the amyloid- 
 ( a ) peptide is believed to play a key 
 pathogenic role in alzheimer s disease ( ad ) . in vivo , a is characterized by a distribution of isoforms , mostly 
 varying in length from 38 to 43 residues .
the most prominent isoform , 
 a40 , typically occurs in a 10:1 ratio to the more 
 amyloidogenic isoform , a42 .
knowledge of the origin 
 of the a isoform distribution , and its dependence on sequence , 
 environment , and cofactors , is critical to our understanding of the 
 etiology of ad .
a is the product of cleavage of app - c99 
 ( c99 ) , the 99 amino 
 acid c - terminal fragment of the amyloid precursor protein ( app ) , by 
 -secretase .
c99 consists of a single transmembrane ( tm ) helix 
 flanked by less structured extra- and intracellular domains .
processive 
 cleavage is initiated in the c - terminal tm helical region and proceeds 
 toward the n - terminus .
cleavage of c99 has been correlated with a number 
 of factors , including peptide sequence and stability of the tm helix .
while 
 the degree of homodimerization of c99 has also been discussed as a potentially important factor in c99 
 processing , the cleavage of c99 dimers has not yet been definitively 
 demonstrated .
environmental influences such as membrane composition , membrane curvature , and the presence of cholesterol may also play critical roles . it has been openly debated whether a quantitative 
 description of 
 c99 homodimerization structure is essential to a complete understanding 
 of the mechanism of cleavage of c99 by -secretase and the genesis 
 of the a isoform distribution .
multhaup and co - workers first 
 recognized that modifications in sequence that reduced homodimer affinity 
 impacted cleavage of c99 by -secretase .
subsequently , studies of homodimer formation in wt and mutant c99 
 congeners have provided support for the view that c99 homodimerization 
 is critical to c99 processing by -secretase and a formation .
however , it has also been argued that c99 homodimerization 
 is weak and may be largely irrelevant in vivo , suggesting 
 that -secretase acts on c99 monomer only as substrate in the 
 production of a. additionally , 
 while tycko and co - workers suggest that the structure 
 of the tm region of c99 depends on membrane composition , recent work by sanders and co - workers supports 
 the view that at least the backbone structure of c99 is largely independent 
 of membrane lipid composition . in contrast
, 
 smith and co - workers suggest that the a product distribution 
 following cleavage of c99 by -secretase may depend on the specific 
 structure assumed by the c99 homodimer , which may depend on sequence 
 and membrane composition .
although 
 there is debate over the relevance of c99 homodimer in 
 the processing of c99 to form a , there is little doubt that 
 c99 homodimer is an essential species in the overall ensemble of c99 
 structures .
the earliest proposed structures for the homodimer 
 of the tm region of c99 ( figure 1 ) were right - handed 
 coiled - coils stabilized by favorable interactions at the interpeptide 
 interface facilitated by the gxxxg motif .
this motif promotes a right - handed crossing in the -helices 
 by providing a good surface for packing and permitting close helix 
 proximity .
in contrast , one recent nmr 
 structure suggests that the structure of gs- c991555 ( c991555 plus two non - native amino acids at the 
 n - terminus ) consists of a left - handed coiled - coil structure stabilized 
 by interpeptide contacts facilitated by a heptad - repeat motif involving 
 g38 and a42 .
a more recent nmr structure of c992355 homodimer finds 
 a right - handed coiled - coil stabilized by interpeptide contacts in 
 the c - terminal region .
depiction of c991555 dimer in a popc 
 lipid bilayer 
 ( left ) and in a dpc micelle ( right ) .
the phosphocholine group is shaded 
 orange ( popc ) or yellow ( dpc ) .
these contrasting results suggest that a number of fundamental 
 questions related to the structure and processing of c99 in the production 
 of a , including the structure of c99 monomer and homodimer , 
 the sensitivity of monomer and homodimer structure to sequence and 
 membrane , and the relevance of homodimer formation to c99 processing , 
 remain open and require further scrutiny . here
we address a number 
 of critical questions regarding the nature of the c99 homodimer strucure 
 and its dependence on membrane or micelle characteristics . what 
 is the structural ensemble of the c99 homodimer in a micelle 
 environment ?
how well do structures of c99 homodimer in a micelle 
 represent the structural ensemble in a lipid bilayer ? to answer 
 these questions , we carried out multiscale simulations 
 of c991555 dimer in popc bilayers and dpc micelles 
 building on our previous successful predictions of the monomer structures .
we used coarse - grained ( cg ) simulations with 
 the martini force field of a broad sampling of the peptide 
 dimer and lipid / surfactant ensemble .
our model is benchmarked against the well - studied glycophorin a 
 ( gpa ) dimer and used to develop novel predictions for the c991555 dimer . following the identification of the 
 predominant structural states 
 through cg simulations , representative conformations in all - atom
molecular 
 dynamics simulations were used to refine the atomistic structure and 
 characterize the detailed peptide  peptide , peptide 
the major finding of 
 our work is that the structural ensemble in a bilayer is heterogeneous , 
 consisting of multiple states , whereas in a micelle only one state 
 is predominantly selected . globally
the curvature of the bilayer and 
 micelle is different , which has a profound influence on the conformational 
 heterogeneity .
more generally , our study demonstrates that the chemical 
 environment imposes a selection on the nature of the app dimer ensemble . 

the environmental selection of the structure is yet another variable , 
 besides sequence , which can affect the plasticity of app , and hence 
 the product distribution upon cleavage by secretases .
initial conditions 
 for the cg 
 parametrization were taken from the experimentally derived structures 
 determined by nmr in dodecylphosphocholine ( dpc ) micellar environment 
 ( pdb 2loh ) using the martinize.py script and the martini 
 2.2 force field for proteins . to build 
 the popc cg systems , two spatially
separated c991555 monomers were overlapped with the pre - equilibrated lipid systems 
 taken from the marrink web site ( http://md.chem.rug.nl/cgmartini/ ) .
all lipid and water residues within 1.5  of the cg peptide 
 were removed . for the dpc cg systems the same dimeric 2loh structure 
 was used .
a dimer was embeded in a pre - equilibrated dpc cg micelle 
 box also taken from the marrink web site .
the cg bilayer system consisted 
 of a c991555 dimer , 256 popc lipids , 3863 water 
 particles , and 6 cl ions to neutralize the system . 

the cg micelle system contained c991555 dimers , 
 54 dpc lipids , 5597 water particles , and 6 cl ions . 

additional cg simulations were performed using 70 and 108 dpc surfactant 
 molecules ( see supporting information ) . for the cg simulations a total of 50 replicas with
1.5 s 
 of md were performed on each system in order to see convergence in 
 the computed distribution of homodimer structures ( see figures s1 and s2 ) .
nonbonded interactions were 
 truncated using shift functions ( between 0.9 and 1.2 nm for lennard - jones 
 interactions and between 0 and 1.2 nm for electrostatics ) . the temperature of the systems was set to 303 
 k using the berendsen weak coupling method with a coupling time of 0.1 ps .
the pressure was set to 1 bar using a 
 semi - isotropic coupling for popc and isotropic coupling for dpc using 
 the berendsen algorithm . a cg 
 structure from each 
 of the different states in the popc bilayer and dpc micelle ( gly - in , 
 gly - side , and gly - out ) was randomly selected and reconstructed into 
 an all - atom representation using pulchra .
the structure with its orientation were resolvated in popc lipids 
 or dpc surfactant using the charmm - gui membrane and micelle builder and modeled using the charmm36 all - atom lipid and protein force 
 field and tip3p water model .
the bilayer systems consisted of the c991555 dimers 
 reconstructed from cg models , 128 popc lipids , tip3p water molecules 
 extending 15  on each side of the bilayer , and 6 cl ions to neutralize the systems .
the micelle systems contained c991555 monomer , 53 dpc molecules , tip3p waters to solvate 
 a box extending 20  from the dpc surfactant and protein , and 
 6 cl ions to neutralize the system . for simulations 
 in the popc bilayer a total of 100 ns of md
were performed on each 
 system ( following minimization and a short nvt and npt equilibration 
 with protein backbone fixed ) .
the pressure was set to 1 bar using 
 a semi - isotropic coupling scheme with lateral and perpendicular pressures 
 treated separately with coupling time 0.1 ps using the parrinello 
the temperature of the system was set to 303 
 k and regulated using the nos 
the nonbonded interactions 
 were truncated using shift functions 
 ( between 0.9 and 1.2 nm for lennard - jones interactions and between 
 0 and 1.2 nm for electrostatics ) .
long - range electrostatic interactions 
 were calculated using the particle mesh ewald ( pme ) method with a fourier grid spacing of 0.12 nm .
the 
 linear constraint solver ( lincs ) method was used to constrain all bond lengths , with a 2 fs integration 
 step .
all - atom simulations in a dpc micelle were carried out under 
 the same conditions using an isotropic coupling scheme to control 
 the pressure .
the simulations were carried out using gromacs 
 ( v4.5.1 ) and the 
 analyses were performed using the gromacs package , the dssp program , 
 and tailored scripts using python and md analysis libraries .
the helanal program was used to calculate the kink angle along the 
 tm helix between residues k28 and v50 .
c991555 homodimer was simulated using cg molecular 
 dynamics in a popc bilayer and dpc micelle ( see figure 2 ) .
multiple independent dynamical trajectories were initiated 
 from the experimentally determined left - handed coiled - coil structure 
 in a dpc micelle .
all simulated replicas 
 were observed to undergo conversion to a distribution that strongly 
 favors right - handed helical packing ( see figure 2 ) .
simulated distributions for a cg model of the c991555 homodimer in popc membrane ( left panel ) and dpc micelle ( right panel ) 
 projected onto the order parameters 4 g and dgg . 4 g is the dihedral angle 
 formed by g29a - g37a - g37b - g29b , where a and b label the two c991555 monomers , 
 and dgg is the interhelical distance between 
 g33a - g33b .
the system sequence is shown below the panels 
 where g29 , g33 , and g37 are highlighted 
 and the tm helical domain is shaded .
the black triangles depict the 
 values of 4 g and dgg obtained from the experimentally derived nmr structure of c992355 .
the structure of the homodimer is conveniently 
 characterized in 
 terms of an interhelical distance , dgg and a dihedral angle , 4 g that differentiates the 
 handedness of the coiled - coil .
the 4 g order parameter 
 is positive for left - handed structures and negative for right - handed 
 structures .
structures stabilized by interpeptide interactions facilitated 
 by the gxxxg repeat region are characterized by small values of the dgg parameter .
the first experimentally 
 derived solution phase nmr structure of 
 the c991555 homodimer in a micelle is characterized 
 by a left - handed coiled - coil geometry with 4 g = 
 + 31 and dgg = 20  , forming 
 an x - like homodimer configuration with minimal interpeptide 
 contact in the g38xxxa42 region .
in contrast , 
 a more recent experimentally derived nmr structure of the shorter 
 c992355 homodimer in a a micelle is characterized 
 by a right - handed coiled - coil geometry with 4 g  
 25 and dgg  14  . the simulated homodimer distribution in 
 popc bilayer and dpc micelle 
 environments are dominated by right - handed coiled - coil conformations . 
 however , the broad distribution of homodimer conformations as a function 
 of dgg ( see figure 2 ) reflects the existence of distinct conformational states , implying 
 considerable heterogeneity in the structural ensemble .
structural ensembles of two c991555 monomers 
 in a popc bilayer , derived from 50 independent cg replica simulations 
 ( see above ) , are presented in figures 2 and 4 .
interestingly , the c991555 homodimer 
 assembled spontaneously on the time scale of a few hundred nanoseconds . 

this suggests that the sampling achieved with the cg model effectively 
 represents the equilibrium homodimer structural ensemble in this single - component 
 popc lipid bilayer .
although there are three characteristic 
 states in the popc bilayer , a significant shift in population between 
 substates is observed in the dpc micelle environment . in particular , 
 while the homodimer in a popc bilayer is predominantly found in the 
 gly - in substate , in the dpc micelle the homodimer is dominanted by 
 gly - side and gly - out conformations .
analysis of the dimer ensemble ( see figures 2 and 4 ) clearly shows the existence of multiple 
 conformational state populations .
crick identifies 
 the location of a residue relative to the axis between the two helices 
 ( figure 3 ) .
smaller values ( between 0 and 60 ) 
 identify residues closer to the dimer interface , while larger angles 
 ( close to 180 ) denote residues on opposite sides of the interface . 
 for the competing structural states ,
we find ( 1 ) gly - in configurations 
 with close interpeptide contacts facilitated by exposure of backbone 
 carbonyls in the gxxxg repeat region ( small dgg , small crick ) , ( 2 ) gly - out configurations 
 characterized by glycine repeats facing the outside of the homodimer 
 interface ( large dgg , large crick ) , and ( 3 ) gly - side configurations characterized by  out - of - phase  
 values of crick ( intermediate dgg , small / large or large / small crick ) .
similar observations have been made in past computational studies 
 of c99 homodimerization employing simplified models .
our results are consistent with those general observations , 
 while providing a more detailed analysis of the homodimer ensemble 
 and its dependence on environment
. definition of crick corresponding to the angle 
 between ( 1 ) the vector connecting the axis points of the two helices 
 and ( 2 ) the vector connecting the c of a given residue 
 to its corresponding -helical axis point .
we define crick to be the average of the g33 and g37 dimer crick angles 
 in order to characterize the g33xxxg37 interface .
most representative structure is a gly - in state 
 for the popc bilayer 
 ( top left ) and gly - out state for the dpc micelle ( top right ) .
simulated 
 distributions for a cg model of the c991555 homodimer 
 in popc membrane ( bottom left ) and dpc micelle ( bottom right ) projected 
 onto the crick angles characterize the relative orientation of peptides 
 within a homodimer .
the black triangles depict the values of crick obtained from the experimentally derived nmr structure of c992355 .
the atomic coordinates 
 of the most representative structures ( gly - in , gly - side and gly - out ) 
 have been deposited as supporting information .
figure 5 shows the distance between residues ak28 and bk28 ( kk28 ) or ak54 and bk54 ( kk53 ) where a and b indicate different monomers .
the 
 two distances represent the separation between the interfacial residues 
 of the tm helices , and is a good measure to characterize the global 
 topology of the dimer structure .
parallel or ll - like 
 structures are characterized by both smaller kk28 and kk54 distances ( 5  ) , -like structures show smaller 
 kk28 distances and larger kk54 distances , x - like structures show large kk28 and kk54 distances ( 15  ) , and y - like structures 
 have small kk54 and large kk28 distances .
simulations 
 in popc adopt predominantly ll - or -like conformations 
 consistent with our previous computational predictions of c992355 homodimer in a membrane environment .
we predicted a -like right - handed helical 
 dimer structure in agreement with solid state nmr studies with a predominantly gly - in orientation between 
 the helices .
in contrast , the dpc micelle simulations show larger 
 populations of y- or x - like structures 
 with a predominantly gly - out orientation between the helices ( figure 4 ) .
distance between ak28 and bk28 ( kk28 ) and ak54 and bk54 ( kk54 ) in popc 
 bilayer ( left ) and dpc micelle ( right ) colored by the most populated 
 4 g : red , green , and blue correspond to gly - in , gly - side , 
 and gly - out conformations for data of cg simulations .
the spot size 
 corresponds to the number of structures for that particular kk28 and kk54 conformation .
the black triangles are 
 the values of kk28 and kk54 obtained from the 
 experimentally derived nmr structure of 
 c992355 . on the lower section the most representative 
 -like ( far left ) and ll - like ( center left ) 
 conformations for the simulations in popc bilayer
x - like ( center right ) and y - like ( far right ) 
 conformations for the dpc micelle environment are shown in red . the 
 k28 and k55 residues are indicated with orange 
 spheres . results of additional cg simulations 
 performed using 70 and 108 
 dpc surfactant molecules are shown in figure 6 .
qualitatively , we see that the x - like strucures 
 are dominant for all micelle sizes . as the number of surfactant molecules 

is increased , there is a broadening of the distribution of x - like states .
these results suggest that the micelle environment 
 suppresses the sampling of gly - in conformations and favors x- and y - like structures , largely independent 
 of the size of the micelle .
left : distance between ak28 and bk28 ( kk28 ) and ak54 and bk54 ( kk54 ) colored by the most populated 4 g : red , green , 
 and blue correspond to gly - in , gly - side , and gly - out conformations 
 for data of cg simulations in 70 dpc surfactant molecules .
the right 
 panel corresponds to kk28 and kk54 for the c991555 system in 108 dpc surfactant molecules .
the spot 
 size corresponds to the number of structures for that particular kk28 and kk54 conformation .
we performed 
 all - atom simulations using charmm36 in popc bilayer and dpc micelle 
 environments , starting from representative cg structures from the 
 gly - in , gly - side and gly - out homodimer conformational states .
experimental data suggest that the average stability of the tm helix 
 is similar for monomeric peptide and peptide associated as a homodimer .
average helicity of each peptide in the micelle 
 and bilayer is shown in figure 7 .
we also show 
 results for the simulated helicity of c991555 monomer 
 in dpc micelle and popc lipid for comparison , along with experimentally 
 derived helicity values for monomeric c99 in a micelle .
the average helicity in the tm domain of the 
 simulated peptide is in good agreement with the experimentally measured 
 helicity , while helicity in the juxtamembrane ( jm ) domain is somewhat larger in the simulated structures . 

this could result from differing size of the hydrophobic core in the 
 micelle versus the bilayer , as the higher water accessibility in the 
 micelle is expected to impact the stability of the helix structure , 
 as well as the differing head groups and interfacial environments 
 of the dpc ( zwitterionic , simulation ) and lmpg ( anionic , experiment ) 
 micelles .
average helicity over the three different states ( gly - in , gly - side , 
 and gly - out ) calculated from all - atom simulations of c991555 homodimer in a popc bilayer ( above ) and dpc micelle ( below ) .
thin 
 black lines show results for helicity of c991555 monomer in the corresponding micelle or bilayer .
the gray shadow 
 shows experimentally determined helicity based on c nmr chemical shift measurements for monomeric c99155 in an lmpg ( lysomyristoylphosphatidylglycerol ) micelle .
structural fluctuations in the kink angle are enhanced and 
 less 
 symmetric in the c991555 homodimer in a dpc micelle 
 relative to the homodimer in a popc bilayer , while fluctuations in 
 homodimeric c991555 are smaller in magnitude than 
 those observed for the c991555 monomer ( see figure 8) .
in particular , in the vgsn region we found more 
 substantial fluctuations in the helicity that can be related to the 
 surface curvature of the micelle environment .
our results 
 indicate that a structural kink appears near g37/g38 in the c991555 monomer in a popc bilayer and dpc micelle , as 
 has been proposed for the monomer structure based on experimental 
 results for c99155 in a lpmg micelle .
a structural kink is also observed near g37/g38 
 in the c991555 homodimer in the dpc micelle environment . 

however , simulations of c991555 in dpc micelles 
 show additional structural kinks in the tm helix .
greater kink angles 
 are induced in an attempt to match the hydrophobic length of the tm 
 helix with the hydrophobic thickness of the small dpc micelle .
gly - in 
 conformations that destabilize the tm - c domain show a large kink at 
 t43 , while gly - out structures that destabilize the tm - n helix show 
 a large kink at g33 .
measurement of the observed kink angle for each peptide 
 of the 
 all - atom simulations of c991555 homodimer in a 
 popc bilayer ( top ) and dpc micelle ( bottom ) .
for the three different 
 systems , gly - in ( dark pink ) , gly - out ( blue ) and gly - side ( green ) , 
 the filled curve represents the difference a 
bkink = akink  bkink , where akink and bkink are the kink 
 angles of peptides a and b , respectively .
the hinge angle in the c991555 monomer is shown 
 in black for popc bilayer and dpc micelle simulations .
densities of the lipid phase of the popc 
 bilayer and dpc micelle were computed using all - atom simulations of 
 the c991555 homodimer ( figure 9 ) . superimposed on the density profiles are distributions 
 of key peptide residues .
importantly , although the density profiles 
 of the lipophilic phase of the bilayer and surfactant micelle are 
 similar , the solvent distributions in the two environments are dramatically 
 different .
in addition , the relative positions of 
 key residues , including the -cleavage site , are significantly 
 shifted relative to the center of the lipophilic phase in the dpc 
 micelle when compared to the popc bilayer .
density distribution 
 of the lipid phases ( shadow ) for the all - atom 
 simulations of the popc bilayer ( above ) and dpc micelle ( below ) . 
 superimposed
are the distributions of c positions 
 of key residues along the z - axis for dimer a ( solid 
 lines ) and dimer b ( dashed lines ) of c991555 for 
 gly - in ( red ) , gly - out ( blue ) , and gly - side ( green ) conformations . 

the number of waters molecules ( nw ) within 
 4  of each amino acid of the dimers are indicated by blue bars . 

the epr power saturation data derived from experimental measurements is shown for comparison ( black dashed line ) . a recent study involving h / d exchange 
 experiments on the c99 peptide , 
 complemented by molecular dynamics simulations of c992855 in a popc bilayer , provided insight into the stability of helical 
 regions of c99 including the tm helix . considering the hinge at g37/38 to be a flexible divider in the 
 tm helix , the n - terminal region of the tm domain ( tm - n helix ) showed 
 enhanced h / d exchange relative to the c - terminal portion ( tm - c helix ) .
our simulation results for the c991555 homodimer in a dpc micelle , in which a dominant  hinge  
 and less stable tm - n domain are observed , are consistent with those 
 experimental and computational results .
studies have indicated 
 that membrane protein structure in micelles 
 and membranes can be similar for certain systems . the gpa homodimer has been extensively studied in micelle 
 and bilayer environments , through both experimental and computational 
 approaches , making it an excellent reference system for understanding 
 environmental effects on the c991555 homodimer . 

homodimerization of gpa in a micelle has been explored using all - atom 
 molecular dynamics simulation , yielding 
 good agreement with known experimental structures .
moreover , simulations 
 employing an all - atom model of the gpa homodimer in dpc surfactant 
 micelle and dmpc / dhpc lipid bicelles found 
 the general topology of the homodimer to be similar in both environments .
in contrast , other studies have noted a dependence of protein structure 
 and association on the differing structural environments of micelles 
 and bilayers , as well as the particular detergent composition of micelles 
 or lipid composition of membrane bilayers . there is substantial evidence 
 that for a particular membrane system , a careful choice of detergent 
 must be made to create a micellular environment in which the protein 
 conformational ensemble is similar to that in a membrane bilayer .
it has been observed that dimerization of gpa 
 can be modulated by detergents , with variations in 
 alkyl chain length and headgroup nature ( ionic , zwitterionic , and 
 nonionic ) potentially influencing helix stability and helix dimerization . 
 as a result , variations in detergent may impact helix dimerization 
 while having little impact on helix stability . finally , it is known 
 that tm helical structure and stability can show a strong dependence 
 on lipid composition in membrane bilayers . as in the case of c99 , dimerization of the gpa tm domain
has 
 been 
 proposed to be a consequence of favorable intermolecular interactions 
 facilitated by gxxxg motif repeats .
we have simulated the sequence 
 of gpa62101 using the same multiscale simulation 
 approach employed in our study of c99 .
the gpa62101 sequence includes a tm helical domain and n - terminal juxtamembrane 
 domain , as is the case in c991555 .
good order parameters 
 for the homodimer structures in gpa ( c99 ) are ( 1 ) dihedral angle gt formed by g18a - t26a - t26b - g18b , where a and b label the two gpa62101 monomers , ( 2 ) interhelical distance dgg between g22a and g22b , and ( 3 ) the crick angle 
 crick of the gxxxg motif .
the gt order parameter is positive for left - handed structures and negative 
 for right - handed structures .
structures stabilized by interpeptide 
 interactions facilitated by the gxxxg repeat region are characterized 
 by small values of the dgg parameter and 
 small values of gt .
experimentally derived nmr structures 
 are found to agree well with the simulation predictions in terms of 
 ( gt , dgg ) .
differences 
 of 15 are observed in the comparison of crick angles between experimentally derived and computationally predicted 
 structures .
the observed differences may result from inherent limitations 
 in the spatial resolution of both the coarse - grained model employed 
 in our study and experimental data . left : simulated distributions for a cg 
 model of the gpa62101 homodimer in a popc bilayer 
 projected onto the order parameters 
 gt and dgg ( top ) , and 
 onto the crick angles crick ( bottom ) .
the panels 
 on the right corresponds to gt and dgg ( top ) and crick angles ( crick ) ( bottom ) 
 for the gpa62101 system in 56 dpc surfactant molecules . 

triangles represent 
 nmr structures derived from gpa62101 in dpc micelles 
 ( 1afo , black ) , gpa7098 in dpc micelles ( 2kpe , green ) , 
 and gpa6198 in dmpc / dhpc bicelles ( 2kpf , red ) .
our simulations results suggest 
 similarities between between the 
 dpc micelle and popc bilayer simulations of gpa62101 and c991555 homodimers , as well as sequence specific 
 effects differentiating the two dimer ensembles .
( 1 ) the strucural 
 distribution of c991555 dimer is signficantly more 
 diverse than in the case of the gpa62101 homodimer . 

( 2 ) in both gpa and c99 homodimers , the structural ensembles are found 
 to be significantly more diverse in the bilayer environment , relative 
 to the micelle , consisting of multiple distinct conformational substates . 
 in the dpc micelle
, only one of the substates tends to be represented 
 as it is selectively stabilized by the micelle geometry and surface 
 curvature .
it is useful to note that while gpa explores gly - side and 
 gly - out homodimer conformations in a bilayer environment , the distribution 
 is substantially more focused and dominated by right - handed gly - in 
 structures than in the case of c991555 homodimer . 

the results of this study , showing a clear dependence of c991555 homodimer structure on micelle and bilayer environment , are consistent 
 with this understanding .
the earliest predicted structures for the c992355 fragment , containing the tm helical domain , proposed a right - handed 
 coiled - coil structure consistent with the gly - in topology described 
 in this work .
a recent nmr structure 
 of the c992355 homodimer in a dpc micelle environment 
 is also a right - handed coiled - coil , although of the gly - out topology .
an earlier nmr structure for the c991555 homodimer in a dpc micelle environment led to the proposal of a 
 left - handed coiled - coil of the gly - out topology .
however , it was noted the homodimer ensemble may well contain 
 right - handed and left - handed coiled - coil structures .
it is expected 
 that both the length and sequence of the c99 fragment are critical 
 to the ultimate homodimer structure . while dimers formed from the 
 wt tm domain alone are almost certainly right - handed coiled - coils 
 in most environments , it is possible that introducing mutations within 
 the tm domain or the addition of the jm domain could lead to a shift 
 in the relative population of one of the various dimer states ( gly - in , 
 gly - side , or gly - out ) or a change in the handedness of the homodimer . 

it has been observed that small changes in sequence can strongly impact 
 binding affinities for tm homodimers .
our study has focused on the 
 c991555 , while the work of the arseniev laboratory is based on gs - c991555 , in 
 which two non - native amino acids ( gs ) have been added to the n - terminus . 

it is possible that the addition of these residues , not included in 
 our computational study , could impact the structure of the jm domain 
 and also the handedness of the resulting homdimer relative to c991555 . in this context , it is important to note that 
 our study focuses on c991555 rather than full - length 
 c99 .
therefore , our results must be considered to provide insight 
 into , but not fully represent , the properties of the full - length peptide .
additional analysis shows good agreement between chemical shifts 
 computed from our simulated homodimer structures and experimentally 
 derived chemical shifts ( see figures s3 and s4 ) .
however , the experimentally 
 derived noe data provide unambiguous support for a left - handed coiled - coil 
 structure of gs- c991555 homodimer in dpc micelle .
this suggests that the chemical shifts are largely 
 determined by the secondary structure of the peptide and local environment 
 and are weak reporters on the nature of the tertiary coiled - coil geometry .
the findings of this paper provide a complete and self - consistent 
 framework for organizing the existing experimental and computational 
 results . for structures of the tm domain represented by the c992355 peptide
, computational and experimental results 
 suggest that in most membrane and micelle environments the structure 
 is a right - handed coiled coil .
the particular homodimer topology will 
 depend on the membrane thickness or micelle size and curvature , with 
 thinner environments ( dmpc bilayer ) and those with substantial surface 
 curvature ( dpc micelle ) selecting gly - out topologies , with thicker 
 environments ( popc bilayer ) selecting gly - in structures .
evidence 
 from this study as well as the available nmr structures 
 suggest that the handedness of the coiled - coil structure adopted by 
 c991555 is ultimately primarily determined by ( 1 ) 
 a preference of the tm domain of the wt protein to form a right - handed 
 coilded coil , ( 2 ) characteristics of the membrane or micelle thickness 
 and surface curvature , and ( 3 ) the secondary structure and relative 
 position of the jm domain . in our study of the wt c991555 in popc bilyaer and dpc micelle
, we have largely assumed that the 
 jm domain is helical , leading to a preference for a right - handed coiled - coil 
 structure .
however , fluctuations in the structure of the jm domain 
 and its orientation relative to the tm domain may lead to a relative 
 stabilization of left - handed coiled - coil strutures .
overall , 
 the homodimer ensemble must be considered to consist of 
 left- and right - handed coiled coils , representing gly - in , gly - side , 
 and gly - out topologies .
the handedness and topology of the dominant 
 homodimer structure will be determined by the protein sequence and 
 the bilayer or micelle .
we find the c991555 homodimer structural ensemble 
 in popc micelles and dpc bilayers consists of multiple conformational 
 states that are structurally distinct and largely characterized by 
 the relative orientation of the peptide helices . a dynamic  hinge  
 near g37/g38 is observed to divide the tm helix , with structural fluctuations 
 being greater in the micelle than in the popc bilayer environment . 

dimerization results in little change in helix stability in the popc 
 bilayer , but a measurable decrease in helix stability is observed 
 in the dpc micelle . although the dimer ensemble in either environment 
 is characterized by multiple conformational states , the dominant structures 
 observed in our simulations in both the dpc micelle and popc bilayer 
 are consistently right - handed coiled - coil structures , supporting the 
 conclusions of earlier experimental and 
 computational studies .
the relative importance 
 of particular states is modulated by the 
 c991555 homodimer environment .
the gly - in substate 
 ( stabilized by interpeptide contacts facilitated by the gxxxg motif 
 repeats ) is predominant in a popc bilayer environment , while the gly - out 
 conformation ( stabilized by interpeptide contacts consistent with 
 a heptad repeat motif including g38 and a42 ) is dominant in a dpc 
 surfactant micelle environment .
our results suggest the dpc micelle 
 environment suppresses interactions mediated by gxxxg repeats in the 
 tm region , leading to an x - shaped structure that best satisfies the 
 boundaries of the surfactant / solvent interface . in this way ,
the environment 
  selects  a predominant substate through membrane thickness , 
 interfacial curvature , and peptide - lipid interactions .
past 
 computational studies of homodimers of the tm domain of c99 
 have noted similar heterogeneity in the homodimer ensemble .
moreover , it has been proposed that the tm domain 
 of c99 may be  processed to the  sites depending on 
 its dimerzation state and on the orientation of the tm helices in 
 the dimers  .
our findings support 
 these prior studies suggesting a role for membrane in modulating the 
 formation of specific c99 homodimer structures for processing by secretases , 
 as well as our interpretation of structures derived in diverse micelle 
 environments .
 the 
 99 amino acid c - terminal fragment of amyloid precursor protein 
 ( c99 ) , consisting of a single transmembrane ( tm ) helix , is known to 
 form homodimers . 
 homodimers can be processed by -secretase 
 to produce amyloid- ( a ) protein , which is implicated 
 in alzheimer s disease ( ad ) . while knowledge of the structure 
 of c99 homodimers is of great importance , 
 experimental nmr studies 
 and simulations have produced varying structural models , including 
 right - handed and left - handed coiled - coils . in order to investigate 
 the structure of this critical protein complex , simulations of the 
 c991555 homodimer in popc membrane bilayer and 
 dpc surfactant micelle environments were performed using a multiscale 
 approach that blends atomistic and coarse - grained models . 
 the c991555 homodimer adopts a dominant right - handed coiled - coil 
 topology consisting of three characteristic structural states in a 
 bilayer , only one of which is dominant in the micelle . 
 our structural 
 study , which provides a self - consistent framework for understanding 
 a number of experiments , shows that the energy landscape of the c99 
 homodimer supports a variety of slowly interconverting structural 
 states . 
 the relative importance of any given state can be modulated 
 through environmental selection realized by altering the membrane 
 or micelle characteristics . 