integral membrane proteins , such as ion channels , receptors , and 
 transporters , perform their vital tasks through complex mechanisms 
 that often involve major structural rearrangements triggered by stimuli 
 such as ligand or substrate binding . the molecular response to the 
 stimuli is propagated across the membrane , connecting the extracellular 
 environment to the interior of the cell . that the membrane environment 
 is involved in the allosteric mechanisms and their regulation is now 
 well - established , but the mechanisms by which this environment participate 
 in the observable and measurable activities of the embedded proteins 
 are yielding only slowly to quantitative understanding . in particular , 
 it has become evident that in order to carry out their tasks membrane 
 proteins take advantage of many structural , thermodynamic , and mechanistic 
 properties of the cell plasma membranes and that in turn the lipid 
 membranes respond dynamically to conformational changes in proteins 
 by locally adjusting their lipid composition , bilayer thickness , and/or 
 curvature . many consequences of such function - dependent 
 cross - talk between proteins and lipids have been identified , including 
 compartmentalization and oligomerization ( often enhanced by specific plasma membrane domains termed rafts ) , which play central roles in the physiological 
 mechanisms of the cell . one difficulty in connecting the physiological 
 observations to 
 the rapidly growing information about the detailed structure and dynamic 
 properties of individual membrane protein molecules is that most structural 
 and many functional assays are conducted in non - native environments 
 in which the role of the plasma membrane and its components ( i.e. , 
 cholesterol and charged lipids ) in modulating structural and functional 
 properties of the integral proteins ( e.g. , ref ( 15 ) ) are not accounted for . thus , most protein preparation methods for studies of integral membrane 
 proteins involve their overexpression , followed by their detergent - mediated 
 extraction from the cell and purification and reconstitution into 
 proteoliposomes . when present in water 
 above certain critical concentrations , the detergents , which have 
 been selected to replace the native environment of these proteins 
 ( i.e. the membrane ) and possess a relatively large polar headgroup 
 and a short hydrophobic tail , self - assemble in aggregates of positive 
 curvature , termed micelles . in such micelles , 
 the polar headgroups of the detergent are exposed to the aqueous solvent 
 whereas hydrophobic tails face each other and thus mimic the membrane 
 hydrophobic environment for an encapsulated protein while excluding 
 aqueous solvent from the micelle interior . although micellar aggregates 
 effectively shield hydrophobic trans - membrane ( tm ) segments of integral 
 proteins from unfavorable polar exposure while bringing polar loop 
 regions into contact with an aqueous phase , they can not capture all 
 of the complexities of the native plasma membranes . indeed , several recent studies have provided dramatic demonstrations 
 of changes in functional properties of membrane proteins due to detergent 
 ( e.g. , refs ( 18 ) and ( 19 ) ) . one such class 
 of membrane proteins studied in detergent environments 
 is the family of neurotransmitter / sodium symporters ( nss ) that is 
 responsible for the removal of extracellular solutes from the synaptic 
 cleft into the presynaptic nerve terminal . the uptake mechanism is energized by the coupling the transmembrane 
 na gradient to the uphill transport of the respective 
 substrate . several x - ray structures of a prokaryotic homologue of 
 nss , the leucine transporter ( leut ) , have identified the centrally 
 located high - affinity substrate binding site , termed the s1 site , and two sodium binding sites termed na1 and na2 . computational and 
 experimental studies have identified 
 a second high - affinity substrate binding site , the s2 site , located 
 in an extracellular vestibule 11  above the s1 site 
 that is essential for the transport mechanisms of leut . in particular , 
 an allosteric mechanistic model of a na - coupled symport 
 was proposed in which intracellular release 
 of the s1-bound substrate is triggered by the binding of a second 
 substrate molecule in the s2 site . remarkably , follow - up studies suggested 
 that experimental conditions and , in particular , a high concentration 
 of n - dodecyl-,d - maltopyranoside ( ddm ) 
 detergent , can obscure the functionally relevant s2 site and result 
 in reduced substrate binding . but the reconstitution of leut , previously 
 preincubated with high concentrations of ddm detergent , into e. coli membranes , showed a full recovery of functionality 
 for the s2 site . interestingly , the loss 
 of the s2 site upon detergent treatment appeared to have a concentration 
 threshold in the range of 0.150.175% ddm . these experimental 
 results illustrate that the function of integral membrane proteins 
 can be modulated by the experimental preparation and the environment 
 in which the protein is studied . to extrapolate the knowledge gained 
 from the experimental explorations conducted under non - native conditions 
 to mechanisms in vivo , it is necessary to understand on the molecular 
 level how detergent micelles form around proteins and what fundamental 
 changes occur when the protein is surrounded by a detergent micelle . the literature discussing molecular models of detergent / protein interactions 
 ( e.g. , refs ( 3039 ) and citations therein ) has not addressed these fundamental questions 
 in a systematic way . to point out the shortcomings associated 
 with the interpretation 
 of membrane protein structure and function in experimental environments , 
 we provide here , to our knowledge for the first time , a detailed molecular 
 view of the leut protein embedded in ddm detergent micelles formed 
 at different detergent / water / protein ratios . this view is offered 
 from extensive atomistic molecular dynamics ( md ) simulations carried 
 out in order to ( 1 ) establish the aggregation number of ddm micelles 
 surrounding leut , ( 2 ) explore the overall organization of the detergent 
 micelle containing the transporter , and ( 3 ) obtain molecular - level 
 insight into the nature and consequences of interactions between leut 
 and ddm . analyzing various protein - to - detergent ( p / d ) number ratios 
 ( i.e. , from 1:160 to 1:300 ) , we show that the aggregation number of 
 ddm in the micelle that surrounds the transporter is strongly dependent 
 on the p / d ratio . moreover , the md simulations of the system at various 
 p / d ratios suggest a mechanism for the dependence of leut substrate 
 binding stoichiometry on detergent concentration . thus , we found that 
 the detergent can penetrate leut through two alternative pathways . 
 as a consequence of such penetration , ddm molecules establish long - lasting 
 contacts with several functionally critical residues located in the 
 s2 site of leut . remarkably , we find that the detergent penetration 
 phenotype is determined by the aggregation number of ddm around leut 
 so that nontransient ddm insertion is observed only in the high - detergent - concentration 
 regime . these results , discussed here in the light of recent experimental 
 findings suggesting the modulation of leut activity by detergent , 
 can explain experimentally observed phenotypes caused by the occlusion 
 of the s2 site in leut at high detergent concentration . for atomistic molecular dynamics 
 ( md ) simulations , we used the x - ray structure of leut with the pdb 
 accession code 3gjd . the transporter in this structure 
 is in the occluded state with leucine ( leu ) at the s1 primary binding 
 site and the two na ions bound at na1 and na2 sites , respectively . thus , the structure also contains detergent n - octyl-,d - glucopyranoside ( og ) at the s2 binding site . the detergent 
 molecule was removed prior to the simulations , leaving the s2 site 
 empty at the beginning of the md runs . the leut residues that were 
 missing from the 3gjd structure ( first four residues on n terminus , last eight residues 
 on c - terminus , and the p132n133a134 stretch in the 
 el2 loop ) were added with modeler . all 
 crystallographic waters were retained in the simulations , and glu112 , 
 glu287 , and glu419 were treated as protonated . the leut model was immersed in a box containing water and 
 ddm detergent molecules . as described in figure 1 ( starting configurations ) and illustrated in figure 2 , some ddm molecules were initially placed in a spherical 
 micelle formation around leut ( see below ) whereas others were placed 
 randomly as monomers outside this central micelle . the starting conditions 
 were varied with respect to the number of ddms in the initial micelle 
 and in the monomers . thus , for the initial set of simulations , the 
 micelle was composed of either 160 or 246 ddm molecules and was surrounded 
 by different numbers of monomeric ddms ( figure 1 ) . accordingly , throughout this work , various simulated constructs 
 are given the designation a / b , where a denotes the initial number of ddms in the central micelle 
 surrounding leut ( figure 2 ) and b is the starting number of monomeric detergent molecules outside 
 this micelle . schematic representation of conditions probed in our all - atom 
 md 
 simulations of leut / detergent complexes : protein - to - detergent number 
 ratios and initial spatial distribution of detergent around leut . the first stage of simulations ( starting configurations ) involved 
 leut surrounded by a ddm micelle consisting of either 160 or 246 detergent 
 molecules . in addition , different numbers of monomeric ddms ( 0 , 54 , 
 or 115 ) were placed randomly outside the central protein / detergent 
 micelle ( figure 2a , b ) . note that conditions 
 with 160 total ddms were probed in two separate md simulations initiated 
 from different random seeds . because all starting configuration simulations 
 resulted in similar numbers of  shell  ddms ( detergent 
 molecules within 4  of a protein , termed shell in methods ) , we initiated a second set of simulations ( resulting 
 configurations ) in which leut with only its shell of detergents ( 120 
 ddms 4  from the protein ) was retained ( chosen from the representative 
 snapshot from starting configuration trajectories ) . this protein / detergent 
 complex was then surrounded by randomly placed monomeric ddms ( 41 , 
 115 , 174 detergents ) , and new md trajectories were accumulated . the 
 time durations for each simulation conducted are given in the respective 
 boxes . ( a ) snapshot of the initial configuration of 
 the 160/115 system 
 ( figure 1 , starting configurations ) . the cubic 
 simulation unit box of 180  linear length contains leut 
 protein ( in cartoon ) , ddm detergent molecules ( in licorice , 160 ddms 
 depicted in gold are in the micelle surrounding leut and 115 ddms 
 in white are in monomeric form outside ) , a water box ( silver dots 
 represent water oxygen atoms ) , and 0.15 m nacl ( yellow and cyan spheres ) . 
 the leucine ligand bound to the leut s1 site and the two na ions are omitted . ( b ) same as in panel a only with water and salt 
 ions removed . ( c ) same as in panel b , only after 140 ns of md simulations . different types of aggregates ( definitions in methods ) are highlighted with arrows and labeled . ( d ) final snapshot of 
 the 160/115 system , after 242 ns of simulations , showing only leut 
 ( cartoon ) and shell ddm molecules ( within 4  of protein ) ( licorice ) . notice strong intermixing of initially micellar ( gold ) and monomeric 
 ( white ) ddms in panels c and d. to build a micelle containing a number a of detergent 
 molecules around leut , we used a multistep algorithm described in 
 ref ( 37 ) . according 
 to this procedure , in step 1 n pseudoparticles were 
 randomly placed on an imaginary sphere surrounding the protein , excluding 
 areas around intracellular and extracellular parts of leut ( figure 2 ) ; in step 2 , the pseudoparticles were replaced 
 with explicit ddm molecules , oriented with their hydrophobic tails 
 facing the center of leut ; and in step 3 , the imaginary sphere ( containing 
 leut and all of the ddm molecules ) was incrementally shrunk subject 
 to concomitant energy minimization to a final radius of 51  . 
 with that , we ensured that in the starting configuration ddm tails 
 were appropriately placed to cover the hydrophobic core of leut while 
 leaving hydrophilic regions of the protein exposed to the solvent . to the box containing the leut - detergent micelle complex ( proteomicelle ) 
 obtained with the procedure described above , we added the desired 
 number of monomeric ddms ( figures 1 and 2 ) positioned randomly outside the central micelle , 
 and the system was then solvated with tip3 waters and ionized with 
 na and cl to achieve an ionic concentration 
 of 0.15 m. the final simulation box had nearly cubic geometry with 
 a linear dimension of 180  and contained 500  000 
 atoms . correspondingly , the detergent concentration was kept above 
 the established critical micelle concentration ( cmc ) of 0.17 mm for 
 ddm in all of the constructs ( table s1 in the supporting information ) . the all - atom md simulations 
 were done with the namd 2.7 package , and 
 the all - atom charmm27 force field , with cmap corrections for proteins and a charmm - compatible force - field parameter 
 set for detergents . molecular constructs 
 were initially equilibrated using a two - phase protocol : ( i ) short 
 energy minimization was carried out during which protein , water , and 
 ion atoms were fixed and the coordinates of only ddm molecules were 
 allowed to evolve freely and ( ii ) 1.5-ns - long md simulations were 
 conducted with the protein backbone harmonically constrained . the 
 constraints were released gradually , in 0.5 ns steps , with decreasing 
 force constants of 1 , 0.5 , and 0.01 kcal/(mol ) . the equilibration procedure was similar to that implemented by 
 others for md simulations of protein / micelle complexes ( e.g. , ref ( 38 ) ) . the 
 simulations implemented pme for electrostatics interactions and were carried out in an npt ensemble under 
 isotropic pressure coupling conditions and at 310 k temperature . the 
 nose - hoover langevin piston algorithm was used to control the target p = 1 atm pressure 
 with the langevin piston period set to 100 fs and the langevin piston 
 decay set to 50 fs . as detailed in the results , the md simulations 
 led to the spontaneous formation of aggregates of different types 
 ( figure 2c ) . accordingly , we distinguished 
 ddm detergents in the following types of aggregates : detergent micelles ( dms ) ddms 
 that are bound to the central micelle as monomers;free aggregates ( fas ) ddms that 
 are monomeric in the solution.ddm shell ( shell )  ddms within 
 4  from leut . ( see figure s1 in the supporting 
 information for a comparison with the results using alternative 
 definitions of the shell . ) ddms 
 that are in the central micelle around leut ; bound aggregates ( bas ) ddms 
 in aggregates that bind to the central micelle ; bound monomers ( bms ) ddms 
 that are bound to the central micelle as monomers ; free aggregates ( fas )  ddms 
 that are part of aggregates in the solution ; free monomers ( fm ) ( see figure s1 in the supporting 
 information for a comparison with the results using alternative 
 definitions of the shell . ) to identify 
 the number of constituent detergent molecules 
 in each type of aggregate defined above , we used an expansion algorithm . thus , to calculate the number of ddms in dm , we first identified detergent 
 molecules within 4  of leut ( ddm shell , figure 2d ) . next , we found all ddm molecules within 4  of this 
 detergent shell , and such an expansion was repeated until the search 
 failed to identify any new ddm molecules . in this procedure , only 
 the atoms in the hydrophobic tails of detergent molecules were used 
 to differentiate between ddms in the protein - surrounding micelle from 
 those that are interacting with this micelle via headgroup atoms . after dm was defined , ddms in bas and bms were counted by selecting 
 detergent molecules whose headgroup atoms were within 5  of 
 the dm and building complexes using the expansion algorithm described 
 above . after all bound aggregates were located , the algorithm identified 
 the ddms in fas and fms by building the remaining aggregates in solution . note that all types of aggregates defined above can , in principle , 
 contain ddm molecules that were initially either part of the central 
 micelle or were monomeric outside the micelle ( figure 2b  d ) . the micellar aggregation number is a description 
 of the number 
 of molecules present in a micelle once the cmc has been reached and 
 is defined as the ratio of micelle concentration over the concentration 
 of monomeric detergent . here , the aggregation 
 number of the leut / ddm proteomicelle was calculated as the dm + ba 
 + bm sum . ( see above . ) to quantify the shape 
 of the micelle around leut , we calculated the eccentricity e of the dm following the procedure implemented 
 in ref ( 34 ) . to this 
 end , we determined the three principal moments of inertia ( i ) of an ellipsoid that encloses the non - hydrogen tail atoms 
 of those ddm molecules that were identified as being part of the dm . using the magnitudes of the smallest principal moment ( imin ) and of the average of all three moments ( iav ) , we then obtained the eccentricity of the 
 micelle as e=1 note that according to this definition , e = 0 for a perfectly spherical micelle . to quantify the extent of detergent 
 penetration into leut in our 
 md simulations , we monitored the time evolution of selected distance 
 measures from different trajectories . specifically , because we observed 
 that ddm is inserted into the transporter and binds in the s2 site 
 ( see results ) , we first screened the residues 
 that have been established from experimental and computational studies to comprise the s2 site in leut ( see also discussion ) : leu25 , gly26 , leu29 , arg30 , gln34 , 
 tyr107 , tyr108 , ile111 , w114 , ala319 , phe320 , phe324 , leu400 , and 
 asp404 . we then tracked the minimal distance from these residues to 
 the nearest detergent molecule in different simulations . the penetration 
 of the detergent molecule was observed in different simulations to 
 follow two distinct pathways ( see results ) , 
 one resulting ultimately in interactions with arg30 and gln34 in transmembrane 
 helix 1 ( tmh1 ) of leut and the other resulting in interactions with 
 phe320 in extracellular loop 4 ( ecl4 ) and leu400 in tmh10 . therefore , 
 we chose to quantify ddm insertion by monitoring the time evolution 
 of the minimal distance ( dmin ) of these 
 four key residues arg30 , gln34 , phe320 , and leu400to 
 the nearest ddm molecule . in a certain md trajectory , therefore , 
 a detergent molecule was considered to be fully inserted ( complete 
 insertion ) into the leut s2 site if dmin between the detergent and any of the above - mentioned four residues 
 was 3  or shorter during at least the last third of the trajectory . 
 if the detergent molecule interacted with either arg30 , gln34 , phe320 , 
 or leu400 ( dmin < 3  ) for a shorter 
 period of time , it was considered to be transiently inserted into 
 the s2 site . in this scenario , the interactions between the detergent 
 and s2 site residues were forming and breaking dynamically ( e.g. , 
 figure 5 ) . finally , if dmin > 5  at all times 
 during the trajectory , then the leut molecule was not considered to 
 be penetrated by ddm . to 
 determine the behavior of the ddm molecule number in a micellar system 
 surrounding leut , we conducted md studies on a series of leut - ddm 
 complexes constructed to explore systematically the effect of protein - to - detergent 
 ( p / d ) number ratios on the nature and dynamics of the resulting complexes . 
 the md simulations of the systems containing from 1:160 ( low detergent 
 content ) to 1:300 ( high detergent content ) p / d ratios , starting from 
 the constructs described as starting configurations in figure 1 in which various numbers of monomeric ddm molecules 
 were added randomly outside the protein / micelle complex ( figures 1 and 2a , b ) . the various constructs 
 converged ( at the simulation times indicated ) to configurations with 
 very similar ddm shells ( defined in methods as the shell ) surrounding the protein . specifically , as shown in 
 figure 3 , we found that in all of the simulations 
 the number of detergent molecules in the shell was 120 . we note that the convergence 
 of the ddm numbers in the shell to similar values in the different 
 simulations is independent of the range explored specifically , from 
 3 to 5  ( figure s1 in the supporting information ) . time evolution ( after initial equilibration phase ) of the number 
 of ddm molecules within 4  of the protein ( i.e. , shell ) in different 
 simulations . the traces were smoothed by the running average algorithm . to investigate further the stability 
 of the identified shell , a 
 second set of md simulations the starting constructs 
 were obtained by extracting leut with its shell ddm molecules ( 120 
 detergents ) from a representative snapshot from the starting configurations 
 trajectories and surrounding this complex with different numbers of 
 monomeric ddms . md trajectories that were 200350 ns long were 
 collected in the environment defined in methods . as illustrated in figure 3 , this second 
 set of md simulations also converged to configurations in which the 
 shell surrounding leut contained 120 ddm molecules . these 
 results suggest that irrespective of the p / d ratio a converged shell 
 of ddms forms around leut . interestingly , however , quantitative analysis 
 of various types of detergent aggregates forming in the md trajectories 
 revealed that the detergent aggregation number , i.e. the number of 
 ddm molecules comprising the entire micelle enclosing leut ( denoted 
 as dm in methods ) strongly depends on the 
 p / d ratio . figure 4a shows the average number of ddm 
 molecules in the entire micelle surrounding leut ( dm ) as well as in 
 aggregates bound to dm ( bmand ba ) and in the entire 
 complex of detergent associated with the central protein / micelle complex 
 ( i.e. , dm , bm , and ba together , which is the proteomicelle aggregation 
 number ) . the averages were obtained from the analysis of the last 
 50 ns of each trajectory . the time trace in figure 4b illustrates the evolution of these aggregates from one particular 
 160/115 simulation , and figure 4c shows , from 
 the same simulation , the evolution of ddm numbers in the free aggregates 
 compared to that in the central protein / micelle . ( analogous plots 
 for other constructs can be found in figures s2s3 in the supporting information . ) ( a ) average number of 
 ddm molecules in the detergent micelle around 
 leut ( dm ) , in the aggregates bound to dm ( ba and bm ) , and in dm , ba , 
 and bm taken together . for each simulated system , the averages were 
 calculated using the last 50 ns segments of the respective trajectories . the dashed horizontal line indicates the average number of ddms ( 120 ) 
 in the detergent shell around leut established from figure 3 . ( b ) time evolution ( after initial equilibration 
 phase ) of the measures from panel a in the 160/115 simulation . ( c ) 
 percentage of ddm detergents in dm , ba , and bm ( blue line ) , in fa 
 ( red line ) , and in fm ( black line ) as a function of time in the 160/115 
 simulation . for definitions of the various types of aggregates , the results in figure 4c show that 
 over 
 80% of 275 ddms present in the 160/115 system become part of the central 
 micelle ( blue line in figure 4c ) . the majority 
 of the detergent that is still in the solution assembles spontaneously 
 into free aggregates ( fas , red line in figure 4c ) , whereas free monomers ( fms ) practically disappear ( black line 
 in figure 4c ) . the interconversion of the different 
 bound aggregates ( ba and bm ) and their melting into the central dm 
 that surrounds leut are illustrated in figure 4b . thus , within the first 50 ns , the number of ddms in dm increases 
 from 160 to 190 and remains at this level until the 
 100 ns time point ( red trace in figure 4b ) . 
 at the same time , the number of aggregates bound to dm remains more 
 or less unchanged ( green trace in figure 4b ) . interestingly , in the subsequent 100150 ns time interval we 
 observe a sudden increase in the number of detergent molecules in 
 dm as a result of the fusion of the bound aggregates with the central 
 micelle ( green curve in figure 4b decreases 
 to 0 ) . after the fusion process is completed ( 150 
 ns ) , the detergent count in dm and in dm + ba + bm remains nearly identical 
 for the remainder of the trajectory ( red 
 and blue traces in figure 4b ) , indicating that 
 practically all bound aggregates ( over 80% of the ddm , see figure 4c , blue curve ) become part of the dm . although 
 the size of the ddm shell appears to be 120 ddm 
 ( as calculated at a 4  distance cutoff ) regardless of the p / d 
 ratio , we found the aggregation number to change significantly in 
 the p / d interval of 1:246 to 1:300 interval , from 2045 ( in the 
 120/174 simulation ) to 2437 ( for the 246/54 system ) , as seen 
 in figure 4a . moreover , these values are substantially 
 higher than the 1453 to 1562 aggregation number range 
 that we find for the three different simulations under low detergent 
 condition ( 1:160 p / d ratio ) . indeed , our results suggest that for 
 the low ddm constructs all of the available detergent eventually aggregates 
 with the central dm around the protein . interestingly , at an intermediate 
 p / d ratio of 1:235 , the aggregation number ( 1865 ) is in between 
 these two regimes . we note that the extensive simulation times 
 that we have reached 
 in the current studies still may not be sufficient for complete equilibration 
 of the dynamic variables discussed in figure 4a ( figures 4b , c and s2s5 in the supporting information ) . however , the examination 
 of a number of control trajectories for the high detergent regime 
 ( figure 1 flowchart ) allowed us to assess with 
 confidence the convergence of the aggregation numbers from below ( 120/174 , 
 120/115 , and 160/115 simulations ) as well as from above ( 246/0 and 
 246/54 simulations ) . on the basis of the average values from these 
 simulations initiated from different starting conditions ( figure 4a ) , we therefore conclude that for the high detergent 
 regime the ddm aggregation number around leut is 22617 . in the 
 low ddm regime , such as the p / d ratio of 1:160 , our results suggest 
 that all available detergents will constitute the dm . in the course of simulations with some of the 
 constructs , we observed ddm molecules inserting into leut . however , when a stable complex 
 was formed , the single ddm molecule penetrated the leut molecule until 
 it ended up interacting with residues in the identified such complete ddm penetration occurred only in the 
 constructs with relatively high detergent content , i.e. for p / d ratios 
 in the 1:246 to 1:300 range ( table 1 ) . in contrast , 
 only transient ddm insertion into leut was observed in simulations 
 of systems with low ( 1:160 p / d ratio ) or intermediate ( 1:235 p / d ratio ) 
 detergent content . we identified two alternative pathways for ddm 
 insertion into leut , one termed  from the side penetration 
 and the other termed  from the top  penetration ( table 1 ) . for the definition of different 
 modes of detergent penetration , see results . different simulations 
 are labeled 
 as a / b , where a and b represent the initial number of ddm molecules 
 in the micelle and in solution , respectively . penetration was observed in several 
 of the md simulations ( table 1 ) , and the path 
 is illustrated in figure 5 for the 160/115 construct . the ddm molecule enters the transporter 
 through an area between the extracellular ( ec ) ends of tmh11 and tmh6 . the first contacts established by the ddm molecule involve residues 
 pro241 , gly242 , and ile245 in tmh6 , trp467 , and val466 in tmh11 ( figure 
 s4 in the supporting information ) . as it 
 penetrates deeper into the transporter , the detergent molecule engages 
 in additional strong interactions with phe405 in tmh10 ( figure s3 ) 
 and eventually with gln34 and arg30 in tmh1 ( figure 5c , d ) . the participation of the latter two residues is especially 
 intriguing because both gln34 and arg30 are situated in the functionally 
 important s2 site of leut . as seen in figure 5a , b , the ddm is stabilized in the 
 s2 site through both hydrophobic and polar interactions as the ddm 
 headgroup engages with the arg30-gln34 pair whereas the tail interacts 
 with hydrophobic residues lining the ec ends of tmh6 and tmh11 . notably , 
 this entry pathway and the identity of residues participating in interactions 
 with the detergent were found to be remarkably consistent among these 
 different simulations , although the extent of the detergent penetration 
 in these trajectories ( figure 5c , d and also methods for insertion criteria ) ranged from completely 
 inserted ( in the 160/115 and 246/54 systems ) to transiently bound 
 ( 120/115 , 246/0 , 120/174 , and 160/0 
 ii simulations ) . from the side entry pathway of a ddm molecule into leut . ( a , b ) 
 representative snapshot from the 160/115 simulation showing ddm detergent tmh6 and tmh11 of the 
 transporter are colored blue and red , respectively , and the key residues 
 are shown in a space - filled representation and are labeled . ( c , d ) 
 time traces of the minimal distance from the inserted detergent molecule 
 to arg30 ( panel d ) and to gln34 ( panel c ) in 246/54 , 160/115 , and 
 120/115 trajectories . penetration from the top of the transporter was observed 
 in the 
 246/54 , 120/174 , and 160/0 i simulations ( table 1 ) . the initial contact was observed to involve the phe235-asp240 
 stretch of ec loop 3 ( ecl3 ) , and the insertion proceeded toward the 
 gly307-ala317 segment in ecl4 ( figure s5 in the supporting information ) . as the ddm molecule penetrated with 
 its headgroup deeper into the transporter , the final set of stabilizing 
 interactions included phe320 in ecl4 and leu400 in tmh10 ( figure 6 ) . the participation of these particular residues 
 in interactions with the inserted detergent is important because they 
 are again , as in the from the side penetration , situated in the functionally 
 important s2 site and leu400 has been directly linked to the functional 
 mechanisms of substrate transport through leut . from 
 the top entry pathway of ddm molecule into leut . ( a , b ) representative 
 snapshots from the 246/54 ( a ) and the 246/0 ( b ) simulations showing 
 ddm detergent ( in licorice ) penetrating leut ( in cartoon ) . different 
 segments of the transporter are highlighted as follows : tmh10 ( blue ) , 
 235240 stretch in ecl3 ( pink ) , 307316 in ecl4 ( red ) , 
 and 317336 ( yellow ) . glu236 and ile314 are shown by the white 
 space fill , and phe320 and leu400 are depicted by the orange space 
 fill . ( c , d ) time traces of the minimal distance from the inserted 
 detergent molecule to phe320 ( panel c ) and to leu400 ( panel d ) in 
 246/0 , 246/54 , and 120/174 simulations . an interesting variation on the mode of binding is observed 
 in 
 the 246/0 trajectory where the inserted detergent molecule is engaged 
 in stabilizing interactions with the same residues as identified in 
 the simulations of the 246/54 , 120/174 , and 160/0 i constructs ( figure 6c , d ) , but it is the hydrocarbon 
 tail that interacts with phe320 and leu400 residues rather than its 
 headgroup atoms ( cf . the 
 convergence of penetration paths and stabilization sites from 
 the various simulations with different constructs indicates the robust 
 nature of these conclusions . moreover , we note that in two trajectories 
 ( 246/54 and 120/174 ) in which the detergent entered the protein from 
 the top , the specific ddm molecule that eventually ended in the s2 
 site of leut came from outside the protein - surrounding micelle , from 
 among those in the monomeric form ( data not shown ) . similarly , the 
 ddm molecule that entered s2 site from the side in the 120/115 simulation ( figure 5c , d ) inserted itself into the protein after diffusing 
 toward leut 
 from the solution . these trends suggest that the md simulations were 
 sufficiently long to allow ddm molecules to diffuse over relatively 
 long distances before reaching the binding sites in the transporter . the dependence of specific measurable properties of leut on the 
 quantitative parameters of the micellar system offers the first detailed 
 molecular perspective on the manner in which detergent solubilization 
 can affect functional phenotypes of this type of membrane protein . the insights result from extensive atomistic md simulations of different 
 regimes of protein - to - detergent number ratios that are commonly used 
 to prepare protein for biochemical / biophysical studies and crystallography . this study followed our recent work that 
 highlighted the effect of the experimental conditions on the activity 
 of leut , in particular , how high ddm concentrations can obscure substrate 
 binding to the functionally important s2 site . taken together , our 
 studies indicate that ddm at high concentrations can occupy the s2 
 site in leut , just like og . these studies 
 emphasize the sensitivity of the s2 site , a site that is yet to be 
 identified with crystallographic approaches in a substrate - occupied 
 state . because the crystallization of membrane proteins requires high 
 protein concentrations that are routinely obtained with centrifugal 
 filtration of purified material in protein detergent mixtures , 
 our studies emphasize a common problem that is associated with the 
 use of protein in detergent - solubilized form . a key observation of 
 the present study is the formation of a converged shell around leut 
 established irrespective of detergent concentration ( within the wide 
 range explored in our studies ) . this nucleus of detergent molecules 
 effectively protects the transporter tm segments from unfavorable 
 hydrophobic / hydrophilic exposure and therefore is key to the stability 
 of the protein . indeed , in control simulations initiated with a nucleus 
 of 100 ddm molecules surrounding leut , the system exhibited instabilities 
 during the trajectory , which resulted in substantial water penetration 
 of the hydrophobic core of the transporter ( data not shown ) . taken 
 together , our findings establish the importance of considering ddm - to - protein 
 number ratios at or above 120 in md simulations of leut proteomicelles 
 ( i.e. , the number required for the formation of a converged shell 
 at 4  radius ) . it is not surprising given the shape of 
 the protein that our simulations 
 show that the proteomicelles forming around leut are nonspherical 
 in shape ( figure s6 in the supporting information ) . the aggregation number of ddm in the micelle that surrounds the 
 transporter depends strongly on the p / d ratio ( figure 4a ) . specifically , in the high ddm concentration regime we 
 identified 22617 detergent molecules in the protein - solubilizing 
 micelle , which is significantly larger than the usual aggregation 
 number for ddm in micelles ( 140 ) ; for low detergent content ( p / d ratio of 1:160 ) , our results indicate 
 the aggregation of all of the available detergent into the leut - binding 
 micelle . we note that the calculated proteomicelle aggregation number 
 ( 22617 ) is not dependent on the simulation box size because 
 there always will exist , irrespective of the available volume , partially 
 formed detergent micelles in the solution together with protemicelles , 
 as observed in our simulations . remarkably , the penetration 
 of a ddm molecule that inserts fully 
 into the transporter , whether it follows the from the top or from 
 the side path described in the results of 
 the md simulations , is found to occur only in the constructs with 
 high detergent content ( p / d ratio range of 1:246 to 1:300 ) ; any penetration 
 observed in systems with low or intermediate ddm fraction is at most 
 transient . because the shell surrounding leut does not depend on the 
 detergent concentration and it is the ddm aggregation number for the 
 system ( and thus the protein - to - detergent number ratio ) that determines 
 penetration , it becomes clear that ddm insertion into leut is not 
 simply related to the interaction with the detergent immediately surrounding 
 the transporter . specifically , we reason that at low detergent 
 concentrations ( at or below 22617 aggregation number established 
 by our studies ) all of the ddm molecules in the system are expected 
 to participate in the stabilization of the proteomicelle . but at higher 
 concentrations , excess ddms , not associated with the proteomicelle , 
 will be present in the solution and will diffuse freely in monomeric 
 and/or in aggregate forms . such dynamics of free detergent increases 
 the probability of random encounters with the protein regions exposed 
 to the solvent and specifically with the large extracellular vestibule 
 of leut , which eventually leads to the observed detergent penetration . the final position of any completely penetrating ddm was found 
 to be the s2 site in the extracellular vestibule of leut and to involve 
 arg30 , gln34 , phe320 , and leu400 in strong interactions with the inserted 
 ddm . specifically , in crystal structures of 
 leut these residues are among those implicated in stabilizing interactions 
 with tricyclic antidepressants ( tcas ) in the extracellular vestibule as well as with og detergent and to bind inhibitors ( such as trp ) . furthermore , highly conserved ionic interactions between 
 arg30 and asp404 in tmh10 have been established as one of the structural / functional 
 hallmarks in nss family proteins that regulate the access of the substrate 
 from the ec vestibule down to the s1 site during the transport cycle . in addition , the importance of 
 leu400 residue in the leut function is highlighted by the phenotypes 
 of leu400-to - ser or leu400-to - cys mutations that impair leu substrate 
 binding in the s2 site . notably , 
 detergent penetration events observed in the simulations 
 occur at p / d ratios that are expected to be realized in the local 
 environments of the solubilized leut proteins during in vitro experiments . thus , the results from the simulations presented here correspond to 
 the experimental observation that the 
 binding of the leu substrate at the s2 site is impaired if leut is 
 preincubated in the presence of high ddm ( 0.3% ) , yielding a protein - to - detergent 
 ratio of 1:225 , but this does not occur at low ddm concentrations 
 ( 0.1% , with a protein - to - detergent ratio of 1:170 ) . a detailed 
 scan through the detergent concentration range revealed that the loss 
 of leu binding occurs abruptly in the interval between 0.15 and 0.175% 
 ddm concentration . in qualitative agreement 
 with these experimental observations , our data indicate the existence 
 of two distinct regimes for ddm concentration , separated by a narrow 
 range of p / d ratios ( from 1:235 to 1:246 ) , that determine detergent 
 penetration . this suggests a mechanistic explanation for the experimentally 
 observed impairment of the s2 site under high detergent conditions 
 that involves the steric hindrance of the s2 site by a penetrating 
 ddm molecule , much like the proposed effect of og on leut when bound 
 in the s2 site . in the low detergent 
 concentration regime , our results suggest that ddm molecules penetrate 
 leut only transiently , therefore leaving the s2 site more accessible 
 for substrate binding . importantly , we stress that because even at 
 the lowest p / d ratios studied here ( 1:160 ) the detergent can still 
 penetrate leut ( albeit transiently ) and given the narrow p / d ratio 
 range that determines the extent of detergent insertion , the data 
 and hypothesis presented above provide a plausible explanation for 
 the findings from the recent functional experiments on leut that were interpreted to suggest the existence 
 of only a single high - affinity s1 site even at low detergent concentrations . furthermore , the results presented here illustrate , on the molecular 
 level , how even small variations in protein preparation with detergent 
 can lead to the differential behavior of the proteomicelle and to 
 differences in processes such as the penetration of detergent into 
 the transporter . we note that to 
 establish unambiguously the conditions concerning 
 ddm penetration behavior and protection by lipids it becomes essential 
 to expand the current md simulations to more complex environments 
 that involve mixed phospholipid - detergent micelles . this is because 
 the treatment of the protein with detergent generally retains a relatively 
 small annulus of lipid molecules that are being extracted together 
 with the protein during the solubilization process . this annulus of 
 lipids can be expected to create a new set of local interactions with 
 the protein and with the detergent , and these could affect the detergent 
 penetration ( especially from the side ) of leut . to establish the specific 
 conditions regarding the lipid core , we are currently extending the 
 studies of leut in mixed micelles by probing different simulation 
 conditions , and the results will be presented in a subsequent publication .
structural 
 and functional properties of integral membrane proteins 
 are often studied in detergent micellar environments ( proteomicelles ) , 
 but how such proteomicelles form and organize is not well understood . 
 this makes it difficult to evaluate the relationship between the properties 
 of the proteins measured in such a detergent - solubilized form and 
 under native conditions . to obtain mechanistic information about this 
 relationship for the leucine transporter ( leut ) , a prokaryotic homologue 
 of the mammalian neurotransmitter / sodium symporters ( nsss ) , we studied 
 the properties of proteomicelles formed by n - dodecyl-,d - maltopyranoside ( ddm ) detergent . 
 extensive atomistic molecular 
 dynamics simulations of different protein / detergent / water number ratios 
 revealed the formation of a proteomicelle characterized by a constant - sized 
 shell of detergents surrounding leut protecting its transmembrane 
 segments from unfavorable hydrophobic / hydrophilic exposure . regardless 
 of the ddm content in the simulated system , 
 this shell consisted of 
 a constant number of ddm molecules ( 120 measured at a 4  
 cutoff distance from leut ) . in contrast , the overall number of ddms 
 in the proteomicelle ( aggregation number ) was found to depend on the 
 detergent concentration , reaching a saturation value of 22617 
 ddms in the highest concentration regime simulated . 
 remarkably , we 
 found that at high detergent - to - protein ratios we observed two independent 
 ways of ddm penetration into leut , both leading to a positioning of 
 the ddm molecule in the second substrate ( s2 ) binding site of leut . 
 consonant with several recent experimental studies demonstrating changes 
 in functional properties of membrane proteins due to detergent , our 
 findings highlight how the environment in which the membrane proteins 
 are examined may affect the outcome and interpretation of their mechanistic 
 features .