structure and dynamics of lysozyme encapsulated in a silica sol−gel matrix

Structure and Dynamics of Lysozyme Encapsulated in a Silica Sol-Gel Matrix

Isabel Pastor,† Maria L. Ferrer, ‡ M. Pilar Lillo, £ Javier Gomez,† and C. Reyes Mateo*,†

Instituto de Biologıá Molecular y Celular, UniVersidad Miguel Hernańdez, 03202-Elche, Spain, Instituto deCiencia de Materiales de Madrid, CSIC, Campus de Cantoblanco, 28049-Madrid, Spain, and Instituto deQuımica Fısica “Rocasolano”, CSIC, 28003-Madrid, Spain

ReceiVed: June 20, 2007; In Final Form: July 30, 2007

Proteins entrapped in sol-gel matrices have been extensively studied during the last 15 years, showing thatmost of them can be encapsulated with retention of their native structure and functionality and with enhancedstability. However, relatively little is known about the structural and dynamical details of the biomolecule-matrix interactions. To achieve this goal, the model protein hen egg white lysozyme (HEWL) has been entrappedin sol-gel matrices prepared from tetraethyl orthosilicate through an alcohol-free sol-gel route, and thephotophysical properties of its fluorescent tryptophans have been determined using both steady-state andtime-resolved fluorescence techniques. By combining fluorescence spectra, quenching experiments, lifetimes,and time-resolved fluorescence anisotropy measurements, we have obtained information on the structure,dynamics, and solvation properties of the entrapped protein. Our results show that the environment of HEWLwithin the silica pore as well as its internal dynamics is similar to that in aqueous solution, except that theprotein showed no or, depending on conditions, very much slower global motion but retained its internalangularly restricted (hindered) segmental rotation upon entrapment. The experiments carried out at differentexperimental conditions indicate that, below the isoelectric point of the protein, a strong electrostatic interactionis established between the protein molecule and the negatively charged sol-gel walls, which is ultimatelyresponsible for the total arrest of the overall rotation of the protein, but without significant effect upon itssegmental rotational relaxation. The electrostatic nature of the interaction is clearly established since eitherreducing the positive charge of the protein (by increasing the pH toward its isoelectric point) or increasingthe ionic strength of the solution (shielding against the attractive interaction) leads to a situation in which theprotein freely rotates within the matrix pore, albeit an order of magnitude more slowly than that in freesolution under similar macroscopic solution conditions, and still retains its segmental rotational properties.

Introduction

Silica sol-gel materials have been shown to form excellentmedia for immobilization of proteins, among them, activeenzymes, and other biologicals macromolecules.1-3 The pastdecade has seen a great deal of progress in the encapsulationof these biomolecules in sol-gel matrices by modifying olderconventional procedures that were designed to afford morebiocompatible environments in such ways as to optimizefunctionality in the resulting bioceramic.4-7 The entrappedbiomacromolecule usually retains its structural integrity andfunctionality and, being accessible to small molecules diffusingintothegel,maybeusedinanalytical2,8-13andbiotechnological1,14-17

applications. Bioencapsulation in silica matrices has also beeninvestigated as an experimental system for testing the effectsof molecular confinement on the structure and stability ofproteins18,19 and for controlling the kinetics of the unfoldingand refolding processes, as well as for trapping and character-izing unstable forms that are likely candidates for transition-state species.20-22 Quite apart from extending the applicabilityof these materials to new applications, there is still a need forsome fundamental questions to be addressed, for example, aboutthe nature of the biomolecule-matrix interactions and how they

might be modulated in a rational way. To help toward achievingthis goal, the rotational motions of different model proteinsentrapped in sol-gel glasses have been analyzed throughdifferent spectroscopic techniques, and the results have beeninterpreted in terms of electrostatic and/or hydrogen bondinteractions between the protein and the silica matrix7,23-25

(which has an isoelectric point, pI, of∼2) or in terms of changesin the effective viscosity sensed by the protein.26-28 It shouldbe noted that the influence of immobilization on the dynamicproperties may vary among different proteins, depending onconstraints caused by heterogeneity of the surface charge densityof the protein and its pH-dependent interaction with thenegatively charged silica matrix. In the case of the greenfluorescent protein mutant GFPmut2, negatively charged atphysiological pH, time-resolved fluorescence depolarizationexperiments indicated that the protein environment inside ofthe silica pores is similar to the one sensed by the protein inthe aqueous solution and that unhindered molecular rotationsoccur.24 On the contrary, for the human serum albumin,significant restriction of its global rotational motion wasobserved, despite its overall negative charge at physiologicalpH.7 This behavior was ascribed to differences in the electro-static interactions between the sol-gel matrix and the individualdomains of the protein. These results are also consistent withthe significantly restricted rotation observed, at neutral pH, formyoglobin (pI∼ 7.2) by fluorescence depolarization27 and forhighly basic horse heart cytochrome c (pI) 9.45) by NMR.23

* To whom correspondence should be addressed. Fax:+34 966 658758. E-mail: [email protected].

† Universidad Miguel Hernańdez.‡ Instituto de Ciencia de Materiales de Madrid.£ Instituto de Quı´mica Fısica “Rocasolano”.

11603J. Phys. Chem. B2007,111,11603-11610

10.1021/jp074790b CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 09/13/2007

It has been suggested that these proteins interact with, or areadsorbed to, the porous walls of the host matrix throughhydrogen bonds or electrostatic interactions which effectivelyhinder the free rotation of the protein.29

With the aim of casting some light on the role of protein-matrix interactions on the structural and dynamic properties ofthe entrapped biomolecules, we have encapsulated the proteinhen egg white lysozyme (HEWL), a small globular, well-characterized enzyme (molecular mass 14.4 kDa), which ishighly positively charged at neutral pH (isoelectric point∼11)and therefore likely to be attracted to the silica surface of thesol-gel glass. HEWL contains six tryptophan residues atlocations 28, 62, 63, 108, 111, and 123, two of which (Trp 62and Trp 108), both partially exposed to the solvent, areresponsible for most of its intrinsic fluorescence.30-32 In thepresent work, HEWL was entrapped in sol-gel matricesprepared from tetraethyl orthosilicate through an alcohol-freesol-gel route, and the photophysical properties of its fluorescenttryptophans were determined using steady-state and time-resolved fluorescence experiments. By combining the resultsfor fluorescence spectra, quenching experiments, lifetimes, andtime-resolved fluorescence anisotropy measurements, we haveobtained information on the structure, dynamics, and averageenvironment sensed by the entrapped protein. Our resultsindicate that, at neutral pH and low ionic strength, there is aqualitative change in the rotational behavior of HEWL entrappedin the pores as compared with that in the bulk aqueous solution,whereas in the latter, global rotation is free and unrestricted,and entrapment in the pores under these conditions completelyinhibits the global rotation, though it does leave the segmentalrotation essentially unaffected. The origin of the completeablation of free global rotation of the protein when in the poresunder these conditions is shown to be mainly electrostatic innature since either reduction of the total positive charge carriedby the protein (by increasing the pH toward its pI) or an increasein the ionic strength of the solution leads to complete removalof this constraint, though the global rotation ensuing is verymuch slower than that in the bulk solution.

Materials and Methods

Chemicals. HEWL (EC 3.2.1.17; 50200 U mg-1) andtetraethyl orthosilicate (TEOS) were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI). Other chemicals wereof analytical or spectroscopic reagent grade. HEWL wasdissolved in a 10 mM, pH 7.4, sodium phosphate buffer preparedwith deionized doubly distilled water. Other buffers were sodiumphosphate buffer (10 mM, pH 7.4, 3 M NaCl) and borate buffer(10 mM, pH 9). The protein concentration was determined fromUV absorbance at 280 nm using an absorption coefficient ofε

) 2.65 mg-1 mL cm-1.33

Immobilization of HEWL in Sol -Gel Monoliths. HEWLwas encapsulated in pure silica matrices prepared through thealcohol-free sol-gel route described in Ferrer et al.5 (2002).Briefly, 4.46 mL of TEOS, 1.44 mL of H2O, and 0.04 mL HCl(0.62 M) were mixed with vigorous stirring at 22°C in a closedvessel. After 1 h, 1 mL of the resulting sol was mixed with 1mL of deionized water and submitted to rotaevaporation for aweight loss of 0.62 g (i.e., 0.62 g is approximately the alcoholmass resulting from alkoxyde hydrolysis). The aqueous sol wasmixed with 1 mL of buffered HEWL (20µM) in a disposablepoly(methyl methacrylate) cuvette. Gelation occurred readilyafter mixing. Following gelation, monoliths were wet-aged inthe phosphate buffer solution at 4°C for 48 h. After aging, themonoliths, of size∼9 × 9 × 19 mm, were removed from the

disposable cuvettes and placed in 10× 10 mm quartz cuvettesin the presence of 500µL of the desired buffer. In most ofexperiments, this buffer was sodium phosphate buffer (10 mM,pH 7.4). For quenching experiments, the same buffer containingdifferent concentrations of acrylamide was used, while for highionic strength and alkaline pH experiments, the buffer wasreplaced by phosphate buffer, 10 mM (3 M NaCl, pH 7.4) andborate buffer (10 mM, pH 9), respectively, and was allowed toequilibrate with the monoliths for 48 h before measurementswere made. Since the pKa of phosphate is known to changedramatically with ionic strength,34 the pH of the NaCl-containingbuffer was always adjusted in the presence of the salt. Blankmonoliths were prepared as above but with the protein solutionreplaced by the working buffer.

Absorption and Steady-State Fluorescence Measurements.Absorption measurements were carried out at 22°C using aShimadzu UV-1603 spectrophotometer (Shimadzu, Tokyo,Japan). Fluorescence measurements were performed in an SLM-8000C spectrofluorimeter (SLM Instruments Inc., Urbana, IL)fitted with Glan-Thompson polarizers. The experimentalsamples (sol-gel monoliths and protein aqueous solution) wereplaced in 10× 10 mm path length quartz cuvettes. The steady-state anisotropy⟨r⟩, defined by

was obtained as a function of temperature by measuring thevertically (parallel) and horizontally (perpendicular) polarizedcomponents of the fluorescence emission with the excitationpolarized vertically. TheG factor (G ) IHV/IHH) corrects forbias in transmissivity between vertically and horizontallypolarized components of the emission introduced by thedetection system. Samples were excited at 300 nm with abandwidth of 4 nm, and the polarized emission was detected at340 nm with a bandwidth of 8 nm. Background intensitiesarising from the sol-gel matrix, which contribute less than 5%to the total signal, were always taken into account and subtractedfrom the measured sample intensities.

Time-Resolved Fluorescence Measurements.The decay ofthe total fluorescence intensity, and those of the parallel andperpendicularly polarized components, were recorded at 20°Cin a single-photon timing system using an experimental setupsimilar to that described previously by Lillo et al.35 In brief,the tryptophan fluorophores of HEWL were excited by meansof vertically polarized light pulses from a Ti:sapphire picosecondlaser (Tsunami, Spectra Physics) pumped with a 5W Nd:YVO4

laser (Millennia, Spectra Physics) and associated with a thirdharmonic generator tuned to 297 nm. Pulses of 1-2 µs widthwere generated at a repetition rate of 4 MHz, giving∼20 µWof average power at the samples. The experimental samples(sol-gel monoliths and aqueous protein solution) were placedin 10 × 10 mm path length cuvettes. Data were stored in 4Kchannels, at a resolution of 11 or 6.1 ps/channel, up to∼5 ×106 total counts. The decay of the total fluorescence intensityIm(t) was recorded at 340 nm with the emission polarizer set atthe magic angle (54.7°) relative to the vertically polarizedexcitation beam. The parallel and perpendicular components ofemission were sequentially recorded at 340 nm by alternatingthe orientation of the emission polarizer every 2 min.

The kinetic parameters of the impulse response fluorescenceintensity decay,im(t) (lifetimes τi and normalized amplitudesRi), were determined by reconvolution and fitting, usingnonlinear least-squares regression methods. The amplitude-

⟨r⟩ )IVV - GIVH

IVV + 2GIVH(1)

11604 J. Phys. Chem. B, Vol. 111, No. 39, 2007 Pastor et al.

weighted lifetimeτj, proportional to the quantum yield anddefined by36

and the average fluorescence lifetime defined by36

were calculated. The anisotropy decay function,r(t), wasdetermined by simultaneous reconvolution and fitting to the twoexperimentally determined polarized components of the fluo-rescence intensity, using nonlinear least-squares global analysisimplemented by GLOBALS Unlimited (Urbana, IL). In thiscase, the analysis consisted of finding the numerical parametersfor r(t) that best fit the two polarized decay functions,iVV(t) )[im(t)/3][1 + 2r(t)] and iVH(t) ) [1/G][ im(t)/3][1 - r(t)], to theexperimental tracesIVV(t) and GIVH(t) upon introducing thelifetime obtained from the magic angle data as a fixed parameter.The general expression used for the anisotropy decay fittingwas a sum ofn exponentials and a constant term37

where

and æi are the rotational correlation times,âi are normalizedamplitudes, andr∞ is the residual anisotropy, containinginformation about the restriction of the depolarizing processes.To perform this analysis, the first channels of the experimentaldecays were not included in the fit in order to avoid differentscattering contributions from the sol-gel monolith amongdifferent samples.

The fits tabulated here both for fluorescence intensity andanisotropy decay represent the minimum set of adjustableparameters which satisfy the usual statistical criteria, namely,a reducedø2 value of <1.3 and a random distribution ofweighted residuals.

Quenching Experiments.Solvent accessibility of the fluo-rescent tryptophans in both solution-free and sol-gel-entrappedlysozyme were analyzed by monitoring the quenching of proteinfluorescence induced by acrylamide. For solution studies, a 10µM HEWL solution was titrated by adding increasing amountsof 5.0 M acrylamide stock solution with continuous stirring,and the fluorescence intensity was corrected by sample dilutionand recorded at 340 nm with a bandwidth of 2 nm (λexc ) 290nm, bandwidth 2 nm). For the quenching studies of theentrapped protein, the diffusion constraints imposed by thedimensions of the monoliths and the restriction to the freediffusion of the quencher through the sol-gel matrix requiredits incubation with the quencher solution for at least 48 h beforeconcentration gradients could be neglected. A different monolithcontaining the same protein concentration was incubated witheach quenching concentration. Data were analyzed accordingto the Stern-Volmer relationship38

whereI0 andI stand for the steady-state fluorescence intensitiesin the absence and in the presence of quencher, respectively,and [Q] is the quencher concentration. Since no significantdeviation was detected in these plots, the “static” quenchingcontribution was considered to account for less than 10% ofthe observed signal, and the Stern-Volmer constant,KSV, wasdirectly obtained from the slope of the linear relationship. Thisparameter represents the weighted sum of the individualconstants of the emitting tryptophan residues and is thereforereferred to asKSV,eff in the text.KSV,eff was used to calculatethe bimolecular rate constant of the quenching process,kQ,eff,from the expressionKSV,eff ) kQ,eff‚τj.38

Results and Discussion

Photophysical Properties of HEWL in Sol-Gel Monoliths.Fluorescence excitation and emission spectra of HEWL insolution and immobilized in sol-gel matrices are shown inFigure 1 for the same protein concentration (10µM). The shapesof these spectra, as well as their maximum intensities, are similarfor the protein in both media, suggesting that the entrapmentefficiency is near unity (i.e., leaching is minimal), and only aslight blue shift (<2 nm) is observed, in both excitation andemission spectra, upon sol-gel encapsulation. This small shiftcannot be attributed to partial unfolding of HEWL since theemission spectrum of the fully denaturated protein is shiftedby 14 nm to the red.39 There are then several possibleexplanations for this behavior. These include slight differencesin the solvation of the native-state surface of the entrappedprotein (solvated by both Si-OH groups and interstitial watermolecules), as compared to their solvation by water moleculesin the bulk solvent, or changes in the mobility of the watermolecules within the sol-gel pore. The later possibility is mostlikely to be correct and has previously been invoked to explainthe blue shift observed in the fluorescence emission spectrumof N-acetyl tryptophanamide (NATA) immobilized in a sol-gel matrix.40 The slower response of the water molecules couldbe due to the presence of Si-OH groups on the porous matrixwall establishing strong hydrogen bonds with individual watermolecules, which may influence their bulk behavior.

Further information regarding the environment of the fluo-rescent tryptophans was obtained from analysis of the fluores-cence decays of the free and sol-gel entrapped HEWL.Experimental decays were best fitted to triexponential functions,and the results, together with theτj and⟨τ⟩ derived from them,are shown in Table 1. A definite assignment of the observeddiscrete lifetime components to the different fluorescent Trpresidues is difficult for multi-tryptophan proteins like HEWL.In this case, it is more useful to compare the averagefluorescence lifetimes⟨τ⟩. These values were found to be similarfor the protein either in buffer solution or encapsulated in thesol-gel matrix but significantly different from that obtainedfor lysozyme adsorbed at a silica/water interface, for which⟨τ⟩decreased from 2.1 to 1.4 ns upon immobilization on silica, thedifference being ascribed to some partial adsorption-inducedunfolding.41 This is not the case for the sol-gel-entrappedHEWL, where a value of 2.2 ns was obtained for⟨τ⟩, suggestingthat the water content within the sol-gel pore is able to maintainthe protein in an essentially native conformation. Similarconclusions were previously reached by Eggers and Valentineby circular dichroism experiments, which demonstrated thatHEWL retains a native-like solution structure following sol-gel encapsulation.19

τj ) ∑ Riτi (2)

<τ> )∫0

∞t × i(t)dt

∫0

∞i(t)dt

)

∑i

Riτi2

∑i

Riτi

(3)

r(t) ) (r(0) - r∞)[∑i)1

n

âi exp(-t/æi)] + r∞ (4)

∑i)1

n

âi ) 1

I0

I) 1 + KSV[Q] (5)

Lysozyme Encapsulated in a Silica Sol-Gel Matrix J. Phys. Chem. B, Vol. 111, No. 39, 200711605

Quenching Experiments. Conformational changes in thetertiary structure of proteins, and especially solvent accessibilityof the tryptophan residues, can also be indirectly inferred fromfluorescence quenching experiments. For this purpose, the effectof acrylamide on the intrinsic fluorescence of entrapped HEWLwas examined and compared with that observed for the freeprotein. For multi-tryptophan proteins such as lysozyme, de-termination of quenching constants would be ambiguous without

selective replacement of Trp residues by nonfluorescent residues.However, in this work, we did not pursue a rigorous quantitativeinterpretation of the quenching data, limiting ourselves tocomparing changes observed in the average fluorescencequenching efficiency upon sol-gel encapsulation. With this inmind, experimental data were analyzed in terms of eq 5. TheStern-Volmer plots were linear, and theKSV,eff values extractedfrom the slopes (Figure 2) were almost identical for both thefree and immobilized protein at (7.2( 0.3) and (7.8( 0.5)M-1, respectively, and similar to the value recently reported ina buffer, for observation at the same emission wavelength, of(6.8 ( 0.1) M-1 at 340 nm.42 The same pattern was observedfor the quenching constant,kQ,eff; values of (5.5( 0.3) × 109

and (6.0( 0.6) × 109 M-1 s-1 were obtained for the free andthe immobilized HEWL, respectively, indicating that acces-sibility of this quencher to the fluorescent tryptophans remainsessentially unaltered upon immobilization. This result supportsthe hypothesis that immobilization does not lead to any majorconformational change, at least in the region of the enzymaticbinding site where the two highly fluorescent tryptophanresidues (Trp 62 and Trp 108) are located.31,43

Dynamics of Sol-Gel-Entrapped HEWL. Steady-statefluorescence anisotropy measurements were made as a first stepto analyze the effect of encapsulation on the dynamicalproperties of HEWL. At a given temperature, the anisotropy ofthe immobilized protein was very similar to that observed insolution (Figure 3). As temperature was increased, the steady-state anisotropies decreased slightly in a similar way for bothsystems. Since the fluorescence lifetimes of HEWL remainedunmodified upon encapsulation, this result suggests that therotational mobilities of the free and encapsulated protein arerather similar. However, it is known that steady-state anisotropyvalues simultaneously contain both structural and dynamicalinformation which cannot be resolved from a simple steady-state experiment.44 To resolve these aspects, we examined thetime-resolved anisotropy decay of HEWL in solution andimmobilized in the sol-gel matrix at 20°C. Representativeexperimental data on the time-dependent decay of proteinanisotropy are presented in Figure 4. Two aspects of these datashould be highlighted from these data, (a) the anisotropy decayfollows very different kinetics for the protein free in solutionand that entrapped in the sol-gel matrices and (b) while free-protein anisotropy, as expected, tends to zero as time increases,a high nonzero residual anisotropy is quite rapidly reached bythe entrapped protein, signifying structural constraints to theextent of its rotation. The results of the deconvolution analysisof these experimental data are listed in Table 2. For HEWL insolution, two rotational correlation times were needed to describethe decay process, a subnanosecond component (around 0.3 ns)and a slower component of 4.5 ns. Since the long rotationalcorrelation time (æ2) was considerably higher than the shortone (æ1), the total anisotropy can be interpreted as the productof two independent depolarizing processes, a first one due tofast restricted segmental motion of the tryptophan residueswithin the protein (r′(t)) and a second one related to the globalprotein rotation45

where

whereS1 is an order parameter characterizing the restricted rangeof internal angular fluctuation of the tryptophan residues, and

Figure 1. Corrected excitation (λem ) 340 nm, bandwidth 2 nm) andemission (λex ) 295 nm, bandwidth 2 nm) spectra of 10µM HEWL inbuffer (s) and entrapped in a sol-gel matrix (- - -).

Figure 2. Stern-Volmer plots for acrylamide quenching of 10µMHEWL in solution (0) and entrapped in a sol-gel matrix (b).

Figure 3. Variation with temperature of the steady-state fluorescenceanisotropy,⟨r⟩, of HEWL in solution (0) and entrapped in a sol-gelmatrix (b). Samples were excited at 300 nm (bandwidth 4 nm), andfluorescence was recorded at 340 nm (bandwidth 8 nm).

r(t) ) r′(t) exp(-t/æglobal) (6a)

r′(t) ) r(0)[(1 - S12) exp(-t/æsegmental) + S1

2] (6b)


the long and short rotational correlation times obtained fromthe fit are respectively related toæglobal andæsegmental(Table 3)by

and

The value ofæglobal is in reasonable agreement with thatreported by Nishimoto et al.46 in a rigorous study in which thefluorescent tryptophans were selectively substituted with non-fluorescent residues. Assuming spherical symmetry for thenative (globular) state of HEWL, this correlation time can beexpressed in terms of the Stokes-Einstein relationship as

whereη is the viscosity of the medium,V is the hydrated volumeof the protein,k is the Boltzmann constant, andT is the absolutetemperature. The volume of the equivalent sphere obtained was1.82× 10-26 m3, which is in agreement with previously reportedvalues.47,48

The segmental correlation time (0.3 ns) reflects an averageof the fast localized motions of the fluorescent tryptophan

residues. The range of angular displacement of these motionscan be derived fromS1 assuming a “wobbling-in-cone” model.49

From this model, the half-angleθ0 of the cone within whichthe segments containing tryptophans freely rotate is given bycosθ0 ) (1/2)[(8S1 + 1)1/2 - 1]. An angle of (20( 1)° wasobtained for HEWL in free solution (Table 3), in agreementwith the data reported by Czeslik et al.,41 and suggests that thesesegments experience relatively little angular displacement withrespect to the protein as a whole during the tryptophan lifetime.The r(0) value obtained was lower than the value of 0.28expected for an immobilized tryptophan residue at the sameexcitation wavelength.50 The existence of an additional ultrafastrotational motion, on the subpicosecond/picosecond time scale,that cannot be observed with our instrument might be one sourceof this depolarization. Another is ultrarapid Fo¨rster resonanceenergy transfer between two closely apposed tryptophan moietieswith their transition dipole moments not aligned parallel to eachother (homoFRET), such as the very close pair Trp 62 and 63in HEWL. Slower homoFRET between the more distant Trp62 and Trp 108 residues has been also suggested for thisprotein.31 If it was the case, the value of the short correlationtime (0.3 ns), which has been entirely related to the segmentalmotion of the tryptophan residues, could be partially effectedfor this process. Nevertheless, the fact that two correlation timesof 0.22 and 4.60 ns have been determined for the anisotropydecay of HEWL, in which Trp 62 was replaced with a tyrosineresidue,46 indicates that this effect would be negligible.

For entrapped HEWL, only one correlation time was obtained,which was very similar, if not indeed virtually identical (seeTable 2), to that of the fast component observed in solution.The most significant change was the loss of the global rotationalcomponent and its replacement by the appearance instead of ahigh residual anisotropy,r∞) 0.14 (Table 2). Analogously tothe model used above for the rotational properties of HEWL insolution, the total anisotropy was interpreted as the product oftwo independent depolarizing processes related to the segmentalmotion of the Trp residues of the entrapped protein and to theoverall rotation of the protein within the matrix pore. Withinthis model, the existence ofr∞ can be explained byæglobal f ∞so thatr(t) ) r′(t) andr∞ ) r(0)S1

2 (eq 6) and, therefore,S1 andthe corresponding half-cone angleθ0 can be extracted from ther∞ value (Table 3). These results suggest that, under theseconditions, the global rotation of the protein is completelyhindered upon sol-gel entrapment, whereas the internal dynam-ics (i.e., the average segmental motions of the tryptophanresidues and the resulting angular displacement of these motions)and the putative homoFRET process mentioned above are notaltered from those obtained for the protein in the bulk solvent.

The isoelectric point of lysozyme is about pH 11, and atneutral pH, this protein is therefore positively charged. Elec-trostatic interactions between the pore wall of the host matrix

TABLE 1: Photophysical Parameters of HEWL (Fluorescence Lifetimes,τi, and Normalized Amplitudes, ri) in Solution andImmobilized in Sol-Gel Matrices under Different Experimental Conditions, T ) 20 °C

mediumR1

(0.01τ1 (ns)(0.1

R2

(0.01τ2 (ns)(0.1

R3

(0.01τ3 (ns)(0.1 ø2 τja (ns) <τ>b (ns)

bufferpH ) 7

0.37 0.3 0.45 1.4 0.17 3.3 1.2 1.3 2.2

sol-gelpH ) 7

0.41 0.3 0.42 1.3 0.17 3.5 1.2 1.3 2.2

sol-gelNaCl 3 M

0.52 0.4 0.32 1.4 0.16 3.5 1.2 1.3 2.2

sol-gelpH ) 9

0.49 0.4 0.34 1.4 0.17 3.5 1.2 1.3 2.2

a Determined from eq 2.b Determined from eq 3.

TABLE 2: Time-Resolved Fluorescence AnisotropyParameters of HEWL (Rotational Correlation Times, æi,Amplitudes, âi, and Residual Anisotropy, r∞) in Solution andImmobilized in Sol-Gel Matrices at Different ExperimentalConditions, T ) 20 °C

medium r(0) â1 æ1 (ns) â2 æ2 (ns) r∞ ø2

bufferpH ) 7

0.18 0.17 0.3 0.83 4.5 0 1.29

sol-gelpH ) 7

0.17 1.0 0.3 0.140 1.18

sol-gelNaCl 3 M

0.16 0.15 0.3 0.85 37 0 1.13

sol-gelpH ) 9

0.16 0.15 0.3 0.85 36 0 1.20

TABLE 3: Parameters (Segmental and Global CorrelationTimes, æi, Order Parameter S1, and Half-Cone Angle,θ0) forthe Fit of the Independent-Two-Motion Model (eqs 6 and 7)to the Anisotropy Decay of HEWL in Solution andImmobilized in Sol-Gel Matrices at Different ExperimentalConditions, T ) 20 °C

medium æsegmental(ns) æglobal(ns) S1 θ0

buffer, pH) 7 0.3 4.5 0.91 20°sol-gel, pH) 7 0.3 0.91 20°sol-gel + NaCl 3M 0.3 30-39 0.92 19°sol-gel, pH) 9 0.3 30-40 0.92 19°

æ2 ) æglobal (7a)

æ1 ) ( 1æsegmental

+ 1æglobal

)-1(7b)

æglobal )ηVkT

(8)


(negatively charged under the prevailing experimental condi-tions) and the protein surface could then be the cause of thetotal arrest of the overall, global rotation of the entrappedprotein. This possibility has recently been suggested by Wheeleret al.23 who showed by NMR spectroscopy that encapsulatedanionic and neutral proteins undergo essentially free rotationwithin the sol-gel pore, while rotation is strongly hindered forpositively charged proteins. To confirm this hypothesis, we havecarried out two different experiments with the goal of reducingsuch electrostatic interactions, one with solvent at high ionicstrength (NaCl 3M) and the other with solvent at pH) 9. Inboth situations, the fluorescence decays of the entrapped HEWLwere best fitted to triexponential functions. The parametersrecovered are shown in Table 1 and were found to be similarto those recovered for HEWL, both in solution and entrappedin sol-gel, at pH 7, in the absence of NaCl. Figure 5A and Bshows the fluorescence anisotropy decay of HEWL in thepresence of 3 M NaCl and at pH 9. From the fit to eq 4, tworotational correlation times were obtained in both cases, asubnanosecond component, similar to the short componentobserved in solution and in the sol-gel system, and a very longsecond component of rotational correlation time around 36-37 ns (Table 2). Since the average fluorescence lifetime of theHEWL tryptophans is only 2.2 ns, it is difficult to extract reliableanisotropy data much beyond∼15-18 ns (∼7-8 lifetimes, i.e.,∼21/2-3 orders of magnitude of decay). Consequently, the

precision of the longer correlation time value recovered islimited. To determine the level of certainty in the correlationtimes obtained, a confidence interval analysis was performed(Figure 6A and B).51 The confidence intervals correspondingto one standard deviation are 30 to 45 ns for the experimentcarried out at high ionic strength and 24 to 41 ns for theexperiment carried out at pH 9. For all of these fits,æ1 was 0.3( 0.1 ns, and values ofâ2 were practically constant, rangingbetween 0.84 and 0.86. Sinceæ2 . æ1, the overall anisotropydecay was again interpreted following the independent-two-motion model (eqs 6 and 7), and the experimentalæ1 andæ2

were directly related toæsegmentalandæglobal, respectively. Thelatter are shown in Table 3, together with the order parameterS1 and the corresponding half-cone angleθ0 calculated fromâ2. A value of (19( 1)° was obtained for this angle in bothsituations, which was very close to that determined in freesolution.

The results presented in Table 3 demonstrate that electrostaticinteractions established between HEWL (positively charged atneutral pH) and the negatively charged porous matrix surfacedrastically affect the global rotation of the protein withoutaltering its conformation and internal local motions. Electrostaticinteractions between phospholipid polar heads and the poroussurface of the host matrix have recently been demonstrated byour group for sol-gel-encapsulated lipid bilayers.52 Theseinteractions seem to play a critical role in the preservation of

Figure 4. Time-resolved fluorescence depolarization of HEWL (A) in solution and (B) entrapped in a sol-gel matrix at 20°C. The smoothanisotropy decay fit according to eq 4 is superimposed on the noisy data. Weighted residuals of the fits are also shown.

Figure 5. Time-resolved fluorescence depolarization of HEWL after 48 h of incubation entrapped in a sol-gel matrix in (A) 10 mM phosphatebuffer containing 3 M NaCl, pH 7.4, and (B) 10 mM borate buffer, pH 9, at 20°C. The smooth anisotropy decay fit according to eq 4 is superimposedon the noisy data. Weighted residuals of the fits are also shown.


membrane structural integrity, causing disruption of the phos-pholipid packing in the gel phase and a noticeable increase ofthe order parameter in the fluid phase, and can be practicallyeliminated using phospholipids with anionic polar heads. In thepresent case of protein entrapment, the electrostatic interactionsare reduced at high ionic strength or alkaline pH, allowing freerotation of the protein within the sol-gel pore. Under theseconditions, however, the correlation times for rotation of theprotein as a whole,æglobal, are approximately 6- to 10-fold longerthan that obtained in free solution. Whether these correlationtimes correspond to the simple Stokes-Einstein relationship (eq8) or not is questionable, as the latter is based on classicalhydrodynamics and requires the hypothesis of a continuous andhomogeneous solvent. In the present case, the aqueous solventis confined within sol-gel pores where different dynamicenvironments coexist.53,54 Noting that changes in the hydrody-namic volumeV, due to changes in the size of the solvationshell and/or the occurrence of intermolecular protein aggrega-tion, are unlikely to be provoked by the caging effect on theprotein in the sol-gel system,19 the Stokes-Einstein relationship(eq 8) with an effective viscosityηeff replacing the normalsolution viscosityη may be appropriate.55 On this assumption,ηeff was found to be (8( 2) cP, considerably higher than the 2cP obtained for entrapped neutral and anionic small moleculesand proteins,56 and was interpreted as arising from the sum oftwo contributions, (a) weak electrostatic interactions which stillremain between HEWL (still with a certain residual positivecharge at pH 9) and the hydrophilic pore walls and (b) the higherviscosity reported for water molecules near the pore walls, whichexhibit dynamics that are an order of magnitude slower thanthat in bulk solution.53,57

Conclusions

In this work, the intrinsic tryptophan fluorescence of theprotein HEWL entrapped in a sol-gel matrix has been exploitedto clarify the role of putative protein-matrix interactions onthe structural and dynamic properties of sol-gel-entrappedbiomolecules. The results of these studies show that (a)immobilization does not lead to any major conformationalchange, at least in the region of the enzymatic binding site,where the two most highly fluorescent tryptophan residues arelocated, (b) the milieu surrounding HEWL molecules inside ofthe silica pore is quite similar to the bulk aqueous solution, and(c) at neutral pH and low ionic strength, the global rotation ofthe protein is completely abrogated upon entrapment. It has beendemonstrated for the first time that strong electrostatic interac-tions between the protein molecule and the negatively chargedsol-gel walls are ultimately responsible for the total arrest ofthis global rotation. The electrostatic nature of the interactionis clearly established since either reduction of the total positivecharge carried by the protein (by increasing the pH toward itspI) or increase of the ionic strength of the solution leads to theremoval of these constraints and allows free global rotation totake place, albeit at a reduced rate.

Acknowledgment. The authors thank the Spanish Ministeriode Educacio´n y Ciencia (MEC) for Grants MAT2005-01004,CTQ2004-07716/BQU, and BFU2006-03905. We are gratefulto an anonymous reviewer whose comments and suggestionsgreatly improved the quality of this manuscript. I. Pastor waspartially supported by the Instituto de Salud Carlos III and bythe Instituto Gil-Albert. We thank G. Bernabeu for excellenttechnical assistance.

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structure and dynamics of lysozyme encapsulated in a silica sol−gel matrix

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