Behavior of Acrylodan-Labeled Human Serum Albumin Dissolved in

Dec 15, 2007 - Buffalo, New York 14260-3000, and Chemical Sciences DiVision, Oak Ridge National Laboratory, P.O. Box. 2008, Oak Ridge, Tennessee ...
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Behavior of Acrylodan-Labeled Human Serum Albumin Dissolved in Ionic Liquids Taylor A. McCarty,† Phillip M. Page,† Gary A. Baker,‡ and Frank V. Bright*,† Department of Chemistry, Natural Sciences Complex, UniVersity at Buffalo, The State UniVersity of New York, Buffalo, New York 14260-3000, and Chemical Sciences DiVision, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6110

Researchers have reported enzymatic reactions performed in ionic liquids (ILs). Currently very little is known about the behavior of biomolecules in ILs in comparison to their better understood aqueous behavior. We report the temperature-dependent behavior of human serum albumin (HSA) that is site-selectively labeled at cysteine-34 (located in loop 1 of domain I) with a single fluorescent reporter molecule (acrylodan, Ac) when it is dissolved in phosphate buffered saline (PBS) or one of three ILs. In PBS, the Ac reporter motion is always coupled to the global HSA protein motion. In the ILs, loop 1 of domain I appears to be almost completely decoupled from the HSA global motion. As temperature increases, neighboring nonpolar amino acid residues and/or IL components apparently become strongly associated with the Ac rotating body. These results show that protein structure and dynamics in an IL can markedly deviate from that which exists in aqueous media. Introduction Biocatalytic reactions can offer efficient, selective, and versatile chemical transformations; however, there are also serious limitations.1-4 For example, biocatalyst stability, selectivity, substrate solubility, and product yield are all factors that require due consideration. In the search for optimizing a given system, researchers have explored a wide variety of alternative reaction conditions and strategies. Strategies based on biocatalyst immobilization,5,6 chemical modification to the biocatalyst,7,8 and/or unconventional/nonaqueous solvent media9-11 are popular approaches. With regard to the nonaqueous solvent approaches, over the years there has been an increase in the use of ionic liquids as the reaction solvent for biocatalytic reactions,12-16 and, more recently, bioassays.17 Ionic liquids (ILs) are organic salts that liquefy below the boiling point of water, typically close to, or even well below, room temperature.10,18,19 ILs are generally based on inorganic or organic anions combined with large (usually asymmetric) organic cations. ILs exhibit a wide electrochemical window, high ionic conductivity, and broad liquid temperature range, lack measurable vapor pressure, and possess excellent chemical and thermal stability. ILs are often discussed as possible replacements for volatile organic compounds (VOCs) in industrial processes and separation technologies. Their potential as “green” solvents is largely associated with their low volatility and high thermal stability. The physical properties of ILs (e.g., density, melting point, polarity, and viscosity) can be tuned by changing the cation and anion partnering.10,20-22 Tuning is highly advantageous because one can, in principle, design an IL for a specific task (extraction, separation, catalytic reaction) simply by manipulating its relevant physicochemical properties. As an example, one can envision customizing an IL for a particular biocatalytic reaction by modifying the anion and/or cation structure to adjust * To whom correspondence should be directed. Tel.: (716) 6456800 ext. 2162. Fax: (716) 645-6963. E-mail: [email protected]. † University at Buffalo. ‡ Oak Ridge National Laboratory.

enzyme selectivity, reaction kinetics, and/or substrate, intermediate, or product solubility. Researchers have already shown that biocatalyst activity, enantioselectivity, thermal or operational stability, and reusability can all be improved in ILs.11-16 Representative examples from the literature include the synthesis of Z-aspartame using thermolysin,23 alcoholysis, ammoniolysis, and perhydrolysis using lipases,24-27 transesterification using R-chymotrypsin,28-30 and ketone reduction using whole cells of Baker’s yeast.31 Additionally, Baker and co-workers32 investigated the first thermodynamic measure of the stability of a protein in an IL and showed the significant stabilization of proteins at elevated temperatures by the IL in comparison to water. However, despite the growing use of biocatalysts in ILs, there is little information on how proteins actually behave when they are dissolved or dispersed within an IL. To explore this issue, we investigate here the behavior of acrylodan-labeled human serum albumin (HSA-Ac) in three different ILs based on the 1-butyl-3-methylimidazolium cation (Figure 1): 1-butyl-3methylimidazolium bis(trifluoromethylsulfonyl)imide ([C4mim][Tf2N]), 1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim][BF4]), and 1-butyl-3-methylimidazolium hexafluorophosphate ([C4mim][PF6]). In the current studies, all ILs contain 2% (v/v) distilled deionized water (ddH2O) as cosolvent. Such an approach has been frequently used in the past to assist in substrate and/or enzyme solubility, with the additional benefit of increasing fluidity and transport kinetics.23,28,29,31,32 HSA was chosen as our model protein because it is widely studied,33-40 has a known and available X-ray crystal structure,41,42 consists of multiple (three) linearly arranged and fairly distinct domains, includes no carbohydrate, and contains a single free cysteine residue.43 This one free thiol residue allows the installation of a single fluorescent reporter molecule at a welldefined position in the protein.34,37,44,45 Acrylodan (Ac, acryloyl(dimethylamino)naphthalene) was selected as our reporter molecule for several reasons. First, it reacts with a high specificity for free thiols, forming very stable covalent thioether bonds, and HSA conveniently has only one such residue available (cysteine-34).46-48 Second, its photophysics are wellknown.46,49,50 Third, its emission spectrum is particularly

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Figure 1. System under investigation: (A) Chemical structures of the ILs used; (B) The tertiary structure of loop 1 in domain I of HSA, showing amino acid residues 27-43 (source: Protein Data Bank, 1HA2). The gray star represents the covalently attached acrylodan (Ac) probe; no particular Ac orientation is implicit. (C) Chemical structure of PRODAN, a nonconjugatable analog of Ac.

sensitive to the physicochemical properties of its local microenvironment.46,49,51 Thus, Ac attached to cysteine-34 within HSA provides a convenient means to study how ILs affect the local microenvironment surrounding a single site within a large, multidomain, and well-understood protein.37 In essence, HSAAc mimics all the key features of an active site within an enzyme. Figure 1B presents the chemical structure of the thiolreactive Ac after modifying its target alongside an X-ray crystal structure of the local microenvironment that surrounds the Ac residue in native HSA-Ac. Figure 1C illustrates the chemical structure for an Ac analog, 6-propionyl-2-dimethylaminonaphthalene (PRODAN), a probe comprising the identical chromophoric unit as Ac. Thus, PRODAN provides a convenient means to decouple the influence of IL structure and system temperature on the reporter molecule per se from their collective influence on HSA which in turn influences the Ac reporter molecule as a result of induced protein conformational changes. The goal of the present research is to explore the effects of IL and temperature on the structure of a specific site within a model protein and compare these results to the same protein

system when it is dissolved in aqueous phosphate buffered saline solution. The steady-state fluorescence spectra are used to evaluate the influence of IL choice and temperature on the average local microenvironment that surrounds the Ac residue in HSA-Ac in comparison to the free probe analog PRODAN. Steady-state fluorescence anisotropy measurements are used to characterize the influence of IL and temperature on the Ac residue mobility within HSA-Ac compared to the probe alone. Theory Fluorescence spectroscopy is an attractive tool for investigating protein behavior in solution52-56 under dilute conditions where protein-protein interactions are minimal. Fluorescence measurements also provide information on changes in protein tertiary structure, subunit affinity, aggregation, and hydrodynamic volume. In the current research, we exploit an extrinsic probe approach to avoid the strong IL autofluorescence background which makes the use of native tryptophan emission a particular challenge.

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The Lippert-Mataga expression (eq 1) illustrates the relationship between a solvent’s physicochemical properties and a luminophore’s electronic absorbance and emission spectra:57 ν A - νF )

(

)

2 2 -1 n2 - 1 (µE - µG) + constant - 2 hc 2 + 1 2n + 1 a3

(1)

In this expression, νA and νF represent the luminophore absorbance and emission maxima (in cm-1), respectively, h is Planck’s constant, c is the speed of light, a is the radius swept out by the luminophore, µE and µG are the luminophore’s excited and ground-state dipole moments, respectively, and  and n denote the solvent dielectric constant and refractive index, respectively. The bracketed term, a function of  and n, is referred to as the orientational polarizability (∆F). ∆F accounts for the spectral shift resulting from the redistribution of “solvent” molecules that surround the luminophore. For Ac and PRODAN the µE - µG term in eq 1 is on the order of 4-5 D and νA is not particularly solvent sensitive.57 Thus, changes in ∆F lead to changes in the Ac/PRODAN emission spectra. Specifically, as ∆F increases (solvent polarity increases), the Ac/PRODAN emission spectra shift red (νF decreases). Luminescence anisotropy measurements provide information on the rotational reorientation of a luminophore and its local partners.58 Steady-state anisotropy measurements reveal information on the aVerage rotational correlation time for all motions experienced by a bound reporter molecule (global, segmental, and local) and thus can only be used as a rough guide to assess perturbations in the overall protein dynamics. Nonetheless, such measurements are able to give information on relative changes in reporter molecule mobility within the cybotactic region, which reflect overall coupling of the reporter molecule motion to that of the protein. The steady-state luminescence anisotropy (r) is determined by measuring the parallel (I||) and perpendicular (I⊥) components of the polarized emission when the sample is excited with vertically polarized excitation:

r)

I|| - GI⊥ I|| + 2GI⊥

(2)

G is an instrument factor that corrects for differing sensitivities of the optics and detection system to the emission polarization. If the luminophore/partner(s) dynamics are described by isotropic rotational reorientation, r is given by the Perrin equation:

r)

r0 1 + τ/θ

(3)

In this expression, r0 is the limiting anisotropy, τ is the excitedstate luminophore lifetime, and θ is the rotational correlation time. The Debye-Stokes-Einstein expression provides a link between the experimental measurable, θ, and the system physical properties,

θ)

ηV RT

(4)

where η is the solvent viscosity, T is the absolute temperature, R is the gas constant, and V is the volume of the rotating unit. Experimental Section Reagents and Chemicals. The following were used: Acrylodan (Ac) and PRODAN (Molecular Probes, now Invitrogen); 1,4-bis (4-methyl-5-phenyl-2-oxazolyl) benzene (Me2POPOP),

and phosphate buffered saline (PBS, 10 mM, pH ) 7.40 ( 0.05) (Aldrich); and HSA (Sigma, fatty acid free). Electrochemical grade [C4mim][PF6] (stated 99+ % pure; < 50 ppm water; < 50 ppm chloride; packaged under argon in Sure/Seal bottles) was supplied by Covalent Associates. [C4mim][Tf2N] and [C4mim][BF4] were prepared as described in the literature.59,60 HSA-Ac Preparation. The bioconjugation of HSA with Ac followed methods described previously by Flora et al.,37 with some minor modifications. Briefly, essentially fatty acid-free HSA was first purified on a Sephadex G-25 column as detailed earlier.37 The so-purified protein was then dissolved at a concentration of 45 µM in 10 mM phosphate buffer containing 137 mM NaCl and 2.7 mM KCl at pH 7.4. An Ac stock solution was prepared in acetonitrile at 5.6 mM and an appropriate aliquot immediately taken and added dropwise over 8-10 min to gently shaken buffered HSA solutions to obtain a final Acto-HSA molar ratio of 1.2:1. We note that the final level of acetonitrile was ∼1% (v/v). Following 6 h of gentle mixing on a reciprocal shaking bath (15 oscillations/min) in the dark at ambient temperature, unreacted Ac was removed by loading the reaction mixture into a 10 000 MWCO Slide-a-Lyzer dialysis cassette (Pierce) and attaching a flotation buoy, followed by dialysis against PBS over a 5 day period at 4 °C, with dialysate exchanges for fresh PBS every 10-12 h. During this period, the buffer reservoir was vigorously stirred using a PTFE-coated octagonal stir bar (600 rpm). As a final purification step, the dialyzed material was serially passed through two freshly packed Sephadex G-25 columns. The purified HSA-Ac was then divided into 0.75 mL aliquots in 2 mL clear polypropylene microcentrifuge tubes and the contents subsequently flash frozen in liquid nitrogen followed by freeze-drying (36-40 h, P < 20 µm Hg) prior to refrigerated storage in a sealed container. The labeling efficiency was determined to be 90 ( 5%. Far-UV circular dichroism (CD) measurements confirmed that HSA fully retained its native secondary structure following these fluorescent probe labeling procedures (i.e., normalized CD spectra for unmodified HSA and HSA-Ac are completely coincident in the 190 to 250 nm window, within the experimental uncertainty of the measurement). Sample Preparation. The HSA-Ac concentration in PBS was kept at 5 µM. The HSA-Ac level in the various IL/2% (v/v) ddH2O mixtures was 3 µM. Viscosity Measurements. All viscosity measurements were performed using a Brookfield DV-II+ Pro viscometer within an inert atmosphere box (Cole Parmer 34790-30). The relative humidity was maintained below 5% by using dry Ar(g) and Sicapent drying agent. The viscometer sample temperature was regulated to (1 °C with a recirculating temperature bath (Brookfield TC-602). Fluorescence Measurements. Steady-state emission spectra were recorded with a SLM 48000 MHF spectrofluorometer. A 450 W Xe arc lamp was used as the excitation source. Singlegrating monochromators served as the wavelength selection devices. The excitation wavelength was set at 360 nm and the excitation and emission spectral bandpasses were maintained at 4 nm. All emission spectra were background corrected using appropriate blanks. Steady-state anisotropy measurements were performed with a SLM-AMINCO model 8100. A 450 W Xe arc lamp served as the excitation source. Double- and single-grating monochromators served as the excitation and emission wavelength selection devices, respectively. The excitation wavelength was set at 360 nm and the emission wavelength was at 500 nm.

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Figure 2. Temperature-dependent viscosities for ddH2O versus the three IL/2% (v/v) ddH2O mixtures.

The excitation and emission spectral bandpasses were maintained at 4 nm. Multifrequency phase-modulation data were acquired by using an SLM 48000 MHF spectrofluorometer. An argon-ion laser (Coherent, Model-Innova 90-6) operating at 351.1 nm was used as the excitation source. Magic angle polarization61 was used for these measurements. Me2POPOP dissolved in ethanol served as the reference excited-state lifetime standard, its lifetime is 1.45 ns.62 For all experiments, the Pockels cell was operated at a repetition rate of 5 MHz. Data was acquired for 60 s over a frequency range of 5-250 MHz (50 frequencies). All multifrequency phase-modulation data were analyzed by using a commercially available software package (Globals WE). Temperature-dependent steady-state and time-resolved fluorescence measurements were conducted between 20 and 90 °C by using a Neslab model RTE-111 refrigerated bath circulator. The samples were allowed to equilibrate for 15 min between temperature adjustments before any measurements were taken. All experiments were performed on at least three occasions. Average results are reported along with the corresponding measurement standard deviations. There was no evidence of photodecomposition under our experimental conditions. Analysis of the Steady-State Emission Spectra. Each steady-state emission spectrum was analyzed by using PeakFit v4.12 (Jandel Scientific). All of the spectra were fit to a single Gaussian profile to estimate the Ac/PRODAN emission maximum. Results and Discussion Solution Viscosities. Figure 2 presents the temperaturedependent viscosity measurements for pure ddH2O and the binary mixtures of [C4mim][Tf2N], [C4mim][BF4], and [C4mim][PF6] that contain 2% (v/v) ddH2O as cosolvent. (Note: Visually, these mixtures remain a single phase across the entire temperature range studied.) At 20 °C, the IL/H2O mixture viscosities are 40-60 times greater in comparison to ddH2O. The IL/H2O mixture viscosities decrease significantly with increasing temperature. The recovered activation energies for viscous flow are as follows: ddH2O, 14.6 ( 0.3 kJ mol-1 (14.3 kJ mol-1);63 [C4mim][BF4]/2% ddH2O, 23.8 ( 0.4 kJ mol-1; [C4mim][TF2N]/2% ddH2O, 24.8 ( 0.4 kJ mol-1; and [C4mim][PF6]/2% ddH2O, 28.2 ( 1.5 kJ mol-1. The latter can be compared to the value of 38.4 ( 0.9 kJ mol-1 we determined earlier in anhydrous [C4mim][PF6],64 illustrating the dramatic

Figure 3. Temperature-dependent steady-state emission maxima for PRODAN and HSA-Ac in PBS and the three IL/2% ddH2O mixtures. The error bar shown is scaled to pertain to the 450-490 nm subdivision of the vertical axis.

“lubricating” influence of water in the IL system and, as a result, the weakened dependence of viscosity on solvent temperature. Steady-State Emission Spectra. The PRODAN/Ac absorbance/excitation spectra are not particularly solvatochromic (results not shown).65 Figure 3 summarizes the temperaturedependent PRODAN (open symbols) and HSA-Ac (solid symbols) steady-state emission maxima in PBS and the three IL/2% (v/v) ddH2O mixtures. PBS. The PRODAN emission occurs at a much longer wavelength, around 30 to 35 nm to the red, in comparison to HSA-Ac at all temperatures studied. This result demonstrates that the local microenvironment surrounding the Ac residue within native HSA-Ac is not as polar in comparison to PRODAN dissolved in PBS, a reflection of the shielding from aqueous solvent afforded by the neighboring amino acid residues in HSA (Figure 1A). As temperature increases, the PRODAN emission continuously shifts blue. The HSA-Ac emission is constant between 20 and 35 °C, suggesting that the average local microenvironment surrounding the Ac residue is not changing significantly over this temperature range. As the temperature increases from 40 to 70 °C, the HSA-Ac emission maximum then blue shifts by ∼ 7 nm. This blue shift is consistent with a change in the local microenvironment surrounding the Ac residue. Above 70 °C, this trend is reversed and the HSA-Ac emission maxima systematically increase. At 20 °C, in the presence of 8 M GdHCl (a strong chemical denaturant of proteins),66 the HSA-Ac emission occurs at 525 nm, close to that of the fully solvent-exposed PRODAN. Together, these results show that the local microenvironment surrounding the Ac residue within HSA-Ac changes with temperature, but the protein does not appear to be fully denatured by temperature even at 90 °C. IL/2% (v/v) ddH2O. The HSA-Ac behavior in the IL systems containing 2% (v/v) ddH2O is considerably different in comparison to that in PBS. These results are summarized in the following sections. [C4mim][Tf2N]/2% ddH2O. The PRODAN emission maximum increases slightly (ca. 2 nm) between 20 and 60 °C and then begins to decrease, falling just slightly below the initial value by 90 °C. Throughout the entire temperature range, the PRODAN emission maximum is far lower in comparison to PRODAN dissolved in PBS, indicating that the [C4mim][Tf2N]/ 2% ddH2O mixture is less dipolar in comparison to PBS. Interestingly, at temperatures of 30 °C or below, the HSA-Ac

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emission maxima in this IL are similar tosalbeit slightly redshifted on averagesHSA-Ac dissolved in PBS, suggesting that the Ac residue is sensing a microenvironment that is quite similar to that experienced in the native protein at these same temperatures. As the IL/water mixture is heated from 25 to 70 °C, the HSA-Ac emission maximum decreases significantly. Above 70 °C, the emission maxima remain temperature independent and are similar to that for PRODAN dissolved in [C4mim][Tf2N]/2% ddH2O. These results imply that as we increase the temperature from 25 to 70 °C, the local microenvironment surrounding Ac evolves, becoming much less polar. The coincidence of the HSA-Ac and PRODAN emission spectra above 70 °C suggests that the local “pocket” hosting the Ac reporter group completely or partially opens up such that the Ac probe now reports from a highly IL-enriched location. At the very least, these data suggest a striking difference in local structure surrounding the Ac-labeled Cys-34 residue. However, it should be kept in mind that HSA contains three domains which are assumed to fold independently.67 Hence, we suggest caution in the (over)interpretation of these results as they apply to the global secondary structure of the protein. [C4mim][BF4]/2% ddH2O. The PRODAN emission maxima in the [C4mim][BF4]/2% ddH2O solution are slightly (2-6 nm) higher in comparison to PRODAN dissolved in the [C4mim][Tf2N]/2% ddH2O system, with the gap between the two solvents falling off with temperature. This result indicates that the [C4mim][BF4]/2% ddH2O mixture is more polar than [C4mim][Tf2N]/2% ddH2O to some extent. As temperature increases, the PRODAN emission maximum in [C4mim][BF4]/2% ddH2O shifts monotonically to the blue. At 20 °C, the HSA-Ac emission maximum is considerably blue-shifted in comparison to HSAAc dissolved in PBS or in [C4mim][Tf2N]/2% ddH2O. This result reveals that the local microenvironment surrounding the Ac residue in HSA-Ac is dissimilar to and strikingly less dipolar than in PBS or [C4mim][Tf2N]/2% ddH2O. In fact, at all temperatures investigated, the HSA-Ac emission maximum in [C4mim][BF4]/2% ddH2O is substantially blue-shifted even relative to PRODAN in the same IL solvent. We note that a comparable blue shift occurred for HSA-Ac entrapped within sol-gel-derived biogels for long storage times of a month or more.40 A similarly blue-shifted emission was also observed for the highly homologous bovine serum albumin (serum albumins are almost completely conserved and BSA and HSA are indistinguishable by most physical criteria) labeled at Cys34 with Ac as a result of conformational changes experienced after sorption at a bare or alkylated (C1 or C18) silane surface.68 Evidently, in our case the Ac residue of HSA-Ac is encountering a microenvironment of appreciably lower dipolarity than the [C4mim][BF4]/2% ddH2O itself, ruling out solvent exposure as a plausible explanation. The HSA-Ac emission maximum remains effectively constant between 20 and 70 °C within the experimental uncertainty, and then increases slightly past 70 °C. Plainly, the local microenvironment surrounding the Ac residue within HSA-Ac dissolved in [C4mim][BF4]/2% ddH2O is surprisingly different from the apparent local polarity in the other two [C4mim]+ based ILs studied here. In either case, the blue-shifted emission is consistent with a decreased aVerage dipolarity or a slowing of the dynamics in the cybotactic region. The possible origin of this distinctive behavior will be considered in a later section. [C4mim][PF6]/2% ddH2O. The PRODAN results in the [C4mim][PF6]/2% ddH2O mixture parallel those for [C4mim][Tf2N]/ 2% ddH2O but are red-shifted by ca. 5 nm on average across the temperature range explored. While these results do indicate

Figure 4. Temperature-dependent emission difference results: (A) HSAAc in IL/2% ddH2O minus HSA-Ac in PBS and (B) HSA-Ac in IL/2% ddH2O minus PRODAN in IL/2% ddH2O.

that PRODAN experiences a higher average polarity in this system relative to the other IL/H2O mixtures studied, the polarity of all three IL/H2O systems falls within a fairly narrow range, an observation that could be anticipated from earlier work involving empirical polarity measurements within [C4mim]+ based ILs. Reminiscent of the results in [C4mim][BF4]/2% ddH2O, PRODAN’s emission maximum in [C4mim][PF6]/2% ddH2O is always higher than that of HSA-Ac dissolved in this IL/H2O mixture. Again, this result implies that the Ac residue within HSA-Ac experiences less polar surroundings compared to the probe feely dissolved in [C4mim][PF6]/2% ddH2O. As the sample is heated from 20 to 65 °C, the HSA-Ac emission maximum blue shifts, showing a minor red shift on subsequent heating to 90 °C. Differential Emission Maxima. The spectral changes observed for HSA-Ac in IL/H2O relative to PBS can be treated to obtain the parameter Γ which can then be used to rank the impact of the different ILs on our model protein. Expressly, the temperature-dependent difference between the HSA-Ac emission maxima in IL/ddH2O and PBS, i.e., Γ ) Em maxIL/H2O - Em maxPBS, provides a convenient method to compare the behavior of HSA-Ac in these solvent systems. Specifically, for Γ above zero, the domain surrounding the Ac residue of HSAAc in the IL/2% (v/v) ddH2O mixture is more polar in comparison to HSA-Ac in PBS. Conversely, if Γ is below zero, HSA-Ac in IL/H2O perceives a lower polarity local medium relative to that in PBS. Figure 4A presents the results of this exercise. At 20 °C in [C4mim][Tf2N]/2% ddH2O, the microenvironment surrounding the Ac residue in HSA-Ac is marginally more polar than in PBS. As the temperature rises, however, Γ

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Figure 5. Temperature-dependent steady-state fluorescence anisotropy for PRODAN and HSA-Ac in PBS and the three IL/2% ddH2O mixtures.

becomes increasingly negative, indicating a lower polarity microenvironment surrounding the Ac residue in HSA-Ac in comparison to HSA-Ac in PBS. Zero-point crossing occurs at a relatively low temperature in this system (∼30 °C). At 20 °C in [C4mim][BF4]/2% ddH2O, the Γ value is far and away the most negative of the IL/H2O mixtures under investigation. This remains the case over the entire temperature range of the experiment. These results are consistent with the Ac residue in HSA-Ac reporting from the most hydrophobic microenvironment in [C4mim][BF4]/2% ddH2O, a somewhat surprising result given the water miscibility of this IL. Interestingly, as the temperature increases, Γ increases in an approximately sigmoidal manner in [C4mim][BF4]/2% ddH2O with an inflection point occurring near 55 °C. In contrast, in the [C4mim][PF6]/2% ddH2O system, Γ is negative and essentially independent of temperature. Finally, as temperature increases, it appears as if the polarities characterizing the microenvironment surrounding the Ac label of HSA-Ac in the IL/water samples converge on one another. According to the structure for HSA proposed by He and Carter,69 Cys-34 is located in a turn between helix h2 and h3 on domain I, and it is only partially protected from (aqueous) solvent. On the other hand, almost nothing is currently known about protein solvation within ILs. Thus, our results appear highly relevant from the standpoint that the temperaturedependence of solvent penetration within a protein structure may apparently be vastly different in an IL compared to the native (i.e., aqueous) milieu. The temperature-dependent spectral difference between the HSA-Ac and PRODAN emission maxima in a given IL/H2O system (∆ ) Em maxHSA-Ac - Em maxPRODAN) provides a measure of the HSA-Ac behavior from a different perspective. Specifically, if ∆ equals zero, this implies that the domain surrounding the Ac residue in HSA-Ac is similar to that for PRODAN in the same IL/ddH2O mixture. Positive and negative values of ∆ insinuate a domain surrounding the Ac residue in HSA-Ac in the IL/ddH2O mixture that is more and less polar, respectively, in comparison to PRODAN in the same IL mixture. Figure 4B summarizes the results for HSA-Ac and PRODAN emission in the IL/H2O systems on this basis. In [C4mim][Tf2N]/2% ddH2O, the microenvironment surrounding the Ac residue in HSA-Ac starts out more polar than for PRODAN, a result of polar residues surrounding Cys-34 coming into close proximity with the Ac reporter group and/or preferential solvation of Ac by a the polar component of the solvent (i.e., water, anion, cation). With increasing temperature, the value of ∆ decays until the Ac residue appears to report

Figure 6. (A) Average temperature-dependent excited-state fluorescence lifetimes and (B) estimated rotating radii for HSA-Ac dissolved in PBS and the three IL/2% ddH2O mixtures.

from a microenvironment comparable to PRODAN’s. This result is consistent with total exposure of the Ac label to the solvent, a likely manifestation of denaturation of the subdomain surrounding cysteine-34 in [C4mim][Tf2N]/2% ddH2O at temperatures of 50 °C and above. At this point, we cannot say a great deal about other domains because the information obtained is from a specific site within domain I. In [C4mim][PF6]/2% ddH2O at 20 °C, the Ac residue in HSAAc and PRODAN appear to report from comparable microenvironments. Once again, this is consistent with complete denaturation of the subdomain surrounding Cys-34. The ∆-T curve shows fascinating parabolic behavior in that, as the temperature increases from 20 to 60 °C, ∆ drops to considerably negative values, followed by an increase toward zero as the temperature further rises to 90 °C. The large negative values of ∆ suggest that the Ac residue in HSA-Ac is encountering a microenvironment of significantly lower dipolarity than the [C4mim][PF6] mixture itself. This is consistent with a contraction in local structure with nonpolar amino acid residues surrounding Cys-34 coming into registry with the Ac reporter or, less likely, preferential solvation by select components of the IL mixture.70 As shown in Figure 1A, there are a number of nonpolar amino acids in the vicinity of Cys-34 including Ala-28, Leu-31, Val40, Leu-42, and Val-43. The phenylalanine residue is particularly hydrophobic with two such residues (Phe-27 and Phe-36) located within the cybotactic region of Ac. The return to PRODANlike spectral maxima at higher temperatures suggests an opening of the pocket housing Ac and full exposure to the IL medium again. In [C4mim][BF4]/2% ddH2O, the Ac residue in HSA-Ac senses the most nonpolar microenvironment of all three IL/

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Figure 7. “Tennis ball” model of HSA showing the three major domains and the disulfide-bridged R-helical subdomains. Panel A illustrates the unfolding process of HSA-Ac in PBS, and panel B compares the aqueous behavior to that postulated in IL mixtures containing 2% (v/v) ddH2O. The numerals indicate amino acid positions, and the horizontal bars and star denote roughly the positions of the many S-S bonds and the location of the Ac residue, respectively. “N” indicates the N-terminus in domain I and “C” is the C-terminal end of the polypeptide chain in domain III.

ddH2O mixtures. Clearly, as discussed above, this is consistent with selective solvation by neighboring nonpolar amino acid residues known to reside in the same loop of domain I as Cys34. As the system temperature increases, ∆ steadily increases, however, it never reaches values consistent with complete solvation by [C4mim][BF4]/2% ddH2O. Steady-State Fluorescence Anisotropy. Figure 5 presents the temperature-dependent steady-state fluorescence anisotropy results for PRODAN and HSA-Ac dissolved in PBS, [C4mim][Tf2N]/2% ddH2O, [C4mim][BF4]/2% ddH2O, and [C4mim][PF6]/2% ddH2O. Turning first to the results in PBS, the steadystate anisotropies for PRODAN dissolved in PBS are much lower at all temperatures than for HSA-Ac in PBS, as expected. This result is a manifestation of PRODAN having a much smaller rotating (hydrodynamic) volume in comparison to HSAAc. The PRODAN steady-state anisotropies decrease slightly

with increasing temperature, a behavior resulting from the decreased viscosity of PBS with increasing temperature (see results for ddH2O in Figure 2). The steady-state anisotropy for HSA-Ac dissolved in PBS remains essentially constant between 20 and 35 °C followed by a significant drop from 40 to 70 °C, leveling off thereafter. Such enhanced average rotational mobility during the thermal unfolding of singly labeled proteins such as HSA-Ac is known to occur as a result of increasing amplitude of the internal motion of the label (Ac) arising from domain unfolding and interdomain separation, as well as shorter rotational correlation times for the whole protein.37 For the entire temperature range studied, the steady-state anisotropies for HSAAc dissolved in PBS remain roughly an order of magnitude higher than the steady-state anisotropies of the free probe (PRODAN) dissolved in PBS. Thus, the Ac reporter motion is never decoupled from the HSA motion(s) within PBS, even at

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90 °C. Nonlinear least-squares curve fitting (SigmaPlot 8.0, Jandel Scientific) of the temperature-dependent steady-state anisotropy data to a two-state unfolding model gave an unfolding temperature (Tun) of ∼60 °C, which is in reasonable agreement with the value 55 ( 3 °C obtained previously for HSA-Ac.37 Several key points are readily apparent from inspection of the anisotropy data in the IL/ddH2O mixtures alongside the PBS results. First, the PRODAN temperature-dependent anisotropy profiles in each solvent depend only on viscosity (i.e., the apparent activation energies are roughly equivalent to the activation energies for viscous flow). Second, contrasting the results in PBS, there is no evidence of temperature-induced “melting” for HSA-Ac in any of the IL/ddH2O mixtures between 20 and 90 °C. Third, the steady-state fluorescence anisotropy for HSA-Ac in PBS is always higher than in IL/ddH2O, despite the fact that the solvent viscosity in the IL/ddH2O mixtures is always significantly greater (10-60-fold) in comparison to PBS. Fourth, the temperature-dependent steady-state fluorescence anisotropies for HSA-Ac in [C4mim][Tf2N]/2% ddH2O and [C4mim][PF6]/2% ddH2O are exponentially activated, but the apparent activation energies (4.7 ( 0.3 kJ mol-1 and 3.73 ( 0.3 kJ mol-1, respectively) are 5.3- and 7.6-fold smaller, respectively, in comparison to the activation energies for viscous flow and for PRODAN reorientation in the same IL/ddH2O mixtures. Thus, the origin of the observed fluorescence anisotropy in these IL/ddH2O systems is consistent neither with free PRODAN nor HSA-Ac. Finally, the temperature-dependent steady-state anisotropy for HSA-Ac dissolved in [C4mim][BF4]/ 2% ddH2O is not exponentially activated. If anything, the anisotropy increases slightly as temperature increases above 60 °C. By means of eqs 3 and 4, which entail several simplifying assumptions of rotor geometry, we can approximate the average radius of the Ac rotating unit and any molecular unit/subunit to which it is coupled. Figure 6A presents the temperaturedependent average excited-state fluorescence lifetimes of HSAAc in PBS and the three IL/ddH2O mixtures. Figure 6B summarizes the effects of temperature and solvent type on the average radius of the species reorienting alongside the Ac reporter. As benchmarks, the average radius of an intact HSA molecule in water is ∼30 Å71,72 and the radius of PRODAN is 4.5 Å.73,74 Given these facts, the most obvious conclusion is that Ac is strongly coupled to the entire HSA protein when HSA-Ac is dissolved in PBS and that the aVerage radius remains essentially constant (25 ( 3 Å). In contrast, the Ac reporter is not as strongly coupled to the HSA protein in any of the IL/ water mixtures. For example, in [C4mim][BF4]/2% ddH2O and [C4mim][PF6]/2% ddH2O at 20 °C, Ac seems totally decoupled from the overall HSA motion. That is, Ac reorients in a manner similar to free PRODAN. In [C4mim][Tf2N]/2% ddH2O at lower temperatures (20 to 50 °C) the Ac reporter is slightly more coupled to the HSA motion. For instance, at 20 °C the apparent radius of the Ac-based entity is nearly twice the value of PRODAN. In all IL/ddH2O mixtures, the apparent volume of the rotating unit increases considerably as temperature increases. This suggests that amino acid residues associate more strongly with the Ac-based rotating body. Conclusions ILs have emerged as an attractive alternative solvent system for carrying out biocatalytic reactions and the results to date have been very promising. Little is known about the influence of IL as solvent on protein structure and dynamics, however.

In this work, which represents the original study of multidomain protein behavior within an IL, we offer preliminary insight into how solvation within an IL influences the behavior of a model multidomain protein: HSA labeled at Cys-34 with the polaritysensitive fluorescent probe acrylodan. Figure 7 summarizes our overall view of HSA-Ac unfolding behavior in buffered solution and in ILs taking into account the entirety of our current results. In PBS, the Ac reporter motion remains largely coupled to the intact HSA protein motion between 20 and 90 °C. Much of the change in the Ac emission between 40 and 60 °C result from unfolding of domain II of the protein, rather than local unfolding of domain I.37 The unfolding of domain I occurs between 70 and 90 °C as is evident from the large changes in steady-state anisotropy (Figure 5) and emission lifetime (Figure 6A). In the IL/ddH2O mixtures, the domain surrounding Cys-34 is clearly very different in comparison to HSA-Ac in PBS. The local loop within domain I already appears somewhat denatured, or at least measurably perturbed, at 20 °C. As the temperature increases from 20 to 90 °C, it appears as if residues of the polypeptide chain in close proximity collapse around the Ac rotor, while still allowing for access to (and solvation by) external IL. Taken together, these results demonstrate that proteins or segments of proteins can behave much differently in ILs in comparison to aqueous buffer. The results of this research have several implications when considering the growing use of ILs as solvent media for performing biocatalytic reactions. Pdrotein behavior is likely to be dependent on both the nature of the protein and the identity of the IL and its level of hydration. It should also be noted that the complexity of the process becomes even more pronounced when the protein contains multidomain structures in which each domain may have the ability to unfold/ refold independently.75 Salt effects are also likely to play a role in the observed behavior.76 Additional experiments involving time-resolution will likely prove essential for unraveling the details of this behavior and form the subject of current work within this laboratory. Acknowledgment This work was generously supported by the U.S. Department of Energy. Literature Cited (1) Roberts, S. M.; Turner, N. J.; Willetts, A. J.; Turner, M. K. Introduction to Biocatalysis Using Enzymes and Micro-organisms; Cambridge University Press: New York, 1995. (2) Faber, K. Biotransformations in Organic Chemistry; SpringerVerlag: Berlin, 1997. (3) Azerad, R. Biocatalysis in organic synthesis. AdV. Org. Synth. 2005, 1, 455. (4) Grogan, G. 9 Biotransformations. Annu. Rep. Prog. Chem., Sect. B 2005, 101, 192. (5) Bornscheuer, U. T. Immobilizing enzymes: How to create more suitable biocatalysts. Angew. Chem., Int. Ed. 2003, 42, 3336. (6) Brena, B. M.; Batista-Viera, F. Immobilization of enzymes. A literature survey. Methods Biotechnol. 2006, 22, 15. (7) DeSantis, G.; Jones, J. B. Chemical modification of enzymes for enhanced functionality. Curr. Opin. Biotechnol. 1999, 10, 324. (8) Davis, B. G. Chemical modification of biocatalysts. Curr. Opin. Biotechnol. 2003, 14, 379. (9) Khmelnitsky, Y. L.; Rich, J. O. Biocatalysis in nonaqueous solvents. Curr. Opin. Biotechnol. 1999, 3, 47. (10) Wasserscheid, P.; Welton, T. Ionic Liquids In Synthesis; WileyVCH: Weinheim, Germany, 2003; pp 1-6. (11) Yang, Z.; Pan, W. Ionic liquids: Green solvents for nonaqueous biocatalysis. Enzyme Microb. Technol. 2005, 37, 19. (12) Kragl, U.; Eckstein, M.; Kaftzik, N. Enzyme catalysis in ionic liquids. Curr. Opin. Biotechnol. 2002, 13, 565.

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ReceiVed for reView August 27, 2007 ReVised manuscript receiVed October 22, 2007 Accepted October 23, 2007 IE071165K