the supramolecular assembly of the -amyloid 
 peptide a 
 to form fibrils and soluble oligomers has been the subject of intense 
 interest and study over the past two decades .
the plaques formed by 
 the 4042 amino acid a polypeptide in the brain are 
 one of the most distinctive physiological features of alzheimer s 
 disease , while the more cryptic soluble a oligomers that also 
 form are now thought to be the primary culprits in the devastating 
 neurodegeneration that occurs . in -amyloid 

the oligomers also 
 appear to involve -sheet formation , but their structures are 
 still largely unknown at atomic resolution . enhanced 
 understanding of the structures and interactions of the oligomers 
 and fibrils offers the promise of preventing and treating alzheimer s 
 and other amyloid diseases .
the highly amyloidogenic central 
 region of a , which includes 
 the hydrophobic pentapeptide sequence lvffa ( a1721 ) has provided an archetype not only for the assembly of a 
 but also for amyloidogenic peptides and proteins in general .
this region is particularly prone to interaction , 
 and peptides derived from a1721 have been 
 found to inhibit the aggregation of full length a. the hydrophobic residues 1721 are flanked 
 by cationic and anionic residues k16 and e22 , making the heptapeptide sequence klvffae ( a1622 ) especially prone to supramolecular assembly to form fibrils and 
 nanotubes .
we recently began using 
 macrocyclic -sheet peptides containing 
 the nonapeptide sequence qklvffaed ( a1523 ) as a model system with which to explore the structures and interactions 
 of amyloid oligomers .
we incorporated the a1523 nonapeptide into a 66-membered ring macrocycle containing template 
 and turn units that help enforce a -sheet structure and block 
 uncontrolled aggregation , and we studied the supramolecular assembly 
 of the resulting macrocyclic -sheet peptides 1 in the solid state by x - ray crystallography and in aqueous solution 
 by nmr spectroscopy .
macrocyclic -sheets 1 contain an a1523 peptide strand 
 connected through two -linked ornithine turn units ( orn ) to a template strand that contains two hao amino acid tripeptide 
 mimics . in the solid state , macrocyclic -sheet 
 peptide 1a forms tetramers , dodecamers , and porelike 
 assemblies of oligomers . in solution ,
macrocyclic -sheets 1 form tetramers that differ in structure from those in the 
 solid state .
the differences between the solid - state and solution - state 
 tetramers are important , because they reveal polymorphism among amyloid 
 oligomers at atomic resolution and illustrate the importance of environment 
 upon oligomer structure . in the current study , we envisioned 
 replacing the template strand 
 with a1523 and determining the structures 
 of the oligomers that form .
attempts to synthesize and study macrocyclic 
 -sheet peptide 2 , which embodies this concept , 
 proved fruitless , yielding only an insoluble peptide hydrogel . incorporating 
 two full strands of a1523 into a macrocycle 
 without a template designed to block aggregation appeared to give 
 a peptide that was highly amyloidogenic . to reduce the amyloidogenicity 
 of the macrocycle ,
we prepared macrocyclic -sheet peptide 3 , which incorporates a single hao amino acid in place of 
 the f19f20a21 tripeptide segment of macrocyclic -sheet 2 , 
 to give an a1523 hybrid strand . to facilitate 
 phase determination through single anomalous dispersion ( sad ) phasing , 
 we incorporated p - iodophenylalanine ( f ) in place of f20 in the a1523 peptide strand . here , we report the x - ray crystallographic 
 structure of macrocyclic 
 -sheet 3 . we compare the solid - state supramolecular 
 assembly to that of the oligomer observed in solution .
we describe 
 a new mode of assembly of a1523 in the solid 
 state  a fibril - like assembly of oligomers  that resembles 
 both fibrils and oligomers .
in 
 the solid state , macrocyclic -sheet 3 forms hydrogen - bonded 
 dimers arranged in a herringbone fashion 
 in offset layers that pack through hydrophobic interactions .
the resulting 
 supramolecular assembly differs substantially both from that which 
 we have observed previously for macrocyclic -sheets 1(8,9 ) and that which others have previously observed for 
 a. macrocyclic -sheet 3 readily formed 
 crystals under sparse - matrix screening conditions with kits from hampton 
 research .
crystals suitable for x - ray crystallography were grown from 
 a 3.5 mg / ml solution with 0.1 m sodium citrate at ph 7.3 , 0.1 m ammonium 
 acetate , and 30% 2-methyl-2,4-pentanediol .
crystal diffraction data 
 were collected on beamline 7 - 1 at the stanford synchotron radiation 
 lightsource ( ssrl ) at 1.00  wavelength to 1.75  resolution . 

the structure was solved in the c2 space group with 
 49% pseudomerohedral twinning to give 
 a model with rfree = 22.0% and rwork = 17.9% ( table 1 ) .
the asymmetric unit contains two molecules of macrocyclic -sheet 3 and two molecules of 2-methyl-2,4-pentanediol .
rfree was computed as rwork using 
 a cross - validation 
 set of 10% nonredundant data .
macrocyclic -sheet 3 crystallizes as a folded 
 monomer in which the a1523 peptide strand 
 and the a1523 hybrid strand form a hydrogen - bonded 
 -sheet .
two conformers of the macrocycle occur in the asymmetric 
 unit , differing in rotamer of f19 and tilt of the hao amino 
 acid .
macrocyclic -sheet 3 forms a hydrogen - bonded 
 dimer , in which the two conformers hydrogen bond to form a four - stranded 
 antiparallel -sheet . the a1523 peptide 
 strands of the macrocycles make up the dimerization interface and 
 are fully aligned , with residues 1523 of one of the macrocycles 
 paired with residues 2315 of the other through eight hydrogen 
 bonds . the side chains of residues q15 ,
l17 , 
 f19 , a21 , and d23 of the a1523 peptide strands and q15 , l17 , and d23 of the a1523 hybrid strands decorate one of the surfaces of 
 the four - stranded antiparallel -sheet dimer ; the side chains 
 of residues k16 , v18 , f20 , and e22 of the a1523 peptide 
 strands and k16 , v18 , and e22 of the a1523 hybrid strands 
 decorate the other surface .
figure 2 illustrates 
 the structure of the hydrogen - bonded dimer and the two surfaces .
we 
 term the two surfaces the lfa face and the vf face for the discussion 
 of the higher - order supramolecular assembly of the dimers that follows .
x - ray 
 crystallographic structure of the hydrogen - bonded dimer of 
 macrocyclic -sheet peptide 3 . ( a ) cartoon illustration 
 of the hydrogen - bonded dimer . ( b ) the lfa face of the hydrogen - bonded 
 dimer , bearing the side chains of residues q15 , l17 , f19 , a21 , and d23 of the a1523 peptide strands and q15 , l17 , and d23 of the a1523 hybrid strands .
( c ) the vf face of the hydrogen - bonded 
 dimer , bearing the side chains of residues k16 , v18 , f20 , and e22 of the a1523 peptide strands and k16 , v18 , and e22 of the a1523 hybrid strands .
the hydrogen - bonded dimers assemble to form an extended -sheet 
 that runs the length of the crystal lattice .
the a1523 hybrid strands form the interfaces between the dimers . at the interfaces , 

the a1523 hybrid strands are not fully aligned , 
 but rather are shifted out of alignment by two residues toward the 
 c - termini . as a result of the shift in alignment , the -strands 
 comprising the -sheets are not orthogonal to the axis formed 
 by the extended -sheet , but rather are rotated approximately 
 20 from orthogonality .
assembly of 
 hydrogen - bonded dimers in the x - ray crystallographic 
 structure of macrocyclic -sheet peptide 3 .
( a ) 
 extended -sheet that runs the length of the crystal lattice . 

( b ) packing of the extended -sheets to form a two - layered structure .
the extended -sheets formed 
 by the hydrogen - bonded dimers 
 pack through the vf faces to form a two - layered structure .
instead , each dimer 
 in one layer sits over the interface between two dimers in the opposite 
 layer .
the hao amino acids come together at the interface 
 between the 
 hydrogen - bonded dimers , tilting alternately upward and downward and 
 stacking to accommodate hydrogen - bonding interactions between the 
 a1523 hybrid strands .
the side chains of the 
 v18 , f20 , and v18 residues create a hydrophobic core that runs along the axis formed 
 by the extended -sheet .
the stacked hao amino acids 
 and the iodine of f20 help fill the void created 
 by the absence of f20 in the a1523 hybrid strand .
the 2-methyl-2,4-pentanediol solvent that crystallizes 
 with macrocyclic -sheet 3 packs alongside the 
 hydrophobic core and further stabilizes the two - layered structure 
 through additional hydrophobic interactions .
( a ) interaction between 
 the hao amino acids at the interface between 
 dimers in the x - ray crystallographic structure of macrocyclic -sheet 
 peptide 3 . ( b ) hydrophobic core formed by the side chains 
 of the v18 , f20 , and v18 residues , between the layers of the extended -sheets in the 
 x - ray crystallographic structure of macrocyclic -sheet 3 . in
aqueous solution , macrocyclic 
 -sheet 3 forms discrete tetramers comprising a 
 sandwich formed by two hydrogen - bonded dimers .
the solution - state 
 tetramer is similar in structure to that which we have previously 
 observed for macrocyclic -sheets 1 .
the dimer subunits form through hydrogen bonding 
 between the a1523 peptide strands , with 
 the -strands shifted out of alignment by two residues toward 
 the c - termini .
the dimer subunits assemble to form the tetramer through 
 hydrophobic interactions between the lfa faces .
we studied the 
 folding and supramolecular assembly of macrocyclic -sheet 3 and homologues 4 , 5 , and 6 by h nmr noesy and dosy experiments on the trifluoroacetate 
 ( tfa ) salts in d2o solution .
macrocyclic -sheet 4 is a homologue of macrocyclic -sheet 3 with phenylalanine in place of p - iodophenylalanine 
 in the a1523 peptide strand ( f20 in place of f20 ) .
macrocyclic -sheet 5 is a double mutant in which the hydrophobic residues v18 and f20 in the a1523 peptide strand are rendered more hydrophilic by hydroxylation : v18 is replaced by threonine and f20 is replaced 
 by tyrosine ( v18t , f20y ) .
macrocyclic -sheet 6 is another double mutant in which the hydrophobic residues 
 f19 and a21 in the a1523 peptide strand are rendered more hydrophilic by hydroxylation : f19 is replaced by tyrosine and a21 is replaced by 
 serine ( f19y , a21s ) .
h nmr studies establish that macrocyclic -sheets 3 , 4 , and 5 form tetramers comprising 
 hydrogen - bonded dimer subunits at low millimolar concentrations .
the three macrocycles exhibit h 
 nmr spectra with similar features ( figure s1 ) .
all three macrocycles show downfield shifting of the -protons 
 characteristic of -sheet structure , magnetic anisotropy of 
 the -linked ornithine pro - r and pro - s -protons characteristic 
 of well - defined turn structures , and 
 upfield shifting of the f19 aromatic resonances characteristic 
 of tertiary and quaternary structure ( figures 
 s1s2 , table s1 ) .
in contrast , 
 macrocyclic -sheet 6 is monomeric at low millimolar 
 concentrations and is less well folded than macrocyclic -sheets 35 , exhibiting less downfield shifting 
 of the -protons and less magnetic anisotropy of the -linked 
 ornithine pro - r and pro - s -protons ( figure 
 s1 ) . in the noesy spectra , 

macrocyclic -sheets 3 , 4 , and 5 exhibit a rich array of noe 
 crosspeaks associated with folding and dimerization ( figures s3s5 ) .
the macrocycles exhibit key noes between 
 -protons associated with folding : k16 and e22 ; v18 or t18 and the proton 
 at the 6-position of the hao residue ; f20 , 
 f20 , or y20 and v18 ; and 
 e22 and k16. the macrocycles also exhibit 
 key noes between -protons associated with dimerization : l17 and d23 ; and f19 and a21 .
table 2 summarizes the key noes observed 
 for each macrocycle ; figure 5 illustrates the 
 structures of the dimers .
500 mhz noesy spectra at 2.0 mm 
 in d2o at 298 k. assignments of k16 vs 
 k16 , l17 vs l17 , 
 e22 vs e22 , and d23 vs d23 are inferred from the observed pattern of noes .
( 3 : x18 = v , x20 = f ; 4 : x18 = v , x20 = f ; 5 : x18 = t , x20 = y ) hydrogen - bonded dimers formed by macrocyclic 
 -sheet peptides 35 in aqueous 
 solution .
key noes associated 
 with dimerization and folding are shown with red and blue arrows . 

( 3 : x18 = v , x20 = f ; 4 : x18 = v , x20 = f ; 5 : x18 = t , x20 = y ) .
dosy studies show that the hydrogen - bonded dimers are subunits 
 of tetramers , which are the stable species in aqueous solution . in the dosy
spectra macrocyclic -sheets 3 , 4 , and 5 exhibit diffusion coefficients of 10.110.7 
  10 cm / s ( table 3 ) .
these values are comparable to those that we have observed 
 previously for similar tetramers and are 0.580.61 times smaller 
 than that of macrocyclic -sheet 6 , which is monomeric . molecular weight calculated for 
 the neutral ( uncharged ) macrocycle .
it 
 is sandwiched through the hydrophobic face that displays l17 , f19 , and a21 , and these residues help create 
 the hydrophobic core of the tetramer ( figure 6 ) .
when f19 and a21 are rendered hydrophilic 
 by hydroxylation in macrocyclic -sheet 6 , the 
 hydrophobic core can not form and the tetramer is disrupted .
in contrast , when v18 and f20 are rendered hydrophilic by hydroxylation in macrocyclic -sheet 5 , the hydrophobic core is unaffected and the tetramer is 
 not disrupted .
illustration of the tetramer formed by macrocyclic -sheet 
 peptides 35 in aqueous solution . 

the tetramer forms as a sandwich - like assembly of two hydrogen - bonded 
 dimers , sandwiched through the lfa faces ( 3 : x18 = v , x20 = f ; 4 : x18 = v , x20 = f ; 5 : x18 = t , x20 = y ) .
the 
 extended layered -sheet formed by the hydrogen - bonded 
 dimers of 3 ( figure 3b ) resembles 
 the structures of amyloid fibrils .
amyloid fibrils 
 consist of extended -sheets composed of networks of hydrogen - bonded 
 -strands running the length of the fibril axis and laminated 
 in pairs through hydrophobic interactions to form two - layered assemblies . in the fibrils formed by a140 , the hydrophobic central and c - terminal regions of the peptide assemble 
 to form extended -sheets that run the length of the fibril 
 axis .
the -strands comprising the -sheets are roughly 
 orthogonal ( 90 ) to the fibril axis . the molecules of a 
 form u - shaped turns , and the hydrophobic central and c - terminal -strands 
 pack in a face - to - face fashion through hydrophobic interactions .
the 
 resulting two - layered -sheets make up the basic fibril structure , 
 further assembling to form four - layered or triangular fibrils consisting 
 of two or three of these subunits . although most of the structures 
 reported for a140 fibrils involve parallel 
 -sheets ,
figure 7a , b illustrates the structures of the parallel and 
 antiparallel two - layered -sheets reported for a140 .
figure 8a illustrates the 
 structure of the component u - shaped turns that make up these two - layered 
 assemblies .
cartoon representations of fibrils formed by a. ( a ) parallel 
 -sheet fibril composed of u - shaped turns in a staggered arrangement , 
 observed for a140 .
( b ) antiparallel -sheet fibril composed of u - shaped turns , 
 observed for the iowa mutant a140 .
( c ) fibril - like assembly of oligomers composed 
 of -hairpins , that we propose from the x - ray crystallographic 
 structure of macrocyclic -sheet peptide 3 .
the 
 green and pink colors represent the central and c - terminal regions 
 of a. cartoon representations 
 of u - shaped turns ( a ) and -hairpins 
 ( b ) composed of a. in the u - shaped turns , the faces of the 
 -strands pack together . in the proposed -hairpins , 
 the edges of the -strands hydrogen bond together .
the green 
 and pink colors represent the central and c - terminal regions of a. the assembly formed by the hydrogen - bonded 
 dimers of 3 ( figure 3b ) differs 
 notably from amyloid 
 fibrils in that it is composed of discrete oligomeric subunits . while 
 the two - layered -sheets of the a140 fibrils contain no subunit larger than the monomer , the fibril - like 
 assemblies formed by macrocyclic -sheet 3 are 
 composed of oligomers consisting of two -hairpin - like macrocycles , 
 hydrogen bonded to form a four - stranded antiparallel -sheet .
the x - ray crystallographic structure of the fibril - like assembly 
 of oligomers formed by macrocyclic -sheet 3 suggests 
 that alternative fibril assemblies of a140 or a142 might also be possible . in a fibril - like assembly of oligomers formed by full - length a , 
 the hydrophobic central and c - terminal regions of the peptide could 
 hydrogen bond to form a -hairpin .
the -hairpins could then further assemble to form layered 
 -sheets through edge - to - edge hydrogen bonding and face - to - face 
 hydrophobic interactions . in this assembly , the -strands comprising 
 the -sheets are not orthogonal to the fibril axis , but rather 
 are rotated approximately 20 from orthogonality . to compensate 
 for this rotation , the -sheets must shift registration by two 
 residues for every two -hairpins .
figure 7c illustrates a structure of this fibril - like assembly of oligomers ; 
 figure 8b illustrates the structure of the 
 component -hairpins .
the solid - state and solution - state 
 structures of macrocyclic -sheets 35 show that a1523 can form both 
 aligned and shifted antiparallel -sheets . in 
 the x - ray crystallographic structure of macrocyclic -sheet 3 , the a1523 peptide strand forms 
 an aligned -sheet , while the a1523 hybrid strand forms a shifted -sheet . in the solution - state 
 structure of macrocyclic -sheets 35 , the a1523 peptide strand forms 
 a shifted -sheet .
( a ) interface between monomer subunits 
 within the dimer of macrocyclic -sheet peptide 3 in the solid state .
( b ) interface between monomer subunits within 
 the dimer of macrocyclic -sheet 3 in aqueous solution . 

( c ) interface between the dimers of macrocyclic -sheet 3 in the solid state . in ( a ) and
( b ) the interface occurs 
 between the a1523 peptide strands ; in ( c ) 
 the interface occurs between the a1523 hybrid 
 strands .
we have previously observed both 
 aligned and shifted antiparallel 
 -sheets involving a1523 in the solid - state 
 and solution - state structures of macrocyclic -sheets 1 . in the solid state ,
macrocyclic -sheet 1a forms tetramers consisting of two hydrogen - bonded dimers sandwiched 
 through the vf faces ( figure 10a ) . in these hydrogen - bonded dimers ,
1a and 1b form tetramers consisting of two hydrogen - bonded dimers sandwiched 
 through the lfa faces ( figure 10b ) . in these hydrogen - bonded dimers ,
the a1523 peptide strands are shifted out of alignment by 
 two residues toward the c - termini .
the fibril - like assembly of oligomers 
 formed by macrocyclic -sheet 3 differs from the 
 solid - state and solution - state structures of macrocyclic -sheets 1 , in that it does not contain discrete tetramers .
instead , 
 each dimer subunit overlaps with two dimers in the opposing layer 
 ( figure 10c ) .
( a ) tetramer of 1a observed in the solid state ( pdb : 4ivh ) .
( b ) tetramer of 1b ( and 1a ) observed 
 in aqueous solution .
( c ) fibril - like assembly 
 of dimers of 3 observed in the solid state .
it is easy to imagine a mechanism , similar to crystallization , 
 by which the fibril - like assemblies of oligomers catalyze oligomer 
 formation . during crystal growth ,
the 
 fibril - like oligomers of macrocyclic -sheet 3 shown 
 in figure 3b must elongate by adding monomer 
 subunits either one at a time or in small groups from tetramers or 
 oligomers present in solution . a similar mechanism of growth
can be 
 hypothesized for the fibril - like assemblies of oligomers that we propose 
 for full - length a in figure 7c , in which 
 the exposed hydrogen - bonding edges and hydrophobic surfaces serve 
 as a template that promotes the addition and folding of monomeric 
 a. the fibril - like assembly of oligomers can then serve as 
 a reservoir of oligomers , dissociating organized dimers , tetramers , 
 or other small homogeneous assemblies of soluble toxic amyloid oligomers . 

such mechanisms for oligomer replication have been seen and discussed 
 previously but have not been observed at atomic resolution .
the x - ray crystallographic structure of macrocyclic 
 -sheet 3 may thus provide a window at atomic resolution 
 into a prion - like mechanism of amyloid oligomer propagation .
the x - ray crystallographic structure of macrocyclic -sheet 3 provides new insights into the supramolecular assembly of 
 peptides from -amyloid , revealing a fibril - like assembly of 
 hydrogen - bonded dimers .
the dimers repeat along the fibril axis , to 
 form an extended -sheet , like in conventional amyloid fibrils . 
 unlike conventional amyloid fibrils ,
the -strands comprising 
 the -sheets are rotated approximately 20 from orthogonality 
 to the fibril axis , and a two - residue shift in alignment of the -strands 
 occurs at the juncture between the dimers .
the dimers 
 are not layered directly over each other , but rather are offset by 
 two strands . as a result , the fibril - like assembly of dimers is not 
 composed of discrete tetramers .
the fibril - like assembly of 
 oligomers formed by macrocyclic -sheet 3 offers 
 the intriguing possibility that full - length a 
 may also be able to form similar assemblies , perhaps consisting of 
 -hairpins formed by the amyloidogenic central and c - terminal 
 regions of a. this model further suggests the provocative hypothesis 
 that fibril - like assemblies of a oligomers might catalyze a 
 oligomer formation and replication .
 a macrocyclic 
 -sheet peptide containing two nonapeptide 
 segments based on a1523 ( qklvffaed ) forms 
 fibril - like assemblies of oligomers in the solid state . 
 the x - ray 
 crystallographic structure of macrocyclic -sheet peptide 3 was determined at 1.75  resolution . 
 the macrocycle 
 forms hydrogen - bonded dimers , which further assemble along the fibril 
 axis in a fashion resembling a herringbone pattern . 
 the extended -sheet 
 comprising the dimers is laminated against a second layer of dimers 
 through hydrophobic interactions to form a fibril - like assembly that 
 runs the length of the crystal lattice . 
 the second layer is offset 
 by one monomer subunit , so that the fibril - like assembly is composed 
 of partially overlapping dimers , rather than discrete tetramers . in 
 aqueous solution , macrocyclic -sheet 3 and homologues 4 and 5 form discrete tetramers , rather than 
 extended fibril - like assemblies . 
 the fibril - like assemblies of oligomers 
 formed in the solid state by macrocyclic -sheet 3 represent a new mode of supramolecular assembly not previously observed 
 for the amyloidogenic central region of a. the structures observed 
 at atomic resolution for this peptide model system may offer insights 
 into the structures of oligomers and oligomer assemblies formed by 
 full - length a and may provide a window into the propagation 
 and replication of amyloid oligomers . 
Introduction
Results
Discussion
Conclusion