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New Reverse Micelle Surfactant Systems Optimized for High-Resolution NMR Spectroscopy of Encapsulated Proteins Zhengshuang Shi, Ronald W. Peterson, and A. Joshua Wand* Johnson Research Foundation and Department of Biochemistry & Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania, 19104-6059 Received May 28, 2005. In Final Form: August 18, 2005 Sodium bis(2-ethylhexyl)sulfosuccinate (AOT) is a surfactant commonly used to encapsulate water soluble proteins within the aqueous core of a reverse micelle. In the context of high-resolution NMR studies of encapsulated proteins the size of the resulting reverse micelle is critically important. We have designed and synthesized a short AOT analogue, 3,3-dimethyl-1-butylsulfosuccinate sodium salt and determined that it is able to form reverse micelles and to encapsulate the protein ubiquitin with high structural fidelity. AOT is often found to significantly destabilize encapsulated proteins, largely through chargecharge interactions between the anionic headgroup and the surface of the protein. Here we demonstrate, for the first time, that proportional mixtures of anionic and cationic surfactants can form reverse micelles that are also capable of protein encapsulation with high fidelity.
Introduction Application of modern NMR spectroscopy to large proteins is inherently hindered by the unfortunate degradation in performance of triple resonance experiments due to the short spin-spin relaxation times arising from slow molecular reorientation. Several approaches have emerged to deal with the various spectroscopic difficulties arising from slow molecular reorientation that, to name a few, include extensive deuteration,1-4 transverse optimized relaxation spectroscopy (TROSY),5-8 and advances in magnetization transfer.9 An additional approach actively seeks to increase the effective rate of molecular reorientation by encapsulating the protein of interest within the protective shell of a reverse micelle and dissolving the resulting particle in a low-viscosity fluid.10 Encapsulation of proteins at micromolar concentrations in high-density organic solvents was extensively investigated in the 1980s as a means to solubilize enzymes for the purpose of large-scale catalysis of reactions.11 In the context of solution NMR, homogeneous preparations of protein at significantly higher concentrations in relatively low viscosity fluids are required.12 These requirements have necessitated the development of new encapsulation * To whom correspondence should be addressed. Telephone: 215573-7288. Fax: 215-573-7290. E-mail:
[email protected]. (1) Venters, R. A.; Metzler, W. J.; Spicer, L. D.; Mueller, L.; Farmer, B. T. J. Am. Chem. Soc. 1995, 117, 9592-9593. (2) Pachter, R.; Arrowsmith, C. H.; Jardetzky, O. J. Biomol. NMR 1992, 2, 183-194. (3) Grzesiek, S.; Anglister, J.; Ren, H.; Bax, A. J. Am. Chem. Soc. 1993, 115, 4369-4370. (4) Gardner, K. H.; Rosen, M. K.; Kay, L. E. Biochemistry 1997, 36, 1389-1401. (5) Pervushin, K. J. Biomol. NMR 2001, 20, 275-285. (6) Pervushin, K.; Riek, R.; Wider, G.; Wuthrich, K. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 12366-12371. (7) Pervushin, K.; Riek, R.; Wider, G.; Wuthrich, K. J. Am. Chem. Soc. 1998, 120, 6394-6400. (8) Salzmann, M.; Pervushin, K.; Wider, G.; Senn, H.; Wuthrich, K. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13585-13590. (9) Riek, R.; Wider, G.; Pervushin, K.; Wuthrich, K. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4918-4923. (10) Wand, A. J.; Ehrhardt, M. R.; Flynn, P. F. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15299-15302. (11) Leser, M. E.; Luisi, P. L. Chimia 1990, 44, 270-282. (12) Wand, A. J.; Babu, C. R.; Flynn, P. F.; Milton, M. J. Biol. Magn. Reson. 2003, 20, pp 121-160.
strategies and apparatus.13-17 In addition to the ability to adjust the effective correlation time of an encapsulated protein by simply varying the bulk solvent,10 the encapsulation of proteins has also been found to be useful in NMR-based studies of protein cold-denaturation18 and the force-folding and stabilization of metastable proteins via a confined space effect.19 The latter may prove to be especially important as it appears that a significant fraction of the soluble proteins of the proteomes of a variety of species are unfolded under standard NMR sample conditions,20,21 suggesting that they are inherently unstable and are force-folded by the stabilizing excluded volume effect present in the cellular milieu.22 Water-in-oil reverse micelles (RM) have attracted significant attention due to their capability to host hydrophilic components in organic solvents.23-26 Reverse (13) Ehrhardt, M. R.; Flynn, P. F.; Wand, A. J. J. Biomol. NMR 1999, 14, 75-78. (14) Flynn, P. F.; Milton, M. J.; Babu, C. R.; Wand, A. J. J. Biomol. NMR 2002, 23, 311-316. (15) Babu, C. R.; Flynn, P. F.; Wand, A. J. J. Biomol. NMR 2003, 25, 313-323. (16) Wu, W. J.; Vidugiris, G.; Mooberry, E. S.; Westler, W. M.; Markley, J. L. J. Magn. Reson. 2003, 164, 84-91. (17) Lefebvre, B. G.; Liu, W.; Peterson, R. W.; Valentine, K. G.; Wand, A. J. J. Magn. Reson. 2005, 175, 158-162. (18) Babu, C. R.; Hilser, V. J.; Wand, A. J. Nat. Struct. Mol. Biol. 2004, 11, 352-357. (19) Peterson, R. W.; Anbalagan, K.; Tommos, C.; Wand, A. J. J. Am. Chem. Soc. 2004, 126, 9498-9499. (20) Yee, A.; Chang, X.; Pineda-Lucena, A.; Wu, B.; Semesi, A.; Le, B.; Ramelot, T.; Lee, G. M.; Bhattacharyya, S.; Gutierrez, P.; Denisov, A.; Lee, C. H.; Cort, J. R.; Kozlov, G.; Liao, J.; Finak, G.; Chen, L.; Wishart, D.; Lee, W.; McIntosh, L. P.; Gehring, K.; Kennedy, M. A.; Edwards, A. M.; Arrowsmith, C. H. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 1825-1830. (21) Peti, W.; Etezady-Esfarjani, T.; Herrmann, T.; Klock, H. E.; Lesley, S. A.; Wuthrich, K. J. Struct. Funct. Genomics 2004, 5, 205215. (22) Dedmon, M. M.; Patel, C. N.; Young, G. B.; Pielak, G. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12681-12684. (23) Luisi, P. L.; Magid, L. J. CRC Crit. Rev. Biochem. 1986, 20, 409-474. (24) De, T. K.; Maitra, A. Adv. Colloid Interface Sci. 1995, 59, 95193. (25) Moulik, S. P.; Paul, B. K. Adv. Colloid Interface Sci. 1998, 78, 99-195. (26) Silber, J. J.; Biasutti, A.; Abuin, E.; Lissi, E. Adv. Colloid Interface Sci. 1999, 82, 189-252.
10.1021/la051409a CCC: $30.25 © 2005 American Chemical Society Published on Web 09/29/2005
New Reverse Micelle Surfactant Systems Scheme 1
micelles are thermodynamically stable, but dynamic surfactant aggregates in an apolar solvent in which polar headgroups of the surfactants are orientated toward the core of the particle whereas hydrophobic tails are directed outward, protecting the inner water core from the apolar medium. Water contained in the cores of the reverse micelle can accommodate a range of hydrophilic molecules such as amino acids,27,28 peptides,29,30 and proteins.23,31-39 Both the electrical and steric properties of the surfactant are an important factor for the formation of reverse micelles.40-42 The hydrophobic tails have an affinity for an organic phase, whereas the polar headgroup has affinity for an aqueous phase. A balance between these affinities dictates in which phase surfactants reside. Sodium bis(2-ethylhexyl)sulfosuccinate (AOT, Scheme 1) is currently the most widely used surfactant for reverse micelle formation.43 However, in the context of NMR spectroscopy of encapsulated proteins AOT has significant limitations: (1) It is an anionic surfactant which mostly limits its application to proteins with relatively high isoelectric points (pI); (2) many positively charged proteins are denatured when they are encapsulated by AOT presumably due to strong electrostatic interactions between sulfonate group of AOT and positive surface charges of proteins, and this interaction cannot be successfully ameliorated by ionic screening without a concomitant degradation in encapsulation efficiency. To relieve some of the restrictions imposed by AOT, we have recently demonstrated that a reverse micelle system formed by the cationic CTAB (cetyltrimethylammonium bromide; see Scheme 2), and a medium-chain alcohol such as hexanol43 can avoid some problems with AOT.17 A central limitation of NMR spectroscopy of proteins in liquids is imposed by the effects of slow molecular (27) Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1990, 94, 64006411. (28) Adachi, M.; Harada, M.; Shioi, A.; Sato, Y. J. Phys. Chem. 1991, 95, 7925-7931. (29) Gierasch, L. M.; Lacy, J. E.; Thompson, K. F.; Rockwell, A. L.; Watnick, P. I. Biophys. J. 1982, 37, 275-284. (30) Thompson, K. F.; Gierasch, L. M. J. Am. Chem. Soc. 1984, 106, 3648-3652. (31) Menger, F. M.; Yamada, K. J. Am. Chem. Soc. 1979, 101, 67316734. (32) Thompson, J. S.; Gehring, H.; Vallee, B. L. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 132-136. (33) Grandi, C.; Smith, R. E.; Luisi, P. L. J. Biol. Chem. 1981, 256, 837-843. (34) Barbaric, S.; Luisi, P. L. J. Am. Chem. Soc. 1981, 103, 42394244. (35) Hilhorst, R.; Laane, C.; Veeger, C. Proc. Natl. Acad. Sci. U.S.A. (Phys. Sci.) 1982, 79, 3927-3930. (36) Luthi, P.; Luisi, P. L. J. Am. Chem. Soc. 1984, 106, 7285-7286. (37) Nicot, C.; Vacher, M.; Vincent, M.; Gallay, J.; Waks, M. Biochemistry 1985, 24, 7024-7032. (38) Zampieri, G. G.; Jackle, H.; Luisi, P. L. J. Phys. Chem. 1986, 90, 1849-1853. (39) Johnston, K. P.; Harrison, K. L.; Clarke, M. J.; Howdle, S. M.; Heitz, M. P.; Bright, F. V.; Carlier, C.; Randolph, T. W. Science 1996, 271, 624-626. (40) Evans, D. F.; Mitchell, D. J.; Ninham, B. W. J. Phys. Chem. 1986, 90, 2817-2825. (41) Chen, S. J.; Evans, D. F.; Ninham, B. W.; Mitchell, D. J.; Blum, F. D.; Pickup, S. J. Phys. Chem. 1986, 90, 842-847. (42) Hou, M. J.; Shah, D. O. Langmuir 1987, 3, 1086-1096. (43) Shinoda, K.; Lindman, B. Langmuir 1987, 3, 135-149.
Langmuir, Vol. 21, No. 23, 2005 10633 Scheme 2
reorientation on intrinsic relaxation rates. The global tumbling correlation time of an isotropically reorienting (spherical) molecule relates to the bulk solvent viscosity and the volume of the molecule through the StokesEinstein relationship:
τm )
ηV kT
The correlation time τm for a spherical particle is linearly related to the viscosity of the solvent. In the time regime of tumbling relevant here, given restricted internal motion, the nuclear spin-spin relaxation time varies approximately inversely with τm. The spin-spin relaxation time (T2) is a critical variable in the performance of modern high-resolution solution NMR experiments44 and with the efficiency of these experiments generally increases with increasing T2. This behavior has been demonstrated directly for ubiquitin encapsulated in AOT and dissolved in a range of alkane solvents.10 The Stokes-Einstein relation also indicates that there is an intrinsic penalty, with respect to tumbling, resulting from reverse micelle encapsulation as the volume of the reverse micelle is determined by the width of the surfactant and the volume of the protein and water pool encapsulated within.12 In line with the above reasoning, one should use surfactants with hydrophobic tail(s) as short as possible in order to reduce the volume penalty of encapsulation. In addition, reverse micelle surfactants often require small molecule cosurfactants in order to form regular spherical structures. Cosurfactants are often highly viscous alcohols such as hexanol, which is employed with the cationic surfactant CTAB to encapsulate acidic proteins with high structural fidelity.17 The presence of cosurfactants such as hexanol can significantly increase the effective viscosity of the solvent should they migrate from the surfactant shell into the bulk phase.45 Hence another important goal is to eliminate alcohol cosurfactant in the cationic surfactant system in addition to substituting CTAB with short analogues. Here we report the design and synthesis of a shortchain analogue of AOT and evaluate its suitability for encapsulation of proteins. In addition, we report the first use of binary mixtures of cationic and anionic surfactant systems to encapsulate proteins. These advances significantly broaden the landscape of available surfactant systems and begin to reveal the underlying features of reverse micelle forming mixtures that are capable of encapsulating proteins with high structural fidelity at (44) Sattler, M.; Schleucher, J.; Griesinger, C. Prog. NMR Spectrosc. 1999, 34, 93-158. (45) Yao, J. H.; Romsted, L. S. J. Am. Chem. Soc. 1994, 116, 1177911786.
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concentrations that are sufficient for application of advance NMR methods.
Shi et al. Scheme 3
Experimental Section Synthesis of the Short AOT Analogue. The synthesis of short AOT followed those of similar molecules by Nave et al.46-48 In detail, fumaryl chloride was added dropwise to a stirred solution of 3,3-dimethyl-1-butanol in dry tetrahydrofuran (THF). The reaction mixture was then refluxed until thin-layer chromatography (TLC) indicated completion. After rotary evaporation of THF, the mixture was dissolved in diethyl ether and washed several times sequentially with 10% HCl and saturated NaHCO3 solutions. The ethereal extracts were dried over anhydrous MgSO4 and filtered, and diethyl ether was removed by rotary evaporation. Vacuum distillation yielded the pure diester, which was yellow oil. This was then dissolved in a 1:1 mixture of ethanol/ water and refluxed with a slight excess of sodium metabisulfite and sodium sulfite to form the dialkyl sulfosuccinate. Soxhlet extraction with ethyl acetate, followed by several centrifugation cycles in methanol, was employed to remove residual sodium salts. The final products were dried in a vacuum oven for at least 24 h and then stored in a desiccating cabinet in sealed vials. The purified surfactant was assessed by 1H NMR (not shown), which gave results consistent with the expected chemical structure. Protein Expression and Reverse Micelle Sample Preparations. Recombinant 15N-labeled human ubiquitin49 and horse cytochrome c50 (cyt c) were prepared as described. Proteins in reverse micelles were prepared by the solid-phase transfer method as described in detail elsewhere.13,15 Briefly, empty reverse micelles without protein encapsulation were prepared by combining the desired surfactant mixtures and the required volume of aqueous buffer to the liquid pentane. This solution was then transferred to a vial containing the lyophilized protein. The proteins were transferred into the reverse micelles phase by gentle shaking and the reverse micelle solutions were transferred to NMR tubes with screw caps. The compositions of each sample and the conditions are specified in the corresponding figure legends. All NMR spectra were collected at 25 °C on a Varian INOVA spectrometer operating at 600 MHz (1H) with a 5 mm triple resonance z gradient cryoprobe. Spectra were processed and analyzed by using the program FELIX (Molecular Simulations, Waltham, MA).
Results
minimize this penalty, we have employed a condensing alcohol that will result in a tail length two carbons shorter than that of the parent AOT molecule. Reverse Micelles Formation by mbAOT. mbAOT fails to form reverse micelles with pure water in pentane but does so with water loadings (molar ratio of water to surfactant) of up to 20 if a 2 M NaCl solution is employed for the aqueous component. Recombinant human ubiquitin can also be encapsulated in mbAOT reverse micelles with essentially complete structural fidelity as judged from the close correspondence of its 15N-HSQC spectrum (Figure 1) to that of the protein in free aqueous solution. The slight differences in chemical shifts of backbone amide 1H-15N resonances of ubiquitin encapsulated in mbAOT and those of the protein encapsulated in AOT largely reflect the difference in ionic strength of the aqueous cores of the reverse micelles (2 M NaCl in the case of short AOT compared to 50 mM NaCl in AOT). Interestingly, the chemical shift differences are even larger between ubiquitin in water with and without high concentrations of salt (data not shown). Reverse Micelles Formed by Mixtures of Anionic and Cationic Surfactants. The size of a reverse micelle is almost unaffected by the absolute concentrations of
Design and Synthesis of Bis(3,3-dimethyl-1-butyl)sulfosuccinate. The formation of reverse micelles by many surfactants is affected by interfacial curvatures.40-42 The curvature is determined by a balance between headgroup forces and the hydrocarbon tail interactions together with geometric and steric constraints of the surfactant(s). We have designed a short AOT analogue, bis(3,3-dimethyl-1-butyl)sulfosuccinate (mbAOT), with branched tails and with primary chain lengths two carbons shorter than those of AOT. The synthesis followed an easy two-step reaction scheme (Scheme 3). The design of the short AOT analogue was guided by two factors. First, branched tails are intended to ensure a molecule capable of forming reverse micelles, which is specified by the surfactant parameter, defined as v/(a0lc) with v denoting the volume of the surfactant hydrocarbon chain, a0 the headgroup area, and lc the effective length of hydrocarbon chain, v/(a0lc) > 1. Second, as discussed above, there is an intrinsic volume penalty for effective molecular tumbling of a protein encapsulated within a reverse micelle, and to (46) Nave, S.; Eastoe, J.; Penfold, J. Langmuir 2000, 16, 8733-8740. (47) Nave, S.; Eastoe, J.; Heenan, R. K.; Steytler, D.; Grillo, I. Langmuir 2000, 16, 8741-8748. (48) Nave, S.; Eastoe, J.; Heenan, R. K.; Steytler, D.; Grillo, I. Langmuir 2002, 18, 1505-1510. (49) Wand, A. J.; Urbauer, J. L.; McEvoy, R. P.; Bieber, R. J. Biochemistry 1996, 35, 6116-6125. (50) Rumbley, J. N.; Hoang, L.; Englander, S. W. Biochemistry 2002, 41, 13894-13901.
Figure 1. 15N HSQC spectrum of ubiquitin in short AOT reverse micelles. The sample was prepared by the solid-phase transfer method as described in the Experimental Section. The concentration of the short AOT is 100 mM; the water loading (the molar ratio of surfactant to water) is 11; the aqueous buffer used is 30 mM sodium acetate, pH 5, containing 2 M NaCl; 20% deuterated pentane was added to the sample for locking; and the final effective concentration of protein in the sample was ∼0.2 mM.
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Figure 2. 15N HSQC spectrum of ubiquitin in short AOT/ DTAB reverse micelles. The sample was prepared by the solidphase transfer method as described in the Experimental Section. The molar ratio of short AOT to DTAB is 60:40, and the total concentration of the short AOT and DTAB is 75 mM; the water loading (the molar ratio of surfactant to water) is 10; the aqueous buffer used is 30 mM sodium acetate, pH 5; 20% deuterated pentane was added to the sample for locking; and the final effective concentration of protein in the sample was ∼0.2 mM.
Figure 3. 15N HSQC spectrum of ubiquitin in AOT/TBMAC reverse micelles. The sample was prepared by the solid-phase transfer method as described in the Experimental Section. The molar ratio of AOT to TBMAC is 55:45, and the total concentration of the short AOT and DTAB is 120 mM; the water loading (the molar ratio of surfactant to water) is 10; the aqueous buffer used is 30 mM sodium acetate, pH 5; 20% deuterated pentane was added to the sample for locking; and the final effective concentration of protein in the sample was ∼0.2 mM.
surfactants and mainly determined by the water loading.51 AOT can form reverse micelles with water loadings up to 55, depending on the surrounding nonpolar medium, the nature and the concentration of the electrolyte, the temperature, and other conditions. However, formation of reverse micelles by the short AOT analogue mbAOT requires as much as ∼2 M NaCl. This seems reasonable since tail-tail as well as tail-solvent interactions contribute favorably to the stability of the reverse micelle but that the head-head repulsions destabilize it. Reverse micelles formed by mbAOT have less favorable combined tail-tail and tail-solvent (pentane in this study) interactions compared to those by AOT and thus the effect of ∼2 M NaCl is to screen repulsions between headgroups. If this is indeed the case, it would be expected that a mixture of short AOT and some cationic surfactants such as DTAB and CTAB could form reverse micelles without use of a high ionic strength aqueous phase. We have found that indeed mbAOT combining with DTAB/CTAB can form reverse micelles with a water load up to 20 at certain molar ratios of the surfactants. We have also found that AOT together with DTAB/CTAB can uptake more water than the short mbAOT with DTAB/ CTAB at equivalent ratios of corresponding surfactants. Ubiquitin can be successfully encapsulated into the mbAOT/DTAB RMs as demonstrated by its 15N HSQC spectrum (Figure 2). The use of the short AOT analogue by itself is limited by the requirement of relatively high concentrations of salt during the process of RM sample preparation. By mixing short AOT with some cationic surfactants such as CTAB and DTAB, in principle, its utility can be expanded. However, the tails of CTAB and DTAB, with a chain of 16 or 12 carbons, respectively, are somewhat longer than those of AOT or the short AOT. Therefore potential gains from the RM system could be canceled in part by the introduction of relatively long tailed CTAB/DTAB. To overcome these potential limitations, we designed a similar surfactant system using AOT or mbAOT com-
Table 1. Reverse Micelle Formation by Mixtures of Anionic and Cationic Surfactants
(51) Zulauf, M.; Eicke, H. F. J. Phys. Chem. 1979, 83, 480-486.
CTAB/DTAB TBMAC/TBMAB
AOT
mbAOT
yes yes
yes no
bined with tributylmethylammonium chloride/bromide (TBMAC/TBMAB, Scheme 2). The tails of TBMAC/ TBMAB are much shorter than CTAB/DTAB with the alkyl chains being only four carbons long, which is also shorter than those of AOT and mbAOT. Unfortunately, we find that TBMAC/TBMAB cannot form RMs together with mbAOT, probably due to the reduced tail-tail interactions resulting from a much reduced tail length of TBMAC/TBMAB though the surfactant system has alleviated repulsive interactions proportionally which exist in the surfactant system by the short AOT only. However, we find that TBMAC/TBMAB can form RMs by mixing with AOT as rationalized by the fact that AOT has more favorable tail-tail interaction with itself or other surfactants in the system (Table 1). Finally, we find that the AOT/TBMAC(TBMAB) RM system is capable of encapsulating ubiquitin with high structural fidelity, as revealed by its 15N HSQC spectrum (Figure 3). Some proteins are denatured when they are encapsulated into RM water cores formed by surfactants such as AOT, probably due to strong interactions between the sulfonate group from AOT and positive charges on the surface of the protein. It is reasonable to conclude that the internal surface of RMs formed by AOT, which has a very high density of negative charges, interacts with the surface positive charge (patches) of an encapsulated protein. The mixture anionic and cationic surfactants are expected to dampen this effect significantly. (52) Douzou, P.; Keh, E.; Balny, C. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 681-684. (53) Brochette, P.; Petit, C.; Pileni, M. P. J. Phys. Chem. 1988, 92, 3505-3511. (54) Battistel, E.; Luisi, P. L.; Rialdi, G. J. Phys. Chem. 1988, 92, 6680-6685. (55) Huruguen, J. P.; Authier, M.; Greffe, J. L.; Pileni, M. P. Langmuir 1991, 7, 243-249.
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Figure 4. 15N HSQC spectrum of cytochrome c in short AOT/ DTAB reverse micelles. The sample was prepared by the solidphase transfer method as described in the Experimental Section. The molar ratio of short AOT to DTAB is 60:40, and the total concentration of the short AOT and DTAB is 110 mM; the water loading (the molar ratio of surfactant to water) is 10; the aqueous buffer used is 20 mM sodium phosphate, pH 7, containing 10 mM NaCl; 20% deuterated pentane was added to the sample for locking; and the final effective concentration of protein in the sample was ∼0.16 mM.
It has been reported that cytochrome c can be transferred into AOT RMs with high efficiency under a variety of conditions.52-56 However, the native structure of protein is not maintained as judged by UV and CD spectroscopy, which is also confirmed by NMR spectroscopy (data not shown). Cytochrome c retains its native state when encapsulated into the reverse micelles formed by a mixture of the short AOT and DTAB (Figure 4). However, the protein’s structural integrity is not entirely maintained in the AOT/TBMAC(TBMAB) RM system, though it is largely nativelike (data not shown). One possible reason is that since TBMAC(TBMAB) has a relatively high solubility in water, it may be present in the water core of the RM and influence the stability of encapsulated protein. Discussion A number of surfactant design studies working toward a variety of goals have been reported. Examples include supporting the formation of RMs in liquid or supercritical carbon dioxide,57 attempting to understand why certain surfactants such as AOT have exceptional ability to form RMs,46-48 and developing efficient RM surfactants for protein extraction and purification,58-64 as well as other purposes.65-67 Here we have tailored surfactant designs (56) Ono, T.; Kawakami, K.; Goto, M.; Furusaki, S. J. Mol. Catal. B: Enzym. 2001, 11, 955-959. (57) McClain, J. B.; Betts, D. E.; Canelas, D. A.; Samulski, E. T.; DeSimone, J. M.; Londono, J. D.; Cochran, H. D.; Wignall, G. D.; ChilluraMartino, D.; Triolo, R. Science 1996, 274, 2049-2052. (58) Ono, T.; Goto, M.; Nakashio, F.; Hatton, T. A. Biotechnol. Prog. 1996, 12, 793-800. (59) Goto, M.; Ono, T.; Nakashio, F.; Hatton, T. A. Biotechnol. Bioeng. 1997, 54, 26-32. (60) Rong, L.; Yamane, T.; Takeuchi, H. J. Chem. Eng. Jpn. 1998, 31, 434-439. (61) Rong, L.; Yamane, T.; Takeuchi, H. J. Chem. Eng. Jpn. 1999, 32, 530-534. (62) Goto, M.; Ishikawa, Y.; Ono, T.; Nakashio, F.; Hatton, T. A. Biotechnol. Prog. 1998, 14, 729-734. (63) Naoe, K.; Nishino, M.; Ohsa, T.; Kawagoe, M.; Imai, M. J. Chem. Technol. Biotechnol. 1999, 74, 221-226. (64) Shin, Y. O.; Vera, J. H. Biotechnol. Bioeng. 2002, 80, 537-543.
Shi et al.
(systems) for the application of high-resolution solution NMR. For this purpose one needs to consider not only the protein encapsulation properties of the RM system but also its solution behavior with respect to the effective tumbling rate of the encapsulated protein. This latter consideration imposes constraints on the bulk solvent viscosity, the size, and, to a limited degree, the shape, of the reverse micelle particle and the long-term stability of the preparation. Due to these considerations, we have designed surfactant systems with branched tails to ensure that their surfactant parameters are larger than 1 as required for the formation of RMs.40-42 In addition the length of the surfactant, which largely defines the width of the surfactant shell, is kept as short as possible to minimize the effective volume of the reverse micelle particle and enhance its rate of diffusional tumbling. With regard to protein encapsulation, the system of the mixture of anionic and cationic surfactants appears to ameliorate the destabilizing effects of charge-charge interactions with the encapsulated protein. The ability to “charge match” can obviously be adapted in a variety of ways to accommodate a broad range of proteins with significantly different properties. The results presented here provide a first step in this direction and open an avenue for future exploration. Though in this contribution the optimized surfactants were based on the simple anionic AOT and cationic CTAB parent molecules, it is important to point out that other surfactant prototypes may be useful. For example, some zwitterionic surfactants such as those derived from amino acids are expected to provide an inner surface environment more benign to proteins and other biomolecules and potentially hold some promise. It still remains an unfortunate fact that protein encapsulation as a tool to study protein structure remains very much a trial and error process. The protein-containing RM is a multicomponent complex molecular assembly with its structure and stability delicately balanced between a variety of interactions and intrinsic physical chemical properties of its constituent components. In this respect, attempting to understand the mechanism of RM formation and protein encapsulation is an obligatory preliminary for expanding applications of the technique to biomolecule structural studies by NMR. The ideal situation is one could choose in advance, on the basis of the fundamental properties of a target protein, the surfactant system that is most likely to provide high encapsulation integrity and efficiency and optimal properties for NMR spectroscopy. The results presented here provide a significant step in this direction. Past studies of protein encapsulation have generally employed average spectroscopic properties such as those provided by UV/vis, IR, and CD spectroscopy to assess the structural integrity of encapsulated protein. High-resolution NMR is both site-resolved and exquisitely sensitive to small local structural perturbations. Indeed, the chemical shift, which is employed here, is also sensitive to electric field effects and may also give a false positive indication for structural variation from the free solution state of the protein. Small chemical shift changes observed for the protein ubiquitin in free solution and encapsulated within AOT reverse micelles were subsequently shown by direct structure determination to not arise from (65) Leydet, A.; Boyer, B.; Lamaty, G.; Roque, J. P.; Catlin, K.; Menger, F. M. Langmuir 1994, 10, 1000-1002. (66) Khoshkbarchi, M. K.; Vera, J. H. J. Colloid Interface Sci. 1995, 170, 562-568. (67) Esalah, J. O.; Weber, M. E.; Vera, J. H. J. Colloid Interface Sci. 1999, 218, 344-346.
New Reverse Micelle Surfactant Systems
significant structural differences.68 Nevertheless, short of a comprehensive structural analysis, the NMR chemical shift remains the most sensitive probe for structural integrity of an encapsulated protein. Using chemical shift based analyses, we have found that many proteins suffer significant structural perturbation when encapsulated within the canonical AOT reverse micelle system. This has prompted us to design current surfactant systems with a hope to discover some that could provide potential advantages over that by AOT or CTAB. Our NMR and CD (data not shown) results have indicated that ubiquitin and cyt c exist in their native state structure within the water core of our designed short AOT/DTAB RMs. For cyt c, we have found that it is denatured in RMs by AOT alone; however, it regains its structural integrity in the designed system reported here. It is difficult for us to pinpoint exact reasons why some proteins tend to denature when they are entrapped in certain RMs. One possible reason is charge imbalance, and the charge matching afforded by the cationic-anionic surfactant mixtures appears to support this as an important consideration. Other possible causes exist and include (i) the concentration of ions inside the water core is potentially very high and could have a destabilizing effect on the protein, (ii) the water activity may change due to the specific RM milieu which may have a profound effect on protein structure as water is considered as an integral part of proteins, and (iii) the small scale of the inner volume of the reverse micelle provides a “confined space” effect on the structure of the protein. We believe there is a salt effect on protein stability; however, we argue that it would have more influence on the encapsulation efficiency rather than on the stability. Regardless, the entire issue is avoided if the chargematched surfactant system is employed, which cannot be achieved for the simple AOT or CTAB systems. As for the second possibility, Politi and Chaimovich69 have demonstrated that the activity of water in AOT RMs is indeed (68) Babu, C. R.; Flynn, P. F.; Wand, A. J. J. Am. Chem. Soc. 2001, 123, 2691-2692. (69) Politi, M. J.; Chaimovich, H. J. Phys. Chem. 1986, 90, 282-287.
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reduced to a significant extent when the water load is below 7.5; however it approaches that of bulk aqueous solution when the water load reaches to ∼10. NMR-based studies of small peptides in AOT reverse micelles have also indicated a significant layer of nonbulk water at the inner surface of the surfactant shell.30 The water loadings of the samples used here are g10; thus, the issue may not apply to our cases. However, it is a general consideration as a main goal is to reduce the volume of the reverse micelle particle as much as possible. Confinement of a flexible polymer chain within a constraining cavity results in the redistribution of conformational states toward those of smaller effective volume.70 Peterson et al. have recently demonstrated this effect in the context of the reverse micelle and that variance of the degree of water loading (the inner volume) of the reverse micelle can be used to “force fold” proteins of marginal stability.19 The generality of this remains to be demonstrated, but the initial result suggests that the confined space does, as theoretically predicted, stabilize proteins rather than destabilize them. The work presented here illustrates the potential of alternative surfactant systems in the context of NMR studies of encapsulated proteins. Further optimization in terms of protein encapsulation efficiency, expanding to a broader range of proteins encoded by the human and other genomes to include the various classes of intrinsically unfolded proteins,71-74 membrane proteins, and proteins of significant size, all with the aim of attaining an effective rotational tumbling time of less than 20 ns. Acknowledgment. This work was supported by NIH Grant GM 62874. LA051409A (70) Zhou, H. X.; Dill, K. A. Biochemistry 2001, 40, 11289-11293. (71) Wright, P. E.; Dyson, H. J. J. Mol. Biol. 1999, 293, 321-331. (72) Uversky, V. N.; Gillespie, J. R.; Fink, A. L. Proteins 2000, 41, 415-427. (73) Iakoucheva, L. M.; Brown, C. J.; Lawson, J. D.; Obradovic, Z.; Dunker, A. K. J. Mol. Biol. 2002, 323, 573-584. (74) Tompa, P. Trends Biochem. Sci. 2002, 27, 527-533.
