the structural and 
 dynamic aspects of proteins have been at center 
 stage of our understanding of the chemical basis of their function 
 for several decades . nuclear magnetic resonance ( nmr ) in solution 
 has contributed significantly to this view and the information inherent 
 in nmr phenomena offers much more . yet , despite tremendous advances 
 in technology , experimental design and analytical strategies , solution 
 nmr spectroscopy of macromolecules remains fundamentally restricted 
 due to its low sensitivity . though state - of - the - art multinuclear multidimensional 
 nmr spectra can be routinely recorded from samples with 0.5 
 mm concentrations , many systems and problems of interest remain inaccessible 
 due to limited solubility and/or limited availability . thus , a further 
 extension of the sensitivity of the nmr method into the low m 
 concentration regime is highly desirable . one approach to increasing 
 the sensitivity of the nmr experiment 
 is to couple the nuclear spins to a reservoir with much higher polarization , 
 such as unpaired electrons ( e/h  660 ) . in dynamic nuclear polarization ( dnp ) the 
 large electron spin polarization is transferred to nuclear spins by 
 irradiation at or near the electronic resonance . originally proposed 
 by overhauser in the context of metals , dnp was subsequently experimentally demonstrated in liquids . elementary models suggested that dnp in aqueous 
 solutions would be inefficient at high magnetic fields and for this 
 reason it was not seriously considered as a method of improving the 
 sensitivity in high - field nmr spectroscopy . however , 
 in the last two decades high field dnp in both liquids and solids 
 has enjoyed a considerable renaissance . in magic angle spinning ( mas ) experiments the mechanisms that mediate 
 dnp are the solid and the cross effects and with sufficient microwave 
 field strengths both lead to signal enhancements >100 at 
 high 
 fields up to 700 mhz for h. in addition , it was recently shown that 
 it is possible to achieve significant enhancements of protons in water 
 at magnetic fields up to 9.2 t via the overhauser effect ( oe ) . despite these successes , application of dnp to enhance the 
 sensitivity 
 of solution nmr first , water absorbs 
 strongly in the microwave region , and can lead to significant and 
 often catastrophic sample heating . second , the skin depth of high 
 frequency microwaves in water limits the penetration of the radiation 
 into the sample . third , transfer of polarization from the solvent 
 to large molecules ( e.g. , proteins ) in solution has up to now not 
 been accomplished . in this paper we propose an approach that deals 
 with the first two challenges and has great potential in overcoming 
 the third . previously , we introduced the idea of encapsulating 
 proteins inside 
 the protective aqueous core of a reverse micelle , which can be prepared 
 in an ultralow viscosity fluid , thereby improving the quality of their 
 nmr spectra . the original goal was to 
 make the entire reverse micelle particle containing the protein tumble 
 with a correlation time that is shorter than in the relatively more 
 viscous water . as we show below , the unique features of this type 
 of sample largely avoids the dielectric loss ( heating ) and penetration 
 issues arising from irradiation with microwave frequencies . in addition , the slower motion of water relative to bulk solution 
 overcomes a previously unanticipated limitation -- namely the short 
 residence time(s ) of water on the surface of protein molecules , which 
 results in inefficient dipolar contact and poor polarization transfer 
 to the protein . in contrast to bulk solution , the residence time of 
 water on the surface of an encapsulated protein is significantly longer 
 and results in excellent polarization transfer . the reverse micelle system also offers great flexibility for introducing 
 the polarizing agent and potentially permits the tuning of the water 
 dynamics to optimize the dnp enhancements . the c55a mutant 
 of flavodoxin from pcc7119 was expressed during growth on minimal 
 media containing nh4cl as described previously . this protein was used for studies where the 
 nitroxide spin label was either free in the aqueous core of the reverse 
 micelle or attached to a lipid embedded in the reverse micelle surfactant 
 shell . the n flavodoxin ( c55a ) with the flavin mononucleotide 
 bound was concentrated to 6.5 mm in 10 mm tris buffer and 100 mm nacl 
 at ph 8.0 for reverse micelle sample injection . to covalently attach 
 a nitroxide spin label to the protein , a surface accessible cysteine 
 mutant of flavodoxin ( c55a , s72c ) uniformly n - labeled 
 flavodoxin ( c55a , s72c ) was expressed and purified as described above 
 except that 1 mm dithiothreitol ( dtt ) was present throughout the purification 
 to prevent dimerization . flavodoxin ( c55a , s72c ) was covalently labeled 
 with n-(1-oxyl-2,2,5,5-tetramethyl - d3-pyrroline-3-methyl)-methanethiosulfonate 
 ( mtsl ) ( toronto research chemicals ) using published protocols . a 10-fold excess of mtsl in acetonitrile was added to a 1 mm solution 
 of n flavodoxin ( c55a , s72c ) in 10 mm tris buffer and 
 100 mm nacl at ph 8.0 . the reaction 
 was allowed to proceed for 16 h at room temperature under argon . solutions of reverse 
 micelles were made with a surfactant mixture containing a 65:35 molar 
 ratio of 1-decanoyl - rac - glycerol ( 10mag ) ( sigma - aldrich , co. , llc ) 
 and lauryl - dimethylamine - n - oxide ( ldao ) ( affymetrix , 
 inc . ) , at 100 mm concentration , 5 mm d-11-hexanol dissolved in d-14 
 hexane with a molar ratio of water to total surfactant molecules ( wo or water loading ) of 20 . ldao and 10mag are combined in the prescribed ratio as 
 dry powders , dissolved in hexane , bath sonicated to promote dissolution 
 and lyophilized in glass vials . lyophilized 
 dry mixtures of surfactants were dissolved in 0.5 ml deuterated hexane 
 and made 5 mm in deuterated hexanol ( 0.3 l ) . an aqueous aliquot 
 equivalent to a water loading of 20 ( 18.2 l ) was injected and 
 then vortexed , resulting in a clear solution . this procedure was followed 
 to prepare reverse micelles containing flavodoxin - mtsl adducts or 
 flavodoxin with tempol dissolved in the aqueous core . in the latter 
 case reverse micelles containing the surfactant nitroxide spin label tempo - pc 
 ( 1,2-dipalmitoyl - sn - glycero-3-phosphocholine ( avanti 
 polar lipids , inc . ) were prepared as above with the additional step 
 of cosolubilizing the tempo - pc with the 10mag and ldao surfactant 
 mixture in final concentrations of 0.6 mm , 65 mm and 35 mm , respectively . an appropriate 
 aliquot was lyophilized in a glass vial and combined with the 10mag 
 and ldao aliquot dissolved in hexane . the dry surfactant mixture was dissolved in 
 500 l of deuterated hexane and 0.3 l of deuterated hexanol . this solution was injected with 18.2 l of buffer or 6.8 mm n flavodoxin c55a , as required , and vortexed until a clear 
 solution formed . n 
 hsqc spectra were collected 
 on an avance iii 600 mhz bruker spectrometer equipped with a tci cryoprobe . two - dimensional spectral acquisitions included 1024 complex points 
 in the h direct dimension and 200 complex points in the n indirect dimension . the sparky graphical analysis software was used 
 to tabulate resonance assignments and associated intensities ( goddard , 
 t.d . and kneller , d.g . paramagnetic relaxation enhancement ( pre ) values were determined 
 from the ratio of hsqc intensities of amide n h correlations in the reverse micelle samples with oxidized 
 ( paramagnetic ) and reduced ( diamagnetic ) nitroxide spin label . pre ratios were normalized to 1.0 using an average 
 scaling factor from the cross peak intensities of the resonances unaffected 
 by the presence of the spin label . the n and h chemical shift assignments for n - labeled flavodoxin ( c55a , s72c ) were mapped from the aqueous 
 flavodoxin assignments . mapped assignments 
 were confirmed by tracing the through space connectivities in a 3d 
 noesy hsqc spectrum collected with a 100 ms mixing time . samples employing 
 tempol in the water core or tempo - pc in the surfactant shell of the 
 reverse micelle were made with n - flavodoxin ( c55a ) . the 
 reverse micelle samples with the spin label covalently attached to 
 the protein were prepared with n - mtsl - n - flavodoxin 
 ( c55a , s72c ) . the n noesy hsqc experiments were 
 collected with 1024 
 complex points in the acquisition dimension , 64 complex points in 
 the n evolution dimension , and 200 complex points in 
 the h indirect dimension . the water flip back pulse was not used in these spectra to maintain 
 the coherence of the water proton magnetization with the protein proton 
 magnetization . the processed 3d spectra were analyzed at the h water plane to measure the relative intensity of the amide 
 noe cross peaks to water relative to the amide diagonal cross peaks . estimates of the effective macromolecular tumbling time of the 
 encapsulated protein were obtained from the n - tract measurements 
 using 40 gradient time increments to describe the decay of the relevant 
  and  n transitions essentially as described elsewhere . exponential decay rates of selected regions of the integrated 
 amide frequencies were fitted using in - house python scripts with al 
 nmr processing . cw epr spectra were acquired on an 
 x - band ( 9.372 ghz ) bruker emx spectrometer . a modulation amplitude 
 of 1 g was employed , and a 100 khz modulation frequency using 1 mw 
 of power . power saturation epr curves were collected to the maximum 
 available power ( 300 mw ) . all reverse micelle solutions tolerated 
 this power . in contrast , aqueous solutions were susceptible to boiling 
 at power levels above 100 mw . four ( 4 ) mm epr tubes were used for 
 data collection of reverse micelle solutions at room temperature . the epr spectra of aqueous solutions were collected in wiretrol 50 
 l capillary tubes ( 0.08 mm i.d . ) dnp 
 experiments employed 
 a bruker elexsys e580 x - band epr spectrometer , which was extended 
 with an ispin - nmr system ( spincore technologies , inc . ) . for observation 
 of h proton nmr spectra were 
 acquired at the magnetic field of the lowest hyperfine line in the 
 epr spectra of the reverse micelles samples . spectral 
 acquisitions included 128 scans with a 10 khz sweep width and 512 
 complex points , and an interscan delay of 4 s. to induce dnp , microwaves 
 were generated at the appropriate frequency with the bruker x - band 
 bridge . the microwaves were then amplified to 10 w using a bruker 
 ampx10 amplifier and directed into the cavity , which was still tuned 
 for continuous - wave epr . the nmr spectra were processed with an exponential 
 multiplication of 10 hz , zero filled once and fourier transformed . for dnp in solution , the dynamics of the polarizing agent - water 
 interaction largely governs the strength of the polarization transfer 
 via the overhauser effect . briefly , the enhancement is usually expressed 
 as:1where s is 
 a saturation factor that describes the efficiency of saturation of 
 the electron zeeman transitions , f is a leakage factor 
 that describes the paramagnetic enhancement of the nuclear relaxation 
 rate over the total nuclear relaxation rate , and  is the coupling 
 factor , which defines the magnetization transfer from the electron 
 to the nuclear spin when the electron spin is saturated . dnp enhancement of the water ( h2o ) and 
 other hydrogen resonances ( hother ) were determined 
 by measuring the total integrated intensity of the unresolved h resonance obtained with ( ion ) and without ( ioff ) saturation of the 
 epr resonance line using samples prepared with h2o and 
 d2o using eq 2 . the relative contributions 
 of the water ( fh2o ) and other ( fhother ) h spins to the spectrum 
 were determined by integration of the high - resolution spectrum obtained 
 at 600 mhz.2 to examine the potential for reverse micelle samples 
 to provide a 
 path to signal enhancement , three types of placement of nitroxide 
 spin radical in the reverse micelle macromolecular assembly were examined : 
 ( a ) attachment to the protein via a cysteine bridge ( mtsl ) ; ( b ) free 
 in the aqueous core in soluble form ( tempol ) ; and ( c ) embedded in 
 the reverse micelle shell using a carrier lipid ( tempo - pc ) ( figure 1 ) . schematic illustrations of the strategies for introduction 
 of nitroxide 
 spin radicals to reverse micelles . ( c ) nitroxide attached to a carrier embedded in the surfactant 
 shell ( tempo - pc ) . here we use mutants of 
 flavodoxin from cyanobacteria anabaena pcc7119 as a test protein . flavodoxins function 
 as soluble electron carriers between redox proteins and contain a 
 noncovalently bound flavin mononucleotide cofactor ( fmn ) that serves 
 as a redox center . n hsqc spectra of ( a ) n flavodoxin 
 ( c55a ) , ( b ) n flavodoxin ( c55a , s72c ) with n mtsl covalently attached , ( c ) n flavodoxin c55a with 
 tempol , and ( d ) n flavodoxin ( c55a ) with tempo - pc . ( e ) 
 the chemical shift differences (  = ( ( nn/h ) + ( h ) ) ) of backbone 
 amide h n resonances of flavodoxin 
 in free aqueous solution and flavodoxin in 10mag / ldao reverse micelles . all residues that could be measured are shown including the site of 
 mutation and ligand attachment ( red arrow ) . very minor chemical shift 
 perturbations are found ( r = 0.999 and 
 < rmsd > = 0.010 ) , indicating that high structural fidelity is 
 maintained 
 upon encapsulation of the protein with spin label in the three labeling 
 scenarios examined ( see figure 1 ) . flavodoxins are characterized by an / 
 doubly wound 
 topology , which consists of a five - stranded parallel -sheet 
 surrounded by -helices on both sides . the fmn cofactor is noncovalently but tightly bound flavin 
 mononucleotide and was studied here in its oxidized ( diamagnetic ) 
 state . the structure and dynamics of this flavodoxin have been studied 
 extensively by both crystallography and by nmr 
 spectroscopy . this protein has also served 
 as a model protein for the development and demonstration of reverse 
 micelle encapsulation . the c55a mutant from cyanobacteria anabaena pcc7119 is used as a parent molecule in order to avoid slow dimerization 
 through an intermolecular disulfide . for direct ligation of the nitroxide radical to the protein , flavodoxin ( c55a , s72c ) was created to provide 
 a readily accessible surface cysteine through which a nitroxide spin 
 radical ( mtsl ) could be attached using standard chemistry . to 
 form the protein containing reverse micelles , we employ a recently 
 developed surfactant system based on the zwitterionic surfactant lauryl - dimethylamine - n - oxide ( ldao ) and the nonioinic surfactant 1-decanoyl - rac - glycerol 
 ( 10mag ) . conditions were chosen to have 
 roughly one nitroxide radical per protein - containing reverse micelle 
 to avoid heisenberg exchange interactions between nitroxide spin radicals . the protein and corresponding spin label were encapsulated within 
 10mag / ldao reverse micelles in hexane with a water loading of 20 . 
 detailed consideration of the n - hsqc spectra of encapsulated 
 flavodoxin in the three scenarios for delivery of the nitroxide radical 
 to the reverse micelle indicates that the structural integrity of 
 the protein is fully maintained ( figure 2 ) . the general strategy that is being 
 followed for the implementation of dynamic nuclear polarization in 
 aqueous solution is to utilize the rapidly fluctuating interaction 
 of solvent water and spin radical to mediate polarization transfer 
 through the oe . the second and equally important polarization transfer 
 between water and the macromolecule of interest will also proceed 
 via a dipole clearly , since sensitivity 
 enhancement is the central goal , it is vitally important that sample 
 size not be overly compromised . unfortunately , the high dielectric 
 loss of standard aqueous samples requires significant reduction in 
 both sample volume and depth . in contrast , 
 solutions of reverse micelles in liquid alkane solvents are relatively 
 transparent to microwaves . though the water core of reverse micelles can have significant dielectric 
 absorption in this frequency region , the overall bulk macroscopic 
 microwave receptivity of reverse micelle solutions is much more favorable 
 than aqueous solutions . this is confirmed here where reverse micelle 
 solutions do not limit the sample diameter or total sample volume . x - band epr spectra of the three nitroxide labeling scenarios are shown 
 in figure 3 . in contrast , aqueous sample volumes 
 and diameters must be kept an order of magnitude smaller for even 
 simple epr spectra to be obtained . epr cw power saturation curves 
 were measured for aqueous mtsl labeled flavodoxin ( c55a , s72c ) in 
 a 50 l capillary and mtsl labeled flavodoxin ( c55a , s72c ) in 
 ldao/10mag reverse micelles in a 4 mm tube . the power required for 
 half - saturation for the aqueous solution was 56 mw and 4 mw for the 
 reverse micelle solution , clearly demonstrating the greater microwave 
 receptivity of the latter . the current strategy in the context of 
 solution oe dnp is to employ optimized coil designs with very small 
 sample sizes on the order of l to nl . this creates at the outset a deficit in signal - to - noise that must 
 be overcome in order for dnp to ultimately prove worthwhile . in this 
 respect , solutions of encapsulated proteins dissolved in low viscosity 
 fluids such as the short chain alkanes largely avoid this issue . x - band 
 epr spectra of the nitroxide spin radical in the three labeling 
 scenarios in 10mag / ldao reverse micelles . ( a ) n - flavodoxin 
 in the aqueous core and covalently attached to n - mtsl 
 ( b ) n - tempol solubilized in the aqueous core with n - flavodoxin . ( c ) n - flavodoxin and n - tempo - pc solubilized in the reverse micelle surfactant shell . the 
 triplet splitting is a result of the spin 1 n - electron 
 hyperfine coupling for n - tempo - pc and n - tempol 
 while the doublet splitting arises from the spin 1/2 n - electron hyperfine coupling of the n - mtsl . the red arrow 
 indicates the frequency for the application of the microwave power 
 for the dnp experiments . the epr spectra indicate that the nitroxide moiety experiences 
 variable dynamics depending on context . a rotational correlation time 
 that is slow or comparable to the applied microwave frequency influences 
 the shape of the observed epr spectrum . to determine rotational correlation 
 times the spectra shown in figure 3 were simulated 
 with easyspin . the motion of the mtsl 
 (   1.5 ns ) attached to the encapsulated protein is 
 slowed relative to tempo - pc embedded in the surfactant shell (  
  0.15 ns ) and tempol free in the aqueous core of the reverse 
 micelle (   0.1 ns ) . this compares to the motion of 
 tempol in free aqueous solution (   20 ps ) . the effective rotational correlation time of 
 encapsulated flavodoxin , was estimated using the n tract 
 experiment to be 12 ns , which 
 is considerably slower than the motion of nitroxide in any of the 
 three labeling scenarios . this has important implications for subsequent 
 optimization of the primary dnp to the water core as discussed below . dependence 
 of paramagnetic relaxation enhancements of the encapsulated 
 protein on the method of nitroxide incorporation . color - coded pres 
 of amide n  h correlations of flavodoxin 
 encapsulated in 10mag / ldao reverse ( a ) pres from 
 the mtsl spin label covalently attached at cys72 ( green dot ) of encapsulated 
 flavodoxin ( c55a , s72c ) . ( b ) pres with tempol in the aqueous core 
 of the reverse micelle with flavodoxin ( c55a ) encapsulated . ( c ) pres 
 with tempo - pc solubilized in the surfactant shell of the reverse micelle 
 with flavodoxin ( c55a ) encapsulated . further analysis also 
 shows the expected presence of paramagnetic relaxation enhancement 
 ( pre ) effects ( figure 4 ) . pres can potentially 
 counter the desired dnp signal enhancement through introduction of 
 line broadening and other relaxation effects . used extensively in 
 the characterization of both static and dynamical features of macromolecular 
 structure , the deleterious effects of the pre here arise primarily from long - range 
 coupling of the electron spin with h spins . in this regard , the placement of the nitroxide radical within the reverse micelle 
 assembly is apparently important . as expected , the mtsl spin label 
 covalently attached to c72 of flavodoxin ( c55a , s72c ) gave significant 
 pres in accordance with expected distant dependence in the region 
 encompassing 15  distances to the spin label ( figure 4a ) . this initial 
 result recommends against employing a covalently attached spin radical 
 in the context of dnp utilizing reverse micelle encapsulation . in 
 contrast , nitroxide radical solubilized within the aqueous core or 
 restricted to the surfactant shell show minimal pre effects ( figure 4b and c ) . dynamic nuclear 
 polarization was obtained by irradiation of the downfield hyperfine 
 transition ( see figure 3 ) . microwave power at 9.4 
 ghz was applied continuously at 10 w during the entire acquisition 
 and the 4 s recycle delay . the 
 signal phase was negative for the spectra with the  microwave 
 on  compared to the  microwave off spectra , 
 confirming that the dnp enhancement is governed by the dipolar - mediated 
 overhauser mechanism . h 
 nmr spectra ( 14.7 mhz ) of the water core of 10mag / ldao reverse micelles 
 with ( blue ) and without ( red ) saturation of the 9.4 ghz epr transition 
 indicated in figure 3 for ( a ) n - flavodoxin covalently attached to n - mtsl and dissolved 
 in the aqueous core ; ( b ) n - tempol solubilized in the 
 aqueous core with n - flavodoxin ; and ( c ) n - flavodoxin in the aqueous core and n - tempo - pc solubilized 
 in the reverse micelle surfactant shell . samples were prepared with 
 a w0 of 20 . to account for variations in line shape , dnp enhancements 
 were 
 calculated using magnetization intensities obtained by integration 
 rather than the maximum of the h resonance . the h nmr spectrum obtained at 14.7 mhz is not resolved ( figure 6 ) . overlay 
 of the 14.7 mhz dnp enhanced h spectrum ( blue , phase inverted 
 for clarity ) onto the 600 mhz h spectrum ( black ) of 400 
 m flavodoxin ( c55a ) , 200 m tempol solubilized in 100 
 mm ldao/10mag reverse micelles at a w0 of 20 . integration values for the 600 mhz spectrum are indicated for 
 the water resonance and the region containing the resonances due to 
 surfactants and alkane solvent . in order to isolate the dnp enhancement of the water core 
 from 
 the dnp enhancement of all other contributions to the unresolved h spectrum , the fractional contributions 
 were obtained by integration of the resolved h spectrum 
 obtained at 600 mhz ( figure 6 ) and the desired 
 enhancements obtained by simple algebra ( eq 2 ) . in this case , free tempol in the aqueous core of the reverse 
 micelle 
 gave dnp enhancements of the water core ( h2o ) of 25  9 without protein encapsulated and 
 34  9 with protein encapsulated . the dnp enhancement 
 for nonwater h spins ( i.e. , those arising from the surfactants 
 and alkane solvent ) was small ( hother  2 ) 
 indicating that its hydration shell will undoubtedly dominate polarization 
 of the protein . the value for hother was subsequently 
 used to obtain the dnp enhancements for the water core in all samples . 
 for comparison , mtsl attached to 
 flavodoxin gave an h2o of 34 
  9 and tempo - pc anchored in the reverse micelle 
 surfactant shell the largest enhancements of the water core ( h2o = 70  18 without protein encapsulated 
 and 93  23 with protein encapsulated ) . the dependence 
 of the dnp enhancement on water loading ( wo ) in the reverse micelle assembly was also evaluated using 400 m 
 tempol in the absence of protein . the enhancement factors are ( within error ) insensitive to the presence 
 or absence of encapsulated protein in the reverse micelle , indicating 
 the details of interaction between polarizer and water molecules relevant 
 to dnp are not greatly perturbed by the presence of the protein . it 
 should be noted that the concentrations of water and surfactants is 
 10-fold larger than that of the protein , thereby 
 masking dnp of the protein itself . these initial results suggest 
 that productive dnp enhancements 
 of the water h spins can be obtained in the reverse micelle 
 system without compromising sample volume . they also begin to suggest 
 that inclusion of the nitroxide label by association with the reverse 
 micelle surfactant shell or having it free in the aqueous core of 
 the reverse micelle is preferable to anchoring the spin radical to 
 the protein . other considerations discussed below reinforce this view . efficiency 
 of transfer of magnetization between encapsulated protein 
 and the water core at 14 t. ( a ) semilog plot of the ratio of the intensity 
 the water noe cross peak relative to the amide diagonal resonance 
 of a 3d n noesy hsqc for flavodoxin ( c55a ) encapsulated 
 in ldao/10 mag reverse micelles at ph 8.0 in red and aqueous flavodoxin 
 ( c55a ) at ph 8.0 in blue the noe mixing time was 100 ms for both spectra . 
 approximately 90% of the amide hydrogens of encapsulated flavodoxin 
 show noes to water . fewer sites show noes to water in the aqueous 
 condition with the intensity ratio reduced by 12 orders of 
 magnitude compared to the reverse micelle spectrum . ( b ) the h water plane of the 3d n noesy hsqc spectrum of flavodoxin 
 ( c55a ) encapsulated in ldao/10 mag reverse micelles at ph 8.0 . ( c ) 
 mapping of the noe intensity ratio of the water cross peak to the 
 amide diagonal peak onto the three - dimensional structure of flavodoxin 
 ( pdb code flv1 ) . the color bar indicates white through 
 dark blue for stronger noes with red indicating resonances that display 
 significant hydrogen exchange with water . these amide hydrogens are 
 located at the edges of secondary structure elements and in loops . a critical component of a possible 
 strategy for dynamically polarizing macromolecules such as proteins 
 is to utilize solvent water as a reservoir of excess polarization . there are at least two potential mechanisms 
 for transfer of nonequilibrium magnetization from water to a protein 
 molecule : hydrogen exchange with solvent and direct dipolar contact 
 between the spins of water and those of the protein . unfortunately 
 in this context , hydrogen exchange can be significantly slowed within 
 the reverse micelle . on the other hand , the motion of water within the reverse micelle is also significantly 
 slowed , relative to bulk aqueous solution , which leads to longer residence times of hydration water at the 
 surface of the protein and more effective dipolar contact with the 
 protein . these two mechanisms lead to excess magnetization 
 residing at spins located at or near the surface of the protein which 
 is then envisaged to flow throughout the protein via the well - known 
 spin diffusion phenomenon . the initial 
 transfer of polarization from water to the protein is therefore critical 
 to the basic approach . the transfer of polarization from water to 
 protein was monitored at 600 mhz ( h ) by recording a three - dimensional n - resolved noesy experiment of encapsulated flavodoxin ( c55a ) 
 using a 100 ms mixing time . at ph 8 , both the free and encapsulated 
 proteins show a large number of cross peaks with the water resonance , 
 which arise from a combination of direct and relayed noes ( figure 7 ) . at ph 5 , the hydrogen exchange - mediated transfer 
 is reduced , but the noes from the rm cavity water to the protein remain 
 largely unaffected ( not shown ) . the amide polarization ratios of flavodoxin 
 ( c55a ) at ph 8 demonstrated in figure 7a show 
 12 orders of magnitude - enhanced polarization for the protein 
 in reverse micelles compared to the same protein in aqueous solution . the general suppression of hydrogen exchange within the reverse micelle 
 is apparently amply compensated by more efficient dipolar contact 
 between water and the protein , thereby preserving the feasibility 
 of moving polarization from the water of the reverse micelle core 
 to the protein . the initial results 
 presented here 
 suggest that the use of reverse micelle encapsulation holds promise 
 for the implementation of dnp in liquids . in particular , solutions 
 of reverse micelles avoid limitations in sample size due to dielectric 
 heating . it should be pointed out that this advantage is anticipated 
 to persist into the thz region . though the water core of the reverse 
 micelle has a modest absorption peak in the subterahertz region , the 
 overall absorption of reverse micelles solutions is relatively limited 
 for water loadings applicable to high - resolution nmr of encapsulated 
 proteins . thus , dielectric heating is 
 not anticipated to limit the application of this approach at higher 
 magnetic field strengths . in addition , the relatively slow motion 
 of water in the hydration layer of encapsulated proteins renders polarization 
 transfer to the protein efficient . these observations indicate that 
 use of reverse micelle encapsulation can overcome two of the main 
 barriers facing the use of dynamic nuclear polarization in solution . nevertheless , several important issues remain to be resolved . beyond 
 the practical improvement in instrumentation , two fundamental parameters 
 governing the dnp effect need to be explored in the context of the 
 reverse micelle . the enhancement factor depends upon the coupling 
 factor (  ) , the leakage factor ( f ) and the 
 saturation factor ( s ) ( eq 1 ) . it has been suggested that in bulk solution the coupling factor 
 is limiting , because the saturation and leakage factors can be usually 
 made close to 1 . the saturation factor 
 can certainly be optimized to approach 1 for even the large sample 
 volumes for the reverse micelle system where application of sufficient 
 gigahertz power is not an appreciable limitation . it is thus important 
 to consider the dependence of the coupling factor and the leakage 
 factor in order to further optimize the reverse micelle for maximum 
 enhancement . the leakage factor has a dependence on the exchange 
 of magnetization 
 of water hydrogens close to the spin label ( bound waters ) and the 
 hydrogens in the rest of the water core . the leakage factor is expressed 
 as:3with t1 describing 
 the longitudinal relaxation time of the water hydrogens in the presence 
 of the spin label , and t10 , the longitudinal 
 relaxation of the water hydrogens in the absence of the spin label . the water loadings typical for high - resolution protein nmr in reverse 
 micelles ( w0 = 1020 ) result in 
 an effective concentration of spin radical on the order of 50 to 100 
 mm , when the concentration of the spin radical is calculated relative 
 to the volume of the water core . this promotes a higher contribution 
 of paramagnetic relaxation to the total relaxation and thereby tends 
 to push the leakage factor to unity . the reverse micelle system offers 
 the ability to optimize the relaxation characteristics through manipulation 
 of the water loading ( see below ) and the number and location of polarization 
 agent molecules provided . for nitroxide radicals , which have 
 no contact contribution to the 
 dnp effect , the coupling factor may be expressed as:4with r1para = k[7j(s,c ) + 3j(s,c ) ] and the correlation time for the dipolar interaction 
 ( c ) is defined as c = ( r1 + s1 + m1 ) , where 
 r , s and m are 
 the reorientation time of the nitroxide , the electron relaxation time , 
 and the nuclear lifetime , respectively . here k and 
 the spectral density , j(, ) , both depend 
 on the precise details of the motion modulating the interaction . the 
 functional form of r1para creates a double 
 dispersion for the coupling constant reaching a maximum at low frequency 
 and dropping to zero at high frequency . thus , the faster motion of 
 water required to maximize the primary dnp effect is counterbalanced 
 by the slower motion of water needed to enhance polarization transfer 
 to the protein . as noted above , the nature 
 of water dynamics within a reverse micelle can be manipulated , and so there is the opportunity to optimize 
 the overall dnp process by fine - tuning the reverse micelle sample . the observed dependence of the dnp enhancement of water on the water 
 loading supports this view as it is well - known that the dynamics of 
 the water in the reverse micelle core are strongly influenced by its 
 size at low water loadings . thus , the choice of the water loading 
 value may require a compromise between the effective correlation time 
 of the protein and the dynamics of the water most optimal for dnp . layered on this consideration is the fact that the net enhancement 
 is scaled by the ratio of the magnetic field at which dnp is performed 
 and that of detection . clarification of this issue will ultimately 
 determine whether both the dnp and the subsequent nmr experiment can 
 be carried out at the same field or if physical sample shuttling between 
 a lower magnetic field , where dnp will occur , and a higher magnetic 
 field , where the high - resolution nmr experiment is performed , will 
 be required . the conditions used 
 here for the reverse micelle encapsulation 
 with the nitroxide spin label incorporated are a promising beginning 
 for the application of the dnp enhancement to proteins in solution 
 nmr . the enhancement of 90 fold at the water resonance can 
 be evaluated in terms of eq 1 as discussed above . the ratio of gyromagnetic ratios of the electron and proton equal 
 to 660 is the absolute maximum achievable . the saturation factor ( s ) is 1 based on the epr saturation curves explored 
 by the cw epr . the leakage factor ( f ) was estimated 
 at high field to be 0.6 . adjusting the water loading , the 
 type and concentration of polarizing agents , and so on can potentially 
 optimize the leakage parameter . taking the saturation and leakage 
 factors at face value , the coupling factor (  ) is suggested 
 to be 0.2 , which is significantly below that estimated for 
 nitroxide in bulk aqueous solution . the 
 coupling factor potentially has the most room for improvement and 
 depends on the ps dynamics of the polarizer and water molecules ( eq 4 ) . from the line shape analysis of the cw epr spectra 
 ( figure 3 ) , it is evident that the dynamics 
 can be tuned by the placement of the spin radical in the reverse micelle 
 and with the correlation time of the reverse micelle particle . it 
 is also well - known that the dynamics of the water core of reverse 
 micelle are highly dependent upon water loading . in summary , further increases in the efficiency of dnp 
 of the water core can be potentially achieved through further exploration 
 of sample conditions as well as through continuing improvements in 
 instrumentation . although encapsulation of proteins and nucleic 
 acids with structural 
 fidelity and high nmr performance within reverse micelles was introduced 
 some time ago , it has not seen general acceptance 
 and use . a perceived difficulty in determining suitable encapsulation 
 conditions and the use of nonstandard apparatus are potential barriers to the method s adoption . recently , we have developed a robust surfactant system that allows 
 for the encapsulation of proteins with a range of sizes ( at least 
 up to 80 kda ) and isoelectric points ( 4 < pi < 11 ) . on the basis of the zwitterionic surfactant 
 ldao and the nonioinic surfactant 10mag , high structural fidelity 
 and excellent effective macromolecular tumbling times could be achieved . although solutions of encapsulated proteins dissolved in the low viscosity 
 short - chain alkanes have more favorable dielectric properties than 
 simple aqueous solutions , they are more limited in the concentration 
 of proteins that can be achieved . with some exceptions , effective 
 concentrations of proteins are generally restricted to less than 200 
 m . fortunately , in contrast to aqueous samples , the low conductivity 
 of reverse micelle solutions does not degrade the performance of high - q 
 cryogenic probes . nevertheless , by taking 
 advantage of the favorable properties of solutions of molecules encapsulated 
 within reverse micelles , one can anticipate that dnp can overcome 
 this modest limitation in concentration and ultimately provide a significant 
 gain in the sensitivity of solution nmr spectroscopy . in summary , the results presented here demonstrate that the reverse 
 micelle encapsulation strategy is a useful starting point for the 
 development of procedures for dynamic nuclear polarization of biomolecules 
 in solution nmr .
despite 
 tremendous advances in recent years , solution nmr remains 
 fundamentally restricted due to its inherent insensitivity . 
 dynamic 
 nuclear polarization ( dnp ) potentially offers significant improvements 
 in this respect . 
 the basic dnp strategy is to irradiate the epr transitions 
 of a stable radical and transfer this nonequilibrium polarization 
 to the hydrogen spins of water , which will in turn transfer polarization 
 to the hydrogens of the macromolecule . 
 unfortunately , these epr transitions 
 lie in the microwave range of the electromagnetic spectrum where bulk 
 water absorbs strongly , often resulting in catastrophic heating . furthermore , 
 the residence times of water on the surface of the protein in bulk 
 solution are generally too short for efficient transfer of polarization . 
 here 
 we take advantage of the properties of solutions of encapsulated 
 proteins dissolved in low viscosity solvents to implement dnp in liquids . 
 such samples are largely transparent to the microwave frequencies 
 required and thereby avoid significant heating . 
 nitroxide radicals 
 are introduced into the reverse micelle system in three ways : attached 
 to the protein , embedded in the reverse micelle shell , and free in 
 the aqueous core . 
 significant enhancements of the water resonance 
 ranging up to 93 at 0.35 t were observed . 
 we also 
 find that the hydration properties of encapsulated proteins allow 
 for efficient polarization transfer from water to the protein . 
 these 
 and other observations suggest that merging reverse micelle encapsulation 
 technology with dnp offers a route to a significant increase in the 
 sensitivity of solution nmr spectroscopy of proteins and other biomolecules .