pathological amyloid fibrils are formed 
 by the misfolding and self - assembly 
 of proteins and peptides such as a40/42 in alzheimer s 
 disease ( ad ) , -synuclein in parkinson s disease ( pd ) , 
 islet amyloid polypeptide ( iapp or amylin ) in type ii diabetes mellitus , 
 and human 2-microglobulin ( h2 m ) 
 in dialysis - related amyloidosis ( dra ) . despite the distinct 
 amino acid compositions of amyloid proteins ,
the self - assembled fibrils 
 adopt a universal and underpinning cross- molecular structure 
 composed of arrays of ribbonlike -sheets running parallel to 
 the long axis of the fibrils . the structural basis of these 
 filamentous aggregates
needs to be investigated to provide a mechanistic 
 understanding of their role in pathological events and to develop 
 therapeutic strategies against protein aggregation diseases .
one avenue 
 toward this end is the determination of the molecular structure of 
 the final fibril aggregates .
magic angle spinning ( mas ) nmr spectroscopy 
 has demonstrated its indispensable role in elucidating the backbone 
 conformations , supermolecular organization and registry of interstrand 
 arrangements of amyloid fibrils , which otherwise are inaccessible 
 by most common techniques .
indeed , models have been established for 
 a number of amyloid fibrils primarily based on mas nmr analysis of 
 fibrils formed in vitro , including a(140 ) , -synuclein , sup35p , human prion protein , and other protein sequences .
in addition , mas nmr 
 and cryo - electron microscopy ( cryoem ) were used to determine the complete 
 high - resolution structure of three polymorphs of amyloid fibrils formed 
 by a peptide from transthyretin ( ttr105115 ) .
two amyloid fibril components , 99-residue h2 m 
 and its truncated variant n6 that lacks the n - terminal six 
 amino acids , are found in osteoarticular 
 amyloid deposits in dialysis - related amyloidosis ( dra ) .
full - length 
 h2 m is remarkably intransigent to fibril assembly 
 at physiological ph and temperature in the absence of cosolvents or 
 other additives . a number of factors , 
 including ph , metal ions , and biologically relevant molecules including 
 collagen , glycosaminoglycans , lysophosphatidic acid , and nonesterified 
 fatty acids induce the fibril formation of h2 m in vitro .
for example , at ph 2.5 , predominantly unfolded h2 m protein associates rapidly in vitro to form amyloid 
 fibrils .
in contrast with the requirement 
 for denaturing or destabilizing conditions to induce fibril formation 
 of the wild - type protein , n6 readily forms fibrils in vitro from an initially  folded  monomeric 
 state at ph 6.27.2 .
most recently , even 
 trace amounts of n6 ( 1% ) have been found to facilitate the 
 fibril formation of the natively structured wild - type protein in vitro at ph 6.27.2 .
the possession of trans - p32 in native n6 
 rationalizes , in part , the ability of this protein to form amyloid 
 on the basis of its structural similarity to the transient folding 
 intermediate ( it ) identified as a key precursor in amyloid 
 assembly of h2 m .
these findings , together with the natural occurrence 
 of n6 in fibrils in vivo , have resulted in 
 increasing attention on this variant , despite the 
 absence of a consensus as to whether the truncated protein originates 
 prior to , or post , fibril assembly in vivo .
therefore , h2 m and n6 provide an interesting 
 pair of proteins by which to study the mechanisms of amyloid assembly 
 at a fundamental level .
since the identification of h2 m as an amyloid 
 protein more than 20 years ago , numerous biochemical and biophysical 
 studies have investigated the structure and dynamics of the protein 
 under different solution conditions .
x - ray crystallography and solution 
 nmr have provided high - resolution structures of the native , monomeric 
 wild - type protein , which shows a -sandwich fold consisting 
 of seven antiparallel -strands , stabilized by a single interstrand 
 disulfide bond .
other studies focusing on the characterization of 
 precursors ( i.e. , the native monomer and its partially folded intermediates ) , 
 fragments , mutated variants , and oligomers of the wild - type protein , 
 have improved our understanding of the nature of the self - assembly 
 mechanisms of h2 m into amyloid fibrils .
however , due to the complexity of the cross- superstructure 
 and the insoluble and noncrystalline nature of these amyloid assemblies , 
 atomic - level information on structures within the fibril architecture 
 remains elusive .
a limited number of pioneering studies have been 
 conducted ; for example , iwata et al .
have successfully determined 
 the tertiary structure of a 22-residue segment of h2 m ( s20k41 ) within amyloid fibrils primarily by using mas 
 nmr , while eisenberg and co - workers have 
 focused on different 7-residue peptides , from the h2 m sequence , that form 3d crystals .
however , 
 fibrils formed from a short peptide fragment are insufficient to represent 
 the structural features of the intact protein since the remaining 
 residues not included in the s20k41 fragment have been found 
 to be crucial in the assembly of the intact protein into fibrils using 
 epr , mutagenesis , cryoem , and solution nmr .
the identification of the fibril cores , and therefore residues that 
 are crucial in the fibril assembly , was investigated by h / d exchange and limited proteolysis experiments .
both techniques provide a global profile of protein segments , showing 
 solvent protection or exposure , and the distribution of preferential 
 proteolytic sites . however , neither of these approaches addresses 
 the residue - specific conformational composition of the fibril core . 

thus , information elucidating the backbone rearrangement occurring 
 on the pathway of amyloid assembly from the native structure to fibrils , 
 is still missing .
therefore , an atomic - level structure of full - length 
 h2 m and n6 in their fibril forms is necessary 
 in order to understand the hierarchical assembly of these elementary 
 building blocks into the complex fibril architecture imaged by cryoem .
we have recently reported the mas nmr 
 characterization of full - length 
 h2 m fibrils formed at ph 2.5 , resulting in the prediction of torsion angles for 40 residues 
 of this 100-residue protein ( the recombinant protein contains an additional 
 n - terminal methionine , denoted here as m0 ) .
these results suggested 
 at least five segments of -strands in the fibril structure . 

the resonance assignments also revealed that h31p32 peptide 
 bond adopts a trans - conformation in h2 m fibrils , consistent with cis - to - trans isomerization of this residue being an important initiating 
 event in fibril formation .
however , a 
 clear picture of the secondary structural content of h2 m fibrils requires complete assignment of the backbone resonances 
 of the protein in fibrillar form .
in addition , no detailed structural 
 studies of the fibrils formed from n6 have yet been performed . 
 here
we present the assignment of backbone resonances of h2 m and n6 fibrils ( 80% and 88% complete , respectively ) 
 using a combination of variously isotopically labeled samples and 
 a set of multidimensional nmr techniques at 750900 mhz .
the 
 resulting atomic - level comparison of the secondary structure within 
 the fibrils formed from these proteins reveals structural differences 
 that explain their ability to copolymerize at neutral ph .
biosynthesis 
 and purification of h2 m and n6 followed protocols as 
 described previously .
briefly , 
 recombinant proteins were expressed in bl21(de3 ) plyss e. coli in the presence of hcdm1 minimal media .
three 
 different isotopically labeled samples were prepared for each protein , 
 including one uniformly n , c - labeled protein 
 and two site - directed c- and uniformly n - labeled 
 proteins .
these three protein samples were produced in minimal media 
 enriched with 1 g / l nh4cl and using either 
 2
g / l d - glucose - c6 ( named as u - h2 m or u-n6 ) , [ 1,3-c]-glycerol ( 1,3-h2 m or 1,3-n6 ) or [ 2-c]-glycerol ( 2-h2 m or 2-n6 ) .
all isotopes were purchased from cambridge 
 isotope laboratories ( andover , ma ) and used without further purification .
the h2 m and n6 fibrils were prepared by 
 incubation in a 96-well plate ( corning incorporated , costar ) in a 
 bmg fluostar optima plate reader at 37 c with constant shaking 
 at 600 rpm .
fibril growth was performed using 0.5 mg / ml soluble protein , 
 0.02% ( w / v ) nan3 and different phs and salt concentrations , 
 i.e. 10 mm sodium phosphate buffer containing 50 mm nacl at ph 2.5 
 for h2 m and 50 mm mes buffer containing 120 mm nacl 
 at ph 6.2 for n6 .
the h2 m and n6 fibrils 
 were harvested after incubation for approximately 14 or 7 days , respectively . 

the fibrils were centrifuged at 14,000 g for 20 min 
 and characterized by negative stain transmission electron microscopy 
 ( em ) .
the fibrils were prepared without seeding , and the consistency 
 of the fibril type was confirmed by analysis of nmr chemical shifts .
the hydrated fibrils were 
 ultracentrifuged for 24 h at 300000 g to pack the 
 pellet into 3.2 mm bruker zirconia rotors ( bruker biospin , billerica , 
 ma ) .
the packed hydrated fibril samples have negligible water loss 
 as monitored by the h signal of h2o .
mas nmr 
 experiments were performed on a custom - designed 750 mhz spectrometer 
 ( courtesy of dr .
david j. ruben , francis bitter magnet laboratory , 
 cambridge , ma ) , and bruker 800 and 900 mhz spectrometers ( h frequency ) .
complete experimental details for the multidimensional 
 mas nmr experiments are included in the supporting 
 information ( si ) .
briefly , three different kinds of 1d c experiments were conducted , including dipolar - coupling 
 based cross - polarization ( cp ) , direct polarization ( dp ) , and scalar - coupling 
 ( j)-based inept .
two - dimensional ( 2d ) homonuclear c  c correlations were recorded using radio frequency - driven 
 recoupling ( rfdr ) , either in a broadband or band - selective manner .
c correlations were achieved by z - filtered transferred - echo double - resonance 
 ( zf tedor ) and proton - assisted insensitive 
 nuclei cross - polarization ( pain - cp ) .
c experiments were performed for sequential assignments , 
 including the conventional n  c 
c experiments , i.e. ncocx , 
 ncacx , and conca , and the most recently designed tedor - cc experiments .
zero filling and lorentzian - to - gaussian apodization for 
 each dimension 
 were applied before fourier transformation .
a 
 line broadening of 3060 hz was used for all 2d and 3d experiments . 

peak identification and assignment were performed with sparky ( t. 
 d. goddard and d. g. kneller , sparky 3 , university of california , 
 san francisco ) .
protein structures were visualized in pymol ( the pymol 
 molecular graphics system , version 1.5.0.4 , schrdinger , llc . ) . 

the assigned n / co / c/c chemical shifts were used as input 
 for the talos+ program to predict backbone torsion angles (  , 
  ) .
obtaining homogeneous samples of 
 amyloid fibrils is an essential priority to ensure high - resolution 
 spectra that enable structural analysis .
figure 1 shows negative stain em ( a , b ) and 2d mas nmr spectra ( c  h ) 
 of h2 m and n6 fibrils , revealing the sample 
 homogeneity as well as spectroscopic differences of the two fibril 
 types .
em images of negatively stained preparations of h2 m ( ph 2.5 , 50 mm nacl ) and n6 ( ph 6.2 , 120 mm nacl ) 
 show a predominantly homogeneous population of long , straight fibrils 
 with no amorphous aggregates present , consistent with previous results . in order to examine the conformational homogeneity of the fibrils 

further , we recorded 2d mas nmr spectra using rfdr and zf - tedor sequences 
 selective for one - bond c  c ( figure 1c  e ) and c  n ( figure 1f  h ) couplings .
the spectra 
 exhibit excellent resolution , in which c and n line widths are 0.5 ppm and 0.9 ppm for backbone c and n resonances , respectively , and 
 0.35 ppm for side - chain methyl carbon peaks for both samples . despite the similarly high degree of conformational homogeneity 
 of the h2 m and n6 fibril samples
, a comparison 
 of the spectra reveals differences in the number of cross peaks and 
 their resonance positions .
for example , the spectrum shown in figure 1e of the n6 fibrils displays all 9 serine c-c cross peaks , whereas two are 
 absent in the spectra of h2 m .
similarly , all three 
 glycine residues ( g18 , g29 , and g43 ) are present in the backbone n 
c correlations of n6 fibrils , 
 but only a single , strong cross peak ( g43 ) and a weak one ( g29 ) appear 
 in spectra of h2 m fibrils ( figure 1h ) .
the presence of the additional cross peaks in the spectra 
 of n6 fibrils qualitatively suggests a more rigid backbone 
 in the truncated variant .
furthermore , those cross peaks displaying 
 low intensity ( e.g. , s88 of h2 m ( figure 1e ( blue ) ) and s11 of n6 ( figure 1e ( red ) ) or peak broadening ( g43 of n6 ( figure 1h ( red ) ) suggest that these residues are in relatively 
 dynamic local regions in the fibril structure , i.e. flexible terminals , 
 turns , or loops .
the fact that the fibrils of h2 m and n6 differ in the position of their n / c/c 
 resonances suggest possible structural differences , which are likely 
 the result of the different lengths of the protein sequences and the 
 different phs ( 2.5 and 6.2 ) employed in the fibril growth .
however , 
 to rigorously compare the conformational differences between h2 m and n6 fibers , the secondary structure needs to be 
 determined from complete assignments .
negative stain electron micrographs ( em ) of ( a ) h2 m and ( b ) n6 fibrils ( scale bar 100 nm ) and their mas 
 nmr spectra of ( c  e ) c  c and 
 ( f  h ) c  n correlations .
( c  e ) 
 one - bond rfdr spectra of u  c , n - h2 m ( blue ) and u  c , n-n6 
 ( red ) .
the cross sections of s52 are shown in ( e ) to illustrate the 
 peak intensity and line width .
( f  h ) one - bond zf tedor spectra 
 of 1,3-h2 m ( blue ) and 1,3-n6 ( red ) .
( c  e ) 
 and ( f  h ) were acquired at 900 and 800 mhz h frequencies , 
 respectively .
assignments in spectra are residue - specific and are 
 based on 2d and 3d experiments . in figure 2 , we illustrate c cross - polarization ( cp ) and direct polarization ( dp ) spectra of 
 h2 m and n6 fibrils at 313 k. the efficiency 
 of magnetization transfer in dipolar - based cp experiments largely 
 depends on the rigidity of the sites , while dp spectra sample regions 
 which exhibit short c t1 s .
the overall 
 cp enchantment factor ( cp ) is around 2.12.5 
 for both fibril samples , which is comparable to the values found for 
 the largely immobile protein dsbb , protein 
 g b1 domain in microcrystals and pi3-sh3 
 amyloid - like fibrils .
in contrast , inefficient 
 cp enhancement ( cp = 0.7 ) was found for 
 the largely mobile -synuclein fibrils at 273 k. we note that the overlaid 1d c 
 spectra of n6 fibrils with ( black ) and without ( red ) h  c cp transfer , in figure 2b , show similar spectral features .
we observed inept signals at 313 
 k for h2 m fibrils , indicative of subnanosecond backbone 
 motions ( figure 2a ) , as assigned previously to arise from spin systems ( identified from 
 the through - bond tobsy spectra ) as the n - terminal seven residues , 
 miqrtpk .
n6 fibrils , truncated at k6 , show no inept intensity 
 ( figure 2b ) .
these observations exclude the 
 possibility that the two proteins possess large reorientational dynamics 
 in their amyloid forms at the temperature employed ( 313 k ) . comparison 
 of 1d c spectra of ( a ) h2 m and ( b ) 
 n6 fibrils at 313 k using cross - polarization ( cp , 
 top ) , direct polarization ( dp , middle ) and inept ( bottom ) . in the 
 case of n6
we superimposed the traces from dp ( red ) and cp 
 ( black ) to illustrate that the spectral features are largely preserved . 

each 
 cp and dp spectrum was recorded with 16 scans , while each inept spectrum 
 required 64 scans .
all spectra were collected at 13 khz mas frequency , 
 100 khz h tppm decoupling , and at 800 mhz h frequency .
c cp contact time 
 was 1.5 ms , and the recycle delay for the dp and inept spectra 
 was 55.5 s. we next aimed to determine 
 the secondary structures of h2 m and n6 fibrils 
 using mas nmr spectra , and the initial step is the assignment of the 
 individual resonances in the protein sequences . in this study , we 
 employed two established strategies to complete the resonance assignment . 

first , we performed a set of one - bond and multibond 2d c  c and c 
n 
 correlation experiments to identify the spin systems and to establish 
 partial inter- and intraresidue connections . samples of h2 m and n6 fibrils with uniform c , n - labeling or labeling with n and 2-c1-glycerol or 1,3-c2-glycerol 
 ( see materials and methods ) were used .
the sequential assignment process involves the use of one - bond c  c and n  c correlation experiments to identify residues with characteristic 
 chemical shifts and specific labeling patterns in 2- and 1,3-samples , 
 as discussed below .
the inter - residue multibond correlation spectra 
 were used to identify the connectivity of individual residues with 
 the immediately neighboring residues , giving a number of sequential 
 assignments .
those residues assigned in 2d spectra then served as 
 anchor points to facilitate the backbone assignments that map the 
 sequential connectivity .
the match of sequence - specific assignments 
 obtained in the comprehensive set of 2d and 3d spectra minimizes the 
 ambiguity in the trial assignments .
uniform c , n - labeling is the customary initial step in the spectral 
 assignment process since it generally yields spectra with high signal - to - noise 
 ratio .
however , the simultaneous labeling of all carbon sites results 
 in significant cross peak overlap , a problem that is exacerbated for 
 relatively large proteins and protein assemblies .
this problem stimulated 
 the use of sparse labeling strategies using [ 1-c]-glucose , 
 [ 2-c]-glucose , [ 2-c1]-glycerol 
 or [ 1,3-c2]-glycerol as the sole c source .
the reduced number of labeled sites can greatly simplify 
 spectra ; for example , [ 2-c1]-glycerol labels 
 the c site for residues including g , s , w , f , y , a , v , and 
 l. in contrast , these residues have c labeling at co and c sites for 
 protein samples prepared from e. coli grown on [ 1,3-c2]-glycerol . as illustrated 
 in si figure 1a  c , 
 h2 m fibrils labeled with [ 2-c1]-glycerol or [ 1,3-c2]-glycerol have relatively 
 higher c and c intensity , respectively , in agreement 
 with the expected labeling pattern .
concurrently , the c line width is reduced due to the abolition of one - bond c  c dipolar and scalar couplings in the specifically 
 labeled samples .
the removal of the one - bond dipolar couplings also 
 attenuates dipolar truncation from homonuclear dipolar couplings , 
 resulting in better recoupling efficiency between inter - residue spins ( see the expanded region at approximately 15 
 ppm in si figure 1b and c ) .
figures 3 , 4 and 5 illustrate the identification 
 of some of the spin 
 systems , as well as partial sequential connectivity in 2d spectra 
 of h2 m and/or n6 fibrils . using the serine 
 residues as an example ( figure 3a ) , 1,3-h2 m and
1,3-n6 samples contain c - labeled 
 co and c carbons , and only the c sites are labeled in 
 samples prepared from growth on 2-glycerol .
all nine serine residues 
 of n6 fibrils have been successfully identified from cc 
 cross peaks in a one - bond rfdr spectrum of u-n6 ( figure 3d ) .
their n  c and cc 
 correlations appear in one - bond zf tedor spectra ( figure 3b ) and in the multibond rfdr spectrum of the 1,3-sample 
 ( figure 3c ) , respectively .
other residues including 
 pro , gly , and thr show fingerprint chemical shifts in 2d n 
more examples can also 
 been found in figure 4c and d , e.g. all three 
 glycines in n6 fibrils were identified on the basis of the 
 cross peaks of the upfield n and c 
 chemical shifts ( figure 4b )
. identification of serine 
 residues of n6 fibrils using 2d 
 mas nmr and variously labeled samples .
( a ) c - labeling 
 scheme of serine using [ 2-c]-glycerol ( red ) or [ 1,3-c]-glycerol ( green ) as the carbon source .
( b ) one - bond zf - tedor of [ 2-c - glycerol]-n6 . 

( c ) multibond rfdr of [ 1,3-c - glycerol]-n6 using 
 an 11 ms mixing period .
( d ) one - bond rfdr of u  c , n - labeled n6 recorded using a 1.6 ms mixing 
 period .
spin systems of all nine serine residues were identified by 
 their characteristic downfield c and c chemical shifts . 

( a ) schematic illustration of the backbone walk that 
 can be obtained through a set of inter - residue c  n and c  c correlations by 
 using 2-h2 m and 2-n6 , which has mostly alternating c enrichment .
( b ) multibond zf tedor spectra of 2-n6 , 
 showing representative n(i)c/(i  1 ) connections of s55-f56-s57 ( blue lines ) and 
 s28-g29-f30-h31-p32 ( violet lines ) .
( c ) multibond zf tedor spectra 
 of 1,3-n6 , showing the n(i)-c/(i-1 ) connectivity of d53-l54-s55-f56-s57-k58 
 ( blue lines ) and h31-p32-s33-d34 ( violet lines ) .
( d ) broad - band rfdr 
 showing the c(i)-c(i  1 ) connectivity of s55f56s57-k58 
 ( blue lines ) and s28-g29-f30-h31-p32-s33 ( violet lines ) .
( e ) band 
 selective - rfdr of 2-n6 , showing the c(i)-c(i  1 ) 
 correlations .
( b , c ) and ( d , e ) were acquired on 800 and 900 mhz spectrometers 
 ( h frequency ) , respectively .
two - dimensional mas nmr has been used successfully to accomplish 
 backbone and side - chain assignment . here
we show 
 that the combination of variously labeled samples of h2 m and/or n6 fibrils and 2d nmr experiments has enabled 
 the identification of amino acid spin systems and their sequential 
 connectivity for both h2 m and n6 fibril samples , 
 despite being 100 and 94-residue proteins , respectively .
figure 4a illustrates four inter - residue correlations that 
 can be established to connect the assignment of atoms in neighboring 
 residues . in the 2-h2 m and 2-n6 protein samples , n  c and c  c correlations including one - bond n(i)c(i  1 ) and 
 multibond n(i)c/(i  1 ) , c(i)c(i  1 ) and c(i)c(i  
 1 ) correlations can be established by using zf tedor with short or 
 long mixing times , and rfdr experiments , respectively .
unbroken blue 
 and violet lines in figure 4b  e guide 
 the partial or complete connectivity of consecutive segments comprising 
 residues s55 to k58 and s28 to d34 of n6 fibrils , respectively . 

different lengths of the mixing time were used to correlate 
 one- or multibond spins as described in the experimental details in 
 the supporting information . to avoid dipolar 
 truncation from one - bond spin pairs and to allow efficient detection 
 of the coupling of distant spin pairs , the use of both the 2- and 
 1,3-glycerol labeled samples is required .
sequential assignments from 
 s28 to d34 of n6 fibrils were established from n(i)c(i + 1 ) and n(i)c/(i  1 ) correlations in figure 4b and 
 c , respectively .
the c labeling at c sites 
 for the majority of residues in 2-n6 facilitates the detection 
 of such weak dipolar coupling , which otherwise is difficult to detect . 

we used long - mixing rfdr ( rfdr = 16.2 ms ) to establish 
 the inter - residue cc 
 correlations .
a low - power ( 12.5 khz ) rectangular  pulse was 
 used in the dipolar recoupling to selectively excite the aliphatic 
 carbons , which has been shown to provide better efficiency . besides the sequential cc correlations
, the connectivity of adjacent residues in the 
 spectra of n6 fibrils was also established from c(i)c(i  1 ) contacts ( figure 4e ) .
representative 
 sequential assignments of n6 fibrils from 
 3d n  c - c correlation 
 experiments .
( a ) the inter- or intraresidue magnetization transfer 
 pathways in conca ( red ) , ncacx ( blue ) and ncocx ( green ) .
n chemical shifts where each 2d plane is truncated are listed in 
 black squares .
the spectra were acquired using u[c , n - labeled]-n6 fibrils on a 750 mhz spectrometer ( h frequency ) .
a representative strip plot for the same segment 
 of h2 m fibrils is shown in si figure 2 . in order to overcome the difficulty of peak overlap in 2d 
 spectra 
 required to obtain near - complete assignments of h2 m and n6 fibrils , the extension to one more spectral dimension 
 is necessary .
two categories of 3d experiments , distinguished by the 
 n  c magnetization transfer , were performed to obtain unambiguous 
 sequential assignment .
the first category of experiments , including 
 ncocx , ncacx and conca , utilizes band - selective specific - cp to transfer 
 magnetization between n and co , or between n and c. taking the 3d ncocx experiment for example ( as illustrated 
 by the green route in figure 5a ) , the magnetization 
 was initiated from the amide h of residue i and transferred to the directly bonded n via cp .
subsequently , 
 a specific cp mixing sequence is utilized to transfer the magnetization 
 from n to c of its preceding residue i  

ncacx correlates the intraresidue 
 backbone to side - chain carbons of residue i , while 
 conca realizes the connectivity of residue i to its 
 succeeding neighbor i + 1 .
reasonably good transfer 
 efficiencies of 3545% were obtained , which again suggests 
 the high rigidity of the majority of the protein backbone of both 
 h2 m and n6 fibrils .
figure 5b shows representative strip plots 
 of the c  c planes of the three 3d 
 ncc spectra of n6 fibrils , providing an indication of the spectral 
 quality .
the plot consists of strips from three 3d spectra : ncocx 
 ( green ) , ncacx ( blue ) , and conca ( red ) .
the sequential connectivity 
 from s52 to k58 is established by n / co / c/c as well as 
 side - chain carbons . residues including ser and thr
the side - chain c chemical shifts can also serve as an identifier of residues 
 including lys , arg , glu , gln , and ala .
examples of the side - chain 
 assignment walks in ncocx ( green ) and ncacx ( blue ) spectra include 
 well - resolved peaks of d53 c ( 178.1 ppm ) , l54 c/1/2 
 ( 30.2 ppm , 27.6 ppm , 25.0 ppm , respectively ) , and k58 c// 
 ( 25.4 ppm , 29.9 ppm , 42.3 ppm , respectively ) .
sequential connectivity 
 for the same region is observed in 2d spectra as guided by blue lines 
 in figure 4 , providing additional verification . 

the same connectivity for h2 m fibrils is obtained , 
 as illustrated in si figure 2 , showing 
 similarly good resolution and intensity .
the cis - to - trans isomerization of the h31p32 peptide bond in 
 h2 m is intimately involved in the backbone rearrangement 
 required to initiate fibril formation , suggesting that isomerization 
 of the main - chain at residue 32 is mechanistically crucial in fibril 
 assembly .
the c chemical shift of proline has been utilized as a reliable sensor 
 to identify the bond conformation of x - pro .
for example , the chemical shift difference between c and 
 c ( c/ ) is normally less than 5 ppm for trans - x - pro but larger than 10 ppm for cis - conformers .
a common difficulty of assigning 
 proline in conventional 3d n  c  c spectra is the weak 
 intensity due to its lack of an n  h group .
it therefore precludes the assignment of the preceding residue as 
 well , usually causing the incomplete mapping of the secondary structure . 

we have recently developed a new 3d experiment , tedor - cc , specifically 
 to resolve this problem .
as shown in figure 6a , the initial magnetization was from the cross - polarization 
 of h  c or h  co , instead of h 
n in the 
 3d spectra illustrated in figure 5a , ensuring 
 the signal of proline residues .
co 
 and n  c transfers in tedor - cc were achieved using dipolar 
 recoupling -pulse trains , without requiring high stability 
 for the long and simultaneous irradiation of all h , n , and c channels in the specific - cp mixing . 

the representative strip plot of 3d tedor - cc spectra of u-n6 
 fibrils is shown in figure 6b .
reliable connectivity 
 from g29 to s33 was established via the well - matched co , c , 
 and c chemical shifts in distinct ncocx and ncacx spectra . 

two - dimensional planes showing full correlations of all carbons of 
 p32 are included in si figure 3 .
representative 
 sequential backbone walks from s28 to s33 in 3d 
 tedor - cc spectra of n6 fibrils .
( a ) simultaneous transfers 
 of n(i)c(i  
 1 ) and n(i)c(i ) . 

the initial magnetization in tedor - cc is from c  h cp , in contrast to the n  h cp in conventional 3d n  c  c experiments , providing the optimal enhancement of proline 
 intensity .
( b ) 2d c  c ( f1f3 ) 
 planes of the 3d tedor - cc spectrum of n6 fibrils .
n chemical shift ( f2 ) for each 2d plane is indicated in black squares . 

one - dimensional cross sections are shown for c peaks 
 in ncacx spectra in green .
the spectra 
 were acquired using the u-n6 fibril on a 900 mhz spectrometer 
 ( h frequency ) .
c correlation spectra of 2- 
 and 1,3-samples , 
 using the specific patterns of c - enrichment , as shown 
 in figure 7 .
n  c cross peaks 
 of p32 and p72 are present in the one - bond tedor spectrum of u-n6 
 ( figure 7b ) , in contrast to the absence of 
 c peaks in 1,3-n6 ( figure 7c ) , 
 which agrees well with the labeling pattern of proline shown in figure 7a . the presence of c peaks in the multibond 
 tedor spectrum of 1,3-n6 ( figure 7d ) , 
 while absent in the spectrum of the 2-sample ( figure 7f ) verifies the identification of the spin system of proline . 
 the set of 2d spectra in figure 7 helps to 
 sequentially assign the two proline residues as well .
histidine has c enrichment at c for the 1,3-n6 and c 
 for 2-n6 , resulting in p32n  h31c cross peaks 
 in the one - bond tedor spectrum and p32n  h31c in multibond 
 spectra in figure 7c and figure 7f , respectively .
the unambiguously assigned chemical shifts 
 of p32 in n6 fibrils , together with our previously reported 
 values of the chemical shifts of this residue in the native monomer 
 of n6 , and both native and fibril 
 conformations of h2 m , are shown 
 in si table 1 .
specifically , c/ 
 of p32 is 4.34.9 ppm for native and fibrillar n6 and 
 fibrillar h2 m , while for native h2 m which contains cis - pro32 c/ 
 it is 10 ppm .
more rigorously , we compared 
 the c , c , and c chemical shifts of p32 in n6 
 fibrils to folded proteins with known x - pro conformations , confirming 
 assignment of the isomeric status of p32 in the different samples 
 ( si figure 4 ) . residue - specific assignment 
 of p32 and p72 of n6 fibrils 
 from 2d zf tedor spectra of proteins labeled at all n 
 sites and varied c sites by using u[c]-glucose , [ 1,3-c1]-glycerol or [ 2-c2]-glycerol as carbon sources .
( a ) c - labeling scheme of pro , his , and thr residues by using [ 2-c2]-glycerol ( red ) or [ 1,3-c2]-glycerol ( green ) as the carbon source .
one - bond zf tedor 
 of ( b ) u-n6 , ( c ) 1,3-n6 , ( d ) 2-n6 .
all spectra were 
 acquired at an 800 mhz h frequency . the disulfide bridge linking c25 and c80 functions as an 
 essential 
 constraint to maintain the hydrophobic core of native h2 m and n6 . to investigate whether 
 this s  s bond is retained in the fibrils formed from h2 m and n6 , we assigned the chemical shifts of their 
 cysteines . si figure 5 shows spectra of 
 2d n  c
c experiments for the 
 assignment of c80 . as a third spin - assisted recoupling ( tsar ) technique , 

pain - cp utilizes second - order recoupling and yields efficient long - range n  c correlations .
taking s52 for example , it established correlations with 
 the nearby residue i  1 ( e50 and h51 ) and i  2 ( d53 and l54 ) .
unambiguous assignment of c80 
 is obtained from the connectivity of a79-c80-r81-v82 in both 2d and 
 3d spectra ( si figure 5 ) .
the chemical shift of c is a good indicator of whether 
 the cysteine is oxidized or reduced .
specifically , 
 a chemical shift value of 3448 ppm indicates the existence 
 of a s  s bond , while a more upfield value ( 2234 ppm ) 
 suggests a reduced cysteine . as shown 
 in si table 1 and si figure 6 , the c values of c80 are 43 ppm and in the 
 middle of the distribution of c chemical shifts of oxidized 
 cysteine for both h2 m and n6 fibrils , suggesting 
 the existence of the disulfide bond .
the absence of c25 resonances 
 is likely due to the chemical shift degeneracy in the fibrils of both 
 h2 m and n6 , ruling out direct analysis of 
 its c shifts .
the combination of multidimensional mas nmr techniques 
 and site - specifically labeled samples has greatly facilitated the 
 sequential assignment of backbone atoms of the fibrils formed from 
 h2 m at acidic ph ( commencing from an acid unfolded 
 state ) and from folded n6 at ph 6.2 .
for h2 m , approximately 80% of the backbone resonances were assigned , including 
 73 residues from cp - based experiments ( i.e. , 2d c / n  c and 3d n
 c  c correlation experiments ) and 6 from 
 inept - based c  c tobsy .
the remaining 21 residues , corresponding to 
 amino acids in the two terminal regions , are unassigned since these 
 resonances are missing in the mas nmr spectra . for the truncated variant 
 n6 , 82 residues out of 94 residues , or 88% , were all assigned 
 from cp - based mas nmr spectra .
the missing resonances of these fibrils 
 are likely due to the intermediate backbone motion on the microsecond 
 to millisecond time scale that has been observed for regions of membrane 
 and amyloid proteins
. alternatively , dynamic disorder of protein segments could also result 
 in loss of signal intensity due to homogeneous broadening .
the assigned 
 resonances served as input into talos+ to predict the backbone torsion 
 angles (  ,  ) , as plotted in figure 8 .
secondary structures of h2 m and n6 fibrils 
 were determined by the predicted torsion angles and shown on the top 
 of the plot .
the h2 m fibrils contain seven -strands , 
 located in the central region ( residues k19 to s88 ) of the protein 
 sequence .
interestingly , these strands appear at similar positions 
 for the n6 fibrils , in spite of slight differences in the boundaries 
 of each segment . additionally , the n6 fibril structure contains 
 two additional -strands in the n- and c - terminal regions , a 
 significant difference from the fibril form of the wild - type protein 
 which contains a dynamic n - terminal region .
the absence of assignment 
 of residues in the c - terminal region of the h2 m 
 fibrils , however , precludes comparison of the structure in this region 
 in the two fibrils types .
secondary structure predictions of ( a ) h2 m and 
 ( b ) n6 in their fibril forms based on talos+ analysis of assigned 
 chemical shifts .
talos+ predicted backbone dihedral angles ( phi , blue 
 squares , psi , red circles ) , with error bars based on the 10 best database 
 matches .
the predicted secondary structures are shown at the top of 
 ( a ) and ( b ) ( -strands , filled boxes ; turn or loop , curved lines ; 
 not assigned , dashed line ) .
the white box in ( a ) depicts the seven 
 residues present in the spectrum of inept - based j - tobsy .
site - specific c enrichment 
 protocols have been applied 
 extensively to elucidate the structures of insoluble proteins using 
 mas nmr , including microcrystalline proteins , protein assemblies , 
 membrane proteins , and protein models of amyloid fibrils . by using a combination of u- 
 and 2- and 1,3-glycerol labeled samples , we have assigned > 80% 
 of 
 the residues of fibrils formed from h2 m at ph 2.5 
 and n6 at ph 6.2 and conducted secondary structural analysis 
 of the two fibril forms .
fibril formation of native , monomeric 
 h2 m is highly dependent on the solution conditions .
the fact that this protein forms fibrils under 
 acidic conditions but stays natively folded at neutral ph implies 
 that unfolding of the native protein is a required step in its assembly 
 into amyloid fibrils .
indeed , a significant backbone rearrangement 
 in the assembly of h2 m into fibrils has been suggested 
 in many studies using solution nmr , epr , h / d exchange , and limited 
 proteolysis .
the mas nmr analysis presented here enables a direct 
 comparison of the secondary structure content of the monomeric and 
 fibrillar forms of h2 m and n6 spanning > 80% 
 of the protein sequence , and provides the first analysis of fibrils 
 formed from n6 , showing distinct changes in the backbone structure 
 between the monomeric and fibril forms for both proteins ( figure 9 ) .
taking natively folded h2 m , 
 for example , its -sandwich structure is composed of two antiparallel 
 -sheets , one represented by the a- , b- , e- , and d - strands , 
 and the other by the c- , f- , and g - stands ( figure 9a ) .
one of the largest differences 
 between the monomeric and fibrillar structures occurs within the loop 
 regions of h2 m in the native form , including b 
c , 
 d  e , and f  g loops ( figure 9b ) , 
 which become part of the -strands in fibrillar h2 m . specifically , the d 
e loop in the native h2 m protein forms noncovalent contacts with the mhc i heavy 
 chain and is dynamic in the native monomeric protein .
our results indicate that the native d- and 
 e - strands are extended in the fibril form by incorporating residues 
 initially in loops or dynamic regions into -strands , which 
 lie in the fibril core .
this validates the 
 hypothesis in many structural studies of monomeric h2 m which suggest the potential of these regions to assemble into amyloid 
 fibrils .
g 
 loops has also been observed in partially folded h2 m , and these residues are also involved in 
 the formation of -strands in the fibrils studied here .
all 
 these differences for h2 m , together with similar 
 observations for n6 , suggest significant structural changes 
 occur in the monomer - to - fibril transition for both proteins .
moreover , 
 our results indicate that d- , e- , and f - strands of h2 m are extended in length in the fibril form . despite the presence 
 of seven -strands in both monomeric native h2 m and its fibrillar form ,
the precise location of the strands differs 
 significantly , suggestive of significant structural differences between 
 the secondary structure of the monomeric and fibril forms . moreover , 
 considering that the h2 m fibrils are formed from 
 an acid unfolded state at ph 2.5 that lacks secondary structure ,
the 
 results indicate that substantial refolding accompanies self - assembly 
 during the fibril formation of this protein at acidic ph .
although 
 the -strands have shifted in location or extended in length 
 in the fibril forms of h2 m and n6 , the chemical 
 shift analysis presented here suggests that the disulfide bridge involving 
 residues c25 and c80 is preserved in both h2 m and 
 n6 fibrils .
this finding concurs with previous studies that 
 identified the s  s bond as remaining intact in fibrils formed 
 from h2 m and n6 in vitro(32 ) and in vivo .
the requirement for an oxidized s  s 
 bond for formation of h2 m fibrils in vitro(90,109111 ) suggests its significant 
 role as a fundamental interaction in providing tight intramolecular 
 contact that presumably rigidifies the monomer in fibrils .
for example , 
 katou et al . and smith et al . have shown that the reduced h2 m protein , in which the only disulfide bond is abolished , forms curved 
 and flexible fibrils different from the long straight fibrils formed 
 at acidic ph .
( a ) similar -sandwich structures of h2 m ( pdb : 2xks(31 ) ) and n6 ( pdb : 2xku(31 ) ) monomer in their native forms .
( b ) comparison of the secondary structures of h2 m and n6 in their native and fibril forms .
the secondary structures of h2 m 
 and n6 monomers were taken from a solution nmr study by eichner 
 et al .
the h / d exchange plot at the bottom 
 is generated from data of h2 m fibrils formed at 
 ph 2.5 by skora et al . and hoshino 
 et al . , where filled and open
green rectangles 
 indicate residues with greater or less than 60% remaining intensity 
 after exchange at pd 2.5 for 78 days at 4 c and 25 c , 
 respectively .
n6 can form fibrils 
 at neutral ph , without the acid - induced unfolding required for formation 
 of fibrils from the wild - type protein in the absence of cosolvents 
 or other additives .
such distinct amyloidogenicity 
 can be rationalized , in part , by the different behaviors of the two 
 proteins in monomer and fibril forms .
for example , the requirement 
 for the cis - to - trans transition 
 of the h31p32 bond in h2 m fibril assembly 
 and retention of the trans - h31p32 isomer 
 in fully assembled fibrils was previously reported ( figure 9a ) . in the current 
 study
, we identified the trans conformation of h31p32 
 in n6 fibrils via chemical shift analysis , the same conformer 
 as in its monomeric form .
proline cis - to - trans isomerization , a process usually 
 accompanied by conformational rearrangement in a variety of proteins , 
 has been proposed to be a  switch  to trigger the assembly 
 of h2 m amyloid fibrils based on the observation 
 of a trans - p32 folding intermediate on the fibril 
 formation pathway .
the identification of the trans - form of h31p32 in both h2 m and n6 
 fibrils supports this view . from a thermodynamic point of view , p32 
 in native h2 m is trapped in a cis conformation by favorable hydrogen bonds and hydrophobic contacts 
 in the native protein .
therefore , partial unfolding of the monomeric 
 structure at acidic ph or by adding denaturants , cosolvents , or cu ions becomes necessary for fibril formation of the wild - type 
 protein .
the resulting backbone rearrangement , particularly the increased 
 conformational dynamics of the n - terminal residues , enables the cis - to - trans isomerization of p32 . the dynamic structure of the n - terminal 18 residues 
 in h2 m fibrils renders them invisible in dipolar - coupling - based 
 mas nmr spectra , while these residues have been observed in j - based n 
h hsqc spectra , suggesting high flexibility 
 of this region on the nanosecond time scale .
low - temperature experiments are necessary to slow or quench the 
 rate of the backbone motion in order to complete the assignments of 
 the terminal residues of h2 m fibrils . by contrast 
 with the dynamic terminal regions of fibrils formed from h2 m
, we show here that n6 fibrils possess a short -strand 
 within each terminal region of the sequence in a similar location 
 to the a- and g - strands in its native structure ( figure 9b ) .
how these strands pack in the fibrils remains to be determined , 
 although retention of a native - like overall topology is highly unlikely , 
 given the incompatibility of the -sandwich fold with a cross- 
 architecture .
the predicted backbone structure of h2 m in the amyloid fibrils studied here contains seven -strands 
 in the region from k19 to s88 , indicating an approximately 70-residue 
 fibril core .
this is in good agreement with the core region suggested 
 by previous h / d exchange and limited proteolysis 
 experiments ( figure 9b ) .
our data further show that the rigid core of h2 m fibrils is constrained by an experimentally observed s 
the high -strand content found in the fibril 
 core ( 55 of the 70 residues have  and  angles consistent 
 with a -strand ) provides opportunities for extensive intermolecular 
 hydrogen bonds between stacked monomers , forming a rigid and stable 
 -sheet core typical of amyloid .
the results presented here provide direct identification of residues 
 in h2 m and n6 amyloid fibrils , as well as 
 the location of -strands in the core region , which are essentially 
 inaccessible by other techniques of structural analysis .
such a finding 
 is supported by the observation of different degrees of dynamics throughout 
 the protein sequence .
for example , residues in the n - terminal 18 amino 
 acids are absent in dipolar - based spectra of fibrils formed from h2 m and instead were identified in spectra of j - based solution - nmr 
 experiments .
the extensive motion of c - terminal 
 residues has also been found by studies using epr .
intriguingly , many features of the fibril core of h2 m are conserved in fibrils formed from n6 , except that 
 the latter fibrils have -strands in the n- and c- terminal 
 regions ( figure 9b ) .
recently , we reported 
 the biophysical characterization of copolymerized h2 m and n6 fibrils in which the 
 two proteins copolymerize in heterofibrils in a 1:1 molar 
 ratio .
the similar core - forming residues in the central region of 
 both proteins , defined by the occurrence and position of -strands , 
 provide a prerequisite for determining the intermolecular hydrogen - bonding 
 patterns between the two protein components of the copolymer , and 
 may provide a structural rationale for why these two proteins copolymerize 
 so efficiently . further investigation of the intermolecular packing 
 of the homo- and heteropolymeric fibrils and a comprehensive comparison 
 of their fibril morphology will provide mechanistic understanding 
 of the role of the naturally occurring truncation variant in the assembly 
 pathway and the extent to which the fibril architecture differs in 
 the different fibril forms .
in 
 summary , we have determined the location of the -strand 
 domains of amyloid fibrils of h2 m and n6 
 by utilizing a variety of c / n - labeling strategies 
 and combining these with multidimensional mas nmr techniques at high 
 magnetic fields .
the results reveal that approximately 70 residues 
 comprise the core of h2 m fibrils , distributed into 
 seven -strands and rigidified by the c25c80 disulfide 
 bond .
by contrast , n6 fibrils contain an additional two -strands 
 that extend the core region to 87 of the 94 residues in this protein 
 sequence .
the relatively more rigid termini of the truncated variant , 
 together with the finding of its natively trans - p32 
 in monomeric and fibril forms , contrasts with the cis 
trans isomerization required for fibril formation of native h2 m , and provides a rationale for the enhanced ability of n6 
 to form fibrils .
the assignments ( > 80% of the protein sequence 
 complete 
 for these 100 and 94 residue proteins ) provide a valuable foundation 
 for further investigation of the intermolecular packing between monomers 
 in these different fibril forms and to elucidate the extent to which 
 the structural architecture of the fibril forms differs . to assign 
 the remaining residues whose resonances
are absent from current spectra , 
 we are performing experiments at liquid - nitrogen temperature to quench 
 the backbone dynamics , in combination with dynamic nuclear polarization 
 ( dnp ) techniques for sensitivity enhancement .
together this information will inform development of 3d models for 
 the fibril architectures of these different 2 m fibril 
 structures .
such information is essential for understanding how and 
 why fibrils develop in dialysis - related amyloidosis and to develop 
 future strategies to prevent amyloid deposition and disease .
 amyloid 
 fibrils formed from initially soluble proteins with diverse 
 sequences are associated with an array of human diseases . in the human 
 disorder , dialysis - related amyloidosis ( dra ) , 
 fibrils contain two 
 major constituents , full - length human 2-microglobulin 
 ( h2 m ) and a truncation variant , n6 which 
 lacks the n - terminal six amino acids . 
 these fibrils are assembled 
 from initially natively folded proteins with an all antiparallel -stranded 
 structure . here , 
 backbone conformations of wild - type h2 m and n6 in their amyloid forms have been determined 
 using a combination of dilute isotopic labeling strategies and multidimensional 
 magic angle spinning ( mas ) nmr techniques at high magnetic fields , 
 providing valuable structural information at the atomic - level about 
 the fibril architecture . 
 the secondary structures of both fibril types , 
 determined by the assignment of 80% of the backbone resonances 
 of these 100- and 94-residue proteins , respectively , reveal substantial 
 backbone rearrangement compared with the location of -strands 
 in their native immunoglobulin folds . 
 the identification of seven 
 -strands in h2 m fibrils indicates that approximately 
 70 residues are in a -strand conformation in the fibril core . 
 
 by contrast , nine -strands comprise the fibrils formed from 
 n6 , indicating a more extensive core . 
 the precise location 
 and length of -strands in the two fibril forms also differ . 
 
 the results indicate fibrils of n6 and h2 m have an extensive core architecture involving the majority of residues 
 in the polypeptide sequence . 
 the common elements of the backbone structure 
 of the two proteins likely facilitates their ability to copolymerize 
 during amyloid fibril assembly . 
Introduction
Materials and Methods
Results
Discussion
Conclusion