43 As neutron scattering on deuterated samples probes essentially the dynamics of nonexchangeable hydrogen atoms of the protein, the correlation between neutron and dielectric results supports the idea that process II involves side-chain dynamics highly coupled with solvent dynamics. Finally, it is remarkable that at temperatures higher than 230 K the protein process II, although occurring on a different time scale, has the same temperature dependence as the hydration water process I. This is clearly shown in Figure 10 where, following a suggestion by Swenson and co-workers,19 we report the relaxation times relative to the protein process II as a function of those relative to the hydration water process I, for the sample at h ) 0.3 where there is a large superposition of measured temperatures. At T > 230 K, a straight line of slope 1 is observed, thus highlighting the fact that cooperative relaxations in the hydration shell are required for the protein large scale cooperative motions, as expected for “solvent slaved” relaxations.44 At temperatures below 230 K, a good fit of log(tauII) vs log(tauI) can be provided by a linear plot with slope
0.85, equivalent to a power law tauII ) A · tauI0.85, thus suggesting a decoupling of dynamical behavior of the two relaxation processes at low temperature. Interestingly, the exponent 0.85 is reminiscent of the breakdown of the Stokes-Einstein relation for the translational diffusion coefficient in supercooled water approaching the glass transition, as recently suggested.45 This would support the idea that the large scale relaxations of side chain in the protein surface are coupled to the activation of translational dynamics of adsorbed water.46 Relaxations in Mb Hydrated Powders: Process III. A third relaxation process is distinguishable in both met-Mb powder samples (process III in Figure 2, dotted circles and dotted squares in Figures 4c and 4d), much slower than the others and showing an Arrhenius temperature dependence (the absence of points at high temperatures for met-Mb at h ) 0.5 is due to the high conductivity signal which makes difficult to clearly identify the peak position). Relaxation times and activation energy are similar in the two samples: this can be explained by attributing this relaxation to inner protein motions. Consistently, the dielectric strengths for this process increase with increasing temperature. In agreement with this conjecture, in our previous work11 we reported that elastic neutron scattering data (mainly sensitive to the dynamics of inner H-atoms of protein in the 100 ps time scale) relative to the same samples do not show any drastic differences between the two met-Mb powder samples. Relaxations in Encapsulated Mb. As previously noted, dielectric behavior in the encapsulated proteins is quite “simpler” than in powders (compare Figures 2 and 3). The only relaxation process observed in our frequency-temperature experimental window is an Arrhenius process, whose activation enthalpy, obtained from the slope of Arrhenius trend, is approximately the same for both hydration levels, ∆H ≈ 70 kJ/mol (see Figure 7b and Table 1). Note that the slope is also very similar to that
Figure 9. χ2 values relative to process II as a function of Tf (see text). Black circles: met-Mb powder at h ) 0.3. Black squares: met-Mb powder at h ) 0.5. The arrow indicates the region where the temperature dependence starts to deviate from VFT behavior.
Figure 10. Relaxation times relative to the protein process II as a function of those relative to the hydration water process I, on a logarithmic scale. Data are relative to the met-Mb powder sample at h ) 0.3. The dashed line represents a straight line of slope 1, the dasheddotted one a straight line of slope 0.85.
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of the slow Arrhenius process observed in the powder samples and in pure water encapsulated in silica hydrogels6 (see Figure 8 and Table 1). Concerning the physical origin of this process, we note that relaxation times are too large to be assigned to some dynamical process of water molecules: rates of dielectric relaxation in water, even in the case of crystalline phase or in confined geometries, have never been observed in the time scale of this process. This supports the attribution of the process to some local, β-like, protein relaxation analogous to process III observed in hydrated powders. Consistently, the temperature dependence of dielectric strength ∆ε for this process (see Figure 7c) shows the typical trend of β-relaxation, where ∆ε increases with increasing temperature. Interestingly, this relaxation in the confined protein is slowed down with respect to the powder, the extent of slowing down depending on hydration and being more pronounced at low (h ) 0.3) hydration level. There is a remarkable agreement with data obtained, in the 100 ps time scale, with elastic neutron scattering on the same samples.11 Although relaxation times are slowed down by silica gel encapsulation, the slopes of the temperature dependence remain unaltered; more than that, they are almost coincident with the slope relative to the local β-like relaxation observed for pure water encapsulated in similar silica hydrogels6 (although, as expected, relaxation times for encapsulated water are much faster). This is again a clear manifestation of the “solvent slaving” effect described by Frauenfelder and co-workers:44 β-like solvent relaxations induce local protein motions so that the activation enthalpy (i.e., the slope of the Arrhenius temperature dependence) is identical. On the other hand, actual relaxation times of protein processes remain much slower because of the protein + hydration shell acting as an entropy reservoir. A pioneering study47 on dielectric relaxation of water adsorbed to crystals of met-Mb supports this scenario: it was shown that the temperature dependence (i.e., the slope in the Arrhenius plot, not the time scale) of relaxation rate of adsorbed water is nearly identical to the temperature dependence of the conformational fluctuation rate of the protein as measured by the Mo¨ssbauer effect. The most interesting finding arising from our data is that metMb confined in silica does not show (at least within the resolution of our measurements) the two faster relaxations observed in the powder. The absence of such relaxations in gel is a direct evidence of the main effect of confinement in silica at low hydration, i.e. the hindering of collective processes of both solvent in the hydration shell and protein. This behavior can be explained by considering the protein-water system as a hydrogen bond network arranged into clusters: collective fluctuations of this network furnish the physical mechanism to couple conformational protein motions with water relaxations. This is the way solvent structure and protein dynamics are correlated. Havenith and co-workers48 have recently found that molecular motions (involving fluctuations of hydrogen bonds network) of a very large extent of water molecules are affected by the presence of protein, at the picosecond time scale; their data suggest that only a coherence between protein and water fluctuations can preserve the collective motions of solvent. In the presence of a rigid and poorly interacting confining structure (like the surfaces of silica pores), the coherence/cooperativity of solvent motions and, as a consequence, solvent-coupled protein dynamics is expected to be strongly affected and, at a low hydration levels, the collective motions involved can be effectively destroyed. On the contrary, in Mb powders the confining agent, i.e. the protein, is able to correctly propagate fluctuations in the hydrogen bonds network, even at a very low
Schiro` et al. water content, where dimensions of solvent confinement are comparable with those in silica. The degree of softness of the host matrix (i.e., the confining agent, in a generalized sense) where protein + hydration water is embedded seems to be an essential parameter to guarantee the preservation of hydration water-protein coupled fluctuations. Conclusions The principal results of the present work are as follows: (1) The dielectric behavior observed in met-Mb powders is characterized by three different relaxation processes: (I) a VFT-like process attributed to hydration water dynamics, (II) an intermediate process attributed mainly to motions of polar side chains on the protein surface showing a crossover between VFT and Arrhenius temperature dependence at T g 230 K, the same temperature range where neutron scattering11,42 (although in a different time scale) indicates the activation of large scale protein motions, and (III) a slow Arrhenius process mainly related to β-like relaxations of inner amino acid side chains in the protein (2) Only the slow Arrhenius relaxation process is observable in the sol-gel encapsulated met-Mb (corresponding to process III in the case of the powder sample discussed above); it shows a dependence on hydration level that can be interpreted in terms of slaving model of protein dynamics as proposed by Frauenfelder and co-workers.44 Dielectric spectroscopy data highlight a clear and specific effect of silica encapsulation on protein dynamics, although water content and dimensions of confinement are comparable to those of powder systems. We think this peculiarity can be explained in the following way: confinement of protein-solvent systems inside a rigid matrix affects the coherence/cooperativity of solvent motions and, as a consequence, solvent-coupled protein dynamics.48 This is clearly evidenced by dielectric spectroscopy which gives a direct picture on the relaxation dynamics nature: cooperative relaxations observed in met-Mb powders are not present in met-Mb encapsulated in silica gel. Acknowledgment. We thank Dr. S. E. Pagnotta for help with dielectric spectroscopy measurements, Dr. Gianluca Di Cara and Prof. Ida Pucci Minafra for lyophilization of deuterated Myoglobin, and Dr. G. Bellavia for help with the calorimetric measurements. This work has been supported by a grant from the University of Palermo (ex 60% funds). References and Notes (1) Minton, A. P. J. Biol. Chem. 2001, 276, 10577. (2) Ellis, R. J.; Minton, A. P. Nature 2003, 425, 27. (3) Ellerby, L. M.; Nishida, C. R.; Nishida, F.; Yamanaka, S. A.; Dunn, B.; Valentine, J. S.; Zink, J. I. Science 1992, 255, 1113. (4) Shibayama, N.; Saigo, S. J. Mol. Biol. 1995, 251, 203. (5) Cupane, A.; Levantino, M.; Santangelo, M. G. J. Phys. Chem. B 2002, 106, 11323. (6) Cammarata, M.; Levantino, M.; Cupane, A.; Longo, A.; Martorana, A.; Bruni, F. Eur. Phys. J. 2003, E12, S63. (7) Levantino, M.; Cupane, A.; Zimanyi, L. Biochemistry 2003, 42, 4499. (8) Schiro`, G.; Cupane, A. Biochemistry 2007, 46, 11568. (9) Schiro`, G.; Cupane, A.; Pagnotta, S. E.; Bruni, F. J. Non-Cryst. Solids 2007, 353, 4546. (10) Schiro`, G.; Sclafani, M.; Caronna, C.; Natali, F.; Plazanet, M.; Cupane, A. Chem. Phys. 2008, 345, 259. (11) Schiro`, G.; Sclafani, M.; Natali, F.; Cupane, A. Eur. Biophys. J. 2008, 37, 543. (12) Curtis, J. E.; Dirama, T. E.; Carri, G. A.; Tobias, D. J. J. Phys. Chem. B 2006, 110, 22953. (13) Rupley, J. A.; Careri, G. AdV. Protein Chem. 1991, 41, 37.
Dielectric Relaxations in Confined Hydrated Myoglobin (14) Careri, G.; Geraci, M.; Giansanti, A.; Rupley, J. A. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 5342. (15) Careri, G.; Giansanti, A.; Rupley, J. A. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 6810. (16) Pizzitutti, F.; Bruni, F. Phys. ReV. E 2001, 64, 052905. (17) Pagnotta, S. E.; Gargana, R.; Bruni, F. Phys. ReV. E 2005, 71, 031506. (18) Jansson, H.; Bergman, R.; Swenson, J. J. Phys. Chem. B 2005, 109, 24134. (19) Swenson, J.; Jansson, H.; Bergman, R. Phys. ReV. Lett. 2006, 96, 247802. (20) Swenson, J.; Jansson, H.; Hedstro¨m, J.; Bergman, R. J. Phys.: Condens. Matter 2007, 19, 205109. (21) Pawlus, S.; Khodadadi, S.; Sokolov, A. P. Phys. ReV. Lett. 2008, 100, 108103. (22) Khodadadi, S.; Pawlus, S.; Sokolov, A. P. J. Phys. Chem. B 2008, 112, 14273. (23) Cerveny, S.; Schwartz, G. A.; Alegrı´a, A.; Bergman, R.; Swenson, J. J. Chem. Phys. 2006, 124, 194501. (24) Capaccioli, S.; Ngai, K. L.; Shinyashiki, N. J. Phys. Chem. B 2007, 111, 8197. (25) Ngai, K. L.; Capaccioli, S.; Shinyashiki, N. J. Phys. Chem. B 2008, 112, 3826. (26) Chen, S. H.; Liu, L.; Fratini, E.; Baglioni, P.; Faraone, A.; Mamontov, E. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 9012. (27) Liu, L.; Chen, S. H.; Faraone, A.; Yen, C.-W.; Mou, C.-Y. Phys. ReV. Lett. 2005, 95, 117802. (28) Swenson, J. Phys. ReV. Lett. 2006, 97, 189801. (29) Cerveny, S.; Colmenero, J.; Alegrı´a, A. Phys. ReV. Lett. 2006, 97, 189802. (30) Pizzitutti, F.; Bruni, F. ReV. Sci. Instrum. 2001, 72, 2502.
J. Phys. Chem. B, Vol. 113, No. 28, 2009 9613 (31) Havriliak, S.; Negami, S. Polymer 1967, 8, 161. (32) Liu, S. H. Phys. ReV. Lett. 1985, 55, 529. (33) Sartor, G.; Hallbrucker, A.; Mayer, E. Biophys. J. 1995, 69, 2679. (34) Steinbach, P. J.; Brooks, B. R. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9135. (35) Lounnas, V.; Pettitt, B. M. Proteins 1994, 18, 133. (36) Doster, W.; Bachleitner, A.; Dunau, R.; Hiebl, M.; Lu¨scher, E. Biophys. J. 1986, 50, 213. (37) Gottfried, O.; Edvards, L.; Wu¨thrich, K. Science 1991, 254, 974. (38) Pal, S. K.; Peon, J.; Zewail, A. H. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 1763. (39) Ho¨hne, G. W.; Hemminger, W. F.; Flammersheim, H. J. Differential Scanning Calorimetry; Springer-Verlag: Berlin, 2003. (40) Johari, G. P.; Aji, D. P. B. J. Chem. Phys. 2008, 129, 056101. (41) Khodadadi, S.; Pawlus, S.; Roh, J. H.; Garcia Sakai, V.; Mamontov, E.; Sokolov, A. P. J. Chem. Phys. 2008, 128, 195106. (42) Doster, W.; Cusack, S.; Petry, W. Nature 1989, 337, 754. (43) Wood, K.; Fro¨lich, A.; Paciaroni, A.; Moulin, M.; Ha¨rtlein, M.; Zaccai, G.; Tobias, D. G.; Weik, M. J. Am. Chem. Soc. 2008, 130, 4586. (44) Fenimore, P. W.; Frauenfelder, H.; McMahon, B. H.; Parak, F. G. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16047. (45) Mazza, M. G.; Giovambattista, N.; Stanley, H. E.; Starr, F. W. Phys. ReV. E 2007, 76, 031203. (46) Tarek, M.; Tobias, D. J. Phys. ReV. Lett. 2002, 88, 138101. (47) Singh, G. P.; Parak, F.; Hunklinger, S.; Dransfeld, K. Phys. ReV. Lett. 1981, 47, 685. (48) Ebbinghaus, S.; Kim, S. J.; Heyden, M.; Yu, X.; Heugen, U.; Gruebele, M.; Leitner, D. M.; Havenith, M. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 20749.
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