solution nuclear magnetic 
 resonance ( nmr ) spectroscopy is a powerful 
 technique for studying the structures of macromolecules under a variety 
 of conditions and their internal dynamics on a wide range of time 
 scales .
sample preparation is often a 
 key factor in being able to obtain high - quality nmr data on complex 
 macromolecular systems .
methods for encapsulating proteins within 
 the aqueous core of a reverse micelle , and then dissolving the micelle 
 in low viscosity fluid , were introduced in the late 1990s to help 
 overcome the 
since then , reverse micelles have been used to study integral and anchored membrane proteins , as well 
 as soluble proteins and nucleic acids of marginal stability .
encapsulation has also 
 enabled studies of various biophysical properties of protein including 
 cold denaturation , hydration and internal motion .
10mag / ldao reverse micelle 
 surfactant system for solution nmr studies 
 of encapsulated proteins and nucleic acids .
( a ) chemical structures 
 of the nonionic / zwitterionic surfactant system 10mag / ldao compared 
 to the currently used ionic surfactants aot or ctab .
( b ) cut - away 
 view of a space - filling model of flavodoxin encapsulated in a 10mag / ldao 
 reverse micelle .
( c ) the direct injection method for sample preparation 
 is illustrated using flavodoxin bound to its brightly yellow cofactor 
 flavin mononucleotide .
conditions : 300 m flavodoxin in 150 
 mm 10mag / ldao surfactant at a molar percent ratio of 65%:35% , 15 mm 
 hexanol and water loading ( w0 ) of 20 . a critical issue when using reverse 
 micelle techniques in nmr studies 
 is that the surfactants must be able to efficiently encapsulate the 
 macromolecule with high structural fidelity while also allowing the 
 use of low viscosity solvents such as liquid pentane , propane or ethane . 
 thus , the overall size of the surfactant and the electrostatic characteristics 
 of the headgroup are critical for maintaining the native structure 
 while also providing the desired reduction in molecular reorientation 
 time for the encapsulated macromolecule .
several studies have identified 
 reverse micelle surfactant systems that meet the strict requirements 
 of high - resolution reverse micelle nmr , but a surfactant 
 system of general utility has not yet been described . currently , the 
 two commonly used surfactants in reverse micelle nmr are the anionic 
 bis(2-ethylhexyl)-sulfosuccinate ( aot ) and the cationic hexadecyltrimethylammonium bromide ( ctab ) , where the latter uses hexanol as a cosurfactant 
 ( figure 1 )
. applications of aot in this context 
 are limited because few proteins have been encapsulated with high 
 structural fidelity , presumably due to the denaturing effect of the 
 anionic headgroup .
possibly 
 due to the help of the nonionic cosurfactant hexanol  and has 
 been used to encapsulate various proteins with high structural fidelity 
 and high quality nmr spectra .
nevertheless , the search 
 for optimal encapsulation conditions for a given protein has been 
 somewhat ad hoc , which has likely limited general 
 application of this approach .
thus , there is a need for a highly robust 
 reverse micelle surfactant system that allows for efficient discovery 
 of encapsulation conditions for a given macromolecule with high structural 
 fidelity . here
, we describe the development of a novel surfactant 
 system that can be generally applied to proteins and nucleic acids 
 thus allowing for high - resolution nmr studies of a wide - range of encapsulated 
 biological macromolecules .
the experimental requirements imposed by 
 reverse micelle nmr spectroscopy led to identification of a binary 
 surfactant system consisting of the nonionic 1-decanoyl - rac - glycerol ( 10mag ) and the zwitterionic lauryldimethylamine - n - oxide 
 ( ldao ) . by combining a zwitterionic headgroup with a relatively short 
 contour length tail ,
the 10mag / ldao surfactant systems provides both 
 high biocompatibility and fast molecular tumbling for the encapsulated 
 macromolecule .
the performance of 10mag / ldao was evaluated by 
 comparing the nmr 
 properties of a variety of reverse micelle encapsulated biological 
 macromolecules with their properties in free aqueous solution .
it 
 is also demonstrated that 10mag / ldao reverse micelles provide an excellent 
 membrane mimetic for studying lipid - modified , membrane associated 
 proteins by solution nmr . an improved procedure that greatly simplifies 
 sample preparation by avoiding the high protein concentrations previously 
 required for preparing reverse micelle samples
proteins 
 and trna were isotopically labeled and purified as described 
 in the supporting information ( si ) .
mixtures of up to four different 
 surfactants featuring nonionic , ionic , or zwitterionic head groups 
 and different types of linear , branched , or cyclic hydrophobic tails 
 were screened for their ability to form stable reverse micelle solutions 
 in the absence of protein .
hexanol , which is typically present 
 as a stabilizing cosurfactant in reverse micelle preparations , was 
 not included in this initial screen .
the 
 buffer for testing empty reverse micelle formation was 20 mm sodium 
 phosphate ( ph 7.0 ) with 50 mm nacl .
bromophenol blue ( 0.2 mg / ml ) was 
 added to the buffer as a visual aid to detect reverse micelle formation
. 
 the molar ratio of water to surfactant , also known as the water loading 
 or w0 , was typically tested from 10 to 30 .
positive surfactant 
 combinations were defined as those showing a single clear blue phase 
 and the absence of any precipitate with a w0 of at least 
 20 .
the 10mag / ldao 
 surfactants were used in a molar percent ratio of 65%:35% and at a 
 total surfactant concentration of 75150 mm as noted .
the concentration 
 of the cosurfactant hexanol depended on the total 10mag / ldao surfactant 
 concentration and on the type of bulk alkane solvent used ( pentane 
 or ethane ) .
pentane samples were prepared with 1020 mm hexanol 
 at 75 mm surfactant and 2030 mm hexanol at 150 mm surfactant . 

ethane samples were typically prepared with 3050 mm hexanol 
 at 75 mm surfactant and 6070 mm hexanol at 150 mm surfactant . 

reverse micelle samples in liquid ethane were prepared using specialized 
 apparatus from daedalus innovations , llc ( aston , pa ) .
ldao , 10mag , 
 and hexanol were dissolved in 300 l of d12-pentane 
 and transferred to the mixing chamber ( 1.65 ml nominal volume ) of 
 a daedalus innovations rm synthesizer unit .
a volume of concentrated 
 protein solution yielding the desired final w0 was added 
 and the mixing chamber was sealed .
pressurized liquid ethane was pumped 
 into the mixing chamber with an isco 65d ( lincoln , ne ) syringe pump 
 while the sample was being stirred .
the sample pressure was increased 
 until a distinct phase transition from a cloudy suspension to a clear 
 solution was observed .
this generally occurred in the range of 275450 
 bar though higher pressures were sometimes required for macromolecules 
 of larger size and/or at higher concentrations .
after completion of 
 mixing , the sample pressure was adjusted to 1420 bar above 
 the observed encapsulation ( transition ) pressure .
the sample was then 
 transferred to a high - pressure 3.6 mm 
 i.d .
nmr cell rated to 1 kbar ( daedalus innovations , llc , aston , pa ) 
 for further analysis by nmr . the final sample contained 80% ( v / v ) 
 ethane and 20% ( v / v ) pentane . for samples prepared by the injection 
evaporation 
 method , 
 water , alkane solvent , and to some degree volatile surfactants such 
 as hexanol are removed under a gentle stream of n2 gas . 

the pentane solvent and volatile components other than water are replenished , 
 as indicated by h nmr spectra , to rebalance the surfactant 
 mixture .
nmr spectra were obtained using bruker 
 avance iii nmr spectrometers equipped with 5 mm cryogenically cooled 
 triple resonance probes .
standard two- and 
 three - dimensional heteronuclear nmr pulse sequences were used to assess 
 spectral perturbations .
effective molecular 
 reorientation correlation times ( m ) were estimated using the n - trosy - based ( tract ) 
 relaxation experiment . to determine m , multiple portions of the amide ( or imino ) 
 region were integrated and that which gave the highest measured m is reported .
error was estimated from the 
 variance in integrated area for duplicate relaxation delay points . 

this error was far smaller than that imposed by the rigid body approximation 
 assumed in the tract analysis . in order to more rigorously compare 
 the tumbling of encapsulated mbp to that in aqueous solution , n t1
, t2 and heteronuclear noe experiments were collected at 750 and 600 mhz . for comparison of n t2 values , t2 relaxation of aqueous mbp
was collected at 750 mhz using a trosy - based 
 pulse sequence.t1 and t2 experiments were collected 
 using six delay times with two collected in duplicate for error analysis . 

rotational correlation times were determined by fit to an isotropic 
 tumbling model using in - house software .
spectra were processed using felix ( accelrys , san deigo , ca ) or 
 in - house nmr processing software ( al nmr ) .
the sparky software package was used for spectral analysis and plotting 
 ( goddard , t.d . and kneller , d.g .
a systematic screen was undertaken to identify promising 
 surfactant mixtures for reverse micelle nmr , where various mixtures 
 were tested for their ability to form stable  empty  

the screen consisted of mixtures of up to 
 four different commercially available surfactants featuring nonionic , 
 ionic , or zwitterionic headgroups and different types of linear , branched , 
 or cyclic hydrophobic tails ( si table s1 ) . 

this screen identified a binary surfactant mixture of 1-decanoyl - rac - glycerol and lauryldimethylamine - n - oxide ( 10mag / ldao ) 
 as having highly desirable properties for encapsulation of proteins 
 ( si figure s1 and table s2 ) .
the nonionic monoacylglycerol head groups of 
 10mag were expected to suppress denaturing ionic interactions between 
 the surfactant shell and the surface of an encapsulated macromolecule 
 ( figure 1b ) .
these properties 
 suggested that 10mag / ldao should faithfully encapsulate target macromolecules 
 independent of their net surface charge .
in addition , the relatively 
 short linear hydrophobic tails of 10mag and ldao ( 10 and 12 carbons , 
 respectively ) were anticipated to favor fast molecular reorientation 
 of the reverse micelle particle and thus to enhance their performance 
 in nmr spectroscopy . in a secondary screen
we fine - tuned the 
 molar ratio of the components 
 of the 10mag / ldao reverse micelle system for optimal nmr performance . 
 the molar ratio of water to surfactant is particularly important because 
 larger proteins require larger encapsulation volumes to form a stable 
 reverse micelle . nevertheless , the total amount of water should be 
 limited to minimize the size and achieve the fastest reorientation 
 of the reverse micelle particle .
optimization of both the 10mag / ldao 
 molar ratio and the amount of hexanol was required to achieve the 
 desired range of water loading ( w0 ) ( supplementary figure 2 ) .
. a molar ratio of 65%:35% of 10mag : ldao at a total surfactant concentration 
 of 75  150 mm accommodated water loadings from 5 to 40 , which 
 is sufficient to fully hydrate proteins up to 100 kda .
the 
 required hexanol concentration ranged from 0 to 100 mm depending on 
 w0 and the alkane solvent employed .
a total of seven globular , water - soluble proteins 
 were tested to determine the general utility of the 10mag / ldao surfactant 
 system for reverse micelle nmr .
all seven of these proteins were successfully 
 encapsulated in 10mag / ldao mixtures in pentane with minimal optimization 
 required . w0 and the total surfactant and hexanol concentrations 
 were optimized to maximize encapsulation efficiency ( highest encapsulated 
 protein concentration ) .
the seven tested proteins range from 8.5 to 
 81 kda and have isoelectric points between 4.5 and 11 .
five of these 
 proteins , ubiquitin ( ub , 8.5 kda , pi  6.8 ) , cytochrome c ( cyt c , 11.7 kda , pi  11 ) , flavodoxin ( flv , 18.8 kda , 
 pi  4.5 ) , maltose binding protein ( mbp , 40.8 kda , pi  
 5.2 ) , and malate
synthase g from e. coli ( msg , 81.4 kda , pi  5.9 ) have been previously encapsulated 
 using the ionic surfactants aot or ctab , or a triple surfactant mixture 
 also containing tetraethylene glycerol monododecyl ether ( c12e4 ) .
in addition , two proteins were examined that are large by routine 
 solution nmr standards and have not previously been studied by reverse 
 micelle nmr : arginine kinase from the horseshoe crab l. polyphemus ( ak , 40.2 kda , pi  6.3 ) and 
 human aldo - keto reductase 1c2 ( akr , also known as type iii 3-hydroxysteroid 
 dehydrogenase , 36.7 kda , pi  7.1 ) .
reverse micelle samples 
 were prepared by the direct injection method , where a specific volume of concentrated aqueous 
 solution of protein is directly pipettd to an alkane solution of surfactants 
 and mixed by vortexing ( figure 1c ) .
ub , cyt c , and flv were used to examine how the charge of the protein 
 affects the efficiency of encapsulation .
the ph of the reverse micelle 
 water core was adjusted so that the net charge of the encapsulated 
 protein was negative for flv and positive for ub and cyt c. setting the ph of the aqueous core of reverse micelles is critically 
 important for achieving optimal encapsulation .
the ph of the water 
 core is dominated by the surfactants and only marginally influenced 
 by the buffer employed since there are a hundred or more zwitterionic 
 head groups and only a handful of buffer molecules per reverse micelle . 

methods for adjusting the effective ph inside the water core of a 
 reverse micelle sample have been described elsewhere .
all three proteins were encapsulated in pentane at final 
 concentrations of 200 m with w0 = 10 ( ub , cyt c ) and w0 = 12 ( flv ) .
all reverse micelle solutions were clear , showed no 
 visible precipitate , and gave excellent n hsqc spectra 
 ( si figure s3 ) .
comparison of the 
 backbone chemical shifts for the protein in aqueous 
 solution and encapsulated in the reverse micelle was used as a quantitative 
 measure of structural integrity . for flavodoxin
there are negligible 
 differences between the amide h and n chemical 
 shifts in aqueous solution and encapsulated in 10mag / ldao ( histogram 
 in figure 2a ) . a more extensive backbone chemical 
 shift analysis ( hn , c , c , c , n ) showed 
 that reduced cytochrome c is encapsulated in 10mag / ldao 
 with little apparent structural perturbation ( figure 2b ) .
three proteins  akr bound to nadp , mbp 
 bound to maltose , and apo - ak  were used to assess the ability 
 of 10mag / ldao to encapsulate larger proteins .
comparison of the resolved 
 cross peaks of the n hsqc spectra obtained in pentane 
 and in aqueous solution indicates that the three proteins were encapsulated 
 in their native state ( si figure s4 ) .
ak 
 was more extensively examined using triple resonance spectroscopy 
 of a sample prepared in ethane , which afforded better relaxation properties 
 and narrower lines .
an excellent chemical shift correspondence was 
 found , demonstrating the high structural fidelity of the encapsulated 
 protein ( figure 2c ) .
finally , msg ( 81 kda ) 
 was used with minimal optimization required to demonstrate the application 
 of the 10mag / ldao mixture to even larger proteins .
the successful 
 encapsulation of seven globular proteins of diverse physical characteristics 
 with only minimal specific optimization demonstrates that 10mag / ldao 
 represents a major step forward in the general applicability of the 
 reverse micelle encapsulation methodology .
encapsulation of diverse 
 proteins with high - structural fidelity 
 in the 10mag / ldao surfactant system .
proteins of distinctly different 
 net surface charges and molecular weights retain their bulk solution 
 structure as evidenced by minimal chemical shift (  ) perturbations 
 upon encapsulation .
( a ) n hsqc spectrum ( left ) of encapsulated n - flavodoxin ( 18.8 kda , pi  4.5 ) in pentane acquired 
 at 500 mhz ( h ) .
histogram of gyromagnetic ratio weighted h and n chemical shift differences relative to 
 flavodoxin in free aqueous solution ( right ) . 

( b ) n hsqc spectrum ( left ) of encapsulated horse c , n - cytochrome c ( 12 kda , pi 
  11 ) in pentane acquired at 600 mhz ( h ) .
histograms 
 of weighted h and n ( middle ) and c , c , and c ( right ) 
 chemical shift differences relative to the protein in free aqueous 
 solution .
( c ) n hsqc spectrum ( left ) 
 of encapsulated c , n - arginine kinase ( ak ) 
 ( 40.2 kda , pi  6.3 ) in ethane acquired at 750 mhz ( h ) .
correlation of amide h and n and carbonyl c chemical shifts for ak . encapsulated cyt c and flv were dissolved in pentane , and ak was dissolved 
 in ethane at 380 bar .
a central 
 advantage of the reverse micelle encapsulation strategy is the ability 
 to manipulate the nmr relaxation properties of the macromolecule by 
 actively decreasing the molecular reorientation time ( m ) through the use of low viscosity solvents .
though n - pentane is often initially employed to explore sample 
 conditions for encapsulation , the  volume penalty  imposed 
 by the reverse micelle particle ( water core plus surfactant shell ) 
 often outweighs gains in tumbling .
lower viscosity liquid alkanes 
 require preparation under pressure . reverse micelle solutions prepared 
 with liquid butane or propane are often sufficient to overcome this 
  volume penalty ,  thereby providing shorter molecular 
 reorientation times for large proteins .
although only moderate 
 pressure is required to liquefy ethane at room temperature ( 47 bar ) , 
 considerably higher pressures are needed to support homogeneous and 
 stable nmr samples of encapsulated proteins in condensed gases ( typically 
 250500 bar ) .
the  volume penalty  is evident 
 for small proteins even when encapsulated in the ultralow viscosity 
 ethane , as shown by the higher m values for encapsulated ubiquitin , cytochrome c , and flavodoxin 
 shown in figure 3 .
n - tract 
 estimates of effective molecular reorientation 
 times ( m ) of six proteins 
 and trna encapsulated in 10mag / ldao reverse micelles 
 in liquid ethane (  ) or free in aqueous solution (  ) . 

error bars , as propagated from the quality of the relaxation 
 fits , are smaller than the size of the symbols shown . as addressed 
 in the text , the primary source of error in the tract analysis is 
 the inherent assumption of no internal motion .
the optimal sample conditions obtained in pentane were generally 
 transferable to ethane although higher concentrations of the cosurfactant 
 hexanol were often required ( see si for 
 detailed sample conditions ) .
hexanol significantly lowers the pressure 
 required for forming stable reverse micelle samples , and the resulting 
 reduced pressure lowers the viscosity and therefore reduces the molecular 
 reorientation time . after the sample 
 is fully formed inside the mixing chamber it is transferred to a specialized 
 nmr cell .
n hsqc spectra of 
 these proteins show excellent agreement with aqueous samples ( figures 2c , 4a , 5a , and si figure s1 ) .
the encapsulation 
 pressures required for preparation in ethane ranged from 275 to 480 
 bar , which are well below that typically required to significantly 
 perturb the structure and dynamics of folded proteins .
long - term stability of the ethane samples at 
 room temperature ranged from more than 4 months for flv , to 6 weeks 
 for msg , mbp , cyt c , ub , and 1 week for ak .
the samples 
 of akr in ethane were not stable for more than a day .
determination 
 of optimal encapsulation conditions in ethane required optimization 
 of hexanol concentration , w0 , and encapsulation 
 pressure for each of the proteins tested . in general , optimal conditions 
 were identified after only a few ( 5 or less ) tests . once optimal 
 conditions were identified , these proved to be highly reproducible 
 ( m  2 ns ) .
subsequent to these tests , several other soluble , globular proteins 
 have been tested for encapsulation in 10mag / ldao mixtures . to date
, 
 only one has been identified that does not encapsulate in 10mag / ldao . 

encapsulation of integral membrane proteins in 10mag / ldao mixtures 
 has not yet been examined .
( a ) n hsqc spectra are shown of 
 mbp in aqueous solution 
 and encapsulated in 10mag / ldao reverse micelles dissolved in liquid 
 ethane .
spectra were collected to have comparably high signal - to - noise 
 for direct comparison of line widths .
( b ) one - dimensional normalized n cross sections ( aqueous : dashed lines , rm : solid lines ) 
 of three cross peaks ( red arrows in a ) are shown to illustrate the 
 narrowed line widths as a result of improved tumbling upon encapsulation . 

( c ) n t2 values were measured 
 for 278 and 291 sites in the encapsulated ( black bars ) and aqueous 
 ( white bars ) mbp samples , respectively , at 750 mhz .
a histogram illustrates 
 the overall improvement in relaxation behavior of the encapsulated 
 protein as a result of improved tumbling .
the difference in average t2 values ( 35 and 46 ms for aqueous and encapsulated 
 protein , respectively ) corresponds to a 31% improvement and correlates 
 well the 32% decrease in rotational correlation time .
( a ) n - trosy spectra are shown of 
 msg in aqueous solution 
 and encapsulated in 10mag / ldao reverse micelles in liquid ethane . 

these spectra were collected with the same total acquisition time 
 and show comparable signal - to - noise despite the reverse micelle sample 
 containing approximately one - third of the protein as that of the aqueous 
 sample .
( b ) h cross sections of exemplary ( red arrows 
 in a ) cross peaks are shown for aqueous ( red ) and encapsulated ( black ) 
 msg .
the n - tract experiment , which measures the relaxation rates of the  
 and  spin 
 states of the amide n , was used to estimate the protein s 
 effective molecular reorientation time ( m ) .
although this method systematically underestimates the true molecular 
 reorientation time ( m ) , it provides a rapid method 
 for comparing the tumbling of proteins in reverse micelle and aqueous 
 solutions .
the error that results from the rigid body assumption inherent 
 in the tract analysis is far greater than the error resulting from 
 the fits to the relaxation profiles . in general , the errors resulting 
 from the quality of the relaxation fits are on the order of the sizes 
 of the symbols shown in figure 3 . comparing 
 the m values shows that macromolecules larger 
 than 20 kda can be made to tumble faster than in aqueous solution 
 when encapsulated and prepared in liquid ethane ( figure 3 ) .
for example , encapsulated mbp and msg showed approximately 
 50% reduction in m . however , 
 it is important to note that , because the tract experiment underestimates 
 the true rotational correlation time , the actual advantage may be 
 more modest . to examine this ,
the rotational correlation time of mbp 
 encapsulated in 75 mm 10mag / ldao was determined with a full suite 
 of n - relaxation experiments to be 15.5 ns .
this corresponds to a markedly slower tumbling 
 behavior than indicated by the tract analysis ( 8.9 ns ) , but a similar 
 discrepancy was noted previously for the tract estimation of tumbling 
 for aqueous mbp . based on this 
 more rigorous measurement of encapsulated mbp tumbling 
 in ethane , the true improvement in rotational correlation time ( 15.5 
 ns for rm versus 23 ns for aqueous solution ) is 32% .
it should also be noted that some trade - off 
 exists between optimal 
 encapsulation efficiency ( i.e. , concentration of protein in the final 
 sample ) and molecular tumbling . in general , increasing the amount 
 of surfactant in the reverse micelle system permits encapsulation 
 of higher concentrations of protein , but the tumbling improvement 
 at high surfactant concentrations is slightly reduced and there is 
 the potential for partial alignment of reverse micelle systems at 
 high surfactant concentrations . as an example , the optimal sample conditions for mbp in ethane 
 were determined to be at 75 mm total surfactant with 100 mm hexanol . 
 because ak is more difficult to concentrate in aqueous solution , higher 
 surfactant concentrations ( 100150 mm ) and higher water loading 
 ( w0 = 20 versus 12 for mbp ) were required 
 for this protein in order to achieve appropriate encapsulated concentrations 
 ( 80100 m ) for efficient collection of nmr data . as 
 shown in figure 3 ,
the tumbling of mbp in ethane 
 is better than that of ak due to this difference in surfactant conditions . 

the spectral improvements in tumbling are demonstrated in figure 4 for mbp encapsulated in ethane as compared to the 
 aqueous protein .
the n hsqc spectrum of encapsulated 
 mbp shows improved resolution due to narrowing of the resonance lines . 

this narrowing can more clearly be seen in the one - dimensional spectra 
 given .
in addition , the distribution of n t2 values is shifted markedly toward longer relaxation 
 times for the encapsulated protein , demonstrating a general improvement 
 in the relaxation properties of the encapsulated protein .
this t2 advantage not only translates to improved 
 line widths but also provides substantial gains in coherence transfer 
 efficiencies in multidimensional nmr experiments .
it is also important to consider that the sensitivity 
 of a modern 
 cryoprobe with rm samples is markedly better than that with aqueous 
 solutions due to the low conductivity of the reverse micelle solution .
this results in improved signal - to - noise per 
 unit of protein in the encapsulated sample . in combination with the 
 improved tumbling and resultant line narrowing and improved coherence 
 transfer ,
for example , the spectra 
 shown in figure 5 demonstrate that comparable 
 signal - to - noise is obtained for encapsulated msg as compared to the 
 aqueous condition using the same amount of signal averaging ( i.e. , 
 identical collection time ) but less than half the protein concentration 
 of the aqueous sample . in summary , the successful encapsulation of 
 a diverse set of globular proteins up to 81 kda in ultralow viscosity 
 ethane shows that 10mag / ldao is a general surfactant system for comprehensive 
 reverse micelle nmr .
the direct injection method ( figure 1c ) is 
 the simplest approach for preparing homogeneous solutions of encapsulated 
 macromolecules .
such solutions are usually prepared under water - limited 
 conditions to obtain the desired water loading ( w0 ) and fast molecular tumbling .
thus , the concentration 
 of protein in the injected solution defines the final concentration 
 of encapsulated protein .
if the protein can not be sufficiently concentrated , 
 the direct injection method will fail to provide a sufficiently high 
 concentration of encapsulated protein to allow facile multidimensional 
 nmr spectroscopy .
for example , using a 1 mm protein stock solution 
 would result in a final concentration of encapsulated protein of only 
 18 m with w0 of 10 and 100 mm surfactant . to address this limitation ,
a method was developed that allows 
 fewer soluble macromolecules to be encapsulated by employing successive 
 rounds of injection of a dilute solution followed by evaporation of 
 excess water .
the injection  evaporation method is illustrated using 
 a solution of 500 m flavodoxin and performing seven rounds 
 of protein injection and water evaporation ( figure 6 ) .
the injection  evaporation method of encapsulation eliminates 
 the requirement for millimolar protein concentrations .
( a ) reference n hsqc spectrum of 500 m flv in aqueous solution . 

( b ) n hsqc spectrum of 200 m encapsulated flv 
 dissolved in pentane prepared by direct injection using 6.3 mm flv 
 ( bottom ) and prepared by seven rounds of injection  evaporation 
 using a 500 m flv stock solution ( top ) .
( c ) the effective tumbling 
 rate of flv can be greatly increased by subsequent solvent exchange 
 to ethane .
all data were recorded at 25 c and at 500 mhz ( h ) and processed identically .
the first round of protein injection yielded an encapsulated 
 flavodoxin 
 concentration of 30 m .
in this example , this allows for a 
 12-fold reduction in the starting protein concentration ( 6.3 mm ) . 

the n hsqc spectra of encapsulated flavodoxin prepared 
 by injection  evaporation or direct injection were identical 
 ( figure 6b ) .
furthermore , the flavodoxin sample 
 prepared by the injection  evaporation method could be redissolved 
 in liquid ethane after partial evaporation of the pentane solvent 
 ( figure 6 ) .
thus , the advantages of the injection  evaporation 
 method can be combined with the improved molecular tumbling offered 
 by ultralow viscosity ethane .
post - translational lipid 
 modification of proteins , such as attachment 
 of myristate or isoprene groups , is responsible for a variety of specialized 
 functions including localization at membranes or hydrophobic molecular 
 switch - like behavior .
exposure of the 
 hydrophobic lipid to water can lead to poor solution behavior that 
 severely compromises structural studies .
consequently , high - resolution 
 structures of lipid - modified proteins in the putative membrane - anchored 
 state obtained using standard nmr spectroscopy or crystallography 
 are surprisingly rare .
it was recently shown that myristoylated recoverin 
 and myristoylated hiv matrix protein could be stably encapsulated 
 in ctab / hexanol reverse micelles in the lipid - extruded state . however , ctab is cationic and has electrostatic 
 properties different from those of natural lipid bilayers , and this 
 mismatch may become limiting .
in contrast , the 10mag / ldao surfactant 
 system more closely resembles the lipids of biological membranes . 

the zwitterionic ldao headgroup mimics the phosphatidylcholine headgroup , 
 and 10mag is a glycerol group esterified to a fatty acid .
the 
 23 kda myristoylated human recoverin ( rec[+myr ] ) , which acts as a 
 ca sensor in the visual system , was used to test if 10mag / ldao could support lipid - anchored proteins . 

calcium - loaded rec[+myr ] was efficiently encapsulated at 
 100 m in 10mag / ldao in both pentane and ethane at a water loading 
 of 12 .
the n hsqc spectra of encapsulated rec[+myr ] were 
 very similar to spectra of natively folded , ca - bound , 
 myristoyl - extruded protein ( see refs ( 5 and 40 ) and figure 7 ) .
the n - tract measurements 
 showed much faster molecular tumbling in ethane than in pentane ( m = 11.7 and 18.8 ns , respectively ) .
interestingly , the m values suggested that two rec[+myr ] molecules were encapsulated 
 in occupied reverse micelles .
a similar observation was made in the 
 previous study of rec[+myr ] encapsulated in ctab / hexanol , suggesting that the protein in the lipid - extruded 
 state is a dimer .
10mag / ldao 
 provides a membrane mimetic environment for nmr spectroscopy 
 of membrane - anchored proteins .
the dashed 
 line box highlights two cross peaks that are assigned to two gly residues 
 involved in stabilizing the ca - bound , myristoyl - extruded 
 protein conformation .
sample prepared in ethane at 
 100 m rec[+myr ] and w0 = 12 in 
 100 mm 10mag / ldao at 450 bar .
dnas and rnas represent a significant 
 challenge to solution nmr spectroscopy because they are built from 
 only four different nucleotides and the proton and carbon resonances 
 are highly overlapped due to their narrow chemical shift ranges .
the 27 kda natively modified valine trna ( trna ) from e. coli(46 ) was employed here to evaluate the 10mag / ldao surfactant 
 system for nmr studies of nucleic acids .
the structure and dynamics 
 of trna have been extensively studied , and it therefore serves as a model system for medium - sized 
 rnas .
n - labeled trna was encapsulated at 
 100 m in 10mag / ldao reverse micelles in both pentane and ethane 
 using the procedures developed above for proteins .
the imino region 
 of the n hsqc spectrum of encapsulated trna is indicative of a natively folded macromolecule using a buffer 
 that included 5 mm mg ( figure 8) .
interestingly , amino resonances were sharper and consequently 
 better 
 resolved for the trna encapsulated in ethane ( at relatively low water 
 loading w0 = 9 ) than in free solution 
 ( boxes in figure 8) .
this is partially related 
 to the reduced effective molecular reorientation time ( m = 7.0 and 9.0 ns , for the encapsulated and aqueous trnas , 
 respectively ) .
in addition , many a , c , and g amino protons are in 
 the intermediate exchange regime in aqueous solution due to either 
 rotation about the c 
n bond , which has partial double bond 
 character , or through the effects of hydrogen exchange with water . 

hydrogen exchange chemistry is slowed in the reverse micelle , and the corresponding reduction in hydrogen exchange rates will 
 tend to sharpen the amino hydrogen resonances . furthermore , the relatively 
 high effective viscosity of the water core of reverse micelles would also tend to slow conformational exchange processes toward 
 the slow exchange time regime .
conversely , the imino protons are in 
 the slow exchange regime in bulk solution , and their resonances are 
 largely unaffected by the slowed hydrogen exchange chemistry or the 
 increased viscosity of the reverse micelle water core
n hsqc spectra of the imino region of trna acquired 
 at 500 mhz ( h ) ( a ) aqueous solution at 200 m with 
 16 scans per fid and ( b ) encapsulated in 150 mm 10mag / ldao and dissolved 
 in ethane at 450 bar at w0 = 9 at 100 
 m with 32 scans per fid .
the dashed line boxes highlight amino 
 cross peaks , folded in the n chemical shift dimension , 
 showing somewhat improved signal - to - noise for the encapsulated rna 
 compared to rna tumbling free in aqueous solution .
the trna was prepared 
 in 10 mm sodium phosphate ( ph 6.8 ) , 80 mm nacl , 5 mm mgcl2 , 0.1 mm edta .
the effect of slowed hydrogen exchange chemistry on the line 
 shape 
 of exchangeable hydrogens has been observed for encapsulated proteins 
 before , particularly in the ability to resolve noe cross peaks to 
 hydroxyl hydrogens of serine and threonine residues .
these hydroxyl hydrogens exchange far too rapidly in bulk solution 
 for such signals to be observed , but they are visible upon encapsulation . 
 at water loadings less than 20 , the water dynamics and hydrogen 
 exchange rates are considerably slower than in bulk water .
many rnas require divalent ions for proper folding and function , 
 with mg generally being the physiologically relevant 
 ion .
the zwitterionic character of the 
 10mag / ldao surfactant system means that neither the rna nor the metal 
 ions required for correct folding / function of the macromolecule are 
 likely to interact to any significant extent with the surfactant . 
 furthermore ,
since nucleic acids all carry a large number of negative 
 charges , they will have similar overall electrostatic surface charges , 
 and encapsulation conditions that work for one nucleic acid , such 
 as trna studied here , should be readily applicable to 
 other nucleic acids .
a new binary surfactant 
 system for high - resolution solution nmr 
 studies of encapsulated biological macromolecules dissolved in low 
 viscosity fluids has been developed .
the performance of 10mag / ldao 
 in reverse micelle nmr has been validated with respect to encapsulation 
 efficiency ( yield ) , fidelity , and molecular tumbling behavior using 
 a set of seven proteins and a trna .
10mag / ldao therefore represents 
 a single surfactant system that can encapsulate with high structural 
 fidelity a broad range of different target macromolecules with molecular 
 weights at least up to 81 kda .
this simplifies the process of preparing 
 reverse micelle nmr samples , which is often plagued by trial and error 
 searches for optimal encapsulation conditions .
it has been found that 
 10mag / ldao may be employed with minimal a priori assumptions 
 about the properties of a given target protein or rna to obtain significant 
 reductions in molecular reorientation times at ambient temperature . 

results presented here indicate that 
 proteins in the 40 kda range are rendered completely accessible to 
 the full battery of triple resonance spectroscopy without the limitations 
 imposed by extensive deuteration or use of the trosy effect .
it should 
 also be noted that the dielectric properties of reverse micelle solutions 
 are more optimal for cryogenic probe technology than standard aqueous 
 samples and can provide a 50100% gain in sensitivity .
the ability of 10mag / ldao to faithfully 
 encapsulate a range of 
 protein sizes and charge states clearly originates from the favorable 
 physicochemical properties of the two surfactant headgroups  a nonionic monoacylated glycerol group in 10mag and a zwitterionic amine - n - oxide group in ldao . 

the nonionic surfactant content ( 65 mol % 10mag ) combined with the 
 zwitterionic character of ldao mixture reduces charge  charge 
 interactions in much the same spirit as the triple surfactant system 
 derived from mixtures of anionic , cationic , and neutral polyether 
 surfactants . here
, however , the relatively 
 short contour length of the ldao and 10mag minimizes the size of the 
 resulting particle and thereby maximizes the effective tumbling time 
 of the encapsulated macromolecule . the surfactant combination of 10mag 
 and ldao also provides a very favorable environment for encapsulating 
 proteins of poor solution properties , such as lipid - modified proteins . 

because all nucleic acids have 
 an overall negative electrostatic surface , it is likely that encapsulation 
 by the 10mag / ldao surfactant system will generally not be sensitive 
 to the precise sequence of the rna ( or dna ) .
finally , the injection  evaporation 
 method allows for the preparation of reverse micelle samples using 
 relatively dilute protein solutions and represents a significant advantage 
 as it avoids the need for concentrated aqueous solutions of the target 
 macromolecule . in conclusion ,
10mag / ldao provides many aspects of 
 an optimal surfactant system for solution nmr of encapsulated proteins 
 dissolved in low viscosity fluids .
it relieves the experimental problems 
 that exist with the currently employed surfactants aot , ctab , and 
 triple surfactant mixtures .
we anticipate that the 10mag / ldao surfactant 
 system will find broad utility in structural and biophysical studies 
 of encapsulated proteins by high - resolution reverse micelle nmr .
 an optimized reverse micelle surfactant 
 system has been developed 
 for solution nuclear magnetic resonance studies of encapsulated proteins 
 and nucleic acids dissolved in low viscosity fluids . 
 comprising the 
 nonionic 1-decanoyl - rac - glycerol and the zwitterionic 
 lauryldimethylamine - n - oxide ( 10mag / ldao ) , this mixture 
 is shown to efficiently encapsulate a diverse set of proteins and 
 nucleic acids . 
 chemical shift analyses of these systems show that 
 high structural fidelity is achieved upon encapsulation . 
 the 10mag / ldao 
 surfactant system reduces the molecular reorientation time for encapsulated 
 macromolecules larger than 20 kda leading to improved overall 
 nmr performance . 
 the 10mag / ldao system can also be used for solution 
 nmr studies of lipid - modified proteins . 
 new and efficient strategies 
 for optimization of encapsulation conditions are described . 
 10mag / ldao 
 performs well in both the low viscosity pentane and ultralow viscosity 
 liquid ethane and therefore will serve as a general surfactant system 
 for initiating solution nmr studies of proteins and nucleic acids . 
Introduction
Experimental Section
Results and Discussion
Conclusions