gsls are endocytosed , trafficked , and sorted through 
 early and late endosomal compartments on the way to the lysosome , 
 where specific enzymes sequentially cleave sugar groups , eventually 
 producing ceramide , which is finally deacylated to sphingosine .
more than 10 different enzymes and five accessory 
 proteins are involved in this important process .
one of those accessory 
 proteins , the gm2 activator protein ( gm2ap ) , is essential for stimulating 
 the catabolism of neuronal gangliosides by extracting ganglioside 
 gm2 from intralysosomal vesicular membranes .
the resulting gm2ap  gm2 complex interacts with -hexosaminidase 
 a ( hex a ) for hydrolysis of the terminal sugar to yield ganglioside 
 gm3 .
activator proteins often function by binding to glycosphingolipids 
 and forming aqueous  soluble complexes , thereby providing an 
 aqueous  soluble enzyme access to a lipid generally confined 
 to a hydrophobic environment .
genetic mutations that alter proper function of hex a or gm2ap can 
 inhibit the crucial degradation pathway , thus resulting in an accumulation 
 of gm2 causing neuronal cell death and lysosomal storage diseases 
 such as tay sachs or ab variant gangliosidosis .
gm2ap is a non - homologous member of the family of 
 proteins called 
 sphingolipid activator proteins ( saps ) .
. four members of this family , saps a  d , 
 contain the characteristic  saposin fold  that consists 
 of four or five -helices anchored by three disulfide bonds . 

the structure of gm2ap , however , consists of eight -strands 
 with a tertiary -cup topology containing four structural disulfide 
 bonds .
in addition to stimulating gm2 degradation , gm2ap has been 
 proposed 
 to act as a general lipid transporter because , in vitro , gm2ap has been shown to bind and extract gm2 from micelles , transfer 
 glycolipids , phospholipids , and fluorescently labeled lipids between 
 donor and acceptor vesicles , bind and inhibit platelet activating factor , and present cd1 molecules to antigens . in order to carry out its function as a lipid transporter
we proposed a 
 model of membrane partitioning based on results of sedimentation assays 
 that showed 15% of gm2ap molecules remaining on the bilayer surface , 
 implying that gm2ap is undergoing exchange on and off the bilayer 
 surface . from modeling of data from sedimentation 
 assays
, we concluded that gm2ap establishes exchange equilibria of 
 a minimum of four species : gm2ap in solution , gm2ap on the bilayer 
 surface , gm2ap  lipid complex on the bilayer surface , and gm2ap  lipid 
 complex in solution .
membrane interactions have also 
 been studied by others using langmuir monolayers , and these data suggest 
 that gm2ap is surface associated without deep penetration into the 
 bilayer .
we also showed that the orientation 
 of gm2ap on zwitterionic bilayer surfaces is such that the mobile 
 external loops that line the opening to the lipid binding pocket are 
 positioned at the bilayer surface .
( a ) the three 
 unique monomers ( a , b , c ) of apo gm2ap ( pdb i d 1g13 ) showing the different 
 conformations of the loop regions at the cleft entrance .
the four 
 disulfide bonds are shown in yellow , the apolar loop ( v54w63 ) 
 is shown in red , the reverse turn ( s130t133 ) is shown in green , 
 and the disordered strand ( v122p129 ) is shown in blue . taken 
 together , the reverse turn and disordered strand are collectively 
 referred to within the text as the flexible loop .
black spheres in 
 monomer a represent the c positions of the reporter sites chosen 
 for sdsl epr experiments .
select amino acid side chains are shown 
 in monomers b and c in stick format , demonstrating how the altered 
 conformations in these regions modulate the size of the entrance to 
 the lipid binding cavity .
additionally , 
 the two conformations have altered orientations of some of the amino 
 acid side chains , including l126 , l128 , and w131 , which alternate 
 between pointing in toward the protein interior and extending out 
 toward the solution .
( b ) sdsl labeling scheme showing the resultant 
 r1 chemical side chain of a cysteine covalently modified with the 
 mtsl spin label .
the models of gm2ap ( pdb 
 i d 1g13 ) from 
 x - ray crystallography reveal that 
 the volume of the hydrophobic cavity is roughly 6 times larger than 
 the volume of a ceramide moiety ( 500  ) .
in addition , 
 on the basis of x - ray structure analysis of five different crystal 
 forms , large differences in the diameter and area of the opening to 
 the lipid binding cavity were detected and attributed to flexibility 
 of loop regions that decorate the rim of the cavity .
these regions ( highlighted in figure 1a ) 
 are of particular interest for this study , as their mobility may be 
 related to function .
the loops are located on opposing sides of the 
 entrance to a prominent hydrophobic cleft whose width varies substantially 
 among the different crystal structures .
, spanning residues v54w63 , 
 protrudes into the solution and has a relatively stable conformation 
 in all crystal structures examined due to its involvement in crystal 
 lattice contacts . on the opposing side , 
 a reverse turn ( s130t133 ) containing w131 exhibits two alternative 
 conformations . in structures where the cleft is wide open and bound 
 to lipid ( pdb i d 1pub ) , this loop is flipped out with w131 exposed to the solvent .
the second conformation of this loop is seen 
 in structures where the cleft is closed , and the loop is tucked in 
 toward the interior of the cleft , burying w131 and allowing van der 
 waals contacts to exist between residues on either side of the cleft 
 ( l128 , p129 , l132 and i72 , i66 ) ( pdb i d 2agc(11 ) and pdb 
 i d 1g13(10 ) ) .
given that this mobile loop is preceded by 
 an extended disordered chain segment ( v122p129 ) , the mobility 
 of this entire region ( v122t133 ) can modulate the width of 
 this cleft , and therefore the size of the circumference of the cavity 
 ( figure 1 ) , allowing for lipid ligand to enter 
 into the binding pocket .
the question arises as to whether the 
 features of the protein that 
 are seen in the crystal structure are in conformational exchange in 
 solution and how these various conformations are related to function , 
 as protein mobility and dynamics are often related to function . for the case of gm2ap
, the multiple conformations in the crystal 
 structure , which in effect modulate the size of the entrance to the 
 cavity , may suggest that a conformational change is necessary for 
 extracting lipid ligand from vesicle surfaces , or that the conformations 
 will be modulated in the halo ( lipid bound ) or apo protein .
another 
 possible role for the conformational flexibility is in conformational 
 entropy to allow gm2ap to partition with the vesicle surface or to 
 interact with hydrolases , such as hexa , in gm2 hydrolysis . to investigate 
 the local structure and mobility of the apolar and mobile loops of 
 gm2ap in solution , we utilized sdsl epr , which is a powerful spectroscopic 
 tool used to study conformational changes in proteins as well as to characterize local backbone motion .
mtsl 
 was purchased from toronto research 
 chemicals , inc . unless otherwise stated , all other reagents were from 
 fisher scientific and used as received .
recombinant 
 wild - type gm2ap and cysteine constructs generated for epr experiments 
 were expressed , purified , and spin labeled as described previously .
properly folded protein was separated 
 from aggregated and misfolded protein by applying it to an s-200 gel 
 filtration column equilibrated with 50 mm sodium phosphate , 150 mm 
 nacl , 2.5% glycerol ph 7 .
function of each of the labeled constructs , 
 defined as the ability to extract both gm2 and a fluorescently labeled 
 lipid , was demonstrated in an earlier study of 
 gm2ap constructs , where power saturation sdsl epr spectroscopy was 
 used to determine the orientation of this protein on lipid bilayer 
 surfaces . for site
l126c , sample homogeneity 
 was further confirmed by mass spectrometry analysis and hplc analysis , 
 where a single elution peak arose from protein samples collected after 
 sec and then injected into a c18 column eluted with an acetonitrile 
 and water gradient . continuous wave x - band epr spectra 
 for single and double spin labeled gm2ap constructs were collected 
 on a modified bruker er-200d spectrometer with an er023 m signal channel , 
 an er032 m field control unit , and a loop gap resonator ( medical advances , 
 milwaulkee , wi ) .
spectral scans were collected over the range of 100 
 gauss ( g ) and were recorded with protein samples in sealed round capillaries , 
 0.60 mm  0.84 mm  100 mm ( fiber optics center , inc . ; new 
 bedford , ma ) , with 3.16 mw incident power and optimized modulation 
 amplitudes .
gm2ap spectra were recorded for each of the cysteine variants 
 in solution ( 50 mm tris 
typical protein concentrations 
 were near 200 m , and 832 scans were collected for each 
 sample .
all spectra shown are baseline corrected and integral area 
 normalized using labview software ( gift from wayne hubbell ) .
pulsed 
 epr experiment distance measurements for double labeled gm2ap constructs 
 were performed on a bruker e580 spectrometer with an md-5 resonator 
 with the four - pulse deer sequence as described previously .
deer data were analyzed with deeranalysis2011 software , available 
 online at http://www.epr.ethz.ch/software/index , and in - house matlab dsim as described previously .
because the average distances determined from pulsed experiments 
 for double labeled sites in gm2ap were found to be near 20  , 
 low temperature cw epr experiments were also performed for distance 
 determination .
spectra were acquired as 200 g scans at 140 
 c with 2 w incident microwave power . for easy comparison 
 of the degree of line broadening , the spectra are normalized to equal 
 areas and are plotted on the same y - scale .
the d1/d0 ratio is defined 
 as the height of the high and low field transitions over the height 
 of the center field transition and can be related to distances from 
 measurements of kcsa . for variable 
 temperature cw epr experiments over the range 540 c , 

the temperature was controlled by flowing nitrogen gas through a copper 
 coil that was submerged in a refrigerated water bath ( thermo scientific 
 neslab rte-7 digital one ( 25 to 150  0.01 c ) ) 
 containing a 40% ethylene glycol ( v / v ) solution .
the cooled nitrogen 
 gas flowed from the copper coil to a quartz dewar ( wilmad - labglass , 
 buena , nj ) that surrounded the loop gap resonator , where the sample 
 was allowed to equilibrate at each temperature for at least 20 min 
 prior to data collection .
for low temperature cw distance measurements , the copper 
 coil was submerged in liquid n2 .
the flow rate and length 
 of insulated tubing was optimized to obtain a steady temperature of 
 140  5 c .
epr spectra of r1 constructs 
 were simulated using the microscopic order macroscopic disorder ( momd ) 
 model of freed and colleagues that is freely available at www.acert.cornell.edu .
double integral area normalized epr spectra 
 were regenerated with either a one- or two - component simulation .
experimental 
 epr spectra were fit with the momd model following the procedure described 
 by columbus et al . with the following values for the a and g tensors : axx = 6 ,
ayy = 6 , azz = 37 , gxx = 2.0089 , gyy = 2.0021 , gzz = 2.0058 , and diffusion tilt angles fixed at d = 0 , d = 15 , and d = 0. an axially symmetric diffusion 
 tensor was used , with values r and r where1the asymmetry parameter and effective mean 
 correlation time ,  , were calculated using23if a single - component fit did not adequately 
 converge , a two - component model was utilized . in cases where two spectral 
 components were necessary for adequate fitting , to simplify the fitting 
 procedure , the order parameter , c20 , for 
 the mobile component was set to 0 .
the values that were allowed to 
 vary to obtain a least - squares fit were gib0 ( inhomogeneous line width ) , azz , r , r , and c20 .
the fractional components determined from 
 spectral simulations of the nitroxide line shapes were analyzed in 
 terms of a conformational equilibrium , as has been done previously 
 for other protein systems .
the equilibrium 
 expression4is defined for the unfolding of the loops , 
 where f1 is the fraction of the more mobile 
 component and f2 is the fraction of the 
 less mobile component .
values for the thermodynamic entropy , s , and enthalpy , h , 
 of this conformational change were obtained by utilization of the 
 vant hoff equation5 experiments 
 were conducted at 
 20 c on a 600 mhz nmr spectrometer equipped with the 1 mm superconducting 
 coil probe ( uf - amris ) .
n labeled gm2ap was overexpressed 
 in bl21(de3 ) cells grown in minimal media supplied with 0.1% ammonium 
 chloride and purified as described previously .
the final sample contains 0.15 mm gm2ap 
 and 20 mm naoac at the desired ph . to change the buffer ph ,
the protein 
 was loaded to a gel filtration column ( g25 resin ) pre - equilibrated 
 with the desired ph ( 4.8 , 5.6 , 6.9 ) with 20 mm naoac . for the gm2 
 binding experiment , the spectra
were collected before and after incubation 
 with a final concentration of 0.6 mm gm2 ( in 20 mm naoac buffer ) at 
 room temperature .
the resulting spectra were processed with nmrpipe and sparky ( goddard and kneller , sparky 3 , ucsf , 
 san francisco , ca ) . because 
 little to no conformational change
was observed in the epr / nmr measurements , 
 the formation of the gm2ap : gm2 complex was confirmed for samples where 
 gm2 micelles were added .
specifically , protein : gm2 complexes were 
 purified by size chromatography and the presence of gm2 in the protein 
 fraction was detected via a rescorcinol assay as described previously .
for both a60r1 and l126r1 , differential scanning calorimetry showed 
 an increase in tm of 0.9 c of the 
 thermotropic unfolding for the halo protein compared to the unliganded 
 protein , thus further confirming that gm2 was indeed bound to the 
 protein .
it is noteworthy that the absolute values of tm differed for the two constructs , giving an indication 
 of the relative stability of the modified proteins relative to each 
 other ( table s1 , supporting information ) .
all md simulations 
 were performed using the amber suite of programs on a potential energy 
 surface described by the ff99sbildn force field .
conformers a and c of the crystal structure of the gma2 protein 
 ( pdb i d : 1g13 ) provided a starting point for our simulations that would help develop 
 an understanding of the conformational ensembles sampled by the protein . prior to running simulations , charged amino 
 acids were modeled on the basis of their protonation states calculated 
 using the h++ protonation state server .
the proteins were solvated in a periodically replicated rectilinear 
 box of explicit spc / e water molecules , providing an 8  solvation layer around the solute molecule . 

the solvated protein was prepared 
 for production simulations using a well - defined protocol .
the protein was first energy - minimized over five stages and then 
 equilibrated over two stages using a simulated annealing - like approach . 

in the first stage of energy minimization , all water molecules and 
 explicit ions were allowed to relax , while all protein atoms were 
 restrained with a strong harmonic potential . in subsequent stages , 

additional parts of the protein were gradually allowed to relax in 
 solvent . in the second stage of minimization , all hydrogen atoms were 
 allowed to relax .
side chain groups and backbone amide groups followed 
 this in the third and fourth stages , respectively .
finally , in the 
 fifth stage , the entire protein was energy minimized along with the 
 solvent molecules and ions .
after energy minimization , the system 
 was slowly heated to 300 k over 100 ps of md for the canonical ensemble 
 ( nvt ) , while the solute was kept restrained with a weak harmonic potential . 
 in the final stage of equilibration ,
all restraints were removed from 
 the protein and 1 ns of md was performed at 300 k for an isothermal 
 and isobaric ( npt ) ensemble .
after equilibration , md simulations of 
 both equilibrated systems were propagated for 275 ns in the npt ensemble 
 at 300 k. a time step of 2 fs was employed , and trajectory information 
 was collected every 1000 steps .
a langevin thermostat with a collision frequency of 1 ps was employed 
 to maintain the temperature of the system .
the shake algorithm was utilized to constrain heavy atom bonds to 
 hydrogen atoms , and long - range electrostatic interactions were calculated 
 using the particle mesh ewald method . in all , 550 ns of md data were collected to provide reasonable statistics 
 in understanding the nature of conformational sampling by gma2p .
ten single 
 cys variants of gm2ap were generated and labeled with 
 mtsl ( referred to within as r1 ) at the sites shown in figure 1a .
six of the chosen spin labeled sites are located 
 in the flexible and apolar loops ( a60r1 , i66r1 , i72r1 , l126r1 , s130r1 , 
 and n136r1 ) and were chosen to investigate the conformational flexibility 
 using epr spectroscopy .
the remaining four cys variants of gm2ap ( v54r1 , 
 l87r1 , t90r1 , and s115r1 ) were generated to probe sites thought to 
 be either structured ( -helices and -sheets ) or unstructured 
 loop regions of the protein .
figure 2 shows 
 the nitroxide spectra recorded at ambient room temperature for gm2ap 
 in solution under basic ph and acidic ph in the presence of gm2 micelles . 

the epr line shapes at each site are consistent with those expected 
 on the basis of the local structure and dynamics reflected in the 
 x - ray structures of gm2ap .
this conclusion is draw upon literature 
 reports showing that the r1 line shape correlates with protein structural 
 components and b - factors .
of the six sites in 
 the flexible loops and disordered strand regions , a60r1 , l126r1 , and 
 n136r1 have line shapes consistent with fairly mobile and solvent 
 accessible sites on proteins ; however , 
 from the shoulders in these spectra ( indicated with arrows ) , it is 
 clear there are two spectral components ( discussed further below ) . 

for example , the spectra from site l126r1 are narrow and intense , 
 reflecting a higher degree of motional averaging .
this site is located 
 in a flexible strand with very high crystallographic b - factors .
sites 
 i66r1 , i72r1 , and s130r1 have broadened epr spectra with structure 
 seen in the high field resonances , which indicate that these spin 
 labeled sites reside in more structured regions of gm2ap . in numerous 
 x - ray structures of gm2ap ,
the side chains of i66 and i72 can be seen 
 to point in toward the hydrophobic cavity , so it is likely that , at 
 these sites , the spin label motion is restricted by neighboring amino 
 acid side chains as well as the limited space of the cleft leading 
 to the lipid binding pocket .
further , the line shape from site s130r1 
 is broadened and more reflective of a spin label attached to an alpha 
 helical region of a protein , and some of the crystal structures of 
 gm2ap place this residue in a helical structure .
stack 
 plot of epr spectra from six sites in the apolar and flexible 
 loops of gm2ap .
the black traces are for samples prepared 
 in basic ph ( 8.0 ) , whereas those in gray were collected at ph 4.8 
 in the presence of 4 molar excess gm2 micelles .
note the spectra obtained for i72r1 in the 
 presence of gm2 showed the most change , but this effect results from 
 protein instability that leads to protein precipitation over time , 
 with increased broadening of the spectral line shape .
the epr spectra obtained under acidic conditions 
 in the presence 
 of gm2 micelles are very similar to those obtained under basic ph 
 conditions , suggesting that little to no conformational change has 
 occurred in the presence of ligand . in efforts to further characterize 
 possible conformational changes of the loops upon gm2 ligand binding , 

the expected experimental values based on conformers in the 
 crystal and mmm ( http://www.epr.ethz.ch/software ) evaluations of mtsl rotamers range between 16 and 30  .
low 
 temperature epr spectra were collected and analyzed for distances 
 across the binding cleft .
as can been seen in the spectra in figure 3a , very little to no differences were detected in 
 the spectra upon ph change and addition of ligand . the average distances 
 obtained from analysis of the spectral line shapes using the empirical d1/d0 parameter are given in table 1 , 
 and values are all between 18 and 20  2  .
a slight increase 
 of 2  in the average distance was seen for i66r1/l126r1 upon 
 addition of gm2 micelles . because the average distances are near 20 
  , which is the upper limit of distances readily detected by 
 this method , pulsed double electron
 electron 
 resonance ( deer ) spectroscopy was also performed for these 
 sites .
the background corrected deer echo curves are shown in figure 3b . although mmm modeling of side chain conformers 
 predicted the possibility of larger distances for these sites , deer 
 data also reported average distances near 20  , which is unfortunately 
 a lower distance cutoff for accurate analysis of deer distance distribution 
 profiles .
nevertheless , the shapes of the echo curves are consistent 
 with an average distance of 2023  , and show that no 
 major conformational changes of these loops occur upon binding gm2 
 ligand .
the lack of distinct oscillations in the deer echo curve indicates 
 a high degree of conformational flexibility , which is discussed in 
 more detail below .
( a ) low temperature integrated 
 absorption and derivative cw epr 
 spectra of doubly labeled gm2ap constructs i72r1/s130r1 and i66r1/l126r1 
 showing no change in conformation upon addition of gm2ap .
( b ) background 
 corrected deer echo modulation traces for doubly labeled gm2ap samples 
 showing again no change upon addition of gm2 .
distances from both 
 cw and pulsed epr were estimated to be within the 1822  
 range and are further discussed within the text . to further investigate how ph and ligand altered the average 
 conformation 
 of gm2ap , uniformly n labeled protein was generated for 
 solution nmr hsqc measurements .
figure 4 shows 
 hsqc spectra of gm2ap at ph 6.9 ( purple ) , ph 5.6 ( red ) , and ph 4.8 
 ( blue ) and upon addition of gm2 at ph 4.8 ( red = with gm2 ; blue = 
 absence of gm2 ) .
the data show that changes in ph cause a few resonances 
 to shift or disappear , likely as a result of changes in local electrostatic 
 potentials .
the binding of gm2 also does not induce shifts in most 
 of the resonances ; again , a few peaks are seen to shift or disappear . 

assignments for gm2ap are lacking , as the broadening of some peaks 
 indicates intermediate exchange , and we have had low signal intensities 
 in some triple resonance experiments needed for assignments .
future 
 nmr studies are aimed at mapping the specific regions in gm2ap that 
 are altered during ph changes and interactions with ligands ; however , 
 these experiments are beyond the scope of this manuscript at this 
 time . here , the data simply indicate that no major conformational 
 changes occur in gm2ap upon complex formation with gm2 under acidic 
 conditions
. the nmr and epr results are also consistent with cd analyses , 
 which show no detectable changes in the spectra when ph is altered 
 or gm2 micelles are added .
hsqc nmr spectra of uniformly labeled n gm2ap ( a ) 
 at ph 6.8 ( purple ) , ph 5.6 ( red ) , and ph 4.8 ( blue ) and ( b ) upon addition 
 of 4 molar excess gm2 micelles ( red ) at acidic ph 4.8 ( blue ) . 

final samples each contain 150 m gm2ap and 20 mm naoac . for 
 the gm2
binding experiments , the spectra were collected before and 
 after incubation with 0.6 mm gm2 at room temperature .
overlay of area normalized experimental epr spectra ( black ) 
 and 
 momd simulated spectra ( dashed gray ) of the 10 spin labeled gm2ap 
 constructs .
all spectra have 100 g sweep widths and were collected 
 at ambient room temperature .
( a ) spectra for sites that are located 
 in the flexible loops required two components for adequate fitting 
 between the experimental and simulated spectra .
these two spectral 
 components are shown , with the more immobile spectrum in black and 
 the mobile spectrum , where c20 = 0 was 
 assumed , in gray .
( b ) adequate agreement between the experimental 
 and simulated line shapes was obtained for sites v54r1 , l87r1 , t90r1 , 
 and s115r1 ( bottom ) using only a single spectral component .
in addition to the above - mentioned 
 six sites in gm2ap , four additional sites were chosen for investigations 
 of conformational heterogeneity .
the epr line shapes of all 10 r1 
 labeled gm2ap constructs in basic solution with the overlaid simulations 
 are shown in figure 5 .
the epr spectra were 
 simulated using the momd model of freed and colleagues . using this method ,
a single component was not 
 sufficient to adequately fit sites in the apolar and flexible loops 
 ( a60r1 , i66r1 , i72r1 , l126r1 , s130r1 , and n136r1 ) .
for these sites , 
 we utilized a model where one component was immobile in the intermediate 
 time regimes , whereas the other component had fast - limit mobility 
 where we assumed that this mobile component had c20 = 0 ( the order parameter ) , as was done previously by 
 others .
as can be seen in figure 5a , the two components have dramatically different line shapes , with 
 one having narrow , isotropic - like features , corresponding to a highly 
 mobile site and the other a more broadened line shape corresponding 
 to a site with more restricted motion . given the presence of two distinct 
 spectra , we can conclude that the conformational exchange is slow 
 on the ns time scale .
these two spectral components are reflective 
 of a more mobile and a more immobile spin label environment , consistent 
 with the alternative conformations seen for these flexible regions 
 in the gm2ap x - ray structures .
these alternative 
 spin label conformations may reflect simply spin label conformational 
 states or protein conformational states .
to further investigate the origin 
 of the multiple - component spectra 
 for sites located in the flexible loop regions of gm2ap , data were 
 collected as a function of temperature over the range 1035 
 c in 5 c increments .
a similar approach has been utilized 
 to characterize a conformational change in the transmembrane sequence 
 of the transducer domain of n. pharaonis halobacterial 
 transducer of rhodopsins ii ( nphtrii ) in lipid membranes . for gm2ap ,
l126r1 in the disordered strand and 
 i66r1 adjacent to the apolar loop were selected for the variable temperature 
 epr experiments .
if these regions of the protein are in conformational 
 exchange , the equilibrium populations of the two components should 
 change in a linear fashion with inverse temperature .
the epr line 
 shapes obtained at each temperature were then simulated using the 
 momd model to extract fractions of populations of the two components . 

representative epr spectra and momd simulations of line shapes for 
 l126r1 and i66r1 at select temperatures are shown in figure 6a .
changes in the epr line shapes were seen for 
 l126r1 with increasing temperature ; however , no consistent changes 
 in the i66r1 line shapes were observed .
the percentages of the mobile 
 and immobile components were obtained from the line shape fittings 
 for l126r1 and i66r1 and are plotted as a function of temperature 
 ( figure 6b ) .
the plot of component percentages 
 for i66r1 did not show a temperature dependent change , possibly indicating 
 trapped conformers of the spin label and not protein conformational 
 exchange .
this interpretation of the data is consistent with the crystal 
 structure , which shows little positional change of the backbone of 
 the segment containing i66 in the three monomers a , b , and c ( 1g13 ) 
 and the presence of an arg - trp cation pi interaction that may prevent 
 this apolar loop from undergoing marked conformational exchange of 
 the backbone ( figure 1 ) .
therefore , the origin 
 of the two - component spectra at this site may arise from different 
 rotameric states of the spin label .
( a ) representative epr spectra of gm2ap 
 l126r1 and i66r1 as a function 
 of temperature ( c ) .
overlain experimental spectra ( black ) and 
 momd simulations ( red ) are shown .
each simulation required two spectral 
 components for an adequate fit to the experimental data .
( b ) plots 
 of the relative percentages of the mobile and immobile components 
 obtained from the spectral fits as a function of temperature ( k ) . for l126r1 , changes in the epr 

line shapes as well as in the relative 
 percentages of the more mobile ( f1 ) and 
 more immobile ( f2 ) components were observed 
 as the temperature was varied .
simulations for a select data set can 
 be found in figure s1 ( supporting information ) . at low temperatures ,
with increasing temperature , 
 the percentage of the mobile component increased and the percentage 
 of the immobile component decreased until almost equal populations 
 of the two components were seen at 3035 c .
the 
 natural logarithm of the ratio of the fractions of the two components 
 exhibited a linear dependence with inverse temperature ( figure 7 ) .
the results were collected from 
 two independent experiments performed on two separate samples prepared 
 from different protein expression , refolding , spin labeling , and purification 
 preparations .
both data sets and simulations were plotted 
 together , and the slopes of the lines were reproducible within error . 
 the solid line shown is a linear fit of all the data taken together 
 with values of 7.8  0.8 for the slope and 25.8  
 2.8 for the intercept , from which the enthalpy and entropy of the 
 conformational change were calculated to be 65  6 kj / mol and 
 215  23 j/(mol k ) , respectively . from the values of f1 and f2
determined 
 from the spectral fits , the value of the equilibrium constant , k , at 298.15 k was 0.7 , indicating that both conformations 
 are easily accessible at physiological temperatures .
a and b , the conformation 
 of the loop is such that the side chain of l126 appears to point toward 
 the protein interior , whereas it extends out into the solution in 
 monomer c. the two spectral components determined from fitting with 
 momd simulations are consistent with these different environments 
 of the spin label at this site .
the immobile component may arise from 
 a fraction of protein conformers having l126r1 pointing in toward 
 the lipid binding cavity , where the motion of the spin label can be 
 restricted by neighboring amino acid side chains as well as by the 
 limited space in the cleft of the cavity entrance .
the mobile spectral 
 component is consistent with the strand conformation seen in monomer 
 c , where the spin label would extend out into solution and have a 
 higher degree of rotational freedom .
linear regression of the ratio of the 
 fractions of mobile ( f1 ) to immobile
( f2 ) populations plotted for l126r1 as a function 
 of inverse temperature . 

two separate epr data sets were collected from different protein sample 
 preparations , and the simulations were performed independently .
the solid line 
 is the linear regression from both data sets taken together . from 
 the slope of the line ( 7.8  0.8 ) and the intercept ( 25.8 
  2.8 ) ,
the enthalpy and entropy of loop motion were found to 
 be 65  6 kj / mol and 215  23 j/(mol k ) , respectively .
the 
 value of the equilibrium constant k at 298.15 k was 
 calculated to be 0.7 . in an effort to understand the conformational dynamics of the protein
, 
 we collected 275 ns of md data from each of our simulations starting 
 from the distinct conformations observed in monomers a and c of the 
 crystal structure ( pdb i d 1g13 ) of gma2 .
we find that both monomers maintained their 
 fold over the course of these simulations while undergoing sizable 
 conformational changes in disordered regions , in good agreement with 
 crystallographic b - factors ( figure s2 , supporting 
 information ) . in each simulation
, the protein initially sampled 
 conformations similar to its starting point prior to sampling conformational 
 ensembles that were not similar to either starting crystal structure 
 geometry ( figure s3 , supporting information ) .
nevertheless , further analyses reveal that there is considerable 
 overlap ( figure s3 , supporting information ) among the calculated population distributions of root - mean - square 
 deviation ( rmsd ) values calculated for protein backbone atoms over 
 both simulations , and a combined trajectory with respect to the crystal 
 structure conformation of monomer a , suggesting both simulations sample 
 similar conformational space .
the beta - sheet regions of gm2ap 
 arrange to form a hydrophobic cavity that changes over the course 
 of our simulations .
as such , the volume of the cavity described by 
 the protein backbone provides a useful metric that describes the conformational 
 changes of gm2ap , which avoids alignment issues common in rmsd calculations . 
 toward this end
, we analyzed the volume of the protein cavity using 
 the recently developed trj_cavity utility that helps identify and 
 quantitatively characterize cavities observed over the course of a 
 md simulation ( figure s4 , supporting information ) .
default program parameters were employed 
 for the grid and nearest neighbor search for a four - sided cavity . 

our analysis of cavity volumes finds that gm2ap once again samples 
 similar conformational ensembles in both simulations .
we find that , 
 although the cavity volume differed in the two crystal structure conformations 
 of the protein , there was much similarity in the volumes sampled by 
 the protein in our calculations ( figure s4 , supporting 
 information ) . in close agreement with our epr data and rmsd 
 data , these volume analyses further indicate that crystal - packing 
 effects likely influenced the distinct protein conformations observed 
 in the crystal structure .
this is not all together surprising , as 
 crystal packing effects have been known to influence the protein structures , 
 such as in the case of the paradigm zinc - sensor protein czra . taken together , these results 
 indicate that the monomer conformations observed in the crystal structures 
 of gm2ap likely represent trapped states .
although the gm2ap structure 
 would sample these higher - energy conformations from time to time , 
 it would predominantly sample an ensemble of conformations having 
 the characteristics of both conformers in solution .
( a ) root mean square 
 fluctuation ( rmsf ) profiles of md trajectories 
 from simulations of monomer a and monomer b. these values were calculated 
 for protein backbone c carbon atoms for each residue .
( b ) ribbon 
 diagram of monomer a where the color coding represents the range of 
 rmsf values as follows : the color code used is gray ( 0.01.0 
  ) , yellow ( 1.01.5  ) , purple ( 1.52.0  ) , 
 blue ( 2.02.5  ) , green ( 2.53.0  ) , and red 
 ( 3.04.0  ) .
we next investigated the mobility of protein residues from 
 our 
 md simulations to build an understanding of the role of residue mobility 
 and the associated changes in protein structure in gm2ap function . 
 toward this end , we calculated the root - mean - square fluctuations ( rmsfs ) 
 for backbone c carbon atoms of each residue for structures 
 obtained from both simulations ( figure 8) . 

in particular , we find that regions predicted in gm2ap 
 to have high mobility or disorder from crystallographic b - factors 
 ( figure s2 , supporting information ) were 
 also highly mobile in both simulations , allowing it to sample multiple 
 conformations . in favorable agreement with epr data ,
results show 
 that a60 , i66 , i72 , l126 , and s130 residues and their neighboring 
 residues are mobile in these simulations ( figure 8) . to better characterize the conformational sampling 
 at these sites
, 
 we performed a clustering analysis on the protein conformations sampled 
 over the course of the simulations . in this calculation ,
all protein 
 conformations from md simulations were first aligned against the crystal 
 structure conformation of monomer a. the implementation of the 
average 
 linkage  clustering algorithm in ambertools was then used to 
 cluster l126 , a60 , i66 , i72 , n136 , and s130 residues individually . in favorable agreement with the epr data ,
our 
 calculations suggest that these residues likely exist in distinct 
 major and minor conformational states ( figure 9 ) .
( a ) percentage of protein structures observed bearing distinct 
 conformations of l126 , a60 , i66 , i72 , n136 , and s130 residues over 
 550 ns of md data .
these percentage populations were calculated for 
 all conformations sampled in our simulations starting from the crystal 
 structures of monomers a and c. all snapshots of protein structures 
 were first aligned to the crystal structure of monomer a based on 
 their backbone atoms .
( b ) ribbon diagrams showing the positions of the l126 side 
 chain in representative structures obtained from the clustering analysis . 
 in state 1
, the side chains point toward the protein interior , whereas , 
 in state 2 , the side chains were seen to point out toward the solvent .
figure 9b shows representative structures 
 for the l126 side chain from this clustering process .
remarkably , 
 the orientations of the side chains are reflective of the two states 
 observed in the crystal structures of monomers a and c , where one 
 side chain points in toward the protein interior and the other is 
 more solvent exposed and likely more mobile . for i66 , a similar trend 
 is also observed : one is buried with close contact to w131 , whereas 
 the other state is not interacting with w131 . for sites i72 and n136 , 
 the two clustered states also differed in that one state is more buried 
 and the other more solvent exposed . for s130 ,
the states differed 
 in the degree of h - bonding that the serine participated in with a 
 neighboring loop . for a60
, however , the methyl side chain remains 
 solvent exposed in both clusters and no significant differences in 
 side chain packing are observed .
the states reported from the clustering 
 analysis for a60 represent different orientations of this extended 
 apolar loop .
these conformations likely result from promiscuity / heterogeneity 
 in e58 side chain interactions to either form a hydrogen bond with 
 n136 or a weak anion interaction with the aromatic 
 ring of w63 .
clustering analysis of the epr control sites v54 , t90 , 
 s115 , and l87 reveals only a single cluster for t90 and v54 .
two states 
 with populations of 85/15 ( % /% ) for both l87 and s115 are observed . 
 as can be seen in figure 8 , both of these sites 
 reside in well - defined secondary structural elements ( l87 in an -helix 
 and s115 in a -strand ) .
md results , however , show these elements 
 to have moderate mobility ( color coded yellow in figure 8) .
the clustering results for these two sites reflect different 
 positions of the helix / strand with no variation in the side chain 
 rotamers . taken together , our calculations support the results obtained 
 from epr investigations , showing that conformational exchange exists 
 in solution and that the x - ray structures likely represent this solution 
 conformational heterogeneity .
gm2ap has eight native cysteines that form four disulfide bonds essential 
 for the stability of exposed loop regions .
we were able to engineer 
 additional cysteines at various sites and express , purify , and isolate 
 homogeneous samples of spin labeled gm2ap .
analysis of the epr spectral 
 line shapes and momd simulations for spin labels in the disordered 
 chain segment and both loops are consistent with the various conformations 
 seen in x - ray structures .
multiple conformations of the mobile loop in solution were detected by epr and md simulations , indicating 
 that the conformations seen in the crystal structure are present in 
 solution and may play a functional role in ligand binding or interactions 
 with lipid vesicles .
sdsl and nmr hsqc titrations show minimal structural 
 perturbations upon addition of gm2 ligand or with changes in ph .
future 
 work is focused on assessing the role loop flexibility plays in gm2ap 
 function .
 gm2ap 
 has a -cup topology with numerous x - ray structures 
 showing multiple conformations for some of the surface loops , revealing 
 conformational flexibility that may be related to function , where 
 function is defined as either membrane binding associated with ligand 
 binding and extraction or interaction with other proteins . here 
 , site - directed 
 spin labeling ( sdsl ) electron paramagnetic resonance ( epr ) spectroscopy 
 and molecular dynamic ( md ) simulations are used to characterize the 
 mobility and conformational flexibility of various structural regions 
 of gm2ap . 
 a series of 10 single cysteine amino acid substitutions 
 were generated , and the constructs were chemically modified with the 
 methanethiosulfonate spin label . continuous wave ( cw ) epr line shapes 
 were obtained and subsequently simulated using the microscopic order 
 macroscopic disorder ( momd ) program . 
 line shapes for sites that have 
 multiple conformations in the x - ray structures required two spectral 
 components , whereas spectra of the remaining sites were adequately 
 fit with single - component parameters . for spin 
 labeled sites l126c 
 and i66c , spectra were acquired as a function of temperature , and 
 simulations provided for the determination of thermodynamic parameters 
 associated with conformational change . 
 binding to gm2 ligand did not 
 alter the conformational flexibility of the loops , as evaluated by 
 epr and nmr spectroscopies . 
 these results confirm that the conformational 
 flexibility observed in the surface loops of gm2ap crystals is present 
 in solution and that the exchange is slow on the epr time scale ( > ns ) . 
 
 furthermore , md simulation results are presented and agree well with 
 the conformational heterogeneity revealed by sdsl . 
Introduction
Experimental Procedures
Results
Conclusions