Amyloid Structure: Models and The o ret i cal Con sid er ations in Fi brous Aggregates +

Transcription

1 Jour nal of the Chi nese Chem i cal So ci ety, 2002, 49, Invited Pa per Amyloid Structure: Models and The o ret i cal Con sid er ations in Fi brous Aggregates + Da vid M. Mor gan a, Da vid G. Lynn a *, Ami S. Lakdawala b, James P. Snyder b and Den nis C. Liotta b a De part ments of Chem is try and Biology, Emory Uni ver sity, 1515 Pierce Drive, At lanta, Ga 30322, U.S.A. b De part ment of Chem is try, Emory Uni ver sity, 1515 Pierce Drive, At lanta, Ga 30322, U.S.A. In celebration of the many con tri bu tions to pep tide chem is try from K.-T. Wang s lab o ra tory, this manu - script ex plores the struc ture of the A (10-35) fi bril. This cen tral seg ment of the A pep tide of Alz hei mer s Dis ease self-assembles into well-ordered paracrystalline ar rays that seem to con tra dict many of the ac cepted par a digms es tab lished for sol u ble, glob u lar pro teins. Here we ex ploit ini tial mo lec u l ar mod el ing ef forts to as - sist in un der stand ing these ap par ent con tra dic tions. The emerg ing struc ture is one of a large self-assembly of -strands, whose sta bil ity is de pend ent on large spa tial and tem po ral fluc tu a tion about a cen tral core, giv ing rise to what can be best de scribed as a dy namic and fluid tube-like mi celle. This fi bril struc ture main tains fea - tures that are dis tinctly dif fer ent from those of ei ther syn thetic or bi o log i cal fi brils, and these dif fer ences have pro found im pli ca tions for bio med i cal in ter ven tion in am y loid dis eases as well as for the de sign of selfassembling nanoscale fi brils. IN TRO DUC TION As our meth ods of anal y sis con tinue to im prove, un - usual sec ond ary struc tures within pro teins are rec og nized. These struc tures are of ten viewed as de riv a tives of the rec og - nized -he li cal and -sheet struc tures, but they range from the rare to the very com mon within known pro teins. In creas - ingly, these struc tures are prov ing to be crit i cal to our un der - stand ing of pro tein struc ture and func tion. For ex am ple, the con tro ver sial -he lix, whose ex is tence and sta bil ity have been de bated for de cades, 1 now ap pears to ex ist on av er age once in ev ery pro tein and may be spe cif i cally as so ci ated with ac tive sites or conformational switch re gions. 2 Am y loid fi bril dis eases, in clud ing Alz hei mer s dis ease, the spongiform encephalopathies, var i ous sys temic amyloidoses, and di a be - tes, are based on an other un usual struc ture that has gen er ated enor mous in ter est. 3 The con tri bu tions of many sci en tists, most no ta bly those of Pro fes sor K.-T. Wang, to pep tide chem - is try and pro tein syn the sis, has made it pos si ble to syn the size these struc tures and study their prop er ties. In this pa per we dis cuss the com bined use of spec tro scopic and com pu ta tional meth ods to fur ther un der stand the self-association of pep - tides and pro teins into am y loid fi brils. Am y loid is a clin i cal term de scrib ing un usual pro - teinaceous tis sue de pos its. The prop er ties of these de pos its in clude in sol u bil ity, large mo lec u lar weight ag gre gates, re - sis tance to pro te ol y sis, and re sis tance to chem i cal de na tur - ation even un der strong con di tions such as boil ing de ter gents. Along with these prop er ties, a de fin ing char ac ter is tic of am y - loid is as so ci a tion with the hy dro pho bic dye Congo Red. These stained ag gre gates dis play a char ac ter is tic ap ple green bi refrin gence when viewed through crossed po lar iz ing fil - ters. 4 In Alz hei mer s dis ease, a se ries of cleav age events of the am y loid pre cur sor pro tein (APP) gives rise to spe cific short pep tide frag ments, most no ta bly A (1-40) and A (1-42). 5 These frag ments have the po ten tial to ag gre gate and form am y loid de pos its in brain tis sue. 6 At least two lines of ev i dence strongly sug gest that the ini ti a tion of Alz hei mer s dis ease is as so ci ated with the ag gre ga tion of these pep tides. First, mu ta tions within the APP that af fect the length of the pep tides, or that in crease the rate of pro duc tion of the pep - tides, cor re late with the se ver ity of the dis ease. 7 Sec ond, syn - thetic fibrilized A pep tides are toxic to cul tured neu rons. 8 As a re sult of these im pli ca tions, a struc ture of the Alz hei - mer s fi bril has been sought for sev eral de cades. FI BRIL STRUC TURAL MODEL The large mo lec u lar weight, in sol u bil ity, and noncrystallinity of the ag gre gated state of the A pep tides have + Based on the lec ture of Da vid G. Lynn pre sented in K.-T. Wang Bioorganic Chem is try Lec ture s hip Symposium 2001 held on Sep tem ber 28, 2001 in Tai pei.

2 460 J. Chin. Chem. Soc., Vol. 49, No. 4, 2002 Mor gan et al. com pli cated the gath er ing of high-resolution struc tural in for - ma tion. X-ray fi ber dif frac tion anal y ses 9 give a char ac ter is tic cross-beta pat tern with re flec tions cor re spond ing to ~ 5 and ~ 10 Å. These re flec tions have been as signed to the spac ing be - tween the strands in -sheets and to the mean dis tance be - tween stacked -sheets, re spec tively. The cross-beta pat tern sug gests that the long di men sion of the fi ber is com posed of -strands ag gre gated into -sheets and that the per pen dic u lar di men sion is com posed of stacked -sheets. These X-ray re - sults have also been in ter preted to sug gest mac ro scopic or ga - ni za tion of the fi bers into bun dles, and some ev i dence has been pro duced for such an or ga ni za tion. 9a Based on FTIR and cir cu lar dichroism mea sure ments, 10 it was gen er ally be lieved that the fi bers were com posed of anti-parallel -strands. In deed, some au thors have ar gued that the re flec tion at ~ 10 Å in fi ber dif frac tion ex per i ments arises from the re peat dis tance be tween -strands that run in the same di rec tion within -sheets in the fi bril (par al lel to one an other), but which are sep a rated by ~ 10 Å in an anti-par al lel -sheet. 9a,e Re cently how ever, a com pre hen sive and ex per i men - tally con strained model for the am y loid fi ber formed by A (10-35) has been pro posed to be that shown in Fig Ac - cord ing to the model, the fi bers formed by A (10-35) pep - tides con sist of par al lel -strands ag gre gated into par al lel -sheets along the long axis of the fi ber. In the per pen dic u lar di men sion, the fi ber is com posed of six such par al lel - sheets, stacked one on top of the other, each sheet ap par ently also par al lel to the oth ers. As the fi ber length ens, i.e., as the num ber of par al lel -strands added to the -sheets grows, the ar range ment slowly spi rals around the fi ber axis, with a he li - cal half-repeat dis tance on the or der of 1200 Å. One half turn of this slowly twist ing fi bril would be a rect an gu lar spe cies of ap prox i mately 60 Å 80 Å 1200 Å, con tain ing 1440 pep - tides, 6 sheets 240 pep tides 5 Å/pep tide, each hav ing a mo lec u lar mass of 2855 Da. The most well de ter mined fea ture of this model is its par al lel ar range ment of -strands es tab lished by solid-state NMR stud ies. In each ex per i ment, a sin gle 13 C la bel was syn - thet i cally in cor po rated at the car bonyl po si tion of one amino acid within the pri mary se quence of A (10-35), and the pep - tide was al lowed to form fi brils. When fibrillization was com plete, the re sult ing intermolecular dis tance be tween the 13 C en riched car bon yls was an a lyzed us ing the DRAWS solid-state NMR pulse se quence, a se quence which recouples di pole-dipole in ter ac tions be tween like spins. 12 These dis - tance mea sure ments were used to es tab lish that the en tire A (10-35) pep tide stacks in a par al lel, in-register ori en ta tion through out the length of the fi bril as shown in Fig ,12a,13 Re cently, this fea ture of the model was in de pend ently ver i - fied for A (1-40). 14 The mo tif of six, stacked -sheets is also well de ter - mined. Small an gle neu tron scat ter ing ex per i ments per mit ted as sign ment of the mass per unit length of the fi ber to be Da/Å. For a sin gle A (10-35) pep tide sep a rated from its neigh bors by 5 Å, the cor re spond ing mass per unit length would be 572 Da/Å. Thus, the fi bril is com posed of stacked pep tides, how ever at lower ph, the num ber of lam i - nated sheets can be smaller. 11 Less well de fined is the rel a tive ori en ta tion of the in di - vid ual sheets. Apart from the phys i cal rea son able ness of a par al lel ar range ment of the stacked -sheets, which clus ters and hence bur ies hy dro pho bic sur face area, the struc ture is sup ported by a bio chem i cal cross-linking ex per i ment orig i - nally de vel oped by Dudek et al. 15 Cross-linking by tis sue transglutaminase spe cif i cally con denses Q15 and K16 side chains of A (10-35) 13a,11 to give hexameric co va lent ag gre - Fig. 1. Model of the A (10-35) fibril. -strands, stacked into a par al lel -sheet (blue) ori ented per pen - dic u lar to the prop a ga tion axis, length en ing the as sem bly in 5 Å in cre ments. These sheets are stacked six deep (green) with 10 Å spac ing be - tween the sheets. The en tire ar range ment twists about the he li cal axis such that one half turn is com prised of ap prox i mately 240 pep tides, or about 1200 Å, in each sheet. Fig. 2. Dis tance mea sure ments within the A (10-35) fibril. A schematic representation of two A (10-35) pep tide strands ar ranged in a par al lel - sheet. The ver ti cal bars in di cate both the po si - tion of 13 C-substituted amino ac ids from dif fer - ent pep tide prep a ra tions, as well as the mea - sured dis tances be tween them in the fi bril.

3 Am y loid Struc ture: Models and Con sid er ations J. Chin. Chem. Soc., Vol. 49, No. 4, gates. Be cause Q15 and K16 are ad ja cent to one an other in pri mary se quence and pro ject from op po site sides of an ex - tended -strand, the side chains are too far apart to be linked intramolecularly. Like wise, if such -strands as sem ble into an in-register par al lel -sheet, all Q15 and K16 side chains are lo cal ized on op po site faces of the -sheet. There fore, cross-linking must oc cur be tween sheets, rather than within the same sheet. Al though anti-parallel stack ing of these sheets ap pears pos si ble, par al lel stack ing would better se - ques ter hy dro pho bic sur face area, max i mize con tact by min i - miz ing the va can cies be tween sheets, and place Q15 and K16 of ad ja cent sheets in prox im ity. MODEL EVALUATION The model of the A (10-35) fi ber con tains a num ber of fea tures in con sis tent with known glob u lar pro tein sec ond ary and ter tiary struc tures. First, the size of the as sem bly is ex - tremely large and re quires a very fa vor able Gibbs En ergy of self-assembly. Sec ond, the model in volves par al lel -sheets, which are gen er ally agreed to be less sta ble than their anti-parallel coun ter parts. At least in the ab sence of com pen - sat ing in ter ac tions, the par al lel -sheet im plies an gled hy dro - gen bond vec tors, rather than the near co-linear ar range ment that is pos si ble in anti-parallel struc tures. 16 Third, the model pre dicts that the twist within each par al lel -strand is small, whereas the twists ob served in -strands oc cur ring in pro - teins can be quite large. In na tive pro teins con tain ing antiparallel -sheets, the twist as so ci ated with move ment along a -strand tends to sep a rate ad ja cent strands from one an other, lim it ing the num ber of con stit u ent amino ac ids that can hy - dro gen bond. Like wise, par al lel -strands are usu ally formed by stretches of amino ac ids that are rel a tively dis tant from one an other in pri mary se quence, and of ten the in ter ven ing se quence forms a sec ond ary struc ture that sta bi lizes the par - al lel -sheet, but lim its its length. Such ar range ments are readily ap par ent in the par al lel -bar rels, for ex am ple, in triosephosphate isomerase. 17 The A (10-35) -strands in am - y loid are com posed of 26 amino ac ids, a much larger num ber than found in glob u lar pro teins. Finally, the model pre dicts that like charges as sem ble in space close to one an other, an as so ci a tion that is ex pected to be en er get i cally un fa vor able. In light of the above-mentioned en er getic re stric tions, the model has been eval u ated computationally us ing a 6 6 block of the fi bril - a six-stranded par al lel -sheet of the A (10-35) in one di men sion and six such sheets stacked in a par al lel ar ray in the per pen dic u lar di men sion-constructed in Sybyl6.7. The start ing struc ture aligned all back bone amides in the same plane, and con strained dis tances be tween pairs of car bon yls to those dis tances pre sented above in Fig. 2. Side chains were ac com mo dated within the 10 Å gap of the par al - lel lam i nated sheets. The sys tem was solvated us ing TIP wa - ters. An im mo bile bound ary of sol vent 8 Å wide was placed 30 Å from the sur face of the as sem bly to pro vide a bounded sol va tion shell. Thus, an in ner fluid shell of wa ter (22 Å wide) sur rounded the am y loid model. Since a de scrip tion of the dy nam ics and flex i bil ity of the am y loid fi bril was sought, the use of an outer rigid wall of wa ter sur round ing a fluid in - ner bath was con sid ered ad e quate to re flect ex per i men tal prop er ties in the in te rior of the pep tide fi bril. Mo lec u lar dy nam ics (MD) sim u la tions with the Kollman all-atom force field and charges (NTV con di tions) were run ini tially at 20 K, and then in creased step wise to 50 K, 100 K, 200 K, and 300 K. At each tem per a ture, the sys tem was al lowed to reach equi lib rium, as evinced by the ap pear - ance of har monic mo tion among the vibrationally cou pled side chains. Sub se quently, the sys tem was sub jected to MD for an ad di tional 65 ps at 300 K af ter which time the back bone OC-CO and CO-HN dis tances were mea sured. The four - sheets lo cated in the cen ter of the sys tem, that is, those not in di rect con tact with the near est wa ter shell, were used for anal - y sis. Over all, the mea sured back bone OC-CO dis tances from the sim u la tion cor re sponded to the ex per i men tal solid-state 13 C- 13 C dis tance mea sure ments. Res i dues at the ter mi nal re - gions of any strand are lo cated closer in space to ad ja cent strands than the res i dues in the mid dle re gion (res i dues 27 to 34) where lon ger dis tances were ob served (Fig. 3). Spe cific Fig. 3. In te rior -sheet 2 af ter 52 ps of mo lec u lar dy nam ics at 300 K. Yel low dashed lines rep re sent back bone hy dro gen bonds, de fined as Å.

4 462 J. Chin. Chem. Soc., Vol. 49, No. 4, 2002 Mor gan et al. dis tor tions were ap par ent in the vi cin ity of these cen tral res i - dues. For ex am ple, there ap pears to be a gen eral ex pan sion of the inter-peptide dis tances, con sis tent with, and pos si bly a re - sult of, the lon ger dis tances ob served by NMR and used ini - tially to con strain this re gion. More over, these dis tor tions are char ac ter ized by a loss in the reg u lar ity of back bone con for - ma tion of the kind ob served in more ter mi nal re gions of the se quence. While other re gions of the pep tide strands within a sheet were seen to bend and buckle al most in tan dem with one an other, in the cen tral re gion of the sheet the con for ma tion of one strand seems to be un cou pled from the con for ma tions of those that sur rounded it. These dis tor tions are par tic u larly ap - par ent along the 30 GSNKG se quence, and may re flect the pres ence of a glycine-channel cre ated by the ab sence of side chains at glycine, each of which oc cur on the same face of each sheet. In the sim u la tion, this chan nel al lows for sol - vent mol e cules to pen e trate the pro tein lay ers over time to solvate the -strands. What is driv ing these dis tor tions? A search of the Pro tein Data bank for pro teins with par - al lel -sheets that do not twist to form a -bar rel iden ti fied phosphoglycerate kinase (PhP). While the sheet is rel a tively pla nar, the lengths of the PhP -strands stretch to only 4-5 res i dues. Silk is known to con sist of stacks of rel a tively pla - nar anti-parallel -sheets, but again strand length is on the or - der of 5-6 res i dues. There fore, the en er getic cost of form ing very long ide al ized -strands ap pears to be pro hib i tive. In or der to better un der stand the conformational en er - gies, the ap prox i mate cost of ex pand ing the length of a par al - lel -strand was mod eled with short strands and min i mized us ing MacroModel 6.5 with GB/SA sol va tion and the AM - BER* force field. 18 -strands were built from frag ments of 2, 3, 4, 5 and 6 res i dues from the core KLVFFA se quence (amino ac ids 16-22) of the am y loid pep tide. First, the strands were con strained to adopt ideal par al lel -strand ge om e try, and min i mized. Next, the con straint to main tain ideal par al lel -strand ge om e try was re moved, and the sys tem was reoptimized. The en ergy dif fer ence be tween the un con strained and min i mized pep tide and the con strained and min i mized pep tide was de ter mined as shown in Fig. 4. For pep tide lengths of 2, 3, 4, 5, and 6 res i dues, these en ergy dif fer ences were 5.2, 6.0, 8.8, 11, and 13 kcal/mol, re spec tively, with the un con strained sys tem be ing more sta ble in all cases. Al - though the en ergy dif fer ences be tween the con strained and un con strained ge om e tries are a func tion of the spe cific amino acid res i due, the ob served en ergy re quired to main tain an ide - al ized ge om e try across this se quence is ap prox i mately lin ear with chain length. The av er age value, rep re sent ing the cost of length en ing a par al lel -strand with ideal ge om e try by one res i due, was cal cu lated to be 2.1 kcal/mol. How do these dis tor tions af fect the H-bonding along the strands? Es ti mates of the ex tent of back bone hy dro gen bond ing were made us ing the def i ni tion that only a CO-HN dis tance be tween Å qual i fies. While groups or blocks of H-bonds were iden ti fied, re gions where more than a few hy - dro gen bonds ap pear con sec u tively along the back bone are rare. Fur ther, the lo ca tion of these back bone hy dro gen bond blocks were found to vary with time in a seem ingly ran dom pat tern. How ever, at any given time step in the sim u la tion, the to tal num ber of hy dro gen bonds ap pears con stant. For ex - am ple, af ter 50 ps of equil i bra tion at 300 K, an av er age of 33 hy dro gen bonds oc curs be tween -strands and that num ber re mains con stant over the next 6 ps for the four in ter nal sheets (Ta ble 1). Fig. 5 shows a view along the length of two ad ja cent strands within a sheet. Where hy dro gen bonds oc cur, they de - fine an ap prox i mate plane, but a num ber of un sat is fied hy dro - gen bond ing part ners have arisen by out-of-plane twist ing. Al though the ex act rea son for the twist ing dis tor tion is un - known, one pos si bil ity is that such move ment re lieves the di - hed ral pres sure of main tain ing the par al lel -strand ge om e Peptide Length (residues) Fig. 4. Cost of ideal par al lel -strand ge om e try vs. pep tide length. The length of the -strand was var ied from two res i dues to six within the core am y loid se quence KLVFFA (res i dues 16-21). Ge om e try optimizations (us ing AM BER* and GB/SA sol va tion) were per formed on the small pep tides ini tially with the back bone am ide con - strained to the MacroModel 6.5 ide al ized - strand ge om e try and, then again with all con - straints re leased. The ap par ent cost of main tain - ing the ideal -strand ge om e try is given by the slope, 2.1 kcal/mol/res i due (lin ear re gres sion, R 2 = ).

5 Am y loid Struc ture: Models and Con sid er ations J. Chin. Chem. Soc., Vol. 49, No. 4, Table 1. Variation of the Number of Backbone Hydrogen Bonds in the Internal -Sheets as a Function of Time Laminated Sheet* Time (picoseconds) Average (per sheet) Total (per ps) * Sheets 1 and 6 excluded due to possible solvent effects. try. Since mo tion within one sheet clearly af fects ad ja cent sheets, via the sheet-to-sheet side chain pack ing, cou pled mo - tions within the fi ber are ex pected. Such cou pled mo tions may help to ex plain how, as pre sented in Ta ble 1, the to tal num ber of hy dro gen bonds pre dicted within the fi ber re main con stant, even though their po si tions change with time. As a re sult, strand en er gies ap pear to limit the num ber of res i dues pos si ble in stretches of reg u lar par al lel -strand struc ture, but it might be the move ment of these blocks of short stretches of hy dro gen bond ing that si mul ta neously per mits global fi bril sta bil ity and the lo cal flex i bil ity re quired to form long reg u lar Fig. 5. The hy dro gen bond plane as viewed down two ad ja cent -strands. The yel low lines in di cate the po si tion of the H-bonds and show the vari a - tion of the rel a tive po si tion of the am ide plane of one strand with re spect its intra-sheet part - ner. The vari able po si tion of the side chains is most ap par ent in this view above and be low the in di cated H-bonding plane. struc tures. In ad di tion, it was found that a sig nif i cant num ber of hy dro gen bonds oc cur be tween pep tide back bone and sol vent mol e cules within the fi ber. Ad di tionally, these sol vent mol e - cules were ob served to move within the struc ture. Thus, sol - vent not only serves to fill spaces be tween -strands, as in the re gion from G 30 to G 34 dis cussed above as a glycine chan nel, but may also pro vide ad di tional el e ments of sta bil ity by hy - dro gen bond ing pep tide side chains within the fi ber. On the ba sis of this mod el ing, the am y loid fi bril ap pears dy namic and flex i ble, par tic i pat ing in time-dependent break age and for ma tion of back bone hy dro gen bonds and gen er at ing dis - tinct re gions of reg u lar and ir reg u lar struc ture. How can bur ied charges be ac com mo dated? As stated above, the par al lel ar range ment of -strands within the am y loid fi ber brings like charges close in space. How ever, ionic de ter gents and other ionic amphiphiles clus - ter like charges as their hy dro pho bic por tions are desolvated, and the charged sur faces of cells or lipid ves i cles are much larger and more highly charged than am y loid pep tides. In this re gard, how se ri ous of an ob jec tion to the model is ac tu ally rep re sented by the fact that it pre dicts the clus ter ing of like charges? Am y loid fi brils are less amphiphilic than the de ter gents and some of the charged side chains are in ter nal. Am y loid for ma tion is gen er ally known to be ph de pend ent, to pro ceed rel a tively slowly un der strongly acidic con di tions, 19 likely re - flect ing a re quire ment to deprotonate ba sic sites within the se quence be fore fibrilization can en sue. The A (10-35) se - quence cer tainly con tains acidic and ba sic res i dues, and Burkoth 20 showed that A (10-35), with 3000 MW poly - ethyleneglycol at tached at the C-terminus, at tains the great - est ex tent of -char ac ter at ph 5.6. As the ph approachs the pi, pep tide-peptide con tacts and self-association be come in - creas ingly pos si ble with A (10-35). This ob ser va tion is con - sis tent with typ i cal pro tein be hav ior, as na tive pro teins are of - ten found to ag gre gate or pre cip i tate in the vi cin ity of their pis. Thus, protonation and deprotonation events may be ma - jor con tri bu tors to the pro cesses by which am y loid sta bi lizes po lar and charged spe cies, and may also be im por tant in the fibrilization path way. In ad di tion, the sim u la tions re ported here sug gest an other method by which po lar and charged groups can be ac com mo dated within the in te rior of the fi ber. As noted above, sol vent mol e cules were iden ti fied within the pro posed glycine chan nel in the cen tral re gion of the fi ber, and ob served to move through out va can cies within the struc - ture. The pres ence of in te rior sol vent mol e cules could sta bi -

6 464 J. Chin. Chem. Soc., Vol. 49, No. 4, 2002 Mor gan et al. lize the bur ied po lar and charged groups, par tially screen ing them from the en er getic con se quences of their se ques tra tion. Spe cific hy dro gen bond ing can both en cap su late the charged groups or bridge among them by vir tue of H-bonded sol vent net works. Fur ther more, al though the sim u la tions did not ex - plic itly in volve salts, ionic spe cies are cer tainly pres ent phys - i o log i cally and in ex per i men tal am y loid prep a ra tions, and these could be readily car ried into the fi ber as it in cor po rates sol vent. DIS CUS SION As we move into the next phase of pro tein struc ture and func tional anal y sis, an un der stand ing of un usual con for ma - tions and as sem blies takes on in creas ing im por tance. Spec - tro scopic and com pu ta tional meth ods have now ad vanced to the point where large nanoscale self-assemblies can be an a - lyzed and struc tur ally de fined. In this pa per, the model for the A (10-35) fi bril, de vel oped from spec tro scopic anal y ses, has been pre sented, crit i cisms of its un usual fea tures out lined, and ques tions raised by these fea tures ad dressed with mo lec - u lar dy nam ics sim u la tions. The solid state NMR and neu tron scat ter ing data, made pos si ble by the ho mo ge neous as sem bly of the A (10-35) pep tide, rep re sent global av er ages of the inter-atomic po si tions. Ironically, the NMR re veals that N- terminal and C-terminal por tions of the se quence aligned ap - prox i mately 5 Å from one an other, but the cen tral por tion main tains lon ger dis tances, ap proach ing 6 Å. The NMR ex - per i ments have not yet re solved whether these lon ger dis - tances arise from greater het er o ge ne ity, greater flex i bil ity, or both, within the cen tral re gions of the pep tide. In con trast to the spec tro scopic and scat ter ing mea sure - ments, the sim u la tions an a lyze the lo cal or der in en er getic terms and help to ad dress this het er o ge ne ity in a num ber of ways. First, they in di cate that the cen tral por tions of the se - quence are sub stan tially more flex i ble, and able to adopt many dif fer ent con for ma tions. Sec ond, these sim u la tions have dem on strated that -strand char ac ter oc curs within lo - cal do mains, and spe cif i cally that the cen tral por tion of the pep tide se quence de vi ates sig nif i cantly from a con tin u ous -strand con for ma tion. Fur ther, ex am i na tion of Fig. 4 re veals that lo cal or der does oc curs within the se quence, namely the core hy dro pho bic se quence of KLVFFA is well de fined as a par al lel -strand from one sheet to the next, but those res i - dues pre ced ing it ex hibit greater ir reg u lar ity. Finally, the sim - u la tions dem on strate con tin u ous for ma tion and de for ma tion of -sheet hy dro gen bonds within the fi ber. Just as struc ture is ir reg u lar within the fi ber from one strand to the next, it now ap pears to be ir reg u lar from one time point to the next. Fi ber dif frac tion ex per i ments 3b,9 have in di cated that the re flec tions vis i ble for am y loid pep tides tend to be those of the gross est struc tural de tails of the fi ber; that is, a 5 Å re flec tion cor re - spond ing to the dis tance be tween -strands, and a 10 Å re - flec tion cor re spond ing to the dis tance be tween stacked -sheets. An im pli ca tion of these ob ser va tions is the ex is - tence of dis or der else where within the fi ber, sug gest ing that its in te rior is not well-packed like that of a folded pro tein, but rather of in def i nite con for ma tion and more mol ten-globule like. The tran sient na ture of the hy dro gen bonds ob served in the sim u la tions sup ports the idea of a mol ten-like struc ture ap ply ing to the back bone con for ma tions as well. As greater or ga ni za tion within a struc ture de creases its en tropy, in - creased crystallinity within the fi bril would be en er get i cally un fa vor able. The am y loid fi bers are no to ri ously sta ble, and these dy nam ics sim u la tions show ing sig nif i cant tem po ral and spa tial dis or der sug gest that some of the sta bil ity of the fi ber, and even its fa vor able Gibbs en ergy of self-assembly, may be due to its flex i ble reg u lar struc ture and its con tin u ous re or ga ni za tion with time. As sug gested above, the re gion of the struc ture that is ex pected to be the most dy namic and flex i ble is the side chain pack ing in the sheet-sheet in ter ac tion. Thus far, in for ma tion about this fea ture of am y loid struc ture has been the most dif - fi cult to ob tain largely as a re sult of the in her ent sym me try of the as sem bly. Re cently, how ever, a the o ret i cal treat ment of fi ber for ma tion has pro vided ground work for in ves ti gat ing the is sue of fi ber thick ness. Nyrkova et al. 21 ar gue that the thick ness of a self-assembled pep tide fi ber will be de ter - mined by the in ter play of two fac tors: (i) the in ter ac tion en - ergy be tween -sheets, and (ii) the elas tic de for ma tion en - ergy caused as sheet-sheet in ter ac tions which dis tort the equi lib rium struc tures of the com po nent sheets. They ar gue that a fi ber will in crease in thick ness un til the to tal elas tic de - for ma tion en ergy ap prox i mately equals the to tal sheet-sheet in ter ac tion en ergy. While this the ory does n t pro vide spe cific struc tural pre dic tions for am y loid, it does al low spec u la tion about the re la tion ship be tween fi ber di men sions and the char - ac ter is tics of the pep tides that make up the fi ber. Among these char ac ter is tics are charge, amphiphilicity, and pri mary se quence length. In deed, un pub lished re sults with other trun - ca tion pep tides of A have sug gested that the pri mary se - quence length does par tially de ter mine the over all di men - sions of the fi ber, spe cif i cally by al ter ing its lam i na tion thick - ness. To the ex tent that Nyrkova et al. s the o ret i cal frame - work ap plies, which seems likely since the num ber and char - ac ter of amino ac ids in a self-assembling pep tide are ex -

7 Am y loid Struc ture: Models and Con sid er ations J. Chin. Chem. Soc., Vol. 49, No. 4, pected to de ter mine the in ter ac tion en er gies be tween selfassembling sheets, then the the ory ad dresses an other pub - lished crit i cism of the A (10-35) model. Serpell s pri mary ob jec tion to the model was its in abil ity to ad dress the elec tron mi cro scopic ob ser va tion that the fi bers formed by A (1-40) and A (10-35) pep tides have ap prox i mately the same di men - sions. 9e In an elec tron mi cro graph, the ob served width of a fi - ber arises both from the inter-sheet and sheet-width terms. If the shorter pep tide forms thicker fi bers and the lon ger pep tide forms thin ner fi bers, then the two fi bers might well show the same di men sions, even though rea son ing based on the length of the con stit u ent pep tides alone would pre dict very dif fer ent di men sions. CON CLU SIONS Aside from con sis tency with solid state NMR and neu - tron scat ter ing data, an ap peal ing as pect of the pre sented model is its suc cess in ex plain ing pre vi ously de scribed char - ac ter is tics of am y loid fi brils in gen eral. For ex am ple, the model re quires large scale clus ter ing of hy dro pho bic sur face area, spe cif i cally by bring ing to gether the hy dro pho bic C-termini, con sis tent with the mi celle-like be hav ior of A pep tides. 22 Given that paired A (10-35) fi brils are con cep tu - ally sim i lar to long, rod-like mi celles, the model takes the de - ter gent-like char ac ter of the pep tide one step fur ther, by sug - gest ing that the pep tide s amphiphilicity both de fines some of the gross struc tural fea tures of the fi bril as well as the path - way for its for ma tion. 20 As the model in volves par al lel - strands, like res i dues are clus tered within the fi ber, among them, the po ten tial metal bind ing res i dues H13 and H14, which are pre dicted to form mul ti ple metal bind ing sites within the as sem bled fi bril. The model there fore, also helps to ex plain the bind ing of multivalent metal ions to am y loid pep tides. 23 When taken to gether, the A (10-35) fi brils and pos si - bly am y loid fi brils more gen er ally, can now be un der stood glob ally as a large ar range ment of self-assembled -strands whose sta bil ity is de pend ent on large spa tial and tem po ral fluc tu a tion about a cen tral core, giv ing rise to a struc ture that can be best de scribed as a dy namic and fluid tube-like mi - celle. These fi bers are func tion ally dif fer ent from syn thetic fi bers, at least in part by their abil ity to self-assemble, and struc tur ally dis tinct from bi o log i cal fi brils whose func tion is to both sta bi lize large-scale bi o log i cal or der and me di ate mechano-chemical move ment. These dif fer ences have pro - found im pli ca tions for bio med i cal in ter ven tion in am y loid dis eases as well as for the de sign of self-assembling nano - scale func tional fi brils. Ef forts to fur ther ex tend the mo lec u - lar mod el ing in con junc tion with and as a guide to phys i cal mea sure ments in the de sign and anal y sis of other fi brils are in prog ress. Re ceived Feb ru ary 22, Key Words Mo lec u lar dy nam ics; Par al lel -sheets; Am y loid struc ture; H-bonds. REF ER ENCES 1. (a) Gaffney, B. J. Ann. Rev. Biophys. Biomol. Struct. 1996, 25, 431. (b) Mor gan, D. M.; Lynn, D. G.; Miller-Auer, H.; Meredith, S. C. Bio chem is try 2001, 40, Weaver, T. M. Pro tein Sci ence. 2000, 9, (a) Carrell, R. W.; Lomas, D. A. Lan cet 1997, 350, 134. (b) Sunde, M.; Serpell, L. C.; Bartlam, M.; Fra ser, P. E.; Pepys, M. B.; Blake, C. C. J. Mol. Biol. 1997, 273(3), 729. (c) Prusiner, S. B. PNAS 1998, 95, (a) Kisilevsky, R.; Fra ser, P. E. Crit. Rev. Biochem. Mol. Biol. 1997, 32950, 361. (b) Sipe, J. D. Ann. Rev. Biochem. 1992, 61, (a)kang, J.; Lemaire, H.; Unterbeck, A.; Salbaum, J. m.; Mas ters, C. L.; Grzeschik, K.; Multhaup, G.; Beyreuther, K.; Mul ler-hill, B. Na ture. 1987, 325, 733. (b) Tanzi, R. E.; Gusella, J. F.; Watkins, P. C.; Bruns, G. A. P.; St. George- Hyslop, P.; van Keuren, M. L.; Patterson, D.; Pa gan, S.; Kurnit, D. M.; Neve, R. L. Sci ence. 1987, 235, 880. (c) Robakis, N. K.; Ramakrishnaa, N.; Wolfe, G.; Wisniewski, H. M. PNAS. 2000, 84, (d) Goldgaber, D.; Lerman, M. I.; McBride, O. W.; Saffiotti, U.; Gajdusek, D. C. Sci ence. 1987, 235, 877. (e) Huse, J. T.; Doms, R. W. Mol. Neurobiol. 2000, 22(1-3), (a) Mas ters, C. L.; Simms, G.; Weinmann, N. A.; Multhaup, B.; Mc Don ald, B. L.; Beyreuther, K. PNAS. 1985, 82, (b) Glenner, G. G.; Wong, C. W. Biochem. Biophys. Res. Comm. 1984, 120, (a) St George-Hyslop, PH. Sem i nars in Neu rol ogy. 1999, 19, 371. (b) Murrell, J.; Farlow, M.; Ghetti, B.; Benson, M. D. Sci ence. 1991, 254, 97. (c) Levy, E.; Carman, M. D.; Fernandez-Madrid, I. J.; Power, M. D.; Lieberburg, I.; van Duinen, S.G.; Bots, G. T.; Luyendijk, W.; Frangione, B. Sci - ence. 1990, 248, (a) Yankner, B. A.; Duffy, L. K.; Kirschner, D. A. Sci ence. 1990, 250, 279. (b) Pike, C. J.; Walencewicz, A. J.; Glabe, C. G.; Cotman, C. W. Brain Re search. 1991, 563, 311. (c) Mattson, M. P.; Tomaselli, K. J.; Rydel, R. E. Brain Re - search. 1993, 621, 35. (d) Pike, C. J.; Cotman, C. W. Neu ro -

8 466 J. Chin. Chem. Soc., Vol. 49, No. 4, 2002 Mor gan et al. sci ence. 1993, 56, 269. (e) Lorenzo, A.; Yankner, B.A. PNAS. 1994, 91, (f) Simmons, L. K.; May, P. C.; Tomaselli, K. J.; Rydel, R. E.; Fuson, K. S.; Brigham, E. F.; Wright, S.; Lieberburg, I.; Becker, G. W.; Brems, D. N. Mol. Pharm. 1994, 45, 373. (g) Howlett, D. R.; Jennings, K. H.; Lee, D. C.; Clark, M. S.; Brown, F.; Wetzel, R.; Wood, S. J.; Camilleri, P.; Rob erts, G. W. Neurodegeneration. 1995, 4, (a) Inouye, H.; Fra ser, P. E.; Kirschner, D. A. Biophys. J. 1993, 64(2), 502. (b) Inouye, H.; Kirschner, D. A. Ciba Foun da tion Sym po sia. 1996, 199, 22. (c) Kirschner, D. A.; Abra ham, C.; Selkoe, D. J. PNAS. 1986, 83(2), 503. (d) Kirschner, D. A.; Inouye, H.; Duffy, L. K.; Sinclair, A.; Lind, M.; Selkoe, D. J. PNAS. 1987, 84(19), (e) Serpell, L. C. Biochim. Biophys. Acta. 2000, 1502, (a) Hilbich, C.; Kisters-Woike, B.; Reed, J.; Mas ters, C.; Beyreuther, K. J. Mol. Biol. 1991, 218, 149. (b) Lansbury, P. T.; Costa, P. R.; Griffiths, J. M.; Si mon, E. J.; Au ger, M.; Halverson, K. J.; Kocisko, D. A.; Hendsch, Z. S.; Ashburn, T. T.; Spencer, R. G., et al. Na ture Struct. Biol. 1995, 2, 990. (c) Fra ser, P. E.; McLachlan, D. R.; Surewicz, W. K.; Miz zen, C. A.; Snow, A. D.; Nguyen, J. T.; Kirschner, D. A. J. Mol. Biol. 1994, 244, 64. (d) Fra ser, P. E.; Nguyen, J. T.; Surewicz, W. K.; Kirschner, D. A. Biophys. J. 1991, 60, Burkoth, T. S.; Benzinger, T. L. S.; Ur ban, V.; Mor gan, D. M.; Greg ory, D. M.; Thiyagarajan, P.; Botto, R. E.; Meredith, S. C.; Lynn, D. G. JACS. 2000, 22, (a) Greg ory, D. M.; Benzinger, T. L.; Burkoth, T. S.; Miller-Auer, H.; Lynn, D. G.; Meredith, S. C.; Botto, R. E. Solid State Nuc. Mag. Res. 1998, 13, 149. (b) Greg ory, D. M.; Mehta, M. A.; Shiels, J. C.; Drobny, G. P. J. Chem. Phys. 1997, 107(1), (a) Benzinger, T. L. S.; Greg ory, D. M.; Burkoth, T. S.; Miller-Auer, H.; Lynn, D.G.; Botto, R. E.; Meredith, S. C. PNAS 1998, 95, (b) Benzinger, T. L. S.; Greg ory, D. M.; Burkoth, T. S.; Miller-Auer, H.; Lynn, D. G.; Botto, R. E.; Meredith, S. C. Bio chem is try 2000, 39, Antzutkin, O. N.; Balbach, J. J.; Leapman, R. D.; Rizzo, N. W.; Reed, J.; Tycko, R. PNAS 2000, 97(24), Dudek, S. M.; John son, G. V. Brain Re search. 1994, 651(1-2), Creigh ton, T. E. Pro teins: Struct.ures and Molecular Prop - erties; W. H. Free man and Com pany: New York, Lolis, E.; Alber, T.; Dav en port, R. C.; Rose, D.; Hartman, F. C.; Petsko, G. A. Bio chem is try. 1990, 29, (a) Mohamadi, F.; Rich ards, N. G. J.; Guida, W. C.; Liscamp, R.; Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem. 1990, 11, 440. (b) Still, W. C.; Tempczyk, A.; Haw ley, R. C.; Hendrickson, T. JACS 1990, 112, (c) ucts/ macromodel.html 19. Lomakin, A.; Teplow, D. B.; Kirschner, D. A.; Benedek, G. B. PNAS. 1997, 94(15), Burkoth, T. S.; Benzinger, T. L. S.; Jones, D. N. M.; Hallenga, K.; Meredith, S. C.; Lynn, D. G. JACS. 1998, 120, Nyrkova, I. A.; Semenov, A. N.; Aggeli, A.; Boden, N. Euro. Phys. J. B 2000, 17, (a) Soreghan, B.; Kosmoski, J.; Glabe, C. J. Biol. Chem. 1994, 269(46), (b) Walsh, D. M.; Lomakin, A; Benedek, G. B.; Condron, M. M.; Teplow, D. B. J. Biol. Chem. 1997, 272(35), (a) Bush, A. I.; Pettingell, W. H.; Paradis, D. M.; Tanzi, R. E. J. Biol. Chem. 1994a, 269(16), (b) Bush, A. I.; Pettingell, W. H.; Multhaup, G.; D. Paradis, M.; Vonsattel, J. P.; Gusella, J. F.; Beyreuther, K.; Mas ters, C. L.; Tanzi, R. E. Sci ence. 1994, 265(5177), (c) Kowalik-Jankowska, T.; Ruta-Dolejsz, M.; Wisniewska, K.; Lankiewicz, L. J. Inorg. Biochem. 2001, 86(2-3), 535. (d) Cherny, R. A.; Atwood, C. S.; Xilinas, M. E.; Gray, D. N.; Jones, W. D.; McLean, C. A.; Barnham, K. J.; Volitakis, I.; Fra ser, F. W.; Kim, Y.; Huang, X.; Goldstein, L. E.; Moir, R. D.; Lim, J. T.; Beyreuther, K.; Zheng, H.; Tanzi, R. E.; Mas ters, C. L.; Bush, A. I. Neu ron. 2001, 30(3), 665. (e) Miura, T.; Suzuki, K.; Kohata, N.; Takeuchi, H. Bio chem is try 2000, 39(23),