specific 
 fibril aggregation of more than 20 proteins has been linked 
 to a pathogenesis of many amyloid - associated diseases such as alzheimer s 
 disease , parkinson s disease , huntington s diseases , 
 and prion diseases . moreover
, a formation of amyloid - like fibrils takes 
 place in other maladies associated with protein misfolding , including 
 diabetes type ii , prolactoma , primary amyloidosis , etc .
striking evidence has recently accumulated suggesting that the ability 
 to form amyloid fibrils is not a peculiarity of this small group of 
 disease - related proteins but , rather , the ability to form amyloid 
 is a generic property of the polypeptide chain .
amyloid fibrils are -sheet rich protein aggregates 
 with different morphologies that constitute a phenomenon known as 
 fibril polymorphism.in vitro aggregation 
 of various amyloid associated proteins , such as calcitonin , amylin , insulin , transthyretin , and 
 mouse prion , commonly leads to the formation 
 of two distinct fibril morphologies : tape - like and helical - twisted
. 
 microscopic examination of post - mortem extracted amyloids from brains 
 of patients who were diagnosed with alzheimer s disease also 
 revealed both of these fibril polymorphs .
thus , a controllable formation 
 of one fibril polymorph versus another could possibly slow down fibril 
 development and progression of various amyloid associated disorders . 
 at the same time , the factors leading to one fibril polymorph or another 
 are not currently known .
it has also been observed that , with protein 
 aggregation , many physical and chemical factors , such as ionic strength , temperature , or pressure , determine the fibril structure and morphology . for example , 
 makarava and baskakov found that hamster cellular isoform of mammalian 
 prion protein forms variable fibril polymorphs under different aggregation 
 conditions .
separately , petkova et al . 
 observed that agitation of a peptide solution results in the 
 formation of structurally and morphologically different fibril polymorphs 
 compared to the fibrils formed without agitation .
a few years ago , dzwolak demonstrated that vortexing of 
 insulin solutions during protein aggregation at 60 c causes 
 the formation of fibril superstructures exhibiting negative induced 
 circular dichroism ( icd ) upon binding the achiral dye , thioflavin 
 t ( tht ) .
if , however , the same fibrillation 
 was carried out at a temperature , below 50 c , randomly two differing 
 supramolecular structures were obtained that showed opposite icd ( icd 
 and + icd polymorphs ) .
insulin fibrils that were not 
 agitated , while being formed , did not exhibit any icd signal .
interestingly , 
 the morphologies of the two observed chiral polymorphs although similar 
 showed a tendency for more lateral alignment of filaments for the 
 + icd polymorph compared to the icd polymorph .
this discovery emphasized the importance of utilizing alternative 
 methods beyond classical microscopy , such as afm ( atomic force microscopy ) 
 and sem ( scanning electron microscopy ) , that are capable of distinguishing 
 chiral fibril polymorphs that otherwise appear identical .
recently , 
 it was discovered that vcd is a unique 
 tool that can directly probe supramolecular fibril chirality 
 in solution as well as in dried films .
studies of insulin fibrils indicated that enhanced vcd sensitivity 
 arises directly from the long - range supramolecular chirality of fibril 
 structures at all hierarchical levels .
it was shown that vcd probes 
 deeper levels of fibril supramolecular chiral organization that are 
 not apparent using existing forms of microscopy .
in particular , we 
 found that insulin aggregation at ph 2.4 and higher results in the 
 formation of fibrils that show a strong vcd spectrum with peaks near 
 1554 , 1593 , 1627 , 1647 , 1670 cm , which have a 
 sign pattern ( +  + 
microscopic examination of these fibrils indicated that a majority 
 of them have a left - handed helical twist .
however , if the ph of the aggregation solution is lower than 2.4 , 
 the distribution of fibrils shifts to increasingly 
 flat , tape - like , or binary fibrils as the incubation ph continues 
 to be lowered that under microscopic examination show no noticeable 
 chirality or twist on their surface .
nevertheless , these fibrils show 
 a strong , but often somewhat smaller , vcd with a sign pattern (     +   
 ) 
 that is nearly the mirror - image of  normal  vcd fibril 
 spectrum and is referred to as the  reversed  polymorph . 

the fact that flat tape - like fibrils show a strong reversed vcd signal 
 indicates that they must be composed of right - handed filaments , the 
 chirality of which lies below the limit of afm or sem detection .
our 
 combined vcd - microscopic studies showed that ph determines not only 
 the net handedness of the filaments , precursors of mature fibrils , 
 but also controls their association pathways .
left - handed filaments intertwine , forming left - handed proto - fibrils 
 and mature fibrils that have normal vcd . on the other hand ,
right - handed 
 filaments associate side - by - side , forming flat , tape - like , or binary 
 fibrils .
thus , ph most likely alters protein  solvent interactions 
 or causes protonation of some amino acid side chains , which are lying 
 on the surface of the filaments .
these changes cause variations in 
 the way these filaments bind together to form mature fibrils that 
 either twist together to form braids or align side - by - side without 
 braiding .
in addition , it was discovered that that ph not only determines 
 insulin fibril morphology and net chirality at the stage of the protein 
 aggregation , but also may dramatically change the morphology of mature 
 fibrils and overturn their initial chirality .
the key question we answer in this paper is whether the supramolecular 
 chirality of the filaments is the underlying cause of morphological 
 differences only for insulin fibrils , or whether it is a more general 
 phenomenon that determines the polymorphism of other amyloidogenic 
 proteins and peptides .
a related question is whether the supramolecular 
 chirality of all fibril filaments can be controlled by ph . to address 
 these questions we studied aggregation as a function of incubation 
 ph for hen - egg lysozyme , bovine apo--lactalbumin , het - s ( 218289 ) 
 prion - forming domain from fungus podospora anserina , and a short 11-residue peptide fragment of human transthyretin 
 ( ttr ( 105115 ) ) .
the intrinsic supramolecular chirality of 
 these fibrils was measured by vcd , and in parallel , fibril morphology 
 was characterized by atomic force microscopy ( afm ) , scanning electron 
 microscopy ( sem ) , and cryo - sem .
in addition the secondary structure 
 of each of these amyloid fibrils was determined by fourier transform 
 infrared ( ftir ) and deep uv resonance raman ( duvrr ) spectroscopy .
lysozyme , like insulin , forms opposite chiral 
 polymorph depending on the incubation ph .
hen - egg lysozyme is a widely 
 used model protein for amyloidogenic studies that aggregates at low 
 ph and high temperatures , forming rod - like fibrils .
hen - egg lysozyme ( 60 mg / ml ) 
 was incubated at 65 c for 3 days at different ph values .
the solution was centrifuged at 10  000 rpm for 20 min , and 
 the resulting gelatinous solution , dominated by mature fibrils , was 
 analyzed by ir and vcd ( figure 1 ) .
vcd ( a ) and 
 ir ( b ) spectra of lysozyme fibrils grown at ph 1.0 
 ( blue ) , 1.5 ( green ) , 2.3 ( black ) , and 2.7 ( red ) for 3 days at 65 c .
the strong peak at 1623 cm in the ir spectra 
 of all studied samples indicates a dominance of -sheet structure 
 ( figure 1 , bottom ) .
the peak at 1663 cm that increases in intensity 
 with increasing ph is associated with nonordered or loop structure , and may be related to the rate of fibril formation 
 during the specified incubation conditions .
we found that relatively 
 small variations in the ph of the aggregation 
 solution cause dramatic changes in the vcd spectra of lysozyme fibrils , 
 the largest of which is almost 10 times more intense than the corresponding 
 intense vcd spectra observed for insulin fibrils grown under the same 
 ph values . as is evident from the vcd 
 spectra , lysozyme fibrils show normal vcd at ph 2.7 ( figure 1 ) and higher ( data not shown ) .
fibrils that were 
 grown at low ph ( 1.01.5 ) , on the other hand , show reversed 
 vcd with intensity comparable in magnitude but roughly opposite in 
 sign compared to normal vcd .
fibrils that were grown at ph 2.3 have 
 very weak vcd intensity , with small residual vcd peaks for both normal 
 ( negative , 1610 cm ) and reversed ( positive , 1624 
 cm ) fibrils present .
one therefore concludes that 
 ph 2.3 is close to an equilibrium balance point in the distribution of normal and reversed lysozyme fibrils .
similar vcd spectral patterns 
 were obtained for insulin fibrils grown at the same ph values .
we also investigated whether prolonged 
 aggregation at elevated 
 temperature causes any changes in lysozyme fibrils .
solutions with 
 ph from 1.0 to 2.7 ( 60 mg / ml ) were incubated at 65 c for 5 days , 
 and their vcd and ir spectra were recorded ( supporting 
 information figure s1 ) .
we found that vcd intensity of all 
 fibril solutions , except the very small vcd intensity from fibrils 
 incubated at ph 2.3 , decreased compared to fibrils grown for 3 days 
 at 65 c .
in addition to the decrease of vcd intensity we found 
 that the position of a negative peak changed : lysozyme fibrils that 
 were grown at ph 2.7 had a negative peak at 1610 cm ( 3 days of incubation ) and at 1616 cm ( 5 days 
 of incubation ) .
the same band minimum shifted from 1610 cm to 1623 cm for lysozyme fibrils grown at ph 
 2.3 .
fluid - cell afm and sem were utilized 
 to investigate morphological 
 differences between normal and reversed lysozyme fibrils . both afm 
 and sem ( but not tem ) microscopy can provide information about the 
 sense , or handedness , of fibril helical chirality
afm and sem are complementary microscopic methods 
 for the determination 
 of accurate species dimensions : afm provides accurate height data , 
 while width dimensions may be larger due to a finite tip - width convolution 
 error .
sem , on the other hand , may not be utilized for height determination , 
 but does provides accurate width data .
we 
 found that lysozyme fibrils that exhibit reversed vcd have a flat 
 tape - like morphology without a noticeable twist .
they have a height 
 between 2.5 and 3.0 nm and a width in the range of 25 nm .
some of 
 these fibrils are composed of two side - to - side associated fibrils 
 ( figure 2 , b ) .
reported that these 
 tape - like lysozyme fibrils consist of as many as 17 laterally assembled 
 filaments . according to our data ,
binary 
 lysozyme fibrils have the same height as the discussed tape - like fibrils , 
 while their width is significantly larger ( 45 nm ) .
some of these 
 fibrils exhibit a well - defined left - handed twist ( figure 2 , c ; identified by yellow arrows ) .
they have a height 
 between 6 and 8 nm and a width between 18 and 20 nm . as with insulin , lysozyme fibrils exhibiting normal and reversed vcd have cylindrical 
 left - twisted and flat tape - like fibril morphologies , respectively .
sem ( a , 
 c ) and fluid - cell afm ( b , d ) images of reversed vcd ( a , 
 b ) and normal vcd ( c and d ) lysozyme fibrils grown at 65 c for 
 3 days .
helical twisted fibrils ( all left - handed ) are indicated by 
 yellow arrows .
scale bars are 100 nm . it is interesting that neither for insulin nor lysozyme fibrils 
 exhibiting reversed vcd is there any evidence of right - handed twist 
 on their surface .
one might suspect that fibril dehydration after 
 fixation on copper grids for sem imaging could result in significant 
 deformations of fibril topology . as a result , it is possible that 
 a right - twist of reversed vcd fibrils may become distorted and therefore 
 not apparent on sem images . in order to show that reversed vcd fibrils 
 do not have a right - handed twist on their surface we utilized one 
 of the most preservative microscopic tools : cryo - sem .
cryo - sem shows that reversed vcd lysozyme fibrils have 
 flat - like topology without a right - handed twist .
in fact applications 
 of cryo - sem for studies of insulin reversed vcd fibrils showed similar 
 results , while at the same time this method confirmed the left - handed 
 twist of normal vcd insulin fibrils ( supporting 
 information figure s2 ) .
deep uv resonance raman ( duvrr ) 
 spectroscopy has proven to be an 
 efficient technique for characterizing protein structural rearrangements 
 at all stages of fibrillation .
in particular , duvrr spectroscopy , together with hydrogen  deuterium 
 exchange , has been found to be a powerful method for the determination 
 of fibril cross--core structure .
previously , using duvrr 
 spectroscopy we found that both normal and reversed insulin fibrils 
 have the same spectrum and hence the same cross--core structure , 
 while they have different environments for tyrosine ( tyr ) amino acid 
 residues . in the present study we used 
 duvrr spectroscopy to demonstrate the same similarity of cross--sheet 
 structures of normal and reversed vcd lysozyme fibrils with small 
 differences for the aromatic side - chains between normal and reversed 
 fibrils ( supporting information figure 
 s3 ) .
apo--lactalbumin 
 aggregation results in the formation of only reversed vcd fibrils 
 at 37 c .
the protein -lactalbumin is a small ( 14 
 kda ) metal binding protein present in the whey milk of most mammal 
 species as a regulatory subunit of lactose synthase .
-lactalbumin consists of 123 amino acid residues 
 arranged into two domains , designated  and . the two 
 domains are divided by a cleft , where a calcium ion is located .
reduction 
 of two of four disulfide bonds in -lactalbumin results in its 
 adopting a molten globule conformation .
the molten globule state of -lactalbumin is a well - studied 
 structure that has native - like secondary structure with an absence 
 of specific tertiary packing interactions of amino acid side chains .
apo--lactalbumin is of particular interest since it 
 is homologous to lysozyme , albeit with different aggregating and denaturing 
 properties .
aggregates of apo--lactalbumin 
 form long unbranched fibrils under low ph and high ionic strength 
 ( supporting information figure s4 ) .
previously the secondary structure of apo--lactalbumin 
 fibrils has been extensively characterized in our group . in the current study
we investigate the effect of ph on the formation 
 of different vcd chiral polymorphs .
apo--lactalbumin ( 5 mg / ml ) 
 was incubated at different values of ph in the presence of 150 mm 
 nacl at 37 c for 3 days .
after fibril formation was terminated 
 by centrifugation and temperature reduction , the vcd and ir spectra 
 of the sample were measured .
we found that fibrils grown at different 
 ph values show reversed vcd , with a positive peak near 1624 cm and a negative peak near 1639 cm ( figure 3 ) .
vcd ( top ) and ir ( bottom ) spectra of apo--lactalbumin 
 fibrils 
 grown at ph 1.5 ( blue ) , 2.5 ( green ) , 3.0 ( black ) , and 4.0 ( red ) for 
 3 days at 37 c
. as depicted , apo--lactalbumin aggregation at this 
 concentration 
 over the ph range from 1.5 to 4.0 at 37 c results in an excess 
 of only one chiral polymorph that has reversed vcd supramolecular 
 chirality .
fibrils that were grown at ph ( 2.5 ) have the highest intensity 
 of a peak at 1617 cm
. fibrils that were grown 
 at ph 1.5 have relatively smaller intensity at 1617 cm , while the intensity of a negative peak at 1666 cm is almost the same as for ph 2.5 fibrils .
fibrils that were grown 
 at ph 3.0 have smaller intensity for all vcd bands relative to ph 
 1.5 and 2.5 fibrils centered at 1624 cm .
finally , 
 fibrils grown at ph 4.0 have the smallest vcd intensity , and the peak 
 previously observed at 1617 cm for ph 1.5 and 
 2.5 is shifted to 1612 cm .
afm was used 
 to image fibrils of apo--lactalbumin that were 
 deposited onto a freshly cleaved mica surface .
the afm images revealed 
 both tape - like and left - twisted fibrils at all ph values .
tape - like 
 fibrils had 26 nm width and 5 nm height , while left - twisted 
 fibrils had the same width but were over twice as high ( 12 
 nm ) .
fluid - cell 
 afm images of left - twisted ( a ) and tape - like ( b ) fibrils 
 of apo--lactalbumin grown for 3 days at 37 c .
the amount of left - twisted fibrils decreased with 
 an increase of 
 aggregation ph ( supporting information figure 
 s5 ) .
specifically , they were abundant in a fibril solution that was 
 grown at ph 1.5 , while less frequently observed at ph 2.5 .
finally , 
 in the fibril solutions grown at ph 3.0 and 4.0 left - twisted fibrils 
 were extremely rare ( supporting information figure s5 ) .
at the same time , the tape - like morphology was commonly 
 observed for fibrils grown at all ph values .
het - s ( 218289 ) 
 peptide fragment of prion protein forms opposite chiral polymorphs 
 at different ph values .
it was previously shown that at low 
 ph its c - terminal fragment ( het - s 218289 ) slowly aggregates 
 forming amyloid - like fibrils .
several 
 independent studies have shown that ( het - s 218289 ) aggregation 
 at ph 2.0 leads to a formation of thick left - twisted cables composed 
 of 36 filaments . at the same time , its aggregation 
 at ph above 3.0 causes formation of thin fibrils without a noticeable 
 twist .
mizuno et al . proposed that these 
 thin fibrils ought to have a left - handed twist , while no repeatable 
 twist on the cryo - em provided pictures was evident .
these thin fibrils were proposed to form a  solenoid 
 ( -roll ) with a triangular hydrophobic core .
it is also of interest to note that these two ( het - s 218289 ) 
 fibril polymorphs have different infectivity : fibrils at ph 2.0 showed 
 very little if any infectivity , while fibrils formed at ph above 3.0 
 were found to be highly infectious . on the basis of electronic circular dichroism ( ecd ) 
 and ir spectroscopy , thick left - twisted cables and thin fibrils were 
 found to have different secondary structure .
it was also shown that these two polymorphs have different assembly 
 kinetics , resistance to denaturants , and intrinsic fluorescence . in our study
we investigated supramolecular 
 chirality of het - s ( 218289 ) prion fibrils grown at ph 2.0 , 
 3.3 , and 3.9 ( figure 6 ) . 

it appears that ph 2.0 fibrils , which have a left - handed twist ( figure 6 ) , exhibited very intense normal vcd .
thin fibrils 
 grown at ph 3.3 and 3.9 ( figure 5 ) showed vcd 
 spectra of the opposite sign with 12 orders of magnitude lower 
 intensity .
the normal sign patterns for ph 2.0 fibrils are 1557 ( + ) , 
 1578 ( + ) , 1614 (  ) , 1640 ( + ) , and 1666 ( + ) cm , while the reversed sign pattern for ph 3.3 is 1560 (  ) , 
 1611 ( + ) , 1636 (  ) cm .
het - s ( 218289 ) 
 prion fibrils grown at ph 3.9 exhibit the same reversed sign pattern 
 but have some differing vcd band frequencies : 1557 ( + ) , 1614 ( + ) , 
 and 1660 (  ) cm .
vcd ( a ) and ir ( b ) spectra 
 of het - s ( 218289 ) fibrils grown 
 at ph 2.0 ( blue ) , 3.3 ( green ) , and 3.9 ( black ) for 2 months at room 
 temperature . for better visualization of band positions and intensities 

the insert shows vcd spectra of only ph 3.3 ( green ) and ph 3.9 ( black ) 
 fibrils .
afm images of het - s ( 218289 ) prion fibrils 
 grown in ph 
 2.0 ( a ) and 3.9 ( b ) .
the ir spectra of all studied samples exhibit a peak around 
 1620 
 cm , indicating a predominance of -sheet 
 structure ( figure 5b ) .
it was found that that the position of this peak is slightly shifted 
 in the spectrum of fibrils grown at ph 2.0 ( 1624 cm ) compared to the corresponding ir band for fibrils grown at ph 3.3 
 and 3.9 fibrils ( 1620 cm ) .
moreover , the ir at 
 ph 2.0 has higher intensity for peaks at 1725 and 1671 cmcompared to those of fibrils grown at ph 3.3 and 3.9 .
these differences 
 indicate structural deviations of normal and reversed vcd chiral polymorphs 
 of het - s fibrils . for more detailed structural characterization of 
 their secondary structure we applied duvrr ( supporting 
 information figure s6 ) .
the positions of amide i , ii bands 
 and c-h band are nearly identical in the spectra 
 of both chiral polymorphs , which indicates a high level of similarity 
 of secondary structure for all three fibril ph values . at the same 
 time
, we found that het - s fibrils that were grown at ph 3.9 have substantially 
 higher amount of unordered structure ( a peak 1268 cm ) compared to the ph 2.0 fibrils .
afm was utilized to reveal morphological organization of the analyzed 
 het - s ( 218289 ) prion fibrils .
we found that fibrils grown 
 at ph 2.0 have a well - defined left - handed twist , 40 nm in width and 
 around 3 nm in height ( figure 7 ) . at the same 
 time , fibrils that were grown at ph 3.9 ( reversed vcd ) exhibited no 
 twist on their surface .
they had a shape of flat tapes that have 25 
 nm in width and 2 nm in height .
vcd ( top ) and ir ( bottom ) 
 spectra of ttr ( 105115 ) fibrils 
 grown at ph 1.0 ( blue ) , 1.5 ( green ) , 2.0 ( black ) , 2.5 ( red ) , and 3.0 
 ( violet ) for 2 days at 37 c followed by 14 days at room temperature .
opposite chiral 
 polymorphs are observed for fibrils formed from a small peptide of 
 human transthyretin .
ttr ( 105115 ) is a peptide fragment of 
 transthyretin , a 55 kda protein involved in the transport pathway 
 of thyroxine and retinol in plasma .
although 
 this peptide is itself not implicated in any human disease , fibrillization 
 of the full - length protein has been linked to familial amyloid polyneuropathy 
 ( fap ) and senile systemic amyloidosis ( ssa ) .
transthyretin has a -sheet rich structure and can form amyloid - like 
 aggregates in vivo .
previously , it was demonstrated 
 that ttr ( 105115 ) is able to aggregate in vitro , forming amyloid - like fibrils .
we grew 
 ttr ( 105115 ) fibrils as described previously , at different ph values for 2 days at 37 c followed 
 by 14 days at room temperature .
as shown in figure 7 , the ir spectra of ttr ( 105115 ) feature 
 a strong band at 1623 cm that is characteristic 
 of -sheet structure . as with insulin , lysozyme , and het - s prion 
 fibrils described above
, the vcd sign of ttr ( 105115 ) reverses 
 as a function of small changes in ph during protein aggregation .
ttr 
 ( 105115 ) fibrils formed at ph 2.0 and above show strong reversed vcd , and the intensity of the positive 1624 cm vcd band increases within increase of aggregation 
 ph . similar to het - s fibrils , and opposite insulin and lysozyme , higher 
 ph favors formation of the reversed vcd for ttr ( 105115 ) fibrils . 

the magnitude of reversed vcd intensities observed for these fibrils 
 is nearly the same as those previously observed for reversed vcd insulin 
 fibrils ( formed at low ph ) .
by contrast , ttr ( 105115 ) fibrils 
 grown at ph values 1.5 and 1.0 show normal vcd with 
 their vcd intensities in the range 26 times smaller than the 
 vcd intensities of reversed ttr ( 105115 ) fibrils .
afm 
 was applied to characterize morphologies of normal and reversed 
 vcd ttr ( 105115 ) fibrils ( figure 8) . 

we found that normal and reversed vcd afm images of ttr ( 105115 ) 
 display different morphologies .
ttr ( 105115 ) fibrils that 
 were grown at ph 1.5 are long and have cylindrical shapes . together 
 with long rod - like fibrils we observed large amounts of small spherical 
 species , which may be fibril precursors ( figure 8a ) .
ttr ( 105115 ) fibrils grown at higher ph ( ph 2.5 ) are 
 significantly shorter than fibrils grown at ph 1.5 .
in addition to the diverse morphologies fibrils 
 with normal and reversed vcd , ttr ( 105115 ) fibril polymorphs 
 have different heights : normal ( ph 1.5 ) vcd fibrils are around 5 nm , 
 while reversed vcd ( ph 2.0 ) fibrils are around 9 nm .
afm images of ttr ( 105115 ) 
 fibrils grown at ( a ) ph 1.5 
 and ( b ) ph 2.5 .
more detailed surface analysis of ttr ( 105115 ) fibrils 
 grown at ph 2.5 ( figure 8b ) indicates that 
 some of them are composed of several side - by - side associated proto - fibrils . 

we did not observe any twist either on the surface of this chiral 
 polymorph or on the surface of fibrils grown at ph 1.5 .
flat - like 
 topology of ttr ( 105115 ) fibrils grown at ph 2.0 , 37 c 
 ( here reversed vcd ) was previously confirmed by jaroniec et al .
it was shown that these fibrils are composed 
 of several side - by - side associated proto - fibrils .
the application 
 of duvrr spectroscopy allowed us to elucidate the 
 secondary structure organization of both normal and reversed vcd of 
 ttr ( 105115 ) fibrils ( supporting information figure s7 ) .
both chiral polymorphs of ttr ( 105115 ) fibrils 
 have narrow and intense amide i , ii , and iii bands , which indicate 
 that they share the same cross- core structure .
we found that 
 the intensity of the tyr band is also identical in both normal and 
 reversed vcd ttr ( 105115 ) fibrils , indicating high similarities 
 in tyr local environment of both polymorphs .
with the results presented here , vcd intensity , enhanced by more 
 than 1 order of magnitude over isolated protein levels , has been observed 
 for three additional amyloid fibrils , namely apo--lactalbumin , 
 the het - s ( 218289 ) prion - forming domain , and ttr ( 105115 ) 
 peptide fragment from transthyretin . in a separate publication ,
similarly 
 enhanced vcd has also been reported for a number of polyglutamine 
 ( polyq ) fibrils from q15 to q45 .
also with this paper , control of the sense of amyloid fibril chirality , 
 normal versus reversed , by incubation under relatively small variations 
 in ph , previously reported only for insulin , has been extended to lysozyme as well as apo--lactalbumin , 
 het - s ( 218289 ) , and ttr ( 105115 ) . as with insulin , 

the sign pattern , defined above , for enhanced vcd intensity is ( +  +    +  + ) 
 for normal fibrils , and the nearly mirror - image relative intensity 
 pattern with signs (     +   
 ) 
 for reversed fibrils reflects an underlying level of fibril chirality . 
 of these signed bands ,
the band near 1620 cm , 
 corresponding to the principal ir marker band for fibril protein structure , 
 is the most intense and is the primary marker band for vcd fibril 
 chirality .
the principal focus of this paper is to correlate 
 fibril supramolecular 
 chirality , based on the sign pattern of enhanced vcd , to the two major 
 classes of fibril morphology , either left - twisted or flat tape - like , 
 as revealed by afm or sem imaging .
the details of this correlation 
 from results described in the previous section , as well as the earlier 
 reported results for insulin , are summarized in table 1 for ease of comparison .
for the proteins and peptide 
 fragments reported here , each has a different sensitivity to ph although 
 the sensitivity of lysozyme is similar to that of insulin : normal 
 vcd is observed above approximately ph 2.2 and reversed vcd below 
 that ph .
though , no point of vcd sign reversal is reached between 
 ph 1.5 and 4.0 . if it were to reverse sign pattern , it is predicted 
 to do so only below ph 1.5 when the population of left - twisted fibrils 
 becomes sufficiently dominant relative to flat tape - like , as reasoned 
 further in the next paragraph .
we have found that , at ph 1.5 , the 
 relative populations of reversed tape - like fibrils and normal left - twisted 
 fibrils marginally favor the left - twisted fibrils ( see table 1 and supporting information figure s5 ) ; however , the vcd spectrum indicates in figure 3 that reversed tape - like fibrils are more influential , 
 perhaps because reversed fibrils in this region have intrinsically 
 higher vcd intensity . at ph 2.5 , the reversed vcd increases ,
as the 
 population of normal left - twisted fibrils decreases , in agreement 
 with the idea that these two morphologies have opposite vcd and reduction 
 of left - twisted fibrils permits an overall increase in the observed , 
 net - reversed vcd spectrum . as the ph
is increased to 3.0 and then 
 to 4.0 , the population of left - twisted fibrils becomes close to zero , 
 but unexpectedly the observed reversed vcd decreases dramatically 
 from ph 2.5 to 3.0 and then to a relative small value for ph 4.0 . 

this reduction must solely be due to a reduction in the long - range 
 chirality of the flat tape - like fibrils . from supporting information figure
s5 the observed height distribution 
 of the flat tape - like fibrils narrows considerably to near 6 nm with 
 increasing ph .
one possible explanation for these observations is 
 that as the ph increases beyond ph 2.5 , the stability of the aggregation 
 of right - handed ( reversed ) filaments that form multifilament tape - like 
 fibrils decreases until at ph 4 only the lowest level of fibril structure 
 remains , possibly only individual filaments having no lateral associations 
 of filaments . beyond ph 4 ,
not even the simplest fibril filament structures 
 are stable , and fibrillation of any kind no longer occurs .
thus , although 
 the ph dependence of apo--lactalbumin differs substantially 
 from that of insulin and lysozyme , our understanding of the vcd and 
 afm imaging for this protein still supports the evidence that the 
 sign pattern for vcd correlates without exception to the morphology 
 of the observed microscopic images of the fibrils grown at various 
 ph values . the two peptide fragments , het - s ( 218289 ) 
 and ttr ( 105115 ) , 

show the opposite vcd sign behavior compared with insulin and lysozyme , 
 with normal fibrils appearing at low ph and reversed fibrils at high 
 ph , although in these two cases the level of vcd enhancement differs 
 dramatically . for the het - s ( 218289 ) peptide ,
the low - ph normal 
 vcd is extremely large with high - ph reversed vcd significantly smaller , 
 whereas for the ttr ( 105115 ) peptide the low - ph normal vcd 
 is small and the high - ph reversed vcd is much larger .
one possible 
 influence beyond the value of ph was explored by introducing 
 differing amounts of sodium chloride and thereby changing the ionic 
 strength at a given ph value .
experiments to date have shown no such 
 influence of ionic strength ( supporting information figure s8 ) .
another variable partially explored is the effect of 
 incubation temperature , duration of incubation , and time after incubation . 
 here
variations in vcd intensity have been observed from which one 
 concludes , as is well - known from many previous studies , that fibril 
 formation , development , and stability can depend significantly on 
 these variables . while studying the dependence of lysozyme fibril 
 supramolecular 
 chirality on incubation conditions we found that prolonged fibril 
 exposure at the elevated temperature results in a decrease of vcd intensity .
as previously demonstrated , fibril formation and 
 development is associated with the continuous increase in the vcd 
 intensity of the analyzed solution .
since 
 fibril fragmentation is the opposite process , it should be associated 
 with a decrease in vcd intensity .
therefore , 
 one would expect that the observed decrease of fibril vcd intensity 
 for lysozyme is indicative of fibril fragmentation during extended 
 development .
in fact , this propensity of fibrils has been extensively 
 studied and proposed to be an intrinsic mechanism of fibril self - propagation , 
 since each individual fibril fragment may act as a seed and give a 
 birth to a new full - length fibril . in the results section
, we noted on several 
 occasions that the sign of the major vcd peak appears to shift from 
 the region of 1620 cm to near 1610 cm .
however , there is no corresponding shift in the peak maxima of 
 the corresponding ir bands near 1620 cm .
a highly 
 likely explanation for the large frequency shift away from the vicinity 
 of 1620 cm is that two opposite - signed vcd bands , 
 with peak maxima only a few wavenumbers apart , interfere and give 
 rise to a couplet of opposite signed vcd bands reduced in intensity 
 but separated by a frequency difference related with the widths of 
 the individual opposite signed vcd bands .
such interference and splitting 
 for peak maxima do not occur for the parent ir bands since their intensity 
 contributions reinforce rather than partially cancel . combining 
 these vcd observations with correlated afm images as 
 summarized in table 1
reveals a correlation , 
 without exception , of the normal vcd sign pattern with fibrils displaying 
 a predominantly rounded left - twisted helical morphology and the reversed 
 vcd sign pattern with fibrils having a flat tape - like morphology . 
 for the flat tape - like fibrils
there is no indication of fibril chirality 
 in their afm or sem images . in the case of insulin , this correlation 
 led us to propose that the enhanced , opposite - signed , mirror - image - like 
 intensity pattern of vcd must have its origins at a deeper level of 
 fibril structure than that visible for fibril morphology using afm 
 or sem imaging .
further , we proposed 
 that opposite vcd sign patterns for normal and reversed fibrils originated 
 at the level of individual fibril filaments , the 
 simplest level of fibril cross- core structure , with filament 
 axial helical twists of opposite sense , left - handed for normal fibrils 
 and right - handed for reversed fibrils
. it follows from this proposition that left - twisted filaments form 
 braids of multiple - filament left - twisted fibrils with observable left - twisted 
 morphology , while right - twisted fibrils do not braid and instead adopt 
 a flat tape - like morphology consisting of parallel side - by - side filaments . 
 to our knowledge , imaging the chirality of individual filaments has 
 not been conclusively observed , and consequently , vcd 
 to date is the only known structure probe of fibril chirality at the 
 level of individual filaments .
this point 
 is reinforced by the fact that vcd can follow fibril formation as 
 intensity enhancement from the earliest stages of filament formation , and that the enhanced vcd intensity grows without a substantial 
 change in the relative intensity pattern as filaments first form , 
 then associate and develop into mature fibrils .
further in the case 
 of insulin , on the basis of the observation that reversed fibrils 
 spontaneously convert to normal fibrils upon raising the ph into the 
 normal fibril ph range whereas normal fibrils do not convert to reversed 
 fibrils when the ph is correspondingly reduced to the reversed fibril 
 ph range , we have concluded that normal left - twisted mature fibrils 
 are more thermodynamically stable , at least for insulin , than flat 
 tape - like fibrils .
this correlates with 
 the idea that left - twisted filament - braided mature fibrils are in 
 deeper thermodynamic sinks from which reversal or decomposition requires 
 extremes of temperature or ph .
we have also considered whether 
 there is any correlation between 
 the ph dependence of filament chirality and pi values for the protein / peptide 
 and found that there appears to be little or no correlation between 
 protein pi and ph dependence of filament chirality .
for example , the 
 pi for insulin is 5.305.35 , while for lysozyme the pi is 10.610.9 . 

nevertheless , both of these proteins exhibit nearly the same ph dependent 
 fibril chirality . at the same time , the pi of het - s ( 218289 ) 
 is 9.19 , while it exhibits the opposite ph chirality dependence of 
 lysozyme ( pi 10.610.9 ) .
for apo--lactalbumin the pi 
 ( 4.24.5 ) is very close to the 
 pi of insulin ( 5.305.35 ) ; however , we observed formation of 
 only one chiral polymorph for apo--lactalbumin . finally , het - s 
 ( 218289 ) and ttr ( 105115 ) exhibit similar ph dependence 
 of fibril chirality ( normal at low ph and reversed at high ph ) . nevertheless , 
 their pis are substantially different : 9.19 for het - s ( 218289 ) 
 and 6.09 for ttr ( 105115 ) .
the only correlation might be that 
 the substantial difference in pi s of apo--lactalbumin 
 ( 4.24.5 ) and lysozyme ( pi 10.610.9 ) might be related 
 to the difference in ph chirality dependence for these two isostructural 
 proteins in their native states .
although the focus here is to relate the sign pattern of 
 the enhanced 
 vcd to the major classes of fibril morphology , inspection of the ir 
 absorption spectra for fibrils described in figures 1 , 3 , 5 , and 7 reveals variations in a band in the 16501670 
 region that also shows intensity differences that correlate with vcd 
 sign and fibril morphology . not as dramatic as the vcd sign pattern , 
 there is likely interesting information about the differences between 
 left - twisted and flat tape - like fibrils associated with these ir bands . 
 in the results section for lysozyme we attributed 
 this band to nonordered or loop structure .
an open question to be 
 pursued in future studies is whether this band is associated with 
 a fully formed fibril filament ( cross--core ) or with partially 
 unfolded proteins yet to be incorporated into the filament or fibril 
 structure .
at least for lysozyme and insulin ( not shown here ) , this 
 band may be associated with parts of the protein structure not in 
 the cross--core for which there would be more residues than 
 for the shorter peptides from het - s and ttr .
finally , examination 
 of the structural organization of the opposite 
 chiral polymorphs using duvrr and ir spectroscopy shows that insulin , 
 lysozyme , and ttr ( 105115 ) fibril polymorphs have the same 
 secondary structure . at the same time
, we have found that normal and 
 reversed vcd chiral polymorphs of het - s ( 218289 ) fibrils have 
 small differences secondary structure distributions with the same 
 cross--core structure .
although 
 the primary findings of this paper are drawn from new 
 experimental results , namely vcd spectra and afm / sem images as a function 
 of incubation ph for four protein molecules beyond our previously 
 published findings for insulin , we offer in this section our views 
 of the theoretical , molecular - level implications for what the experimental 
 evidence suggests .
the theoretical 
 model of schweitzer - stenner is the first 
 and simplest computational model 
 of enhanced vcd in fibrils , first observed by ma et al .
the model is based on exciton coupling among 
 amide i transition dipoles arrayed as dual , stacked -sheet 
 ribbons corresponding to the simplest representation of the structure 
 of a single cross - beta core filament .
the strands 
 in this model , only two peptide units long , run perpendicular to the 
 filament axis direction . in order to generate enhanced vcd ,
a twist 
 of 2 was introduced between the strands yielding a long - range 
 gradual helical twist to the filament structure with a one full - turn 
 distance of 180 strands .
it was found that such a structure yields 
 enhanced vcd that corresponds to what is referred to as normal vcd 
 ( negative vcd near 1620 cm with positive vcd to 
 higher wavenumber frequencies ) for a left - hand helical fibril twist . 
 a corresponding 2 intrastrand helical twist in the opposite 
 direction , corresponding to a right - hand helical filament , reversed 
 the sign of the enhanced vcd couplet and corresponds to our reversed 
 vcd .
filament chirality is therefore a very delicate , subtle level 
 of protein chirality that is apparently sensitive to incubation ph . 

most likely , the sensitivity of amino - acid side chains to the aqueous 
 solvent is responsible for this ph sensitivity , and apparently , differing 
 arrays of constituent side - chains , exposed or not to the solvent , 
 results in different ph sensitivity ( high versus low ) for the sense 
 of filament chirality observed .
this is a long - range fibril property 
 that likely can not be predicted , even qualitatively , without a realistic 
 model of protein side chain influence on the sense of helical chirality 
 as a function of ph .
as far as we are aware , the chirality of individual 
 fibril filaments lies below the sensitivity of afm or sem imaging , 
 but can be observed with vcd at the initial and subsequent stages 
 of fibril formation .
it is known from afm 
 and sem images that fibril filaments form higher levels of structure 
 subsequent to the formation of individual filaments .
it has been demonstrated , 
 for example by kinetic studies , that 
 the degree of enhancement of vcd is sensitive to the degree of filament 
 association as fibrils mature and develop .
this is particularly true 
 for the left - twisted morphologies composed of multifilament chiral 
 braids of left - helical filaments and to a lesser degree for the flat 
 morphologies of side - by - side associations of right - helical filaments . 

an important unanswered question is why only left - helical filaments 
 form braids corresponding to the left - twisted fibril morphology as 
 illustrated diagrammatically in figure 9 .
the 
 answer most likely is that left- and right - helical filaments are not 
 mirror - image structures as both consist of l - amino acid residues . 

a model calculation of this property , the appearance of left - twisted 
 braids and nonappearance of right - twisted braids of helical filaments , 
 again involves consideration of the structure and influence of amino 
 acid side chains .
the model of schweitzer - stenner applies only to 
 the filament level of structure and does not include any side chain 
 chirality ( l- or d - amino acid residues ) or filament  filament 
 interactions or braiding . answering
the question of why only left - twisted 
 fibril images and vcd are observed is two levels beyond the schweitzer - stenner 
 ( s - s ) model of enhanced vcd .
one first needs a more realistic model 
 of cross--core filament structure that includes side chain 
 detail , and second , one needs to extend such a model to a new , higher 
 level of fibril chirality , namely the chiral braiding of multiple - filament 
 fibril structure .
diagram illustrating the progression from folded protein 
 to individual 
 straight filament with chirality below the senstivity of afm / sem and 
 on to mature multifilament fibrils with right helical filaments forming 
 flat tape - like fibrils of straight filaments and left helical filaments 
 forming , possibly more stable , left twisted braids of filaments .
the 
 ph dependence may vary between proteins / peptides but not the vcd intensity 
 patterns ( normal left helical filaments versus reversed for right 
 helical filaments ) .
a step in this direction 
 has just been published by welch , kubelka , 
 and keiderling ( wkk ) .
they employ modeling at the level 
 of a full dft calculation with transfer of parameters from smaller 
 peptides to build large -sheet structures .
their model is quite 
 different in dimension from the ( s - s ) model although both use stacked 
 -sheets to simulate the cross--core structure .
the wkk 
 model involves strands that are 10 residues instead of 2 residues 
 wide , but the fibril axis is only 5 strands long rather than in the 
 100-strand range . to induce enhanced vcd , they introduce a twist along 
 the strand direction , not the fibril axis direction , of 15 between 
 residues .
they also explore a rotation of the stacked sheets relative 
 to each of 45 , again a chiral departure from an initial achiral 
 cross- core structure .
these two degrees of chiral freedom 
 may be important as filaments aggregate and braid about a common axis . 

the point needs to be explored as the models move beyond individual 
 filament models and on to multifilament simulations of more mature 
 fibrils .
nevertheless , it seems reasonable that chirality along the 
 filament axis , as emphasized in this paper ( see figure 9 ) , as opposed to the more local strand or intersheet twist 
 chirality , is the form of filament chirality 
 that can be extended most easily to the supramolecular level of filament 
 structure and hence to vcd enhanced by up to 2 orders of magnitude 
 relative to the isolated - protein vcd intensity level . at this 
 stage , even with the recent work of wkk , there are no structural 
 models of interfilament association leading to the formation of different 
 fibril morphologies .
it is our view that the realization of a model , 
 even at the course grain level , that is capable of rationalizing the 
 correlation of left - helical filaments to left - twisted fibril morphology 
 and right - helical filaments to flat fibril morphology , is beyond the 
 reach of near - term possibilities and must await further advances in 
 our understanding of the unusual sensitivity of vcd to filament and 
 mature fibril chirality as well as the many chiral degrees of freedom 
 that may influence fibril morphology .
two further observations 
 relevant to this discussion have been 
 established previously for insulin and remain to be confirmed more 
 systematically for the new fibril results presented here .
as fibrils 
 grow by adding filaments and becoming thicker , the vcd grows from 
 initially observed levels , whereas the ir intensity does not grow 
 in overall magnitude .
this is more dramatically true for the braided left - twisted morphology 
 where an additional order of magnitude of vcd may be observed compared 
 to the initial enhanced vcd of newly formed filaments .
this strongly 
 implies a second level of chirality beyond the filament level that 
 is ir intensity neutral .
second , the left twisted morphology is more 
 stable than the flat tape - like morphology .
thus , the interfilament interactions of the left - twisted braided 
 fibril consisting of left - helical filaments are stronger than the 
 corresponding side - by - side interactions of the flat tape - like fibrils 
 composed of right - helical filaments .
a successful model of fibril 
 morphology and associated vcd intensity should reflect this difference 
 in thermodynamic stability .
the 
 results of the present study , and the absence of any observable 
 chirality by afm for flat tape - like fibrils , extends , beyond the case 
 of insulin , the correlation of the sign of vcd intensity pattern with 
 the two major classes of amyloid fibril morphology , round left - twisted 
 and flat tape - like . also extended by this study ( to fibrils of four 
 new protein or peptide fragments ) is the hypothesis that the near 
 mirror - like opposite - signed vcd patterns associated with these two 
 morphologies arise from the opposite sense of helical twist of individual 
 fibril filaments that comprise observed multifilament , more - fully 
 developed fibrils .
further , for all five analyzed amyloid fibrils 
 documented in table 1 , filament chirality appears 
 to lie below the level of direct detection of afm or sem imaging . 

finally , there is wide variability in the ph dependence of filament 
 chirality , being one sense ( reversed , right - handed at lower ph and 
 normal , left - handed at higher ph ) for the proteins insulin , lysozyme , 
 and apo--lactalbumin , and the opposite sense ( normal , left - handed 
 at lower ph and reversed right - handed at higher ph ) for the peptide 
 fragments het - s ( 218289 ) from prion protein , and ttr ( 105115 ) 
 from transthyretin .
we have investigated whether there is any correlation 
 between ph dependence of filament chirality and the pi of the protein / peptide 
 and found no direct correlation .
thus , we have shown extensive new 
 evidence that supports an affirmative answer to the question posed 
 in the title of this paper .
namely , yes , the chirality 
 of individual amyloid fibril filaments does indeed appear to be the 
 underlying cause of the major morphological differences ( round twisted 
 versus flat tape - like ) in all amyloid fibrils explored to date by 
 vcd .
nevertheless , the protein / peptide fibrils sampled for morphology 
 and vcd is so far limited to five , albeit somewhat diverse , cases , 
 and there well could be surprises and exceptions in future studies . 

a final understanding of the correlations reported here must await 
 sufficiently realistic and accurate modeling of filament and fibril 
 chirality together with the calculation of the associated vcd spectra .
 the unique enhanced 
 sensitivity of vibrational circular dichroism 
 ( vcd ) to the formation and development of amyloid fibrils in solution 
 is extended to four additional fibril - forming proteins or peptides 
 where it is shown that the sign of the fibril vcd pattern correlates 
 with the sense of supramolecular filament chirality and , without exception , 
 to the dominant fibril morphology as observed in afm or sem images . 
 
 previously for insulin , it has been demonstrated that the sign of 
 the vcd band pattern from filament chirality can be controlled by 
 adjusting the ph of the incubating solution , above ph 2 for  normal  
 
 left - hand - helical filaments and below ph 2 for  reversed  
 
 right - hand - helical filaments . from afm or sem images , 
 left - helical 
 filaments form multifilament braids of left - twisted fibrils while 
 the right - helical filaments form parallel filament rows of fibrils 
 with a flat tape - like morphology , the two major classes of fibril 
 morphology that from deep uv resonance raman scattering exhibit the 
 same cross--core secondary structure . here 
 we investigate whether 
 fibril supramolecular chirality is the underlying cause of the major 
 morphology differences in all amyloid fibrils by showing that the 
 morphology ( twisted versus flat ) of fibrils of lysozyme , apo--lactalbumin , 
 het - s ( 218289 ) prion , and a short polypeptide fragment of 
 transthyretin , ttr ( 105115 ) , directly correlates to their 
 supramolecular chirality as revealed by vcd . 
 the result is strong 
 evidence that the chiral supramolecular organization of filaments 
 is the principal underlying cause of the morphological heterogeneity 
 of amyloid fibrils . 
 because fibril morphology is linked to cell toxicity , 
 the chirality of amyloid aggregates should be explored in the widely 
 used in vitro models of amyloid - associated diseases . 
Introduction
Results
Discussion
Theoretical Considerations
Conclusions