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High-resolution NMR spectroscopy of encapsulated proteins dissolved inlow-viscosity fluids

Nathaniel V. Nucci, Kathleen G. Valentine, A. Joshua Wand ⇑Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104-6059, USA

a r t i c l e i n f o

Article history:Received 14 September 2013

Keywords:Reverse micelle NMRProtein encapsulationNucleic acid encapsulationLow viscosity fluidStructural biologyBiophysics

a b s t r a c t

High-resolution multi-dimensional solution NMR is unique as a biophysical and biochemical tool in itsability to examine both the structure and dynamics of macromolecules at atomic resolution. Conven-tional solution NMR approaches, however, are largely limited to examinations of relatively small(<25 kDa) molecules, mostly due to the spectroscopic consequences of slow rotational diffusion. Encap-sulation of macromolecules within the protective nanoscale aqueous interior of reverse micelles dis-solved in low viscosity fluids has been developed as a means through which the ‘slow tumblingproblem’ can be overcome. This approach has been successfully applied to diverse proteins and nucleicacids ranging up to 100 kDa, considerably widening the range of biological macromolecules to which con-ventional solution NMR methodologies may be applied. Recent advances in methodology have signifi-cantly broadened the utility of this approach in structural biology and molecular biophysics.

� 2014 Published by Elsevier Inc.

1. Introduction

Nuclear magnetic resonance (NMR) spectroscopy continues toplay a central role in the characterization of the structure anddynamics of proteins, nucleic acids and their complexes. Over thepast several decades developments in solution NMR techniques,especially triple resonance spectroscopy and supporting technolo-gies, have made the comprehensive structural characterization of20 kDa soluble proteins routine [1]. Solution NMR spectroscopycan provide a detailed view of the solution structure of macromol-ecules and allows unparalleled access to dynamic phenomena. Italso provides a unique avenue to monitor the full structural and dy-namic effects of changes in temperature, pressure, and other solu-tion conditions including the binding of small and large ligands.With the development of multinuclear and multidimensionalNMR, the structures of proteins of significant size and spectral com-plexity can now be efficiently determined. However, increasingmolecular size leads to slower tumbling and correspondingly short-er spin–spin relaxation times (T2). Short T2 values severely limit thepower and flexibility of multiple pulse NMR experiments in at leasttwo ways. The signal-to-noise ratio of a Lorentzian line degradeswith decreasing T2 and the effectiveness of the library of conven-tional multidimensional and multinuclear NMR experiments are

exponentially sensitive to T2 [1]. Accordingly, the standard tripleresonance experiments become unreliable at room temperaturefor proteins larger than 30 kDa and largely fail for proteins above35 kDa in the absence of elevated temperature and/or extensivedeuteration. A variety of approaches have been developed to com-bat the limitations caused by fast transverse relaxation broughtabout by slow molecular reorientation where the combination ofperdeuteration with selective reprotonation [2–4] and the trans-verse relaxation optimized (TROSY) spectroscopy [5,6] has enabledaccess to an array of insights into very large macromolecular com-plexes. Nevertheless, this basic approach does come with costs bothwith respect to the production of material and loss of potentialinformation resulting from extensive perdeuteration.

It was in this context some fifteen years ago that we introduced analternate strategy that focused on attacking the origin of the ‘‘slowtumbling problem’’ presented by large macromolecules [7]. That isthe inescapable fact that the larger the protein is the more slowlyit tumbles in free aqueous solution. Slower macromolecular reorien-tation leads to faster spin–spin relaxation and therefore broader res-onances and poorer coherence transfer in multi-dimensionalexperiments. Briefly, if we consider a heteronuclear spin pair suchas an amide 15N–1H, we find that the spin–spin relaxation rate (R2) is:

R2 ¼1T2

� �

¼ l0

4p

� � �h2c2Nc2

H

6r6

!½4Jð0Þ þ JðxH �xNÞ þ 6JðxHÞ þ 3JðxNÞ

þ JðxH þxNÞ� þx2

NDr2

18½4Jð0Þ þ 3JðxNÞ� ð1Þ

1090-7807/$ - see front matter � 2014 Published by Elsevier Inc.http://dx.doi.org/10.1016/j.jmr.2013.10.006

⇑ Corresponding author. Address: Department of Biochemistry and Biophysics,University of Pennsylvania Perelman School of Medicine, 904 Stellar-ChanceLaboratories, 422 Curie Blvd., Philadelphia, PA 19104-6059, USA. Fax: +1 215 5737291.

E-mail address: [email protected] (A.J. Wand).

Journal of Magnetic Resonance 241 (2014) 137–147

Contents lists available at ScienceDirect

Journal of Magnetic Resonance

journal homepage: www.elsevier .com/locate / jmr


where J(x) is the spectral density at frequency x, r is the effectiveN–H bond length, and the remaining symbols have their usualmeanings. It is useful to express the spectral densities in terms ofthe popular Lipari–Szabo (L–S) model-free treatment [8] to makethe dependence of the spin–spin relaxation rate on the time scaleof macromolecular tumbling clear. For an isotropically reorientingmacromolecule, the L–S model-free spectral density is:

JðxÞ ¼ 25

O2

1þx2s2mþ ð1� O2Þ

1þx2s2

" #; s�1 ¼ s�1

m þ s�1e ð2Þ

where O2, se and sm are the model-free squared generalized orderparameter, effective correlation time and macromolecular reorien-tation correlation time, respectively. Thus, in the absence of com-plete disorder (i.e. O2 ! 0), the spin–spin relaxation will beheavily influenced by the time scale of macromolecular tumbling.

The key ingredient leading to an alternate approach to the slowtumbling problem is revealed by the Stokes–Einstein equationwhich describes the molecular reorientation time of a sphericalparticle in a solvent of much smaller molecules:

sm ¼4pr3g3kBT

ð3Þ

where sm is the rotational correlation time, kB is the Boltzmann con-stant, T is the temperature, g is the viscosity of the liquid, and r isthe radius of the particle. Obviously, the larger the particle is themore slowly it will tumble. Consideration of Eq. (3) suggests threepossible routes to alleviating the slow tumbling of a macromoleculesuch as a protein. A classic approach is to reduce the volume of theprotein by examination of fragments that can be made in variousways. This reductionist strategy, though often advantageous, hasbegun to fall out of favor in modern-day structural biology and bio-physics. Using elevated temperature is an enticing possibility sinceit will increase the thermal energy (kBT) available for molecularreorientation, activate internal motion and reduce the viscosity ofwater all of which will tend to reduce the spin–spin relaxation rate.Unfortunately, however, most biological macromolecules are stableat only modestly elevated temperatures making this tactic of lim-ited utility.

One variable in Eq. (2) remains – the viscosity. The strategy thenbecomes to find a means to ‘‘trick’’ the protein of interest by pro-viding it with a stabilizing hydration shell and dissolving the entireassembly of protein and hydration water in a low viscosity solvent.Methods have been developed to solubilize proteins with minimalwater directly in organic solvents but only at very low concentra-tions that are unsuitable for NMR spectroscopy [9,10]. To protectand solubilize individual hydrated protein molecules, we turnedto the reverse micelle encapsulation technology developed for avariety of applications during the 1980s [11]. The goal was to adaptreverse micelle encapsulation to the stringent requirements ofhigh-resolution solution NMR: maintenance of structural integrity,sufficient concentrations of macromolecule (i.e. near mM) and lowenough bulk solvent viscosity to enhance the NMR performance bysuppression of spin–spin relaxation through increased macromo-lecular tumbling.

2. Reverse micelles for encapsulation of macromolecules

Under appropriate conditions, mixtures of surfactants and asmall volume of water immersed in a bulk non-polar solvent suchas the alkanes will spontaneously organize into spherical reversemicelles (Fig. 1) [12]. Surfactants capable of forming spherical re-verse micelles generally have small polar or charged headgroupsand long and often branched hydrophobic tails. Under conditionsappropriate for solution NMR, each reverse micelle will contain100–400 primary surfactant molecules, one to five thousand

waters, and a single protein molecule. The size of the aqueousnanoscale water core of the reverse micelle is largely influencedby the molar ratio of water to surfactant molecules, which is oftentermed the water-loading or W0. These mixtures can form a varietyof structures ranging from small nanometer scale spherical parti-cles to macroscopic gels. The phase diagram is often complexand dependent on a variety of parameters such as water loading,temperature, pressure, absolute surfactant concentrations and ra-tios e.g. [13]. Reverse micelle encapsulation has long been usedfor investigations of protein structure and function using relativelylow-resolution spectroscopic methods such as ultraviolet–visibleabsorption and fluorescence emission spectroscopy [11,12]. Theconcentrations of encapsulated protein required for these studiesare generally much lower (�lM) than that needed for efficientinvestigation by NMR (�mM).

Protein and peptide molecules encapsulated within reverse mi-celles were first examined using simple solution NMR methodsover two decades ago [14,15]. One-dimensional NMR measure-ments of proteins in reverse micelles offered more conclusive evi-dence of the maintenance of protein structural integrity, indicatingthe potential of this approach for high-resolution solution NMRinvestigations. These early studies largely utilized reverse micellescomprised of the surfactant bis(2-ethylhexyl)sulfosuccinate (AOT)and were typically prepared in the relatively high-viscosity iso-oc-tane. Since the radius of the reverse micelle is substantially largerthan the radius of the protein (Fig. 1), the viscosity of the non-polarsolvent therefore becomes a vital parameter for solution NMR ofthe encapsulated macromolecules. Unfortunately, there are veryfew solvents that are capable of supporting the formation of smallreverse micelles and are of low enough viscosity to overcome thevolume penalty imposed by encapsulation (Fig. 2). The short chainalkanes [7], carbon dioxide [16] and xeon [17] have been examinedin this context.

Fig. 1. Schematic of a reverse micelle encapsulating a protein. Maltose-bindingprotein is shown in cartoon representation (PDB ID 2H25) [72] encapsulated withina reverse micelle. Drawn to scale. The diameter of the protein was measured usingPymol [73]. The diameter of the water core is based on measurements of MBPtumbling in reverse micelles composed of CTAB/hexanol in various solvents [22].The thickness of the surfactant layer is based on estimates for CTAB protein-freereverse micelle systems [57]. A reverse micelle under these conditions containsapproximately 200 surfactant molecules and �3000 water molecules.

138 N.V. Nucci et al. / Journal of Magnetic Resonance 241 (2014) 137–147


Historically, AOT has been widely used presumably because itcan form stable reverse micelles over a broad range of surfactantconcentrations and water content. Unfortunately, efforts to encap-sulate proteins with high structural fidelity in AOT reverse micellesrevealed that this surfactant has a strong denaturing effect on mostproteins [18]. Several other surfactant mixtures have been devel-oped for high-resolution NMR studies of encapsulated proteins.These surfactants and their 1H spectra are illustrated in Fig. 3. Eachof these surfactants contains distinct 1H signals that permit de-tailed analysis of sample compositions such as the precise surfac-tant ratios and measurement of W0. For high-resolution NMRstudies, however, the advantages of AOT are overwhelmed by itsstrong propensity to denature encapsulated proteins. For the pur-poses of protein biochemical and biophysical investigations, thisfeature renders AOT nearly useless for solution NMR investigationsof proteins. Indeed, in our hands, ubiquitin is the only natural pro-tein for which conditions could be found that maintain the nativefold structure encapsulated in AOT reverse micelles.

CTAB has also been widely employed for reverse micelle-basedstudies [11]. This cationic surfactant requires the addition of rela-tively high concentrations of co-surfactant molecules in order toform stable reverse micelle mixtures. The short-chain primaryalcohols are the most common co-surfactants used for CTAB re-verse micelle applications. Because these high-concentration co-surfactants contribute somewhat to the overall bulk viscosity ofthe reverse micelle mixture, low-viscosity short-chain alcoholsare more advantageous for promoting fast tumbling of the reversemicelle particle. Shorter-chain alcohols, however, are required inhigher concentration than longer-chain alcohols, thus a balancemust be struck between high concentrations and a potential degra-dation of the viscosity. Hexanol provides the best general perfor-mance for CTAB reverse micelle encapsulation of proteins withrespect to the overall impact on reverse micelle rotational diffu-sion. The CTAB/hexanol mixture has a considerably lower chargedensity at the water–surfactant interface than AOT, producingexcellent versatility for encapsulation of a wide range of macro-molecules with structural fidelity [18–25]. A practical shortcomingof this reverse micelle system arises from the charged nature of the

surfactant–water interface that produces subtle chemical shift per-turbations of the encapsulated protein that require reassignmentof a minority of amide resonances. Effects on aliphatic resonancesare generally small and compare closely to aqueous assignments[22]. Despite this minor limitation, the CTAB/hexanol mixture

Fig. 2. Calculated effective isotropic rotational correlation time (sm) for encapsu-lated proteins prepared in various non-polar solvents. Based on a spherical particlewhose size is determined by the hydrated radius of the protein plus the thickness ofthe surfactant layer [22]. The hydrated radius was a spherical protein of averagedensity and average hydration [74]. Viscosities were obtained from the US NationalInstitute of Standards and Technology [75]. The viscosity of ethane is pressuresensitive and viscosity over the range of pressures typically used is shown in gray.While specific conditions will produce some variation from these predictions, thesetypes of estimations agree well with experimental determinations for the CTAB/hexanol and 10MAG/LDAO reverse micelle systems [20,22].

Fig. 3. Structures and 1H spectra of select reverse micelle surfactant systems. One-dimensional 1H spectra are shown for each of four reverse micelle surfactantmixtures at W0 of 15. (A) 75 mM bis-2-ethylhexylsulfosuccinate (AOT). (B) 75 mMcetyltrimethylammonium bromide (CTAB) with 450 mM hexanol (HexOH) as co-surfactant. (C) 105 mM tetraethylene glycol monodecylether (C12E4), 30 mM AOT,15 mM dodecyltrimethylammonium bromide (DTAB). (D) 52.5 mM decyl-1-rac-glycerol (10MAG), 22.5 mM lauryldimethylamine-N-oxide (LDAO). Distinct 1H sitesfor each surfactant are indicated by � or # on the chemical structure and on thespectra at the characteristic resonance. In each spectrum, the singlet at far left is thewater signal. No solvent suppression methods were employed in the collection orprocessing of these data. All samples are dissolved in d-12 (98%) pentane(Cambridge Isotopes, Tewksbury, MA).

N.V. Nucci et al. / Journal of Magnetic Resonance 241 (2014) 137–147 139


has been successfully implemented for a wide range of proteinsand a broad spectrum of solution NMR techniques as discussed be-low [21,23–26].

In order to mitigate complications arising from the high chargedensity at the water–surfactant interface, nonionic surfactants of-fer an appealing complement to charged surfactants [18]. C12E4 isthe primary component in a three-surfactant mixture with AOTand DTAB that provides a reduced charge density at the surfac-tant–water interface, thereby minimizing denaturing effects onthe encapsulated proteins and providing the ability to charge-bal-ance the surfactant interface for encapsulation of acidic, neutral,and basic proteins. This surfactant mixture provides excellent ver-satility for encapsulation of globular proteins and yields closeagreement between chemical shifts of encapsulated proteins andthose in bulk aqueous solution. Despite these advantages, however,the length of the C12E4 molecule results in a relatively thick surfac-tant layer and a larger reverse micelle particle, thereby resulting insignificantly slower tumbling. The inclusion of AOT in this mixturealso limits its utility in ultra-low viscosity solvents since relativelyhigh pressures are required, which compromises the desired effect(see Fig. 2).

Recently, a new surfactant mixture composed of LDAO and10MAG was developed that offers low charge-density at thewater–surfactant interface as well as short contour length of thesurfactant layer and amenability to encapsulation in ultra-low vis-cosity solvents (see Fig. 2) [20]. The 10MAG/LDAO surfactant sys-tem permits encapsulation of proteins at least as large as 80 kDawith isoelectric points ranging from 4 to 11. Nucleic acids can alsobe encapsulated. This surfactant system also provides a net charge-neutral surfactant interface that results in very little perturbationof the NMR spectrum of the encapsulated macromolecule. Impor-tantly, 10MAG/LDAO reverse micelles can be readily prepared inultra-low viscosity short-chain alkanes providing a significantrotational diffusion advantage.

3. Sample preparation

The key to the reverse micelle encapsulation strategy is the nat-ure of the sample. In its simplest form, sample preparation involvesmixing of surfactant components in the non-polar solvent followedby injection of an appropriate volume of protein solution toachieve the desired water content [11]. Due to the substantial dilu-tion of the protein that this direct injection method creates, highconcentration aqueous protein solutions (>2 mM) are required toachieve final encapsulated protein concentrations appropriate forefficient collection of NMR spectra (i.e. 100–200 lM). In rare cases,protein-free reverse micelle solutions can even be used to simplysolubilize dry protein.

While many proteins are amenable to the high initial concen-trations necessary for direct injection, the majority of proteins donot tolerate such conditions. Alternative methods for encapsula-tion and concentration of the protein in the reverse micelle solu-tion are available. The phase transfer method involves mixing thealkane solution of surfactants with a large volume of dilute aque-ous protein solution and subsequently allowing the phases to sep-arate [11]. Under appropriate conditions, the protein willspontaneously partition to achieve reverse micelle protein concen-trations appropriate for solution NMR. However, this method suf-fers from poor control of W0, frequently resulting in extremelyhigh water content in the reverse micelle phase that may compro-mise the tumbling advantages of encapsulation. This problem can,however, be mitigated by the addition of dry surfactants to reducethe W0 (i.e. distribute water to reverse micelles not containing aprotein molecule) [27].

The phase transfer method is rather unpredictable and cumber-some. To overcome the limitations of the phase transfer and directinjection methods, we have developed a method that allows diluteprotein solutions to be used to create solutions of encapsulatedprotein at sufficient concentrations and water loadings appropriatefor NMR [20]. Termed the injection–evaporation method, this ap-proach uses repeated rounds of injection of small volumes of aque-ous protein solution followed by partial evaporation. Through thisprocess, dilute protein solutions can be used to produce reversemicelle samples with reasonable concentrations for NMR whileoffering excellent control of the water content of the system. Gen-erally, solutions of encapsulated proteins can be prepared to con-centrations of 0.1–0.2 mM. In other words, though solutions ofreverse micelles can be made to mM concentrations, the loadingof individual protein molecules is limited to 10–25%. Protein load-ing is currently the limiting parameter of the reverse micelle NMRstrategy and remains a central focus for technical development.The injection–evaporation method shows great promise in thisrespect.

Historically the principle barrier to the reverse micelle NMR ap-proach has been the identification of appropriate encapsulationconditions (surfactants, concentrations, W0, etc.) that provide highstructural fidelity with excellent macromolecular tumbling andsufficient long-term sample stability and protein concentration toallow multidimensional NMR studies to be carried forward. As de-scribed above, it is hoped that the identification of the 10MAG/LDAO surfactant system and the associated reverse micelle condi-tion screening protocol will provide a general solution to this prob-lem [20]. The tuning of the water content has proven to be aparticularly important parameter for identifying appropriateencapsulation conditions. Indeed, it seems that the optimal watercontent for encapsulated proteins correlates closely with their hy-drated radius in free aqueous solution [22]. This suggests that thelowest free energy state of the reverse micelle system tends to fa-vor just enough water to satisfy the protein’s hydration layer andtherefore provides an ideal balance between protein structuralfidelity and optimal tumbling.

Optimization and control of pH within the reverse micelle inte-rior is also an important factor in preparation of reverse micellesamples. The contributions of titratable surfactant head groups tothe effective pH of the aqueous interior of the reverse micelle havebeen a controversial and frequently overlooked factor. It must beemphasized that the water core of a reverse micelle that is typi-cally used for NMR studies of encapsulated proteins contains onlya few molecules of buffer. Thus it cannot be assumed that simplyadjusting the pH of the buffer used to solubilize the protein setsthe pH of the water core. In most cases, it is the character of thesurfactants that determines the effective pH. A simple approachfor control and measurement of the pH within the reverse micelleinterior has recently been developed and allows facile adjustmentsin order to set and optimize encapsulation conditions [20,28].

4. Encapsulation in ultra-low viscosity solvents

The tumbling advantage offered by reverse micelle encapsula-tion for large macromolecules fundamentally comes from thelow viscosity of the bulk non-polar solvent. Pentane, which is a li-quid at standard pressure and temperature, offers a viscosity about1=4 that of water and is satisfactory for many solution NMR applica-tions. For larger macromolecules, the volume penalty for encapsu-lation is sufficiently large that there are only a few solvents withlow enough viscosity to effectively overcome it. These includethe short chain alkanes such as butane, propane and ethane[7,29], carbon dioxide [16] and xeon [17]. Carbon dioxide hasroughly the viscosity of propane but suffers from the fact that



the pH of the reverse micelle water core cannot be controlled andis quite acidic [30]. Xeon, with a viscosity approaching that of li-quid ethane, has seen far fewer applications, presumably becauseof a paucity of suitable surfactant systems. The short chain alkanesappear to be the solvents of choice in the context of high-resolu-tion solution NMR of encapsulated macromolecules. Obviously,however, none of these solvents are liquids at STP and elevatedpressure is required. Though the short chain alkanes require onlymoderate pressures to be liquefied at room temperature, experi-ence has shown that considerably higher pressures are requiredto prepare solutions of encapsulated proteins that are optimal forNMR spectroscopy. For example, samples prepared with the10MAG/LDAO surfactant system in ethane require pressures onthe order of 3500–6000 p.s.i. (240–415 bar), depending on the de-tails of the sample. In most cases, conditions that are identified inliquid pentane require only subtle modification for encapsulationin liquid propane or ethane [20].

Preparation of samples at these pressures requires specializedapparatus [31,32]. Samples prepared in pentane or other alkanesolvents that are liquid at STP can usually be used with standardglass NMR tubes equipped with screw cap seals. Butane samplescan be used with heavier walled glass NMR tubes equipped withTeflon needle valves. For propane and ethane, where considerablepressure is required to stabilize solutions of encapsulated proteins,caution requires the use of specialized NMR tubes made from PEEK(for propane) [33] or the more desirable zirconia ceramic (for pro-pane and ethane) [27,32,34]. It is important to note that thoughthese pressures require careful consideration, they are well belowthose required to induce significant structural or dynamical transi-tions in proteins [35,36]. The CTAB/hexanol and 10MAG/LDAO sur-factant systems provide excellent encapsulation efficiency in bothliquid propane and liquid ethane [22,31,34]. Fig. 2 shows the the-oretical tumbling advantages available through the use of the shortchain alkanes. Liquid pentane provides modest tumbling advanta-ges over aqueous solution for proteins of �40 kDa or larger. Liquidbutane, propane or ethane offer the potential for remarkableimprovements in rotational diffusion of encapsulated macromole-cules. Indeed, in principle, liquid ethane could allow a 100 kDaencapsulated protein to be made to tumble as quickly as a10 kDa protein in water. It should be noted that the viscosity of

ethane is particularly sensitive to pressure and this often repre-sents the limiting factor in the use of this solvent for a particularprotein system. For example, CTAB/hexanol reverse micellestypically require protein encapsulation pressures of �3000–5000 p.s.i. [22,31] while the encapsulation pressures for the10MAG/LDAO system are typically 3500–6000 p.s.i. [20] producinga viscosity range of 68–90 lPa s. The effective macromoleculartumbling correlation times of encapsulated proteins have beenexperimentally estimated using the TRACT method [20,22]. Thetumbling advantages achieved are more modest than predicted.These differences likely arise from imperfect optimization, varia-tion of the effective viscosity due to the presence of various co-sol-vents and co-surfactants and deviation from the assumptions of

Fig. 4. Aligned ensemble of structures of encapsulated ubiquitin (1G6J) (blue)determined by NMR [19]. Also shown is the crystal structure of ubiquitin (1UBQ)[45] (green) and the lowest energy conformer of the ensemble determined for theprotein in free aqueous solution (1D3Z) (yellow) [46]. The structures are statisti-cally indistinguishable.

Fig. 5. (A) 15N-HSQC spectrum of arginine kinase in 10MAG/LDAO reverse micelles dissolved in liquid ethane [20]. (B) Comparison of amide 1H and 15N chemical shifts theprotein encapsulated in 10MAG/LDAO reverse micelles versus those in aqueous solution.



the prediction model (perfectly spherical geometry, a constantthickness of the protein hydration layer, etc.). Despite these dis-crepancies, remarkable tumbling advantages can be realized. Forexample, encapsulation of the 40.8 kDa maltose-binding proteinin CTAB/hexanol or 10MAG/LDAO in liquid ethane at �4500 p.s.i.produces a 2.5-fold improvement in tumbling relative to aqueoussolution [20]. Several promising additives that permit lowering ofthe required encapsulation pressure (and with it the viscosity)have also been identified [31].

5. Practical considerations

From a spectroscopic perspective, perhaps the most importantdifference between a reverse micelle sample and a typical aque-ous sample is complexity of the solvent and surfactant mixture.In general, solutions of encapsulated proteins prepared in alkanesolvents have a much lower dielectric than an aqueous sample. Asa result reverse micelle samples are non-lossy and do not signif-icantly diminish the quality factor of the cryogenically cooled

Table 1Proteins and nucleic acids successfully encapsulated in low viscosity alkanes.

MW (kDa) PI Applicable (optimal) surfactant mixtures

Ubiquitin [18–20,22] 8.5 6.8 AOTCTAB/HexOHC12E4/AOT/DTAB10MAG/LDAO

Cytochrome c[18,20,21] 12.3 10 CTAB/HexOHC12E4/AOT/DTAB10MAG/LDAO

Staphylococcal nuclease D+PHS 16.1 9.2 CTAB/HexOHC12E4/AOT/DTAB10MAG/LDAO

T4 lysozyme 18.6 9.8 C12E4/AOT/DTAB10MAG/LDAO

Hen egg white lysozyme 14.4 11.3 CTAB/HexOH10MAG/LDAO

Flavodoxin [20,21] 19 4.2 CTAB/HexOHC12E4/AOT/DTAB10MAG/LDAO

Dihydrofolate reductase (human) [22] 18 4.8 CTAB/HexOH10MAG/LDAO

Aldoketoreductase 1C2 [20] 36.7 7.1 10MAG/LDAOArginine kinase [20] 40.2 6.0 10MAG/LDAOMaltose-binding protein [20,22,31] 40.8 5.2 CTAB/HexOH

10MAG/LDAOeGFPa [21] 54 5.6 CTAB/HexOH

10MAG/LDAOMalate synthase G [20,22] 81.4 5.9 CTAB/HexOH

10MAG/LDAODsRed2+a,b 103 6.2 CTAB/HexOHHIV-1 matrix proteinb [25] 14.7 9.1 CTAB/HexOHRecoverin [25] 26.9 5.7 CTAB/HexOH

10MAG/LDAOKcsAa,b [23,24] 54 10.5 CTAB/DHAB/HexOHtRNAval [20] 27 n.d. 10MAG/LDAOHIV tetraTARb 8.5 n.d. CTAB/HexOHU4 snRNAb 7.4 n.d. CTAB/HexOH

a Sizes listed for these proteins are for the multimeric state. Monomer sizes are: eGFP – 27 kDa, DsRed2+ – 25.8 kDa, KcsA – 13.5 kDa.b These molecules have not been tested in the 10MAG/LDAO mixture.

Fig. 6. Planes of a (H)C(CO)NNH TOCSY experiment [48,49] from uniformly 15N, 13C-labeled maltose-binding protein encapsulated in CTAB/hexanol reverse micelles in liquidethane. Figure modified from [31]. Data were collected at 600 MHz on a Bruker Avance-III TXI cryoprobe at 20 �C. TOCSY mixing times and indirect 15N resonances for thepictured data are indicated.



cryoprobe [37]. In practice, this provides a two- to threefold sig-nal-to-noise advantage, which is particularly important in light ofthe relatively limited concentration of encapsulated protein solu-tions that can currently be achieved. Looking forward, the recentdemonstration that solutions of reverse micelles seem to enabledynamic nuclear polarization in large-volume liquid samplesbodes well for overcoming this concentration limitation in the fu-ture [38].

The alkane solvent eliminates the need for the detailed watersuppression techniques typically required for aqueous samples.The amount of water in a reverse micelle solution is quite small(<2.5 M), which is generally eliminated by the X-nucleus editinginherent in heteronuclear pulse sequences [1]. For homonuclear1H multi-dimensional experiments, simple flip-back approaches[39] remove interference from the water resonance. The alkane

solvent and surfactants are more challenging, as they arecomprised of multiple 1H resonances involved in extensive J-cou-pling. Deuterated short-chain alkanes are available though 1–2%residual hydrogen is present in currently available stock.Deuterated solvent also reduces radiation damping [40]. The highconcentrations of protonated surfactants provide additionalsources of contaminating signals (see Fig. 3). Not all surfactantsare commercially available in deuterated form. 1H resonances ofthe surfactants are most effectively eliminated through the useof the gradient-selected quadrature [41], where the

p2 loss in

sensitivity is generally more than compensated by the reductionof t1 correlated noise arising from incomplete suppression ofsurfactant signals. States-TPPI quadrature methods [42,43] canalso be employed in combination with WET suppression [44].

Fig. 7. Planes of four-dimensional NOESY spectra of myristoylated recoverin in reverse micelles. (A) An indirect 1H, 13C plane at x1 = 7.18 ppm 1H and x2 = 121.7 ppm 15N ofthe 4D 13C-HSQC-NOESY-15N-HSQC spectrum [76] of 225 lM uniform 15N–13C labeled myristoylated revoverin in 75 mM CTAB, 400 mM hexanol in pentane. The 1Hacquisition dimension x1 is collected with 512 complex points, 16 complex points in the x2

15N dimension, 32 complex points in the x31H dimension and 16 complex points

in the x413C dimension. A sweep width of 20 ppm was used in the x4

13C dimension, heavily folded for optimal resolution with minimum overlap. The NOESY mixing timewas 90 ms. The total data collection time was 164 h. The 4D spectrum was processed using Felix processing software (Molecular Simulations) including forward linearprediction of �20% of the 13C x2 complex points. (B) An indirect 1H, 13C plane at x1 = 0.92 ppm 1H and x2 = 42.5 ppm 13C of the four-dimensional 13C-HMQC-NOESY-13C-HMQC spectrum [77] of the above sample. The acquisition dimension x1 was collected with 512 complex points, 32 complex points in the x2

13C dimension, 26 complexpoints in the x3

1H dimension and 20 complex points in the x413C dimension. A sweep width of 20 ppm was used in both the x2 and x4

13C dimensions, folded for optimalresolution. The NOESY mixing time was 90 ms. The total data collection time was 177 h. The spectrum was processed using forward linear prediction of �25% of the 1H x3

complex points.

Fig. 8. Paramagnetic relaxation enhancements of MTSL-labeled flavodoxin (S72C, C55A) encapsulated in 10MAG/LDAO reverse micelles (red circles). Ratios amide 1H–15Ncrosspeak intensities of oxidized MTSL (paramagnetic) and reduced MTSL (diamagnetic) labeled flavodoxin are shown. The strongest PRE is seen at the site of labelattachment (highlighted in red). PREs are color-coded on a ribbon representation of the structure of flavodoxin [78] from red (strong PRE) to white (no PRE). The flavinmononucleotide ligand is shown in stick representation (green). The location of the site of MTSL labeling (C72) is also indicated by the arrow. Adapted from [38].



6. Validation of the reverse micelle NMR method

In the context of structural biology and molecular biophysics itmakes little sense to undertake studies of proteins and nucleicacids if their native structure cannot be maintained in the reversemicelle. The first protein to be encapsulated and examined usinghigh-resolution multi-dimensional NMR methods was the8.5 kDa model protein ubiquitin [7,34]. The structure of this pro-tein was solved using AOT reverse micelles dissolved in liquid pen-tane [19]. The structure of encapsulated ubiquitin was found to beindistinguishable from the X-ray crystallographic [45] and bulksolution NMR structures [46] (Fig. 4). The determination of thestructure of encapsulated ubiquitin provided an initial proof ofprinciple for the reverse micelle encapsulation strategy. The tech-nology has progressed greatly over the past decade. Whereasencapsulation of ubiquitin in AOT produced sufficient chemicalshift (though not structural) perturbations to necessitate de novoassignment, the recently developed 10MAG/LDAO surfactant mix-ture provides excellent correspondence between the NMR spectraof macromolecules in free aqueous solution and encapsulated. Thecorrespondence of the chemical shifts of amide 1H and 15N of the40 kDa arginine kinase encapsulated in 10MAG/LDAO reverse mi-celles dissolved in liquid ethane is shown in Fig. 5.

The development of the various surfactant mixtures describedabove has permitted globular proteins as large as 103 kDa to beencapsulated (Table 1). Nucleic acids have also been successfullyencapsulated [20,47]. In the case of nucleic acids, the confinementof the reverse micelle [47] and reduced rates of hydrogen exchange[20] can significantly improve the spectral quality. The broad rangeof macromolecules for which the reverse micelle method has beenapplied reveals the promise of this approach for solution NMRcharacterization of biological molecules of all kinds.

7. Access to the full catalog of solution NMR experiments

7.1. Assignment of hydrogen resonances and access to the NOE

A central advantage of the reverse micelle encapsulation strat-egy is that large proteins retain the spin relaxation properties ofsmaller proteins through an increased rate of molecular reorienta-tion [7]. In principle this would avoid the need for deuteration andpermit the comprehensive assignment of 1H resonances andemployment of 1H–1H NOEs as desired. For example, theHCC(CO)NH-TOCSY experiments of Montelione and coworkers[48,49] provide comprehensive side chain correlations resolvedon the amide N–H but are highly T2 sensitive and their use is gen-erally restricted to smaller proteins. However, the reverse micelleencapsulation strategy overcomes this limitation as is illustratedfor the 41 kDa maltose-binding protein [22,31] (Fig. 6). Though ef-forts continue to avoid the extensive use of short range NOEs in thedetermination of structures of proteins [50], comprehensive accessto the NOE greatly increases the probability that a high-resolutionmodel can be obtained. It is also apparent that as larger proteinsare made accessible by the reverse micelle method, the spectralcomplexity becomes correspondingly greater. Traditionally forsmaller proteins this is ameliorated through use of three- andfour-dimensional NOESY experiments [1]. Four-dimensional15N-HSQC-NOESY-13C-HSQC and 13C-HMQC-NOESY-13C-HMQC[51,52] spectra of the 27 kDa membrane-anchored protein rec-overin encapsulated in CTAB/hexanol reverse micelles dissolvedin liquid pentane illustrate the ability to obtain NOESY spectra onthese types of samples (Fig. 7). These data were obtained for thisprotein in its membrane-anchored state without the need for highpressure, ultra-low viscosity solvents. It should be noted that therequired collection times for these data sets was comparable to

that required for collection of such data on a typical aqueous pro-tein of comparable size.

7.2. Paramagnetic relaxation enhancement

Paramagnetic relaxation enhancement (PRE) effects have be-come widely used to obtain long-distance restraints and dynamicalinformation [53,54]. PRE data are readily obtained for encapsulatedproteins using the same methods as applied in aqueous solution.Fig. 8 illustrates attenuation of amide hydrogen cross peak inten-sity in an 15N-HSQC spectrum in the presence of oxidized (para-magnetic) MTSL versus reduced (diamagnetic) MTSL attached toresidue 72 of a double mutant (S72C, C55A) of the 19 kDa proteinflavodoxin in 10MAG/LDAO reverse micelles dissolved in liquidpentane [38]. These relaxation enhancements are also mapped toa cartoon of the flavodoxin structure, illustrating the expected dis-tance relationship between the site of the probe and the broadenedamide sites.

7.3. Residual dipolar couplings

Residual dipolar couplings (RDCs) are not only critical to obtain-ing high quality structural models [55] but are also rich in motionalinformation [56]. A variety of mechanisms are available in aqueoussolution to induce the partial alignment necessary to reintroducedipolar couplings. As mentioned above, the phase diagram of re-verse micelle surfactants is complex and contains regions wheresmall reverse micelles suitable for NMR and liquid crystalline com-ponents co-exist [12,57]. In addition, there are regions of the phasediagram for protein-containing reverse micelles where the parti-cles are non-spherical and can have an inherent anisotropic mag-netic susceptibility [26]. These various conditions can bepopulated through manipulation of surfactant concentrations andratios and can result in partial alignment. Sufficient partial align-ment to produce measureable RDCs has been demonstrated for anumber of small proteins [26]. An example is shown in Fig. 9.

8. Additional applications

8.1. Ligand binding

In addition to providing access to the standard solution NMRexperiments for determination of structure and dynamics of mac-romolecules, reverse micelle samples are also amenable to a wide

Fig. 9. Residual dipolar couplings obtained from encapsulated flavodoxin inpartially aligned reverse micelles. Shown is a correlation plot of the predictedRDCs by the crystal structure [78] versus the measured RDCs for the encapsulatedprotein. Also shown is a ribbon representation of the protein oriented in thedetermined alignment tensor. Figure reproduced from Ref. [26].



range of biochemical and biophysical characterization methodstypically applied in aqueous solution. Small molecule binding stud-ies can be routinely carried out with encapsulated macromole-cules. For example, the membrane-associated HIV-1 matrixprotein was encapsulated in its lipid-extruded form and anchoredto the reverse micelle surfactant layer [25]. In this context, the re-verse micelle provided a useful membrane mimetic system that al-lowed the binding of the lipid ligand PI(4,5)P2 to be monitored [25](Fig. 10). Similar experiments have also been used to characterizethe sequestration of the myristoyl group of the peripheral mem-brane protein recoverin [25].

Integral membrane proteins have also been encapsulated andexamined using high resolution NMR [23,24,58]. In a somewhatdemanding case, the reverse micelle provided sufficient structuralsupport to encapsulate the 54 kDa potassium channel KcsA in itsfunctional form [23]. Functional competence was demonstratedby the selective binding of potassium over an excess of sodium,

which was detected by chemical shift perturbations induced bytitration with potassium [23] (Fig. 10). The potassium bindingproperties and the localization of chemical shift changes uponpotassium binding closely mirrored those measured previously inmicelles in aqueous solution [59] where tumbling of the proteinis much more problematic.

8.2. Protein biophysics

The nanoscale water core of the reverse micelles has a numberof characteristics that can be utilized in studies of macromolecularstructure and biophysics. For example, the small volume of thewater core can serve as a ‘‘confined space’’ that promotes proteinfolding by disfavoring extensive (partially) unfolded states [60].Similar effects can be used to stabilize oligomers [61]. The smallscale also suppresses the freezing point of the water core such thatcertain reverse micelle solutions are stable to �20 to �30 �C. This

Fig. 10. Examples of biophysical experiments with encapsulated proteins. (A) Titration of myristoylated HIV-1 matrix protein encapsulated in CTAB/hexanol reverse micelleswith PI(4,5)P2 (mole ratios of PIP:HIV-MA are indicated) [25]. Sites whose amide 1H–15N chemical shifts change appreciably upon binding are highlighted in red on a ribbonrepresentation of the structure [79]. Adapted from Ref. [25]. (B) Titration of the KcsA potassium channel encapsulated in CTAB/DHAB/hexanol reverse micelles with potassiumin an excess background of sodium. Adapted from Refs. [23,24]. The behavior of Thr-72, a key residue in the ion selectivity filter, is also shown. These results demonstrate thefunctionality of the encapsulated channel [23,24]. (C) Pressure perturbation of ubiquitin encapsulated in AOT reverse micelles in liquid pentane. Residues H68 and E64 areshown inset. Pressures shown are 1 bar (blue), 800 bar (green), 1600 bar (yellow), and 2500 bar (red). The reverse micelle retains integrity over this large pressure rangethough the increased viscosity of pentane at high pressure is apparent in the broadening of the amide peaks [71].



permitted the cold denaturation of ubiquitin to be characterized indetail without the need for destabilization through application ofpressure or addition of denaturants [62,63]. The dynamics of water[64] and acid–base catalysis [65] are considerably slowed in thecore of small reverse micelles relative to bulk aqueous solution.

The slowed motion of water in the hydration layer of the encap-sulated protein permits extensive site-resolved detection of theinteraction of water with the surface of the protein via NOE/ROEratios [66]. The suppression of hydrogen exchange chemistry lar-gely eliminates the artifacts that make this approach difficult inbulk solution [67,68]. In the first application of this strategy, slowhydration water was associated with regions of the ubiquitin sur-face that binds other proteins (Fig. 11). This suggests that proteinhydration may be optimized to promote binding by manipulationof the hydrophobic effect i.e. by maximizing the entropy gain ofsurface water displaced by binding [69]. The ability to faithfully de-tect the interaction between water and protein also allowed themechanism of pressure-induced protein unfolding to be examined.Simulations had suggested that water penetrates the core of ubiq-uitin at high-pressure, promoting unfolding [70]. Reverse micellesamples are reasonably pressure resistant, which allowed this tobe examined experimentally. Encapsulated ubiquitin was shownto maintain structural integrity up to 2.5 kbar (Fig. 10) but no inte-rior water was detected [71]. These are but a few illustrations ofthe range of unique applications of reverse micelle NMR in proteinbiophysics.

9. Summary

Reverse micelle encapsulation provides a range of advantagesfor investigation of structural and biophysical characterization ofmacromolecules using solution NMR methods. A primary motiva-tion for using this approach is the ability to make the rotationalcorrelation time of the encapsulated macromolecule shorter thanit is free in aqueous solution by simply using a low viscosity

solvent. A variety of surfactant systems are available for robustencapsulation that preserves the native structure of the proteinor nucleic acid. The recently developed 10MAG/LDAO surfactantsystem is broadly useful and is able to encapsulate macromole-cules with quite variable physical properties without the needfor extensive optimization. In addition to classic structure determi-nation, the reverse micelle NMR systems offer a range of benefits inbiophysical studies of proteins and nucleic acids such as hydration,temperature and pressure dependent phenomena, ligand binding,and confined space effects. The recent observation of substantialnitroxide-mediated dynamic nuclear polarization of the reversemicelle water core encourages the development of the reverse mi-celle NMR method as a generally applicable approach [38].

Disclosure

AJW declares a competing financial interest as a Member ofDaedalus Innovations, LLC, a manufacturer of reverse micelle andhigh-pressure NMR apparatus.

Acknowledgments

This work was supported by NSF Grant MCB-115803 and NIHGrants GM085120 and GM107829.

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