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Preferred peptide backbone conformations in the unfolded state revealed by the structure analysis of alaninebased AXA tripeptides in aqueous solution Fatma Eker Kai Griebenow Xiaolin Cao Laurence A Nafie and Reinhard SchweitzerStenner Departmen iii isityofpii n Rl p an iiian PRINT 3 i R A i Rico IDep chemistry Syracuse university Syracuse NY 244 and Deparmierit of chemistry Drexel university 3m chestnut street Philadelphia PA i9i04 Edited byR h ril Raid in iarif rd CA We have combined Fourier transform IR polarized Raman spec roscopy and yibrational co meas m ts of the amide l band two peptide groups The results were checked by measuring the sorted into three classes Valine phenylalanine tryptophan his tidine and serine minantly adopt an extended rmati Catio i lysine and proline prefer a poly ine Ml iuiii piii M 2004 most relevant structural motif 12 The classical secondary strucr tures such as achelm Brshee a tabilized by a binatio local no ocal ptideesolvent interactions 13 whereas the P11 conformation of nonprolme resd s can exst only in wat r and reflects e ocal prope ity given residue 14717 r m it follows that even short temperaturedependent mixture of PPII and extended Brslrand 39 39 rialanine quot 39 alaninerbased oligopeptides 21 22 PPHrlike wnformalionswere so obtained for EEE DDD and KKK 23 Even the dass39mal alanine dipeptide seems to occ a upy predominantly the PPH confbrr mation 15 On the contrary trivaline mostly adopm an extended h fr 39 1 1m confo n pro lllike structure Alanine methionine glycine and leucine populate 39 39 39 39 HI eSLIItS irr pAc 39 39 ivxtiriesr supportive of Flory39s isolatedpair hypothesis We combined the that Flory39s bolaledrpair hypothesis isvalid for all extended wnr obtained structural propensities o 39 39 39 39 39 qua ra f h similar information about other residues in the literature ie Therefore the local confomrationofan unfolded pept39de strongly nllnamatn nan a i nlnllrirm 39 39 39 39 39 39 39 39 39 39 ble conformations of the monomeric amyloid p peptide Alarch in u state of pepti es can be un erstood in terms o the intrinsic structural propensities of their amino acid residues roteins are often called the workhorses of a living cell because they perform a plethora of functions such as anal i 39 39 39 39 mission of signals It was demonstrated gt40 years ago that the amino acid sequence o a 39 A 39 39t w t formation and function However this view is currently under 39 ca o t discovery of natural disordered or roteins with very well defined functions 1 Such ITTm ac39d residue To address 11139s 39ssue experiment ally we performed a simple hostegiest experiment by analyzing the structure of a series ofAXApepti leswhere representsGV LM K SH P YW and F The solution stnicture of A 39 dev rm pola roscopy as well as vibrational CD VCD ECD measurements 39s applied to obtainthe dihedral angles ofthe 1 model The applicability ofour resulm for a more detailed under possible structures of monomer39m amyloid pept39des in aqueous on based on individual propersitieso the amino acid residues soluti investigated in this study and related studi intrinsically unstructured pr t in be involved in DNARNAeprotein interaction functioning as inhibito nd engers and facilitating the formation and The 391 Y Sed b mm quot3155 Wepmesmm function ofmulti rotein complexes 1 3 Other IU39Ps mediate 5 Pm and quot 39he ms blF quot13quot le 59w has been gulatory posttranslational modification processes suc as d w bed def 25v 26 Bne yv We mm Wquot39 SC 3 phosphorylation and pr lys39 iscovery of natively n W51 13 an f9 quot X F g between me quotquot0 Wm WM f 01de pm 5 d Dymn 3 to pmpose a trrpeptdes by tr rtron rpol and dirougtbond coupling 27 assess the slmclmgmnmon pa Th39s approachwasjustified recently zsbydetailed computational The unfolded state of proteirs and pept39des is still w39dely f m esPn pep mes and WH39m F Peresmwmspmd39 r nnr PivP 39 39 respective dihedral angles are assumed to sample the entire allowed g re 39on of the Ramaohandran space 479 This view re ects the lXegt cosleigt swim 1 p the ra coil odel ofBra who ix simixp WWW treated an unfolded polypeptide like a synthetic exible polymer H errme al and heo l viden tor Vided last 15 yeals for the notion that well defined Thipapeiwaubmitteddiiet y ratkll omePNAS office c n nscan pers39st locally even in isor re ept a Abbreviation ECDelectronicCDiFTFouriernanxformiVCDibratiorialCDiPPll poiyy proiine ii o d proteins 10 In this context the polyproline 11 PPII confomrai nquot uu u as u 39 1 10054710059 i PNAS i lutyszooa i vol i0i i no 27 Wwwpria orggldollo i073pnar OAOZSZZlOl The parameter c describes the degree of mixing between the unperturbed states in and l X1 which ismaximal at c 45 This case requires he unperturbed modes to be accide tally degene erate in and ix are L 39 39 f L 39 L an 39 39 39 39 re nertivel The mixing parameter p can be determined from the intensity ratio RBa IgaIga ofthe two amiieI39 bands in the spectrum of i 39rnn39v 39 7 and 39 39 39 39 39 of ixs and lxgt respectively The correspondirg ratio Rm L u rc a r angles 45 and b which determine the relative orientation of the nentide mun 39 39 39 39 determined R value Raman tersor of the excitonic states ix and lxgt has to be calculated as follows a cospc1 r sinp39 rQ 54 sinp39 tq sin1w5rg to be tr to the coordinate system fthe other one 26 e a a ft xcitonic states be ome dependent on the relatrve orientation of th ptide groups la means of E 2 can be used to calcu ate the isotropic and anisotropic scattering for the two excitonic states as follows E 3 0411 T 2 X Harv apps2 an ways am emf Eq 3 can be used to calculate RBa Bisl3 and Rum vfmsajvfm as a function ofthe mixing parameters p and the dihedral angles 45 and 4 In the next step we use the mixing parameter and the intensity ratio RKR my in the FT IR spectrum to obtain the angle 7 between the trarsition dipole momentsofthe amide 1 mode Scan be calculated as a function of 45 and l 39 are generally obtained In most cases six ofthern can be ruled out The physical dihedral and oriental parameters obtained as described were finally used to simulate the VCD signal of amide I as described in detail in ref 18 Thus VCD serves as a check AVA were customrsynthesized by Peptide Intemational 95 purity AVA and AMA peptides exhibited siguif39mant signal Done tributions to the amide I region from tri uoroacet39m acid TFA h39 Lrvr Here teps 39 39 39 1 M HCl to remove the TFA before the final spedroscopic data acqu39sition NaClOA was ob tained from sigma All chemicals were of analytical grade The 39 39 39 39 Ir 2Mfor IR Raman spectroscopy and VCD and 1 mM for ECD The pH 39 439 A y addirg small aliquots onHCl or NaOZH to obtain the different protonation states of the pepti les r Long 29 to corred the values obtained from pH electrode measurements For the Raman spectroscopy experimenm the solvent contained 01 M NaCiol the 934 cm Raman ban which was used as an intemal standard 30 Spedrwopies We used the same equipment and experimental set ups escribed in ref 18 The Raman spectra were obtained with the 442 nm 65 mW excitation from an E HeCd laser immon Electric Englewood CO The ChiralIR VCD instrumentation Biotools Edmonton Alberta Canada de scription was the same as used in ref 18 Spedral Analysis All IR and Ramanspectra were analyzed by using the program MULTlFlT 31 ywere normalized to the internal standard the 104 band at 934 cmquot To eliminate solvent contributions we m d t ref r ce spect for both anzatio were then subtracted from the corresponding peptxiespectr The int iiesofthe nnalizedp nzed Raman b ds were derived from their b Th a t e voler spondingI spectrum ere sel ly an at they pro es isotropic an iso ensities and the depolarization ratios pwei39e calculated as follows 4 I 1eg 4 lama Y 7 IX P 1y 7 where Ix and Iy denote the Raman scattering polarized parallel and perpendicular to the polarization ofthe exciting laser light is Experimenial and Iiieoreiital Prochol We have measured the FT IR isotropic Raman anisotropic Raman and VCD spectra of a c renmsent VI s M P H KW Y and F For most ofthese peptides we took spectra at acid neutral and alkaline 2H In our analysis we generally preferred the spectra taken at neutral pH bec 39 39 39 L 39 D ectra are nearly symmetr r r C therefore easier to analyze 18 However for AHA 39 39 A L ake 1 to avoid the coexistence solutions obtained from the IR and Raman spectroscopic data In mn t hash 39 39 39 39 45 an Materials and Methods Malerials LralanylLLLglycylLLLalanine AGA LralanylLLLtryptoL phylLLLalanine AWA LLalanylLLLprolylLLLalanine APA L I I w I I I I I m r r ALA LLalanylLLLphenylalanylLLLalanine AFA LralanylLLL 39 39 AYA Rarhem Rinsrienre Inc 39 39 39 39 lealamle LrserylLLLalanine ASA LralanylLLLlysylLLLalanine AKA LL alanylLLLmethbnylLLLalanine AMA LLalanylLLLValinylLLLalanine Ekeveta of different protonation states For AYA and AW only the cation39m state p 1 because uorescence impaired the nalys aman tra a ral and alkaline pH Finally we sel ted the anionic state for APA 2 a th 0 amide 1 bands are better resolved than ectra 39 39 39 A aninnic lt1 ates r can be justified because the protonation states of the terminal pondirosp intheooi39r 18 by the obtained dihedral angles are clasi ed in terms of the conformational letter code suggested by Zimmerman a ul 6 PNAS i lutyszooa i vol 101 i no 27 i moss HIOPNVSICS unou AFAInlnnl Ali Ml 20009 10000 isotropic ecuuenug ALA zwmnrmnlc an rwtueuenrr All 6000 Anna 2000 Anlsmmpic Sca ering erabsnrpllan 12 M be L i M U VbV x We 1500 tssu 1700 15m tssa nan tauo 1700 avanum bar cm 1 Flg 1 lsotropK and anlsotropK Raman and lit amide l pedra of mu lit tutu lnn ml N anHzo from the R and Raman deompoltlon The solid line pe roopl date namelyEfor180 Slt1gtSi110 110 Sw180 andeor110 4 s 740 130 3 b 3 130 Other conformational regions considered by zimmerman ct uZ are not relevant to the present Stu Shuttule Analysis 039 AXA Peptides Fig 1 shows a representative data set depicting the spectra of APA ALA and AFA at 15507 1700 cm i The selfmnsismnt decomposition of the IR and Raman spectra yielded the amide 139 bands AI and AF which are amide 139 vibrations respectively Apparently the corresponding 39 39 39 39 quot t quot I an P39 im that their dihe of those three intensity ratios dral angles are different The amide 139 VCD signals peptides reflect a leftrhanded conformation The Rm Rm and Rue obtained from the spectral x rr Table 1 The c and p values obtained for the three residues each of the Ramachandran plot Fig 2 The respective angles and the transition dipole momeno that were inferred from the integrated IR intensities were d to calculate the VCD s39 nal and an excellent agreement with the experimental spectra were obtained solid lines in Fig 1 The obtained 4 and b angles of APA and AFA are indicative nd a mostly extended Bestrandrlilae conformation in the E region for the AFA Fig 2 ALA however behaves like AAA r on ar a r with a 4 angle of 425 1m coordinate is close to the border between the E and F regions This transition regionquot is illusr trated in Fig 2 Our earlier findings on AAA KAA and SAA 10056 l wwwpnasergegtdetlo1073pnasoaozezzlol suggest that dihedral angles in this region reflect a nearly equal mixture of PPII and Bestrand 18 19 ig plum t e spectra of the above peptides taken at different temperatures ALA and APA show the asymmetric couplet with a minimum at 195 and a maximum at 210220 nm pronounced for APA indicating a larger PPII population The spectra of A do not correspond to any of the reported basis spectra of secondary structures 32 Peptides with aromatic resir coupling between residue and backbone transitions 33 This effect precludes any structural information from this spectrum V pectra of the remaining peptides ere vc spectra n t e respective dihedral R and Raman spectra Thus we found hei 39 quot Iquot 1139 three categories represented by APA PPII F ALA and Bestrand E and AFA Bestrand E AHA ASA AVA AWA x V r x x A conformation in the E region Cationic AKA is predominantly PPII F AGA and AMA are assignable to the border region L I I e lran her with the simulations angles derived from the 1 our c c Into the PPII F All re tr ni p t th obtained dihedral angles are given in Table 1 Fig 2 illustrates the dihedral angles in the we have also added the recently reported dihedral angles for E and D 23 which are both located in the F region Some of these resulo deserve further commeno It has to be noted that the VCD spectrum of AGA displays three bands with Eker ete Table 1 spectroscopic parameters and the obtained dihedral angles of AYA peptides AXA RlR Risa Ranisn p W W AAA39 i52f 043t li4t 022t 0l3t nl207n3t l647n2f AFA i52 046 033 02i 0l3 4401 i701 AGA l35 034 l3l 023 003 4052 l70 AMA l35 045 lie 0l9 009 nil0l039 l70l039 AHA i3 02 M4 035 0l3 nl60l0 l40l0 AHA i55 056 094 025 0l5 nl30l0 l70l0 AVA 05l 093 2 0l2 i655 i405 AKAf l64 053 067 003 007 nl55l0 l45l0 AKA lie 042 l06 0l6 0l7 755i0 l5020 ALA i45 05 l02 029 0l3 4255 l70l0 AVA M5 033 40 034 005 7010 l50l0 APAl l2 03 l 5 0l3 0l5 7755 i505 AWA i 5 03 05V 25 0l3 470 10 150120 ASA l75 03 033 023 nl30l0 l7320 39Taken from ref l3 ZWitterionic SCationic ii could not be eliminated Anionic a negative signal flanked by two positive ones We tested two ss39b nations First we assumed that G could also sample the achelical region This scenario did not yield a 3 Z Secon t G c quadrant of the Ramachandran space Now we o tain a satise 39 e experimental spectrum he ana is oft e spectra of A A was difficult because the VCD spectrum reveals vibrational coupling between the tye rosine ring mode at 1610 cm and the Ccterminal amide 1 mode In contrast to AWA and AFA the AYA ring mode ith that of the amide I mode This ng o the vibrational states and a rum which overlaps with that of amide 1 The simulation of the VCD spectrum does not take this ment which can interactw 39tonic mixi 130 ser F E Phe M9 Len G Asp 160 Tyr Lys Glu Try Pm 140 ValHls 120 D 100 480 150 140 420 4 30 50 M0 Flg 2 Representation ofthe dihedral angles obtainedforthe investigated AxA peptides in the upper left square of the Ramachandran space The Zimmerman code was used to differentiate between the conformational from the transition region discussed in the text Ekef et al 1676 cmquot band of TFA which interaction into account It is therefore not surprising that the negative signal at Al was reproduced only qualitatively Fig 4 We have also measured the ECD spectra for x G V H M K H Y a ted we observed the PPH coup1et for all nonaromatic residues data not shown including also lt2 i g 3 200 210 220 Wavelength nm Fig 3 Temperatuferdependent ECD spectra of APA LA nd AFA mea sured athHl Thelmfementforthepe fafewrdlngwa wc lnsetdisplays the difference spectra As 30kt PNAS l iulys2ooa l vol ioi l no 27 l 10057 HIOPHVSICS Q 9 l 39V 39L u L r k 1 1 AMA A as A F A r 1 34 2 F T i T r 7 l y 39 n0 11 Wavenumherhm H I94 VCDspedraof W5004750mquot theanalysi 39 3 of zwtterlonlt state of the terminal group A AHA and ASA because the terminal alanine residues can depend on the proline context but is instead an intrinsic property be expected to populate PPII at least partially Generally a in accordance with recent theoretical prediction by Pappu and quantitative interpretation is by far more difficult for ECD than coworkers 33 Comparison with the study of Avbelj and Baldwin for VCD spectra because ECD spe r sult from a superpor 39 p They o 39 e L quotL L sition of contributions from all residues whereas VCD spectra angles from the coil library of the Protein Data Bank and plotted mostly reflect coupling between the peptide groups A detailed the frequency curves g for a set of representative amino acid comparative study on the spectra of dipeptides and tripepr residues They showed that these curves could e mposed into tides is necessary to determine the individual residue contribur t Gaussi 39 t 39bu i 39 1 tion to the ECD signal Bestrand They found that these distributions are not consistent with predictions of the randomcoil model of Brant and Flory 7 The PP 39 L gt ing from their data are A gt r ings Discusion 39 L I I u m in F gtgt V resulo First they strongly corroborate the notion that individual data indicate a somewhat higher Bestl39and propensi for F Our amino acid residues have a clear structural preference in aqueous resulo combined with the data in the d39scussed literature clearly solution and that they do not sam le the entire sterically allowed L L L 39L 39L L 39 39 L L part of the Ramachandran space as suggested or example in the that the local conformation refleco to a major extent the individr earlier studies o Scheraga and colleagues 6 and reiterated in a ual propensity of the amino acid The finding is important for the ry recent computational study on prolinerbased peptides 34 L L39 L LL L fr t 39 A Second they provide evidence for the notion that in the absence peptides of any nonlocal interactions the conformation of a po ptide Our ECD spectra Fig 3 indicate that either the extended chain in the socalled unfolded state can be described in terms of Bestrand conformation or a truly random coil state is stabilized a tworstate modelcomprising PPH and an extended Bstrand The at the expense of PPII at higher temperatures 19 22 Our L L WWW k H n I am on the respective residue particularly for the extended Bestrand Bestrand conformation even though some degree of heteroger 35 Third our data support the isolatedrpair hypothesis 36 in 39 L L L L 15 intern tin I b Gru o that APA AVA and cationic AKA resemble P V3 and K with and coworkers 39 u dersc this notion in that it reveals a respect to the conformation of the central residue which indicates substantial Bestrand character of proteins at high temperature able only for zwitterionic AKA in that K is switched to a Bestrand study and related data reported by Kelly et all 37 chellgren conformation in the zwitterionic state apparently because of the and Creamer 40 and Avbelj and Baldwin 35 to predict some Columbic interactions between side chains Our data are in line conformational propertiesofthe amyloidpe tide A8142 To this 39 L L L 39 A v 7 Av r e we assumed the absence o any nonlocal interactions This NH2 peptides in that these experimeno revealed the hierarchy P consumption might be considered unrealistic for such a long gtgt A gt G L M gt V for the PPII propensity ofx 37 Our data L 39L L 39 suggest that the PPII propensity of nonproline residues does not et all 41 revealed weak backbone hydrogen bonding only for the 10058 i wwwpnascrgtgidcilolo73pnasoaozezzlol Ekereta residues E 11 and 326 which were interpreted as indicating a turn or be ike structure ofthe segments D77E11 and 13207326 The of random coil and short Bestrandrlike segnents Large scale structures do not exist Earlier NMR an D data provided compelling evidence that arhelical segnents require the pres he i reagents 42 In the absence of any norllocal interactions the conformational manifold reflects the individual propensities of the side chains which yields the fo owing immerman co e ence for A 1iz D1FVA2FEVE3FVF4E 7R5Fr VD7F738E7G9FE7Y10F7E11F7V12E7H13Er H14EsQ15F7K16F7L17FE7V18E7F19E7F20E7A 21FEVE22FVD23FVVZ4E7G25FE7326E7N2 K28F7G29FEVA 30FEVI31EVI32EVG33FEl34F E7M35 E7V36E7G37FErG38FEVV39EVV40EV I41E7A4 FEThenotation 39 39 tesa 39 ureofPPII and Brstra d The pr 39tionsf wer made ba the recentlyresul conformationo an Es23 We assume thatR asal a Ipr p with V 37 It follows from o possible PPH content is 5er w assume that PPII and dstrand are isoenergetic forA L M and G the conformation with 43 PPII has the highest probab39 ity 39ff e ive r assignment t at the highest I E and 213 8192 di erent conformations coexist e resp ct c mbinatorial entropy is 7 UK corresponding to a free energy contribution of 20 kJmol at room temperature e NMR data 0 ou et ul were interprete as suggesting that the segments L177V187F197FZO and V397V407141 adopt a Brstrand structure 39 39 39 39 prediction Altogether however our prediction suggests that the sorcalled randomrcoil fraction of the peptide inferred from the ins a dynam39 39 f PH ata conta lc mixture 0 and extended residue conformations The existence 0 fa substantial PPH propensity of the amyloid peptide is supported experiments on shorter AB fragmenm Jarvet et al 43 investigated AB 25 by CD and NM39R and 39dentifiedasubstantial PPH population h 39 39 A at a similar conclusion for the longer and more representat39 e entAsr 39 r 39 39 Srinwasm R is Ruse G n 713wva Natl Am Sc USA 91 Ekeveta 139 band profile ofthe an39sotropis Raman spectrum with simulation rious conformations assignable to the leftrhanded quadrant of e An estimation based on the p sities described above yields an average PPH content of 50 forthis peptile By comparing the res ctive Ac values at the 195nm minima ofthe ECD spectra ofPs 100 PPH 2c and Alma 44 we 39 a fraction of 65 t 5 This result ind39mates that our pred39mtion even underestimates the average PPII content which is indeed not unlikel because theoretical 45 and experi has been provided recently forthe notion ine 39 creases with th ber of cause of peptidersolvent interactiom 45 int ancementquot effect has not been taken 0 oomi lel39ation n FT IR Raman VCD and ECD spedroscopy to the central residue confonnation for a series of AXA peptides and proteins in that t ence in aqueous solution of all investigated amino acid residues strorg p p 39 39 a Howeverwe found that G M L and A exhibit similar propersities for PPII and and conformation at room temperature ined our results with structur reported previously to predict poss e co omiatiors amyloid peptide Adria The result of this prediction is in good 39 39 39 Hmrp the knrwllprl p ofthe individual propensity can be used as a very suitable starting point for predicting the structure of unfolde pept39 es and disorr dered proteirs as well as for simulations ofprotein o ing WethankDf MichaelZagorski for prwrdmguswrthaoopy ofthegalley ofref 41 and Thomas Measey for a thorough check of the manuscript This work was supported by National Institutes of Health Center of Blomedlcal Research ellence H Grant P20 RR16439701 to the Pr t w trii tiir Pun ti n with mi mi the University ofPuerto Rico Fondos lnstituoionales para la Investigar egg n n M cm x smug L 20m G 1999 G is Graslum A PNAS i lutyszooa i vol lol i no 27 i 10059 HIOPNVSICS m m Correcllon mmmmmmmmammw mmmmmmnmmwbym mi km mmmnsmm wmm mm m m 21 st mm umNmAw 5a yuan mnsvxmss 4 mm m m m mzmnmmm m mm mamas man 1 2 wwwmmhgml wum 7 u m w nnmmmaw aAzgxmmm me 32 M 3quot I 3 arA11 mhmi w 31 ww MN MMwW uumm mm 5762 mumu carnaln mmmw W JOBNAME AUTHOR QUERIES PAGE 1 SESS 3 OUTPUT Wed Jul 21 085721 2004 balt4zpq7pnaszpqipnaszpqforigzpq5762d04a AUTHOR QUERIES AUTHOR PLEASE ANSWER ALL QUERIES 1 A Au Please contact Peggy Moore e rnail rnoorerncad1nuscorn phone 410 691 6275 fax 410 691 6220 if you have 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