‘l’i ​II: .11)1 ​uN ‘.

1, 0,- ​11 ​IsTod-ti EM ​IsTRY ​AN Ii ​(2 ​YTOCII ​EMISTRt’ ​( ​‘opvrigtit ​i ​1965 ​by ​‘lb 11
E. I). EANES ​.tNI) C
​ . C. (IIENNE1t
istis-liemiciut Society, Inc.
\‘t. ​16, No. 11
J’rish-d ​i,i ​U.S,,-l.

X-IL\\ 1)1 ​1”I’HA(’TION ​STUJ)IES ON \ \1\’LOID i’ILAI\IENTS’

.\(1( 100(11 ​lust ito ​Ic o​ f ​DenIal Rese i (‘(-It (​ 10(1 ​_\u1 ​100(11 ​Instil ​Ut( of ​-1​il/i ​iii iS ​(111(1 ​Met(IlIOli(’ ​Diseases,

_“sa/ional Ins-/il tiles ​of ​Health, Beth(’.s’da, Ma ,“jland

lteceive(i ​for ptll)l icat ​ion Juuie 14,

The filamentous protein component of amyloid-laden tissue was studied by x-ray dif- fraction procedures. The principal features of the
x-ray pattern from nonoriented amyloid material consist of a sharp, intense ring at 4​ .75 A ​ overlaying a diffuse halo at 4.3
A, ​and a broad and less intense ring at 9.8 A ​ hen oriented, the material gives a “cross-a” x-ray pattern. The x-ray
​ .W
findings are interpreted in terms of a “pleated sheet” structure formed by the amyloid polypeptide chain folding in a
regular manner on itself such that adjacent chain segments are laterally arranged in an antiparallel manner. The x-ray
patterns from oriented amyloid suggest further that the a​ xes ​of the chain segments r​ un ​transverse to the filament a​ xis.
Ie(-ordiiig of ​the ​diffract mon diagram, ​the capillary was sealed with wax
before ​mounting ​in the x-ray camera. The l)urmfie(l ​ext racts were ​prepared
l’lie ​ext ​1-arellulal- ntaterial (leJ )osited ​iii ​the )atho1ogic ​tolh(lit ​ion knowis ​as
as
amvloidosis ​is ​(-hai-a(’terize(l ​ill ​all affected tissUes alI(l ​species i y
described ​by ​(ilen ​tier e​ t ai. ​(8), Ivophilized and ​mouiite(l directly ​iii (​ ltlaltz
the ​)leseilce ​of a (list ​met ive filamentous J)1o- teni ​(OIIIJ)OileIlt ​(8, ​13) ​-
(al)illaiies,
The elect ​ron iniri-osro )ic
Sample orient ​at ion was iiudticed ​iii I ​lie purified
(lesrnl)t ​11)115 ​of ​this ​(onIJ)oneist iii ​affected human
filament extracts ​iii one (if two ​ways. The filaments were ​mechanically o​ riented
I issues mnilirate that ​it ​(olISists of ​mll(lmvm(lual
by ​gent 1 tansping the mat ermal packed at ​I ​lie sealed Iip of a 0,3-mm

filansents less than SO ​A ​in width ​and UJ) to at ​least 10,0(X) ​A ​ilk ​length, iapml- lary wit It a palla(liuini wire. The I​ ​ip of the capillary was sqi iared
ofi so I​ ​hat I​ ​lie applied st ress was per- p​ euidi(-tllar ​to a flat surface. ( )rietit
which may aggregate laterally to J)lo(lu(e a fihril measuring ​S1J) ​to ​300 ​A
at iou of fila- meitts was a​ lso ​induced Iw stispeuidiiug them i​ ii ​1 ​drop of
ill ​width. At il-regular intervals ​Iharl-ownig ​or water place(l out a glass slide and allowing
a ​twist of’ ​the filament ​o ruis ​an(l, ​Oil ​ex osure (lie (li-ui) ​to ​evaporal ​p ​Ii) (lu’yuiess. ​The result iuig thick laniella was removed
and mounted mit a capil-
of’ ​the ​filttlIh(’ihts ​to ultrasomuol, transverse ​frac-
Ian’ wit li ​I ​he 1amel lar surface parallel ​1 o ​the axis of I​ ​lie
titres at-c J )i-oduee(l ​without a (onstailt relation caJ)illary.
to these ​J)oiilts of’ ​narrowmg (8). X-ray diffraction The x-ray di ITract mon diagrams were recorded out film wit Ii
nickel-flit er-ed copper radial bit (X ​= ​154 ​A) ​01)1 am tied f​ rom ​a fine focus
studies ​were ​uinlei’takeit ​to ​elu(mdate the crystal- ​l( ​gl’itJ ​)hic ​J)IOJ t(’lt ​mes
I ​ube o​ perat ​ed at 30 kv and 38 ma. Pat ferns of fresh t:itlvophilized amvloid
of ​this ​tli h1(llle ​amyloid pro- deposits were 01)1 amuue(l wit Ii a 57.3-mm
(emit ​iii ​or(lei- to (l(fiuf(’ more fully its ultrast rue- ture, ​The ​present paper diameter I)ebye-Schuerrer l)o% ’der camera. Ac- curate recordiutgs of the
is ​a report of these amyloid difiract mon max-

st ​ll( ​lies. ima ​iii ​purified prepai-at moius were ​made tisiiig a I 14,fi-mm diameter I
)ebye-Scherrer powder ​cam- era, ​The diffraction hues from small arnouiuuls
of
EX1’ERIM ​i-: XTAL PROCEDURE
palladium metal mixed with ​I l​ ie amyloid deposits were used as
Amyloid-laden ​t ​isstle ​was obtained from the I iver and spleen ​of ​human patients and internal calibratiout standards in these latter recordings, The patterns fruits
experi- oriented i​ iimtt i’rial ​were obt ai ted tlsiutg a Chesle’v-Philips microcamera
(4) h​ avitug a specimen ​film (list aui(e of 14.7 mm and equipped with a 100-,i
mental animals afflicted with amvloidosis, Both fresh ​I isstie ​aii(l purified
bore ​glass
​ ere st tidied. ​Fresh amyloidot ic t issue from htimaii spleen
ext racts ​(8) w
and First in a series of papers entitled “Physical
capillary (olliflsator, The unechaiiicallv oriented
liver ​wa.s ​01)1 mu ​iie(l ​post mort em and stored wet ​and i​ n I he cold
(0#{176}C)tint il examined by x-rays. Samples of I his material were and Chemical Properties of Amyloid Fibers.”
prepared for diffrac- specimens were exammuued wit Iithe x-ra beam path

mon ​by s​ uspending ​small pieces of t lie fresh :​ trii - ​loid d ic t​ issue ​in distilled w
​ ater ​and
67
coiuceuit ​rat ​i tug the ​suspension into ​the tip of a 0,7-mm dianseter
3
I ​hiiu walled quartz capillary by ​(Cult rifttgtttioui ​(1000 X g) ​- ​lo prevent
nsomst tire loss iltiritug ​the
674 ​EANES AND GLENNER
1
FIG. ​1. X-ray diffraction pattern of au unoriented sample of purified humaus amyloid filaments. ​FIG. ​2. X-ray diffraction pattern of a mechanically oriented sample ​of humaus amyloid
filaments. ​Direction of mechanical stress is parallel to the short dimension of figure. This stress line is perpendicular to the direction of the incident x-ray beam.
FIG. ​3. X-ray ​diffraction pattern of a lamella of human amyloid filaments prepared by evaporatiuig an aqueous suspension of the filaments on a horizontally flat surface. The lamella was
photographed with ​its broad ​dimension ​face parallel to the ​of the figure. The ​incident ​9.8 ​A l​ ine x-ray splits beam. iusto ​The face normal ​a ​doublet with is approximately the inner member
parallel of the to pair the at 13 louig ​A. ​The origin ​of this splitting is not presently understood. ​The ​darkening around ​the lower left hand edge of
​ ​the central hole ​is ​due to “halatiout” of the
incident x-ray beam.
FIG. ​4. X-ray diffraction pattern of a preparation of amyloid deposits composed of rod-like aggregates of pentagonally arranged globular units.
either parallel or perpendicular to the direction of applied pressure. The lamellar samples were photographed with the lamnellar surface either
parallel or perpendicular to the beam. Both the microcamera and the 57.3-mm diameter camera were used in comparing preparations from tissues from differeust origins. The x-ray
patterns used in
the i​ llustrations were recorded, however, exclu- sively ​with the microcamera.
RESULTS

The dominant ​feature of ​the difTraction photo- graphs from utsoriented specimens of the amyloid filament was a sharp and intense ring at 4.75 ​A (​ ±0.01 ​A) o​ verlaying the inner halo

centered at approximately edge 4.3 ​A o​ f a diffuse

(Fig. 1).
Autother ring, broader and less intense, occurred ​at approxiunately 9.8 A. The position of the sharp
diffraction ring corresponds to ​the value pre- dicted for polypeptide chaiuss arranged in a or

4
pleated sheet coutfiguration (12). The ​spacing of ​4.75 ​A i​ s, however, somewhat larger thaus ​the 4.65-A spacing reported for $-kerat ins (4.65 A) (2).
It e​ quals, however, ​the value calculated for the
“h)ack house’ distance between adjacent chain ​segments when laterally aggregated in a​ n anti-
parallel arrausgemeust, as would be the case if the
pleated sheet is formed by a single polypeptide chaius folded in a regular manner on ​itself ​(12). The ​9.8-A l​ iuse corresponds to ​the “side chain” S )acing between chaiuss of neighboring
sheets (10),
These diffractiois feat tires did not appear to be
specific to fllameuits of ausy one tissue or species. ​They ​were found ​in s​ amples of amyloid filaments ​from a variety of origiuts, ​eg., i​ n the spleeuu ausd
liver of humnaus “primary” and “secousdary” cases,
liver of duck and spleen of mouse, ​and neither cats
these diffraction effects be ascribed to ​alteratiouts
in the structure of the filaments iusduced by the
purification and dryi uig procedures employed as

X-RAY DIFFRACTION STUDIES ​675

they were also present ​in ​the x-ray ​diagrams from ​fresh amyloid-ladeut tissue.
Wheui a mechanically oriented sample of purified human amyloid
filaments w ​ as ​photographed with the direction of mechaisical
stress perpendicular to the beam direction, each diffraction ring
split i​ uito ​two ​diamnetrically opposing arcs (Fig. 2). The
symmetrically disposed arcs of the ​4.75-A s​ paced ​rintg ​were
perpendicular to those of the ​9,8-A r​ ing, ​iuidicative of a “cross-fl”
pattern (2). ​in addition, ​the ​4.75-A s​ paced arcs were positioused
perpen- dicular to the directiout of applied pressure, and the
9.8-A arcs lay along the line of this presssire. Wheui the sample
was positioused so that the x-ray beam was parallel to the line of
stress, a nouiorieuited pat- tern was obtained.
It is not unreasonable to ​assume ​that, ​in ​packiuig the filaments together under
the influeusce of a uuiidirectional force, the axis of each filament
would tend to aligns itself normal to this line of force. If this is
the case, theus the ​4.75-A a​ rcs would correspond to spacings
parallel to the long axis and, consequently, the polypeptide chains
would be aligned perpendicular to this direction.
Similar conclusions can he reached with the x- ray results from the thick
plate produced by the evaporation of an aqueous suspension of
amyloid filaments on the flat surf ace of a glass slide. When such
a lamella was photographed on edge, i​ .e., w
​ ith the beam parallel
to the broad face of the plate, it was found that the photograph
​ rcs
was ​highly oriented with the directiors of the ​4.75-A a
parallel to the plane of the plate and the ​9.8-A a​ rcs perpendicular
to the plate surface (Fig. 3). When the plate was photographed
with its broad face normal to the direction of the x-ray beam, the
pattern was unoriented. It is again reasonable to assume that,
duriiig the evaporation of the sus- pension, the amyloid filaments
would tend to mat with their axes lying randomly i​ n ​the plaute of
the resulting lamella, i​ .e., w
​ ith the axes parallel to the surf ace of
the broad face. If the matting does occur in such a manner, the
x-ray findings would agaius lead to the conclusious that the
4.75-A ​backbone spacing runs parallel to the filameust axis.
The axes of the polypeptide chains would, then, be per-
pendicular to the direction of the axial dimeission of the fiber.
In both modes of orientation, the arcing of the ​9.8-A ​ring appeared to he
​ irse. The
somewhat less pro- notinced than that of the ​4.75-A h
observed arcing of this “side chains” spacing would not even
have beers predicted, however, unless the filament is not strictly
acicular but tends toward a tabular shape with the side chain
groups directed outward from the face of the flat surface. Such a
morpho- logic shape would preferentially orient with its broadest
surface lying in the plane of the mat or
lamella. The reflections from the “side chain” spacing
would, therefore, ten(I to arc with the two maxima directed
normal t​ o ​the plane surface as was ol)served.
The x-ray d​ ata ​from these oriented preparatiouts suggest
theni that the filaments are uiot truly thread-like with
eqitidimnensiotsal cross-sections, but, rather, should l)e more
lath-like in appearance with the folded polypeptide chairs
segmenta extend- ing across the width of the filament. The cross-
sect ionial morphology of the filaments as deduced from the
oriented diffraction data is, theus, con- ​sistent with the pleated sheet
polypeptide con- figuration suggested by ​the observed spacing of 4.75
A ​for the principal reflectioui from the fila- merits.
Preparations from amyloid deposits which, ​upon ​electron
microscopic examination, were found to be composed of the
rod-like arrays of globular uusit structures (3) produced x-ray
patterns essentially amorphous i​ n ​appearance (Fig. 4). The
distinct diffraction line at 4.75 A​ w​ as abserst i​ n ​these pat- tertis.
The only prominent features were two broad and diffuse halos
centered at approximately 10.5 A ​ ​arid 4​ .65 ​A ​with the outer halo
more intense relative to the intier one. This very vague patterus is
generally characteristic of many dried proteins and affords very
little iuisight iusto the structure of this type of globular proteiui.
DISCUSSI
ON
The observed x-ray diffraction properties of the amyloid
filament when interpreted within the framework of the basic chain configuration of the backbone struc- ture imparts a pleated
configurations estab- lished for proteins by Pauliisg and Corey appearance to the sheet with a-carbons lying along the creases
(12) suggest that the polypeptide chains comprising amyloid and the side chains projected alternately above and below the
filaments fold back and forth on them- selves in a regular manner plane of the sheet as one uroceeds along t​ he ​chain. The presence
to form sheet-like structures. In t​ his ​antiparallel arrangement, the of a side chain reflection in the x-ray patterns indicates that the
amino acid residues are, at 3.5 ​A i​ n length, close to being fully sheets ​may be stacked to form a three-dimensional array. The
extended but contracted sufficiently to give the chain backbone a orientation properties of the filament would lead one to conclude,
corrugated appear- ance. With adjacent chain segments regularly however, that the thickness of the stacked array of sheets is less
​ ​apart b​ y ​hydrogen bonding, this corrugated
spaced at 4.75 A than the
676 ​EANES AN!) GLENNER
lateral (liulsensions ​of t​ he mn(lmvidual sheets ​iii the
array.​The above described structural situation re-
quires that the )olypepticle chain reverse direc-
tion at regular iister -als aloisg its length. Un- ​fortunately, ​an a​ i roi ​)riate uiieehaisisuis by which this can be a​ ccomplished has ​yet to l)e eluci(lated with ​certainty.
Nevertheless, several l)ossibilities to ​account for ​the reversal of chain direction can be l)osttilate(1. Ouie attractive possibility utilizes the fact that the prohine
residue when ​in ​a ​cia ​configuration ​can, ​in ​l)rmlscil)le, produce a ​sharp ​bend its a olypeptmde chaits resulting ​in ​a reversal of direction (5). This residue, moreover,
occurs i​ n ​human aunvloid filameists i​ n ​the approxiunate ratio of o​ ne ​to every 16 residues total (8). If the
prohmne residue was distribute(l periodically so that it oi-curn-ed at every 16th position along the poly- peptide chain its a c​ ia ​configuration ​it would l)e i​ n ​a
1)OSitiols ​to act as the agelst necessary to re- verse, by b​ ending, ​the (Iirection of the chain i​ n ​a regular manner.
The above proposal regarding the role of pro- line i​ n chain ​bending is made somewhat iunprob- able, however, by the following observations: (1) prohine i​ n ​a c​ ia
configuratious has not beets seems iii native l)lotemns, (2) the a​ ssuuiiption ​that Prohine
periodically’ reverses the directious of the chain at the position of ​every ​16th residue filanseist width at approximately ​so ​would ​A, s​ et ​the
a value which is much less thais the minimum observed diameter of 75-SO A (5, 13) ​and ​(3) a folded chain cross-a ​protein containing ​110 ​l)rolilie has been
​ hrysopa ​(11), the model which best describes the nature of the folds,
previously ​reported (7, 11). In the case of this latter l)rotein, a silk from the egg stalk of C
requires that glycine be one of the two residues involved i​ n ​each hemsd conformation (7). This model i​ s ​also a d​ mstmct possiblity for explaining ​the cham folding ​in
the ​present case as the amyloid ​filaments have a coissiderable glycine ​content (11 ​residue ​%) (​ 8).
Iunphcmt its the previous (liscllssiofl is the as- ​sumptiols that the pleated sheet is formed by a single oly wptide (hails which regularly folds back ​on i​ tself. The
alternative possibility is that each sheet is formed by the side to side aggrega- tion of a number of short polypeptide chains with each chain lyiisg antiparallel with
respect to i​ ts neighbors. This latter possibility can ​be dis- counted, however, silsce the observed molecular weight of about 20,0002 is several-fold greater
2​G. ​G. Glenner, unpublished data.
than a I)re(hicte(l molecular ‘eight of al)out 2,500
for the short chains.
Fhe cross- 3 exteisded sheet structure is tsot
iulconsl)at ibie with the mon )hologic ai i )earance of single filameusts of huuisaii ansyloid as seems in ​the
electron itsictos oise (5) . ‘l’he uitiforunmty in the ​lateral ​dimension an(l tise itsdcteruninacv in t​ he
length of a Pleate(I sheet stt-u(tule woul(1 allow ​for the develol)unent of rmbl)ols-lm ke unicelles similar ​us forun to ​the ​observed auiivloid filaments. The x-ray unodel may
variable length of these filaments seen i​ n ​electrots uiucrographs (8, 13). The pleated sheet configura- tion would imply that the aunyloid
account, unol-eover, for ​the
filament should be more friable traussverselv as the cohesive forces normal to this direct ​mon ​are predominately due to relatively weak hydrogeis 1)onds. The
tendency, therefore, would l)e foi- the filaments to fracture into smaller lengths.
The cross-a type of (​ liffra(tiolt liattelts ​obtained from the oriented ansvloid filauisents has been rarely reported for naturally occurring proteins. Such x-ray patterns
have 1)eeul PreviouslY de- scribed only for the proteits of bacterial flagella (1), the protein of the egg stalk of C ​ hrysopa ​(7, ​11) and the protein of the matrix of
bovine dental enamel (9). Other l)roteilss cats he induced to give a cross-$ pattermi b​ y subjecting thens ​to highly isonphysmologic stresses as, for exaullh)le,
stretching
poached egg white to twice its initial length (2) or b​ y treating stretched fiber-s ​of’ k​ eratin with ​steaun (6). The preparative p​ rocedure ensployed in ​this study to ol)tans the
aum’loid extract from which the cross-a photograph w ​ as r​ ecorded was relatively mild, however, compared ​to ​the uiseth- ods required to transforun a normally
occurring a or protem into a cross-$ type. ​This ​fact, to- gether with the observation that the ​position of ​the l)rincipal ​4.75-A s​ pacmg is ​unaffected ​by the extractiois
procedure, suggests that the cross-a
patterms accurately represents the diffraction properties The of preseisce the of ​in t​ he ​vim ​4.75-A f​ ilaunents.
spacing its ​the x-ray diffraction diagrams of ​mouse and duck ​aunyloid would indicate that these filauiients ​have t​ he same pleated sheet structure as l)ostulated for the
huunan amyloid. No attempts were unade, how- ever, to obtaims oriented x-ray patterns of mouse and duck amyloid; consequently, predictions about filauisent
morphology ​in ​these cases are not possible. Electron microscopic studies3 would
H. A. Bladen and G. G. Gleusner, unspublished observations.

X-RAY I)IFFRACTION STUI)IES ​677

Ihe attllsot-s wish to express their iut(lebte(htess to Miss (‘at-lie

indicate, ​howevet, that these ansyloids ​are siunilar ​in b​ asm(- uisOl )h1Ologic
Goo(lwimm fot her- ​skilled t​ echitical
features to the humaut
assistance aitil to I​)t’. ​H. H. Higdott, titiveusity
nsatet-m
al. of Texas, Medical Itrautch, Gal vest ott, Tex., for ​supplying the ilttt-k
tissue used ​iii this study.
ACKNO\\’LLDGM
EXTS REFEREN
 CES
1. ​Asthttu- ​, ​W. ​J., ​Beightouu, E. autd Wembtrll, C.: ‘Ihe ​sI ​rtt(-t itt-c
of bacterial flagella. ​Pi’oc. Soc. E.rp. Rio!. Me(l. 9: ​282, 1955. ​2.
Asthury. W. ​J., ​J)ickiutsouu, S. aitd Bailey, ​K.:
The x-ray iitterpretatiouu of deutat uratiout amid the stm-t -t utre of the
​ 351, ​1935. ​3. Bladen, 11. A.,
seed glohtthiuts. ​J3iocheun. .1. 29: 2

Nylen, M. U. and Gleustier, U. ​U.: The ttltrastructutre o​ f

hiiirnauu amyloid as
revealed by the utegative stairtiutg technique. ​.1. (7trasli’tict.
​ . Cheslev, F. ​U.: X-ray diffractiott camera for
​ 49, ​1966. 4
1?es. 14: 4
microtechitiques. ​Rev. Sei. Instrum. 18: ​422, 1947. 5. Edsall, J. T.:
Coiifigirratiotu of certain proteimi
ntolecitles. Au inquiry coutcertiiuig the pres- cut status of
outr kuiowledge. ​J. Polymer Sci. 12: ​253, 1954. ​6. ​Fiiiean, J. B.:
Biological Ultrastructure, ​Aca- d​ emmc Press, New ​\ork, ​1967, p. 119. 7.
Geddes, A. ​J., ​Parker, K. 1)., Atkins, H. 1). T. and Beight out, H.:
“Cross-fl” conformation iii proteiits. ​J. Mol. Rio!. 32: ​343, ​1968. ​8.
Gleuuuier, U. U., Keiser, H. U., ​Bladen, ​II. A., ​Cinatrecasas, P.,
​ auufer, ​J. N. ​and J)eLellis, ​H. A.: ​Amy-
Eaites, H. I)., ​Ham, J. S., K
​ orphologic componettts of httman
lOi(1. VI: A comnparmsout of ​two m
amnyloid deposits. ​.1. Hislochem. (‘ytochem. 16: ​633, 1968. ​9.

Glinscher, M. ​J., I​ 3oiiar, L. C. and Daniel, H. ​J.: The

molecmtlar structure ​of ​the proteiui

matrix of b(uviute dental enamel. ​J. Mol. Rio!. 3: ​541, 1961. 10.
Ketidrew, ​J. ​C.: Structure proteins. I. us ​The
Proteins’, e​ dited by ​H. Neurath amid K. Bailey, Vol. 11,
Academic Press, N ​ ew ​York, 1​ 954, p ​893. 1​ 1. ​Parker, ​K. 1). a​ nd
ltutdall, K. M. :The silk of
the egg-stalk of ​the greeni lace-wing fly. ​Natun-e (London) 179: ​905,
1957. 12. Paitling, L. and ​Corey, ​H. B.: Two rippled-
sheet coutfigurationts of polypeptmde chaints, and a note
about the pleated sheets. ​Proc. Nat. Acad. Sci. U.S.A. 39: ​253,
1953. 13. Shirahansa, T. amid Cohen, A. S.: High-resolnt-
tiors electron microscopic analysis of the aniyloid fibril. ​J. (‘eli Rio!.
33: ​679, ​1967.