pathological protein aggregation is associated 
 with numerous diseases , many of which feature the formation 
 of amyloid aggregates , which 
 are characterized by a parallel , cross- structure .
an example is amyloid -peptide ( a ) , 
 which is the principal protein component of intra- and extracellular 
 oligomers , fibrils , and plaques in alzheimer s disease ( ad ) 
 and cerebral amyloid angiopathy ( caa ) .
notably , specific mutations 
 of a have been shown to lead to severe alzheimer s phenotypes .
a and many other amyloidogenic peptide sequences are natively 
 -helical but unfold to give rise to -strand structures . 

although the a monomer is nontoxic , understanding the factors contributing to helical instability on 
 the monomer level is of fundamental interest in elucidating the initial 
 events in these disease pathways , as well as providing crucial insight 
 into the stabilizing forces in protein folding .
this cooperativity 
 drives unfolded proteins toward the native , folded 
 state to avoid aggregation and amyloid formation .
the 
 folding of -helices is driven in part by cooperativity arising 
 from the alignment of peptide bonds through hydrogen bonding , as the peptide - bond dipole moments enhance each 
 other during helix formation .
the alignment 
 of peptide - bond dipoles also gives rise to a helix macrodipole , with 
 excess positive charge at the n - terminal end of the helix and excess 
 negative charge toward the c - terminus .
the macrodipole has been implicated 
 in helical stability and ligand binding , and induced polarization arising from hydrogen bonding is an important 
 contributing factor to this phenomenon , as recently shown for the folding of an -helical peptide 
 using molecular dynamics simulations in conjunction with a polarizable 
 force field .
folding studies of 
 the a monomer have shown that its transition 
 from a predominantly -helical form to a disordered ensemble 
 is modulated by ph and solution environment . at physiological 
 ph
, acidic groups are predominantly deprotonated , contributing to 
 charge repulsion that can drive the peptide toward a more disordered 
 ensemble .
hypothesized that disorder might originate 
 at the glu22asp23 acidic dyad toward the c - terminal end of 
 the first helix in the a structure , given that the a structure at a membrane  water interface 
 was not completely helical .
positions 22 and 23 in the a sequence 
 are particularly interesting because they are the sites of several 
 mutations that give rise to altered aggregation properties and greater 
 cytotoxicity .
the most widely studied mutant a forms are 
 the d23n  iowa  , e22q  dutch  , e22 g
each 
 of these mutations alters the electrostatic nature of the peptide ; 
 thus , an investigation of the microscopic details underlying their 
 unfolding pathways is of particular interest . in the present 
 work ,
wild - type ( wt ) and mutant a1527 sequences 
 were used in a systematic study of -helix unfolding 
 at the level of the a monomer based on md simulations performed 
 using a polarizable force field based on the classical drude oscillator 
 model .
the a1527 region of the peptide was chosen as it contains three full helical 
 turns while remaining a computationally tractable system for md simulations 
 in explicit solvent , similar to previous studies on fragments such 
 as a1224 , a1228 , and a1326 .
although n - terminal residues have 
 been implicated in modulating the conformational ensemble of a , such fragments serve as useful models for critical regions in a ; 
 the unfolding of the -helix in this central region of a 
 is known to be important in aggregation and toxicity .
given that a is proteolytically cleaved in the membrane from 
 the larger amyloid precursor protein ( app ) and unfolding takes place in solution and at membrane  water 
 interfaces , the effects of solvent polarity were examined using ethanol 
 as a membrane surrogate .
the findings have implications not only for 
 the unfolding mechanism in ad and caa directly but also for factors 
 governing -helical stability in general .
presented 
 are simulations of the unfolding of wt a1527 along with several mutants [ figure s1 , supporting information ( si ) ] performed using a polarizable force field 
 based on the classical drude oscillator model , allowing for an understanding 
 of the role of the induction of dipole moments on helix unwinding . 

in addition , to verify that the observed unfolding mechanism was associated 
 with explicit polarization in the force field , several of the simulations 
 were repeated using the polarizable model and performed with the additive 
 charmm36 force field . to facilitate analysis 
 of the induction of the peptide backbone dipoles in the polarizable 
 simulations , we define the dipole moment along the helical axis , para , , as described in figure s2 ( si ) .
the 
 unfolding of the wt helix in the polarizable simulation proceeded 
 rapidly in both water and ethanol ( figure 1b , f ) , yielding an average -helical 
 content of less than 20% ( table s1 , si ) . 
 in ethanol ,
310-helices were observed , whereas in water , 
 one - turn -helices re - formed transiently , with -helical 
 conformations lost by the end of the 300-ns simulation in both cases . 

helix unwinding in water began when the glu22 and asp23 side chains 
 came in close proximity ( < 4.5  , figure 1a , c ) and , as a consequence of the repulsive 
 interaction , the dipole moment of the asp23 side chain increased by 
 1.5 d ( figure 1c ) . in response , the component of the dipole moments of the glu22asp23 
 and val18phe19 peptide bonds parallel to the helix axis ( para , ) decreased in magnitude before reversing in direction 
 ( figure 1d ) , leading 
 to the unwinding of this turn of the helix .
the reduction in para coincided with the loss of the ( i , i + 4 ) hydrogen bond between val18 and glu22 ( figure 1d ) , as well as the phe19asp23 
 hydrogen bond ( figure s3 , si ) .
the simultaneous 
 change in direction of para and breaking of backbone 
 hydrogen bonds in both cases indicates that the local peptide - bond 
 dipole moments respond quickly to variations in the side - chain dipole 
 moments , leading to alterations in helix hydrogen bonding .
whereas 
 the electrostatic repulsion between neighboring anionic side chains 
 is intuitively unfavorable , the subsequent destabilization of backbone 
 hydrogen bonding and dipole - moment cooperativity up to four residues 
 away , through the hydrogen - bonding network and encompassing an entire -helical 
 turn , emphasizes the specific role of side - chain dynamics on the stability 
 of the -helix through alteration of the peptide - bond dipole 
 moments .
water molecules that bridged the glu22 and asp23 carboxylate groups 
 ( defined as any water with an oxygen atom simultaneously within 3.5 
  of a carboxylate oxygen of each residue ) had an average  
 value of 2.53  0.17 d , slightly above the bulk value ( 2.46  
 0.17 d ) .
this outcome indicates that the repulsion between the two 
 negatively charge side chains is somewhat dissipated by hydrating 
 water .
unfolding of wt a1527 in water and ethanol 
 and response of dipole moments to side - chain dynamics .
atoms are colored 
 by element ( c in gray , o in red , n in blue , h in white ) , lone pairs 
 in cyan , and drude oscillators in green .
( a ) close contact of glu22 
 and asp23 side chains in water that leads to destabilization of the 
 helical structure from a snapshot at 36 ns .
( b ) secondary structure 
 over time in water according to dictionary of secondary structure 
 of proteins ( dssp ) .
( c ) dipole moment of the asp23 side chain over 
 time ( blue ) and minimum distance between glu22 and asp23 carboxylate 
 o atoms ( black ) .
( d ) val18 to glu22 i to i + 4 backbone hydrogen - bond existence ( blue spikes indicate 
 the presence of a hydrogen bond , i.e. , existence = 1 ) and time series 
 for the component of the dipole moment parallel to the helix axis 
 ( para ; figure s2 , si ) 
 for the indicated peptide bonds .
( e ) salt bridge formed by lys16 and 
 asp23 with the resulting distortion of the -helix in ethanol 
 from a snapshot at 40 ns .
( f ) secondary structure over time in ethanol . 
 ( g ) time series of the minimum distance between lys16(n ) and 
 asp23(o1/o2 ) atoms .
any value 
 less than or equal to 3.5  ( red dashed line ) indicates an intact 
 salt bridge .
( h ) time series of para for the indicated 
 peptide bonds , showing the loss of dipole - moment alignment upon stable 
 formation of the lys16asp23 salt bridge .
the dipole moment 
 and distance time series in panels c , d , g , and h are shown as 1-ns 
 running averages for clarity .
a negative value of para indicates alignment of c = o with the helix axis . in ethanol
, the wt a1527 peptide also 
 unfolded rapidly ( figure 1f ) , although the mechanism was different .
helical unwinding 
 began around leu17 and proceeded toward the c - terminal end of the 
 -helix , culminating in a total loss of -helicity by 
 45 ns .
unwinding was initiated when the side chain of lys16 
 formed a stable salt bridge with asp23 ( figure 1e , g ) , straining the backbone and perturbing 
 the leu17ala21 backbone hydrogen bond .
this strain led to 
 a reversal of para in the leu17val18 and 
 phe20ala21 peptide bonds ( figure 1h ) , indicating a loss of dipole alignment 
 and hydrogen bonding .
the association of opposing charges in ethanol 
 is more favorable because of the lower solvent polarity (  = 
 22 ) and resulted in the salt bridge remaining intact for 90% 
 of the simulation ( figure 1 g ) . in water , lys16asp23 interactions were screened 
 by the large dielectric constant of the medium and did not contribute 
 to the destabilization of the helix , as the salt bridge was intact 
 for less than 0.2% of the trajectory . even though lys16 is able to 
 sequester the side chain of asp23 away from that of glu22 to prevent 
 unfavorable side - chain interactions as in the case of the simulation 
 in water ,
taken together , the results 
 of the wt a1527 simulations suggest that 
 its -helical structure is prone to disorder in environments 
 with either high or low polarity .
experiments 
 have shown that these mutants unfold 
 and aggregate more rapidly than the wt peptide , suggesting that 
 factors beyond the side - chain charge 
charge interactions seen 
 in the wt peptide contribute to the instability of the a helical 
 structure . in water ,
the helical turn of residues 2226 in 
 the d23n peptide was lost almost immediately ( figure 2b ) , initiated by the rotation of the 1 dihedral angle of asn23 ( n  c-c-c ) 
 from trans ( t ) to gauche ( g , figure 2a , c ) .
this rotation led to a hydrogen bond between 
 its side - chain amide and the backbone carbonyl group of phe19 ( figure 2a ) , causing a significant 
 decrease in the magnitude of para of the glu22asn23 
 peptide - bond dipole ( figure 2d ) and leading to disorder in this region of the peptide at 
 10 ns . the remainder of the peptide remained helical ( figure 2b ) , and para of the phe19phe20 peptide bond remained aligned 
 with the helix axis out to 160 ns ( figure 2d ) .
subsequently , the remainder of the helical 
 content was lost ( figure 2b ) , coinciding with the loss in magnitude and then a switch 
 in the orientation of para of the phe19phe20 
 peptide bond . in ethanol , this outcome was not observed , and the peptide 
 remained 70% -helical for the duration of the simulation 
 ( table s1 , si ) despite the fact that the 
 asn23 side - chain 1 distribution was nearly identical 
 to that in water ( figure s4 , si ) , indicating 
 that interactions with the phe19 backbone carbonyl were still occurring 
 in ethanol but did not lead to destabilization . in water ,
the average 
 asn23 side - chain dipole moment was 6.27  0.47 d ( average  
 root - mean - square fluctuation ) , whereas in ethanol , it was only 5.17 
  0.45 d. the larger dipole in water allowed the side - chain amide 
 of asn23 to more effectively compete for the backbone hydrogen bond 
 between phe19 and asn23 , leading to unwinding , whereas in ethanol , 
 the smaller side - chain dipole moment did not have this effect . moreover , 
 the transition from t  g in water led to an increase in the asn23 side - chain 
 dipole moment ( inset , figure s4 , si ) , suggesting 
 environment- and conformation - dependent behavior . considering only 
 -helical conformations , in water , the t conformation 
 of asn23 had an average dipole moment of 5.68  0.48 d , whereas 
 rotation to g led to an increase 
 to 5.96  0.46 d. in ethanol , the side - chain dipole moment was 
 insensitive to conformation , being 5.17  0.44 d in t and 5.17  0.42 d in g. dynamics 
 of charge - neutralizing mutants d23n and e22q in water . 

( a ) snapshot of d23n in water at 11.81 ns showing hydrogen - bond formation . 

( d ) time series of para for the peptide bonds involved in hydrogen - bond exchange . 

( e ) snapshot of e22q in water at 124.56 ns showing hydrogen - bond formation 
 leading to dipole enhancement .
g ) time series of para for the peptide 
 bonds affected by the gln22asp23 side - chain hydrogen bond , 
 the formation of which is indicated by black spikes .
( h ) side - chain 
 dipole moment of gln22 , with values for each frame shown as circles 
 and a 1-ns running average shown as a red line .
the para time series in panels d and g are also shown as 1-ns running averages 
 for clarity . side - chain 
backbone 
 interactions also figured prominently 
 in unfolding of the e22q peptide in water .
helical instability from 
 leu17 to phe20 appeared within 40 ns of simulation time ( figure 2f ) .
transient hydrogen - bond 
 formation between the side - chain gln22 amide and asp23 carboxylate 
 early in the trajectory led to short - lived decreases in the magnitude 
 of para of several nearby residues ( figure 2 g ) , with the loss of helix 
 from leu17 to phe20 corresponding to a decrease in para for the val18phe19 and ala21gln22 peptide bonds . 

the remaining helical structure was lost at 124 ns when the 
 gln22asp23 side - chain hydrogen bond ( figure 2 g ) persisted .
this interaction arose when 
 the 1 and 2 ( cccc ) 
 dihedral angles of gln22 were both gauche ( g ) . in this hydrogen - bonded conformation ( figure 2e ) , which persisted for 18 
 ns ( 124142 ns in the trajectory , figure 2 g ) , the side - chain dipole moment of gln22 
 increased by 2 d ( figure 2h ) , an effect subsequently transmitted to the backbone 
 peptide bonds , leading to a reversal in the direction of para of the ala21gln22 and gln22asp23 peptide 
 bonds ( figure 2 g ) . 

this reversal of para was transmitted to the val18phe19 
 ( i  4 ) peptide bond , leading to unwinding 
 of this turn of the -helix ( figure 2f , g ) .
the reversal in para of the gln22asn22 
 peptide bond in water was a direct result of interactions with the 
 gln22 side chain .
figure s5c , d ( si ) shows 
 the relationship between para of the gln22 side 
 chain and para of the gln22asp23 peptide 
 bond . in water ,
the backbone para responded to the 
 fluctuations in the side - chain para ( figure s5c , si ) , which are due to changes in side - chain orientation 
 and concomitant gln22 and asp23 side - chain  side - chain hydrogen - bond 
 formation ( figure s5e , si ) . in ethanol , 

the backbone para was not sensitive to this effect 
 ( figure s5d , si ) , despite conformational 
 variations in 1 that lead to hydrogen bonding ( figure 
 s5f , si ) .
it is also important to highlight 
 the fact that , whereas gln22asp23 hydrogen - bond formation 
 in water resulted in a large dipole response [ increase in the total 
 side - chain  value of 2 d , figure 2h , and 4 d in para , figure s5c ( si ) ] , there was no systematic 
 response in para in ethanol ( figure s5d , si ) .
thus , the backbone para remained aligned with the helix axis , and the majority of the helix 
 persisted throughout the simulation ( figure s5b , si ) .
nmr experiments by masuda et al . showed 
 that , in the e22k 
 a peptide , a salt bridge forms between lys22 and asp23 , promoting 
 the formation of a local bend instead of a helix .
upon formation of the salt 
 bridge at 25 ns ( figure 3b ) , the magnitude of lys22asp23 para decreased , which was transmitted to the neighboring ala21lys22 
 peptide bond ( figure 3d ) , contributing to the loss of the -helical turn that includes 
 lys22 and asp23 ( figure 3a ) . throughout the remainder of the simulation , the lys22asp23 
 salt bridge broke and formed multiple times ( figure 3b ) , leading to sampling of both g and t conformations of 1 of lys22 ( figure 3c ) .
in contrast , in ethanol , the salt bridge persisted ( figure 
 s6d , si ) , with lys22 1 heavily favoring the t conformation ( figure s6c , si ) ; in this case , the helix was stable throughout 
 the simulation ( figure s6a , si ) with only 
 transient deformation due to the lys22 g state ( figure s6b , si ) .
salt - bridge dynamics 
 in e22k a1527 perturb 
 nearby dipole moments in water .
( b ) time series of the minimum distance between 
 lys22 n and asp23 o1/o2 atoms , shown as a 1-ns running average for clarity .
a value of 3.5 
  ( dashed blue line ) was used as an indicator of an intact salt 
 bridge .
( d ) time series of para for the indicated 
 peptide bonds , illustrating their response to the side - chain dynamics . 

( e ) snapshot from 22.30 ns of the simulation in water , with an intact 
 lys22asp23 salt bridge , with the lys22 1 in the t conformation , such that asp23 is sequestered 
 away from the peptide bond .
( f ) lys22 in the g conformation from a snapshot at 25.01 ns of the simulation 
 in water , interacting directly with asp23 , which is brought in close 
 contact with the peptide bond .
table 1 reports 
 the average para values for the ala21lys22 
 and lys22asp23 peptide bonds as a function of lys22 1 in -helical states of the ala21lys22asp23 
 sequence .
in both water and ethanol , rotation of lys22 1 to the g conformation led to 
 a decrease in the magnitude of para in both of these 
 peptide bonds ( table 1 ) , which destabilized the helix .
the reason for this destabilization 
 is that the g state of lys22 1 brought the asp23 carboxylate in close proximity ( 3.98  
 0.48  on average in helical states ) to the carbonyl oxygen of 
 the lys22asp23 peptide bond ( figure 3f ) , whereas the t conformation 
 of lys22 1 sequestered the acid moiety away ( 4.47 
  0.20  , figure 3e ) , although the lys22asp23 side - chain interaction 
 is present in both the g and t 1 conformations .
thus , although the 
 lys22asp23 salt bridge can form in both water and ethanol , 
 the differences in the dielectric constants of the solvents lead to 
 increased opening and closing in the former that allows for interactions 
 of the asp23 side chain with the backbone while the salt bridge persists 
 in ethanol with 1 in t dominating , 
 thereby not destabilizing the helix . unlike in the wt peptide ,
in 
 which water molecules between glu22 and asp23 dissipated some of the 
 ion  ion repulsion , in the case of water - mediated salt bridges , 
 there was no shift in the water dipole moments .
they behaved exactly 
 like bulk water , with an average value of  = 2.46  0.17 
 d. in the case of water mediating interactions between unlike charges , 
 there is no dissipation of the dipole response .
values ( average 
  root - mean - square 
 fluctuation ) calculated only for frames in which ala21lys22asp23 
 were in an -helical conformation .
the a1527 e22 g mutant rapidly 
 unfolded in water ( figure s7b , si ) , whereas 
 unfolding in ethanol proceeded more slowly ( figure s8d , si ) , such that 43% total -helical content 
 was retained during the simulation ( table s1 , si ) . in the e22 g peptide ,
close packing between gly25 and 
 gly22 in water arose because of the absence of side - chain atoms at 
 position 22 ( figure s7a , si ) .
this packing 
 persisted for 50 ns , during which time the -helix was 
 in a distorted but metastable state in which backbone hydrogen bonds 
 deviated from linearity ( figure s7a , si ) .
conversion of these two gly residues between right- and left - handed 
 helical conformations , with peptide - bond dipole moments responding 
 accordingly ( figure s7c , d , si ) , initiated 
 unfolding by perturbing the ala21gly25 backbone hydrogen bond 
 ( figure s7a , si ) .
unfolding then propagated 
 through the backbone hydrogen - bonding network as para reversed along the helix axis ( figure s7c , d , si ) . in ethanol , gly 
gly packing was not observed , 
 although several residues in the e22 g peptide were disordered for 
 the majority of the simulation ( figure s8 , si ) .
the intrinsic flexibility of gly25 allowed asp23 to reorient such 
 that the asp23val24 peptide bond was oriented in the opposite 
 direction of the helix , with para reflecting this 
 change in conformation ( figure s8a , c , si ) and subsequently perturbing nearby peptide - bond dipoles to transiently 
 destabilize the helix at 100 ns ( figure s8c , d , si ) .
the disordered state of asp23 was stabilized 
 by a tight turn around gly25 , allowing nearby amide groups to form 
 favorable interactions with the carboxylate moiety of asp23 ( figure 
 s8b , si ) .
the wt and d23n peptides were each simulated 
 two additional times with the drude model for 100 ns , initiated from 
 the same starting configurations but different velocities , to test 
 the reproducibility of the observed unfolding mechanism .
the results 
 are summarized in figures s9 and s10 ( si ) for the wt and d23n systems , respectively .
all runs produced compatible 
 results ; that is , for the wt peptide , proximity of glu22 and asp23 
 acid groups resulted in spikes of the asp23 side - chain  value , 
 concomitant with the reversal of para along the 
 helix axis and destabilization of backbone hydrogen bonding ( figure 
 s9 , si ) .
similarly , the rotation of asn23 
 into a g 1 state 
 destabilized phe19asn23 backbone hydrogen bonding and resulted 
 in unfolding of the d23n peptide ( figure s10 , si ) .
all of these events occurred within the first 100 ns 
 of each replicate simulation , thus the results described above from 
 the initial simulation are consistently observed and the role of the 
 polarization response is emphasized . to provide additional context 
 for the findings using the drude polarizable force field , we performed 
 the same simulations with the additive charmm36 force field . by definition , dipole response will not be observed 
 in this fixed - charge model ( beyond geometry changes such as bond stretching 
 or dihedral - angle rotation ) , so the additive simulations serve as 
 an important reference for establishing the role of induced polarization 
 in the simulations described above . in water ,
all of the a1527 peptides disordered to some extent ( table s1 and 
 figure s11 , si ) , although the wt , d23n , 
 and e22k peptides were more helical with charmm36 than they were in 
 the drude simulations . in the additive systems , unfolding of all of 
 the peptides proceeded in a similar manner .
the n- and/or c - terminal 
 residues initially became disordered , allowing the side - chain amide 
 groups of gln15 and asn27 to interact with various side chains in 
 the a1527 sequence . in none of these cases 
 did these side chains compete for backbone hydrogen bonding .
instead , 
 hydrogen bonds transiently formed between groups already destabilized 
 and exposed to water , thus stabilizing the unfolded state but not 
 contributing to the unfolding pathway .
this is a nonspecific effect 
 that occurred in all of the simulations , which , in the majority of 
 cases , did not propagate to the rest of the structure as observed 
 in the polarizable simulations .
the wt simulations in water 
 with charmm36 , the glu22 and asp23 
 side chains to come in close contact of < 3.5  at 3.23 ns . 
 however , no perturbation of the helical structure occurred ( figure 
 s11 , si ) , which is assumed to be due to 
 the lack of a change in the asp23 side - chain  value .
the absence 
 of a destabilizing effect by glu22asp23 interactions is inconsistent 
 with experimental evidence that they are responsible for destabilizing 
 helical structure as a function of ph . in the d23n mutant ,
the asn23 side chain heavily sampled the g conformation and formed hydrogen bonds 
 with the phe19 carbonyl oxygen .
however , as changes in the dipole 
 of the side chain were limited to the impact of dihedral - angle rotation 
 ( figure s12 , si ) , helicity was preserved 
 for most of the trajectory ( figure s11 , si ) . as with the drude systems
, the wt and d23n simulations in water 
 were repeated twice more for 100 ns , with nonspecific unfolding occurring 
 in all cases ( figure s11 , si ) .
we note 
 that the additive tip3p water diffuses approximately 3 times too fast , 
 which might enhance the rate at which conformational changes occur 
 in a nonspecific manner . in the e22k simulation , the lys22asp23 
 salt bridge formed only infrequently ( 6.9% of the time ) and
was associated 
 with transient destabilization of the helical turn involving only 
 these two residues ( figure s11 , si ) , as 
 a result of a small change in peptide - bond alignment rather than an 
 electrostatic response .
these results suggest that there is an intrinsic 
 strain on the helical structure associated with the formation of the 
 salt bridge between these neighboring residues , although the charmm36 
 simulation suggests that it is not a strong destabilizing force , whereas 
 the polarizable simulations described above clearly showed propagating 
 effects of dipole reorientation that destabilized the -helical 
 structure .
both the e22 g and e22q peptides disordered rapidly in water 
 with the additive force field ( figure s11 , si ) ; the disordering of the termini led to fast unfolding that was 
 not associated with any specific interactions arising from the mutations . in ethanol , all peptides with the additive force field remained 
 predominantly helical , with generally a larger -helical content 
 than the drude force field ( table s1 and figure s11 , si ) .
n - terminal residues were the only ones to disorder in 
 these simulations , and the helical turns encompassing the sites of 
 mutations remained helical throughout all of the simulations ( figure 
 s11 , si ) . thus , the additive simulations 
 would lead to the conclusion that , in ethanol , there is little to 
 no disordering associated with the mutagenic region of a. taken 
 together , the simulations using charmm36 suggest only a nonspecific 
 fraying at the ends of the a1527 peptide 
 occurs in ethanol and water , with this fraying leading to unfolding 
 in water whereas specific behaviors related to glu22 , asp23 , or any 
 of the mutations , were not observed .
the 
 present study involved a systematic investigation of the underlying 
 contributions to helical unfolding in the amyloidogenic a1527 fragment and examined the effects of both mutations 
 and solvent on the unfolding of this peptide .
similar a fragments 
 have been used to efficiently examine unfolding phenomena . moreover , by studying systems 
 in water (  = 79 ) and ethanol (  = 22 ) , solvent effects 
 can be evaluated and related back to unfolding in aqueous solution 
 or at membrane  water interfaces , as ethanol is a reasonable 
 model of the glycerol region of the membrane  water interface 
 given its dielectric constant .
previous studies have concluded that mutant a peptides have 
 enhanced cytotoxicity , aggregate more rapidly , and have altered fibril structures .
the present work did not seek to resolve the details of 
 these pathways , instead focusing on the early events in the unfolding 
 of the helix that is generally believed to be intact upon liberation 
 of a from the helical transmembrane domain of app .
is a logical 
 initial model , as it was determined in the presence of detergent micelles , 
 thus approximating a membrane  water interface . in agreement 
 with the mechanism hypothesized by coles et al .
, our results indicate that side - chain electrostatic 
 interactions are principally responsible for unfolding of the wt peptide , 
 with glu22asp23 repulsion driving unfolding in water .
this 
 repulsion perturbs the asp23 side - chain dipole that is communicated 
 to the dipole moment of the adjacent peptide bond and propagated through 
 the backbone hydrogen - bonding network to perturb additional peptide - bond 
 dipoles
. salt - bridge formation also plays a role in helical destabilization 
 of the wt peptide in ethanol and in the e22k peptide in water , but 
 in contrast , the salt bridge stabilized the -helicity of e22k 
 in ethanol .
thus , the present results show how side - chain dynamics 
 affect peptide - bond dipole moments , in turn contributing to the instability 
 of -helical structure of the a1527 peptide .
electrostatic interactions between charged side chains 
 are the most important intrapeptide interactions that affect helix 
 stability , suggesting that our findings might also extend to intrinsically 
 disordered proteins , which are often highly charged .
similar to the lys22asp23 dynamics , in the 
 e22q peptide , 
 hydrogen - bond dynamics between gln22 and asp23 led to helical instability 
 in water .
although the formation of this side - chain hydrogen led to 
 a large ( 2 d ) increase in the gln22 side - chain dipole moment , 
 only in water was the gln22 side - chain amide group aligned such that 
 its para opposed the helix axis , causing a similar 
 response in the backbone and subsequent helical unwinding .
the effect 
 on peptide - bond dipoles was not observed in ethanol , suggesting that 
 the polarity of the solvent modulates the susceptibility of the backbone 
 to side - chain dipole dynamics .
hydrogen bonding and competition for 
 native interactions contribute to the helical instability of the d23n 
 peptide in water .
the side - chain amide of asn23 destabilizes its own 
 backbone hydrogen bonding , leading to rapid unwinding in water .
the 
 solvent polarity and subsequent response of the side chain are important 
 factors , as in water , asn23 is more strongly polarized than in ethanol , 
 allowing it to more effectively compete for backbone hydrogen bonding . 

the side - chain response to solvent polarity and nearby dipoles is 
 a feature unique to the drude polarizable model , highlighting the 
 importance of explicit polarization in md simulations , especially 
 mechanistic studies such as this one .
the 
 present work has provided new insights into the driving forces 
 for a unfolding by modeling a peptide fragment that is known 
 to harbor mutations that lead to severe disease phenotypes .
explicit 
 inclusion of induced polarization , an effect that is not frequently 
 included in md simulations , is a critical component of the physical 
 behavior , as evidenced by the different mechanisms of unfolding seen 
 with the additive charmm36 force field .
whereas the additive force 
 field undergoes generic fraying of the n- and c - termini , leading to 
 unfolding in water , polarization effects manifest themselves on the 
 local backbone dipoles , leading to sequence - dependent effects , a behavior 
 that has been suggested previously for other proteins .
the polarizable model also shows clear differences 
 in the mechanism of unfolding as a function of solvent , and the results 
 have implications for understanding a unfolding in different 
 environments , such as the membrane or in aqueous solution .
further 
 studies of full - length a and other amyloidogenic peptides using 
 models that include explicit treatment of electronic polarization 
 in water and membranes would be necessary to more fully describe the 
 unfolding and aggregation pathways , and perhaps mechanisms of membrane 
 perturbation .
the initial coordinates 
 for the a1527 fragment were taken from the 
 first model of the nmr structure of 
 a40 of coles et al .
, in which the fragment forms a continuous -helix , flanked 
 by disordered residues at the n - terminal end and a bend at its c - terminal 
 end . to negate end effects from charged termini ,
the n- and c - termini 
 of the a1527 fragment were capped with acetyl 
 and amide groups , respectively .
the sequence of the wt fragment is 
 ac - nkvlffaedvgsn - nh2 [ see figure s1 ( si ) for additional sequence information ]
. mutations ( d23n , 
 e22q , e22 g , and e22k ) were introduced by deleting conflicting side - chain 
 atoms and rebuilding the missing atoms of the mutated residue using 
 the internal coordinate builder in charmm .
each peptide was solvated in a 43- cubic box of water ( swm4-ndp for drude systems or tip3p for charmm36 
 systems ) or ethanol . to neutralize the 
 net charge of the wt peptide , one na
coordinates and topologies were prepared by charmm - gui using the drude-2013 or charmm36 force fields for proteins .
each system was energy - minimized and equilibrated under an npt ensemble for 1 ns with restraints on all peptide non - hydrogen 
 atoms ( 5.0 kcal mol  ) . given the considerable change in size and electronic nature at 
 position 22 of the e22k mutation , this peptide was equilibrated for 
 an additional 1 ns with restraints on backbone non - hydrogen atoms 
 only , allowing the side chains to reorient to avoid any initial bias 
 from the in silico mutation . following equilibration ,
all polarizable simulations 
 were performed using namd with the langevin integration scheme for 
 polarizable simulations or the standard leapfrog 
 integrator for additive simulations .
neighbor lists were updated within 
 16.0  , and short - range van der waals interactions were switched 
 smoothly to zero over a range of 10.012.0  .
electrostatic 
 interactions were calculated using the particle mesh ewald ( pme ) method , with a 1- grid spacing throughout the unit cell . for polarizable 
 systems ,
a dual langevin thermostat was 
 used to regulate the temperature of the real atoms at 298 k and the 
 drude oscillators at 1 k , using friction coefficients of 5.0 and 20.0 
 ps , respectively .
the langevin piston method was used to regulate the pressure at 1.0 atm with a decay period 
 of 0.1 ps and an oscillation period of 0.2 ps .
bonds involving hydrogen 
 atoms were constrained with shake , and 
 the time step was set to 1 fs . a 
hard - wall  constraint was used to prevent displacements larger than 
 0.2  between the drude oscillators and their parent atoms . for 
 additive systems ,
the langevin piston method was used to regulate 
 temperature and pressure , with the same relaxation times as in the 
 drude simulations .
analysis was performed using facilities in charmm or the gromacs 5.0 interface 
 to the dictionary of secondary structure of proteins ( dssp ) . for dipole analysis , the peptide bond of residue i is defined as the carbonyl functional group ( including 
 real atoms , drude oscillators , and lone pairs ) of residue i , and the n , h , c , and h atoms and connected 
 drude oscillators in residue i+1 ( figure s2 , si ) . the total dipole moments were decomposed 
 in three dimensions to provide insight into their orientations .
the 
 peptides were aligned along the x axis based on a 
 least - squares fit to c atoms , after which dipole moments were 
 calculated .
the x component of the dipole moment 
 is thus parallel to the helix axis and is referred to in the present 
 work as para .
values of para < 
 0 indicate alignment with the helix axis ( figure s2 , si ) .
 amyloid - forming proteins undergo 
 a structural transition from -helical 
 to disordered conformations and , ultimately , cross- fibrils . 
 
 the unfolding and aggregation of the amyloid -peptide ( a ) 
 have been implicated in the development and progression of alzheimer s 
 disease ( ad ) and cerebral amyloid angiopathy ( caa ) . 
 however , the events 
 underlying the initial structural transition leading to the disease 
 state remain unclear . 
 although most cases are sporadic , several genetic 
 variants exist that alter the electrostatic properties of a 
 and lead to more rapid unfolding and more severe phenotypes . in the 
 present study , 
 the enhanced unfolding is shown to be due to the mutated 
 side chains altering the local peptide - bond dipole moments leading 
 to local destabilization of the -helix , as determined from 
 polarizable molecular dynamics ( md ) simulations of wild - type ( wt ) 
 a fragments and several common mutations . 
 the local perturbation 
 of the helix then leads to progressive unwinding of the -helix 
 in a cooperative fashion due to decreases in adjacent ( i  1 ) and hydrogen - bonded ( i + 4 ) peptide - bond 
 dipole moments . 
 side - chain dynamics , subsequent variations in dipole 
 moments , and ultimately the response in the peptide - bond dipole moments 
 are all modulated by solvent dielectric properties based on simulations 
 in water versus ethanol . the polarizable simulation results , along 
 with simulations using the additive charmm36 force field , further 
 indicate that cooperativity due to the alignment of peptide bonds 
 leading to enhanced dipole moments is a fundamental force in stabilizing 
 -helices . 
Introduction
Results
Discussion
Conclusions
Methods