Amide modes and protein conformation

Bivchimica et Bioph~'sica Acta. 1120 ( 19921 123-143 ~) 1992 Elsevier Science Publishers B.V. All rights reserved 11167-4831~/~,12/$(15.(lt1

123

B B A P R O 34158

Review

Amide modes and protein conformation Jagdeesh Bandekar The Chemical and Slnlt'tural Anal)'sis Group. The BO(" (;roup Inc.. Technical Center..$ht'r,:;, Hill, NJ (USA) (Received 17 O c t o b e r It ~ l )

Key words: Infrared spectro~opy: FTIR; Pmlcin conhJrmation: Amide

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

124

II.

Normal coordinate analysis treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. N-Methyl acelamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. N-Muthyl acclamide and protein conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

124 124 126

III.

Some useful standard confl)rmalh)ns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Antiparallel pleated sheet structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. ¢~-Ilelix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ('. /3-Turns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. -y-Turns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

128 128 128 128 131

IV.

Vibrational spectroscopic methods fi)r studying pr~tein confi~rmalion . . . . . . . . . . . . . . . . . . . . A. Raman .~pectro.~opic m e t h o d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Method of Lippert el al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Method of William.,, and Dunker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Method utilizing the amide I l l ' hands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Method of Williams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Method of Bcrjot et al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Method o f Bussian and Sander . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Resonance Raman m e t h o d s o f Spiro el al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. FT-IR spectroscopic m e t h o d s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Melhod of Su,;i et al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. M c t h o d o f D o n g c t al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Method of Lee el al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Methods of P~zolct el al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Method o f Eckert et al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Method of Vcnyaminov cl al . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

132 132 132 132 132 132 132 133 133 133 133 133 133 133 133 133

Abbreviations: A , IR intensity of the a l h normal mode: u-pLA, (,-poly-l-alaninc: APPS, antiparallel pleated sheet structure" Asp, asparlic acid; ATR. attenuated total internal reflection; b. bend: T ,[/3 sr', antiparalld i,i) ~-hclix with n = 5.6: J, ~, ~"~, parallel .,i)/J-helix with n = 6.3, /3-PLA. /~-poly-i.-alanine" BR, bacteriorhodopsim BSA, bovine serum albumin; (', velocity of lighl: d, dch~rmation" itl~/i~r, dipole momcnl derivative: e A, unit vector; eAl:~, unit vector joining A and B going from A to B; FT-IR. Fourier-transform-infrared: GA. gramicidin A: it. uni! height: IGG, Ile-Gly-Gly: ib, in-plane-bend: a L G , a-lactalbumin;/~LG, ~-Iactoglobulin: n, number of amino acid residues per turn of the helix: NCA, normal coordinate analysis Ireatment: oh, out-ot-plane-bend: PED. potential energy distribution: ~, dihedral angle corresl~)nding to rotation about the partial double bond, NC"; ~b, dihedral angle corresponding to rotation about the partial double bond. C"C: Q , . a t h normal coordinate: r, internal cot~rdinat¢; SR. sarcoplasmic reticulum: ~', unit twist: t, torsion; TD('. transition dipole coupling; U V R R , ultraviolet resonance Ramam VCD. vibrational circular dichroism; VGG, VaI-Gly-Gly: w, dihedral angle corresponding to rotation al~mt the CN h~md; II. IR dichroism parallel to sample orientation: _L, IR dichroism perpendicular to sample orientation. Correspondence: J. Bandekar, The Chemical and Structural Analysis Group, The BOC Group Inc., Technical Center, IlX) Mountain Avenue, Murray Hill, NJ 07974, USA.

124 V.

Applicalions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Bacteriorhodopsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Structure and confi)rmation of toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Structure and conformation af gramicidin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Structure of capsid proteins a t viruses and phages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Miscellaneous applicatioos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

134 134 134 134 135 135

VI.

Critical assessment of amide modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

138

VII.

Perspectives for the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

139

VIII. Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

140

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

140

!. Introduction

Infrared spectroscopy (IR) has played a pioneering role in studying the conformations of peptides, polypeptides and proteins [l]. The advent of stable and powerful lasers led to the use of laser Raman spectroscopy for such purpose [2,3]; it also led to the development of Fourier transform methods for data aquisition and reliable digital subtraction procedures. As a result we now have both FT-IR and FT-Raman spectrometels. The availability of fast computers has led not only to FT-processors which can rapidly compute Fourier-transforms, but also can carry out detailed normal mode analysis treatments on model biopolymers [4-6]. On the numerical front, Fourier self-deconvolution techniques [7] and computation of second derivative spectra [8] have proved very useful. Progress in theoretical understanding of such spectra [6,9-12] has made vibrational spectroscopy, i.e., spectroscopy including FT-IR, Raman ( a n d / o r FT-Raman), and normal coordinate analysis treatment (NCA), an integrated and powerful technique for studying polypeptide conformations. What one understands, when one refers to the amide modes of a peptide, is due to the early work of Miyazawa on N-methyl acetamide (NMA) [13]. He worked out the normal modes of NMA and postulated that the normal modes of vibration of a polypeptide backbone are related to those of NMA. in this article we will review IR and Raman spectroscopic results and NCA treatments on amide bands in peptides, polypeptides and proteins. We will, however, not discuss resonance Raman spectroscopy (except when it overlaps with the concept of amide bands), fibrous proteins like collagen or near-infrared spectroscopy. Readers interested in resonance Raman spectroscopy are referred to the book by Carey [14]; for near-lR applications see Ref. 15. Readers interested in work on easily polarizable H-bonds and its applications to proteins are referred to Ref. 16.

11. Normal coordinate analysis treatment (NCA)

II-A. N-methyl acetamide (NMA) NMA derives its importance from the fact that it is the smallest molecule containing a trans peptide group. A study of this molecule, therefore, provides important insights into the nature of the so-called amide modes of a peptide group. A number of normal mode analyses have been done on NMA [13,17-20]. We present here one due to Cheam [6]. The geometry used in this computation is the standard geometry of the ~L~eptidegroup (see Table Ill, Ref. 6) and the force field is the one due to Dwivedi and Krimm [21]. Fig. l shows a schematic diagram of NMA where the CH.~ group is assumed to be a point mass. The local symmetry coordinates used are listed in Table 1. Table II lists frequencies calculated by using Wilson's GF technique [22], observed frequencies and the potential energy distributions (PEDs). In Fig. 2 are shown the cartesian eigenvectors. The main amide modes are listed below. 1. NH stretch. The NH stretch mode (NH s), is a localized mode. However, it is not very straightforward to use, since it usually appears as a doublet (called amide A and B [6]). The other interacting component is either the overtone of the amide It mode or a combination band. Before one can relate this band to protein conformation, one has to carry out the Fermi resonance analysis [23-26].

He C2

X

Fig. i. Schemalic diagram of N-meth'ylacetamide molecule.

125 TABLE I

Local .~'mmetry coordinates o f the peptide group " St = S2 = S.a = Sa = Ss = S~ =

r(CC) r[(C2)N] tIN(Call r(CO) tlNH) [ 2 0 ( C C N ) - OiCCO )

- O(NCO)l/vrd

C C stretch (CC s) CN stretch (CN sl NC stretch (NC s) CO stretch (CO s) N H stretch (NH s) C C N deformation (CCN d)

S7 = [O(CCO)- 0(NCO)l/v~-2 S s = [2O(CNC)- 0[(C2)NH)

- et(C4)NHII/Cg S, = 1O[(C,)NH1- Ol(C~)NHll/v~2 S t o = ~a(CO) sin(CCN) Sit = oJ(NH) sinlCNC)

St, = [r(CCNC)+ r(CCNH) + r(OCNC)+ ¢(OCNtII]/2

CO in-plane-bend (CO ib) C N C deformation (CNC d) NH in-plane-bend (ib) C O out-plane bend ~' (CO oh) NH out-of-plane-bend ~ (NH oh) CN torsion (CN t)

'~ Atom numbering from Fig. I. h Positive: C moves in + Z. c Positive: N moves in - Z .

2. Amide L The amide ! mode is primarily a C=O s band. It does have some contributions from CN s and CCN deformation (CCN d) [see Fig. 2]. 3. Amide IL The amide II mode is an out-of-phase combination of largely NH in-plane-bend (NH ib) and CN s. Smaller contributions come from CO ipb, CC s and NC s. 4. Amide IIL The amide I11 mode is the in-phase combination of NH ib and CN s, with small contributions from CC s and CO lb. 5. Amide IV. The amide IV band is mainly CO ib plus CC s, with a small contribution from CNC d.

6. Amide V. The amide V mode is largely an NH out-of-plane (NH ob) motion with some CN torsion (CN t). it was mainly an NH ob mode in the work of Miyazawa [13] and of Kessler and Sutherland [27]. 7. Amide VI. The amide V! mode is mainly CO ob as far as PED is concerned [see Table II] but N and H atoms are also displaced during this normal mode of vibration [see Fig. 2g], so that its intensity is derived. from this contribution also. 8. Amide VII. Amide VII mode is a mixture of NH ob and CN t [6]. It is mainly CN t mode in Miyazawa's work [13] and is related to the barrier to rotation about the CN bond. Some recent developments from U V R R spectra of model compounds deserve discussion here. Song et al. [28] have drawn attention to a peptide U V R R band, near 1390 c m - ~, which disappears in D 2 0 and may be conformation-sensitive. For NMA, they identified it at a much higher frequency at 1496 c m - ~. It was assigned to the overtone of the NH ob, amide V, mode [see Fig. 3]. However, this band was previously assigned to photo-isomerized cis-NMA [29]. Wang et al. [30] studied this band in resonance Raman spectra of NMA as a function of power. At low power on a flowing sample of aqueous NMA (when cis-NMA species are expected to be absent), this band was absent [see Fig. 4D]. It gained in intensity with power as the number of cisN M A species began to increase, showing that it is indeed produced by photoisomerization [Fig. 4C]. Its frequency is the same as amide I1 band of cis-NMA produced from matrix-quenched high-temperature nozzle [31], The band in caprolactam [Fig. 4A] coincided with this, showing that the above may indeed be due to cis-NMA. The results pointed to another band at 1384 c m - ~ which is not photo-induced but is absent in D 2 0 and, therefore, involves the NH group vibra-

TABLE !1

Obsen'ed attd calcnlated frequenck's of N-methylacetamide Mode

va, ~ (cm - l ) ,

vca~¢ (cm t)

Potential energy distribution b

NH s Amide I Amide II Amide ill NC s CN s, CC s Amide V Amidc IV Amide V l CCN d CNC d Amide VII

3236S c 1653S 1567S 1299M 1096W 881W 725S 627W 600M 436W 289 206

3254 1 646 1515 ! 269 1070 908 721 637 655 498 274 226

NH s (100) C O s (83), CN s (15). CCN d (11) NH ib (49), CN s (33). CO ib (12), CC s (10), NC s (9) N H ib (52), CC s (18), CN s (14). CO ib (1 l) NC s (77). CC s 0 7 ) CN s (31). CC s (17). CO s (16). CNC d (14), CCN d (10) CN t (75). NH ob (38) C O ib (44), CC s (34), C N C d (11) C O ob (85), CN t (13) CCN d (63), CO ib (I 1), CN s (8), NC s (8) C N C d (71), CO ib (19). CCN d (13) N H ob (64). CN t (15). CO ob (12)

S, strong: M, medium: W. weak: Data from Ref. 220. h s, stretch: d, deformation: t. torsion: lb, in-plane-bend. " Unperturbed by Fermi resonance.

126 give the frequencies (related to the peak positions) while the latter are related to IR intensities [30]. IR integral intensity of the a t h normal mode, A,, is given

tions. Since it was too low to be an overtone of amide V band, it was called an 'amide S' band. Quantitative analysis of UVRR spectra of a number of proteins showed this amide S band intensit), to be linearly related to the non-helical content; for 100% a-helix, the intensity extrapolated to zero. This will be discussed later (see section IV-A.7).

by

where N is Avogadro's number, c is the velocity of light and Q, is the a t h normal coordinate, alz/aQ,, is defined by

II-B. N-Methyl acetamide and protein conformation How does one apply results on NMA to study polypeptide and protein conformation? This is done by assuming that the force constants and dipole moment derivatives, O/~/ar are transferable, with only minor refincraents, to polypeptides and proteins. The former

/

alZ /aQ.

=

~~.(a/.t/ari ) L

(2)

......

i

where Lia is the eigenvector matrix defined by S = LQ.

\ (a)

(I)

A,, = ( N = / 3 c 2)[~, /a(2,, 12....

/" (b)

(c)

j. if)

(e) [d)

(i) (g)

L/R

7

J (j)

(It)

(I)

Fig. 2. Normal vibrations of N-methylacetamide: (a) NH s; (b) amide I; (c) amide I!; (d) amide 111;(e) amide IV; (f) amide V; (g) amide VI; (h) amide Vll; (i) 1070; (j) 908, (k) 498; and (I) 274.

127 z

"I

/'/

"

IV\ !

I

I

\

i

m-y

I /L.-"

I

llll/ENUMBERS/cm-'

Fig. 3. IR (upper) and Raman (lower) spectra of NMA in aqueous solution. IR: 1.0 M. run in circle ATR cell (ZnSe rtRI), 1000 scans at 4 c m - I resolution, water spectrum subtracted. Raman: 0.4 M concentration with 0.2 M perchlorate at 7 cm t resolution.

Thus, although a/~/ar values are transferred from NMA, the a # / a Q values for polypeptides will be different for different molecules because of different Li,'s. The second factor that leads to conformation-dependent effects in the spectra is the coupling between

~L0 - 2 0 o h m

"~o

i. . . . . . . . . . . . . . I

I

,,,

%__,I

(

_

"l

_

,.-.,.

~

X Fig. 5. Geometrical parameters in transition dipole coupling interaction.

transition dipoles (TDC). This gives rise to fine structure in the amide I, II and III bands. Since dipole-dipole interaction is dependent on orientation (and distance) between the interacting dipoles [33], the fine structure and relative band intensities contain valuable information on conformation of the polypcptide backbone. The frequency shift, /iv,, in c m - i for the ath mode due to TDC is [32]

-

^

.!%, = (8486191vo)(op /itQo)2 X'~ll = (848619/vo)~_,Li~,Li<,Fii

....

(3)

i.j

where XAB is the geometrical factor, defined by XAa = [ eA"ea - 3( e A"eaB )( eli' eAB)] H e r e eA, e a and eAa are unit vectors in the directions of the dipoles and the line joining their centers, respectively [see Fig. 5]. Fii are the force constants. Note that Av,~ would be zero in the absence of TDC and that all the (7=0 s bands would then coincide. Finally, the integrated intensity, A, in cm mmol-i, is given by [32] Raman

Shift

( c m -1)

Fig. 4. 200-nm excited R R spectra of (A) aqueous caprolactum (3 mM with 0.3 M Na2SO a) and (B-D) aqueous NMA (10 raM, with 0.3) and Na2SO4). Spectrum B was obtained with a stationary sample (cuvene), while C and D were obtained with a flowing sample at high and low laser power levels, estimated to be 41) and 800 MW/cm2/puise, respectively. The peak assignments for the amide modes are indicated.

A = 4225.47[0p/pQ,, ]2....

(4)

Ch"-am and Krimm [34] have illustrated the transferability of NMA dipole derivatives to polyglycine I. This transferability has helped to distinguish between different conformations [6].

128

HI. Some useful standard conformations

T A B L E I!I

Parameters of antiparallel-chain pleated sheet 13-poly(L-alanine)"

IlI-A. Antiparallel pleated sheet structure (APPS) p-PLA The structure of p-PLA is shown in Fig. 6 and its parameters are given in Table 11I [35]. The optical activities of the normal modes of p-PLA are as follows [34]: A Iv(0, 01, Raman; B, Iv(0, rr)l, Raman, IR (11); B2 [v(w, 0)], Raman, IR ( _L): and B 3 [ v ( ' r r , "rr), Raman, IR

(~)].

Table IV lists some frequently used amide modes [36]. Note that observed amide 11 and V bands are very weak, if not absent, in the Raman spectra. They are, however, intense in IR.

III-B. a-Helix a-PLA The structure of a-PLA, as reported by Arnott and Dover [37], is shown in Fig. 7. It has n = 3.62, h = 1.50 /~, r = 99.6 °, d, = -57.4 °, ~b= -47.5 °, and to = 180.0°. Table V lists some useful amide modes [38]. For limitations of space, we will not discuss polyglycine I, polyglycine I1, single- and double-stranded L,D p-helices. Those interested in this work are referred to our review article [6].

III-C. p-Turns Fig. 8 gives a schematic illustration of a p-turn of CH3-CO-(AIa)4-NH-CH 3 [39,40]. Tables VI and VII show the computed amide 1, II and Ill modes of p-turns of types I, II and III and their mirror images. Table VIII lists the results for CH3-O-GIy-AIa-AIaGly-O-CH 3 [39], a model for Z-GIy-Pro-Leu-GIy-OH, a type I p-turn model peptide [41,42]. Table IX lists similar results for Pro-Leu-Gly-NH2, a type II p-turn model peptide [43,44]. Kawai and Fasman [45] used a tetrapeptide, CbzGly-L-Ser-(O-But)-L-Ser-Gly-O-stearyl ester, which is believed to exist as a p-turn of type I. They proposed

Bond lengths (,~): I(C"CI = 1.53 I ( C " H ) = 1.07 I(CN) = 1.32 I ( C H ) = 1.09 I(NC'O = 1.47 I ( N H ) = 1.00 I(C=O) = 1.24 Bond angles (degrees): C " C N = 115.4 C'~CO = 121.0 C N C a = 120.9 CNH = 123.0 All angles about C a and C a tetrahedral Dihedral angles: d~ = - 138.4 °, .~ = 135.7 ° Sheet parameters: a / 2 = 4.73 A b / 2 = 3.445 ]k (fiber axis) Ab = - 0.27 ~, a = 80.0° Hydrogen-bond parameters: I ( H . . . O ) = 1.75 ,~, I ( N . . . O):= 2.73 ,~ O(NH, N O ) = 9.8 ° y ( N H O ) = 164.6' Inter-sheet parameters: c / 2 = 5.27 p = 90.0 ° " From Ref. 35.

that the intensity ratio of the 1695 to 1635 c m - ' bands is indicative of p-turns. Thomas et al. [46] studied Raman spectra of type I! p-turns in cyclo(L-valyl-L-prolyiglycyl-L-valylglycyl)3 (abbreviated cyclo(VPGVG) 31 and poly(VPGVG). The results were in general agreement with the predictions of Bandekar et al. [39,40]. By using the dihedral angle values of four p-turns in insulin [47], Bandekar and Krimm [48] computed the normal modes for the model tetrapeptides. Table X

T A B L E IV

Amide modes of [3-poly-L-alanine Amide

Observed bands

Calculated frequencies a

mode

Raman

A

IR

Bt

B2

B3

! 698 1694W (11) 1669s

1695

1670 ! 632vs ( 3_ )

1630

1555MW ( l )

1562

II

1592 1553VW 1538W

!11

1 335W

1539 1524S (II) ! 330W ( ± )

1528 1332 1317 1305

1311W

1309sh

1299 1243S 1226M

1236 1222S (II)

1231

V

708 706

706S (II, ± ) Fig. 6. O R T E P drawing of antiparallel-chain pleated sheet of ,O-PLA. The CH 3 group is approximated by a point mass.

705 704

A d a p t e d from Krimm and Bandekar [6].

129 TABLE V

Obserred and calcu&ted frequencies of a-poly-t.-alanhw Amide

Observed bands

Calculated frequencies "

mode

Raman

IR

A

1658VS (ll)

1657

1

EI

1655S

E,

1 655 1645 1 549

II ! 543VW

1545VS ( & ) 1519M,sh

1538 1519

!11

1349 1 338M,sh

1345 1317

1 278W 1 271W 1 261W

12711M ( ,L 1 ! 265M,sh

662W

658S ( ± )

660

618S ( ± )

608

I 287 1278 1262

V

675 637 589

" Adapted from Krimm and Bandekar [6].

Fig. 7. O R T E P drawing o f a - P L A .

lists amide 1 and II! frequencies. The amide I frequencies are found to center around two values, namely, 1652 and 1680 cm-~. Observed amide ! bands in the

Raman spectra of single crystals of insulin are at 1658 and 1681 cm -j [49]. Bandekar and Krimm [481 assigned the 1681 cm-~ band to fl-turns and the 1658

T A B L E VI

Calculated amide L IL and IH frequencies and transition dipole coupling inchMed fiJr types l, II arm !il fl-turn., Type 1 Amide I Group ~

T y p e II F r e q u e n c y (cm - =I

Group J

..t#~t~(D)

F r e q u e n c y (cm - ' )

Group ~

d/.tCu(D)

0.0

1).30

0.35

0.45

1676 1673 1671 1666 1665

! 678 1659 1685 1675 1654

1 679 1650 1694 1681 1647

1680 1642 1702 1690 1'640

0.0

0.20

0.27

0.40

5 4 I 2 3

1579 1569 1567 1554 1540

1575 1561 1563 1562 ! 543

1568 1555 1557 1567 i 550

1559 1536 1547 1575 1558

Amide III 1 4 3 2 5

1331 1324 1305 1299 1293

5 2+1 1+2 3+4 4+3

Type III

Ap, ctt(D)

0.0

0.30

11.35

0.45

1676 1675 1674 1671 1665

! 680 1666 1671 1681 1661

1 683 1661 1669 1689 1659

1 686 1656 1666 1693 1657

(1.(1

0.20

0.27

0.40

5 4+ 1 1+4 3 2

1578 1568 1567 ! 563 1 553

1573 1564 1561 1561 1550

! 561 1557 1555 1560 1545

1555 1547 1548 1558 1540

1 4 2+3 3+2 5

i 330 1329 i 303 1297 ! 293

5 3 2+1 1+2 4

F r e q u e n c y (cm - I )

0.0

11.311

0.35

0.45

1676 1674 1 671 ! 667 1 665

1679 1661 1680 1675 1657

1 682 1 649 1687 1680 1652

1684 1643 1694 1686 1648

0.0

0.20

0.27

0.40

5 ! 2 4 3

1578 1563 1550 ! 543 1 536

1569 1562 1554 1545 1536

1561 1560 1559 i 549 1537

i 553 1558 1562 1551 1539

! 4 3 2+ 3 5

1321 1 317 1 303 ! 291 1 286

5 2+1 1+2 3+4 4+3

Amide !!

J#~ff(D)

A#~.,(D)

-I#~.fr(D)

a Group numbers refer to the pcptide groups of Fig. 8. The designation 2 + l indicates thai both groups contribute to the mode, 2 contributing more.

130 0

T A B L E VIII

Calculated atnide moth" frequencies of CH.t-O.GI)'-(Ala)2-Gly-O.CH~ and obsert'ed amide bands of type ! 13-turn Z-Gly-Pro-l.eu-Gly-Oli a Mode

7".:' L, ',-t

C a l c u l a t e d ( c m " ~~

O h s e r v e d b ( c m " t)

v

Group "

Raman

Infrared

5 I 3+ 2 4 2+3 2+1 3 4 I + 2 2 4 3 I 3 4 "~ I 3 I+2 4

1741MS 1689S ! 674W 1 65flS 1644MW -

1 741S 1686S 1673W 1655VS 163qVS 1568MS 1548MS 1 5Z'~M

1 333M 1325W 131hW

1333W 1314W

1 291M -

1 294S 5gqM -

743 688 681 659 647 57~ 562 544 534 391 331 326 313 31111 291 281 6119 583 565 498

Amide I

A m i d e !1

qr/k..,

......

qs.\

X.. qt)'='C~c'x.

Amide III

C'7--Xm' 1% "O Amide V

Fig. 8. Schematic d i a g r a m of a /.C-turn of C H ~-CO-(Ala).t-N|I-Cll ~ with e x t e r n a l h y d r o g e n b o n d s included.

c m - ' band to a-helix plus/3-turns, and concluded that bands at about 1655 c m - ' are not due only to a-helical conformation.

" D a t a f r o m K r i m m a n d B a n d e k a r [6]. " S. strong; M. m e d i u m ; W. weak; V. very. ¢ See fta)tnote a. T a b l e VI. for e x p l a n a t i o n of symbols.

T A B L E VII

Calcuhued amide L IL and I1." ~'qu~,wies and transition dipole coupling inchah'd fi,r typc.s I '. I1' and Iii' [3-mrnx T y p e I' Amide I Group "

T y p e Ii'

-.~ Frequency (cm

t)

G r o u p '~

.l#,.,r(D)

5 2 ! 3+ 4 4+3

T y p e i11' F r e q u e n c y (cm

iI

Group "

A/~,.:u(D)

0.0

0.30

0.35

0.45

1676 1675 I ~72 1669 1 685

1 676 1 677 1671 1676 1657

1676 1678 1 670 1 680 1651

1676 1 680 166q 1684 1 646

2+5 5+2 I 3 4

Frequcncy(cm

t)

d~.rr(D)

0.0

(I.3(I

11.35

0.45

1676 1676 1673 1672 1666

1682 1674 1673 1670 1665

1 685 i 673 ! 673 1 668 1665

1 687 1673 ! 673 1 fi66 1664

5 2 I 3 4

(I.0

0.30

0.35

0.45

1 676 1675 1671 1669 1666

1677 1671 1678 1674 1 658

1 677 ! 66'9 ! 685 I 676 1 654

i 679 1667 ! 691 1 678 1649

A m i d e II

dgcrf(D) 5 I 4 3 2

.lu~rf(D)

0.0

0.20

0.27

(I.40

1 568 1555 1548 1 541 1 536

1 563 I 549 1 531 1545 1538

1553 1543 1529 1550 1542

1 546 1536 1 521 1553 1 544

d~t~rr(D)

0.0

0.20

0.27

0.40

5 I+ 2 4 I +2 3

1 569 1 553 1 548 1536 1 525

1565 1548 1 539 1530 1524

1559 1546 1 531 1523 1 523

! 551 1 541 1 526 1517 1 523

I

1311 1309 1300 1273 1267

0.0

0.20

0.27

0.40

5 I 4 2 3

! 566 1 555 1546 1 542 1537

1 551 1552 1549 1 545 1 535

! 543 1550 1 552 1548 1 534

! 53 I 1 549 1 553 1 548 1532

1

1311 1303 1 288 1 274 1 271

A m i d e Ill

! 4 3+ 2 2+3 5

1318 1311 1290 1273 1 268

" See footnote to Table VI.

4 2+4 3+2 5

4 2+4 2+3 5

131 TABLE IX

Calculated mtd ohserred amide frequencies of ~'pe H [3-mm Pro-LeuGI),.NH2 . Mode

Amide I

Amide I! Amide Ill

Amide V

Calculated (em- ')

Observed h (cm

z,

Group"

Raman

Infrared

! 680 1 664 1658 1568 1545 1375 1354 1335 1328 i 266 1237 699 657 6113 575

2+3 3+ 2 4 2 3 2 3 2 2 3 2 2 2 3 3

i 691MW 1 664sh 16525 1375W ! 351MW 1M 5 M W 1338M 1 271 MS 1241S 695W 647W fil4W 571M

1680VS 1 662M 1650sh 1565sh 1 556VS 1370M 1336M ! 270sh 1241M 687MS 645S hl2MS 5711MS

i)

" From Krimm and Bandekar [61. h See flmlnotc a. Table Vlrl. fi)r explanation of symtads. ~' See fix)tnote b. Table VIII. fiw explanation of syml'mls.

HI-D. y-Turns

TABLE X

Computed amide Iattd I!i frequencies of 13-turn CH~-CO-(,41aLa-NItCtl3 with the aid of dihedral angh's /'or fimr [3-ttmts m insulin a

A7~lO (type IV)

A12-15 (type l i d

B7-10 ltype 11')

B20-23 (type i)

T A B L E XI

Cah'tdated atnide frequencies " of CH-CO-O.-Ala) 3 NH-CH~ b~ .r-ttmt conformations h Mode

Fig. 9 shows a y-turn of CHa-CO-(Ala)3-NH-CH3. Table XI lists the computed amide frequencies [50,51]. These results were applied to the cyclic pentapeptide, cyclo(D-Phe-L-Pro-Gly-D-Ala-L-Pro), whose x-ray struc-

~-Turn

Fig. 9. O R T E P drawing o f CH~-CO-IL-AIaI.~-Nlt-CH 3 model of a "r-turn.

Amide !

Amide Ill

Group

Frequency (cm - t)

Group

1+2 4+5 5+4 3 2+ I 1+2 4+3 5 2+1 3+4 I 5 2 3 4 2+ 1 3+4 5 4+3 1+2

1697 1677 1660 1656 1650 1696 1683 1674 1655 1648 16811 1677 1675 1655 1 650 i 684 1683 1671 1653 1646

1+3 4 2 3+ I 5 I 3+4 2+4 4+3 5 1+3 3+ I 4 2 5 1 3+ 1 4+5 2+3 5+2

" From g r i m m and Bandekar [61.

Frequency (cm - i ) 311 302 296 290 281 310 305 299 289 283 319 311 307 289 281 315 296 290 287 281

y

.rl

7.~

), Group " z, Group c . (cm - l) ,, (cm - h ~ (cm - t) fi84 670 655 653 552 Amide Ii 529 526 5119 Amide 111 3911 1367 1 336 1327 1297" 1282 1 242 * 12Z';*

Amide !

Amide V

11+3 I 2 3 + 11 0 1+3 3+ i 2 I1+ I I I 3 3+2 1 0 3+2

1675 1668 1 656 I 654 1551 1 5411 1527 15 ! 8 1387 1352 1 331 * 1310" 1261" IZ~i4* 1 248 *

3 3 1 I+ 2 2 2 0 0 0

729 719 7117 677 643 570 562

709 706 676 655 608 602 548 517 493

0+3 2 1 3+0 2+1+11 0+2 3 I I1 2 1 2+3+1 0 3 I1+ !

2 I 3 2 I 0 11

1675 1667 16~| I 649 1546 1 54fl 1512 15113 1375 1371 i 35 i 1346 1323 1308" 1268 1243" 1232 * 718 712 706 698 644

Group t0+3 2+ 1 I+2 3 + 11 1+2 I +2 3 0 I 2 3 0 I 2+1 3+ 1 3+2 0 1 2+3 3 0+ I 2

•~ Data from Krimr~ and Bandckar [6]. Only frequencies with an asterisk have a CN s contribution, in all cases appropriate to the peplide group except the 1268 c m - t band of .rL which has a CN(2) s contribution. b Data from Bandekar and g r i m m [51]. T h e numbers refer to the peptide groups of Fig. 9. The designation 0 + 3 implies that both groups contribute to the mode. mode (1 contributing more.

132 T A B L E Xll

Obserced and calculated amide frequencies of tTclo (o-Phe-t.-Pro-Glyo-Ala.t-Pro)

phosvitin. The results one obtains by this method were found to be baseline-dependent [54,55].

IV-A.Z Method of Williams and Dunker 156] Mode

Amide I

Observed (cm - i ) ,,

Calculated h (cm - I )

Raman

Infrared

~,

1685M

1689S

1664S ! 634M 1618W A m i d e I1

A m i d e III

Amide V

1389VW 1 MOW 1327W 1303W 1278M 1273M I !~2S 747W 724VW

666VS 638MS 621S 563sh 542MW 522MS 386sh 341W 319MW 304MW 1280sh 1272MW 1266W 1248MW ! 233W 750M 724W

693 667 659 641) 628 561 549 531 380 336 313 3111 293 286 272 266 249 237 756 731

703 701VW 588MW

700M 653sh 593MW

hgl 655 593

Group" 2 4+0 0+4 I +3 3+ I 4+2 2+4 0 4 2 4 0 ..)

0 0 4 -1

4 21. + Phe) 4 O( + Phe) 4( + Phe) 0 4

From Bandekar and K r i m m [6]. See footnote a. Table VIII. for explanation of symbols. h C o m p u t e d by Bandekar a n d K r i m m [511]. c G r o u p n u m b e r s refer to the peptide groups in Fig. 9.

ture is known [52]. The results are tabulated in Table Xll.

IV. Vibrational spectroscopic methods for studying protein conformation in vibrational spectroscopic studies, two main approaches are utilized. The first is qualitative, and makes use of diagnostic amide bands for identifying different secondary structural components. In the second method, secondary structure is determined on a quantitative basis. We describe below various methods for quantitative determination of secondary structure.

IV-A. Raman spectroscopic methods IV-A. 1. Method of Lippert et al. 153] This method makes use of amide I and 111 Raman bands in H 2 0 and D20. This limits the applicability to water-soluble proteins; it uses peak intensities instead of band areas; it incorporates only three components, namely, a-helix, E-structure and random coil, in the analysis, it may, therefore, not be suitable for proteins containing a significant number of g-turns, like

Six reference spectra corresponding to six component secondary structures were included. This was later superceded by the method of Williams, discussed below.

IV-A.3. Method utilizing the amide !ii' bands [57] This method makes use of amide I!I' bands to estimate the percentages of ,secondary structures in proteins. CH deformation modes are intense in the amide I!1 region, so tha~ it is difficult to delineate amide 111 bands. The amide I11' region is less affected by such problems. The authors pointed out that this method would be useful in estimating the structure of protein domains that are exposed to, or sequestered, from the aqueous environment. But amide ill bands may not be real indicators of protein conformation, since they are non-localized and spread over a broad region [58]. We might have to deal with more than one amide !I1' band and it is unclear which one to choose.

IV-A.4. Method of Williams 15o,601 In the initial method, which is a refinement of earlier work [57], the amide I Raman band in a protein was analyzed as a linear combination of amide ! bands of component structures. Fourteen proteins were analyzed and the correlation coefficients for a-helix, /3sheet, g-turn and random coil components were 0.98, 11.98, 11.82 and 0.35, respectively. The average deviation of these Raman estimates from x-ray data was less than 4%. This method was subsequently modified [59,60] to include amide I!1 band intensities. The correlation coefficients obtained this way were 0.89, 0.80, 0.72 and - 0 . 8 2 for a-helix, g-sheet, g-turns and undefined structure, respectively. Currently, this Raman method is the most widely used.

IV-A.5. Method of Berjot et al. 1611 This method uses amide I Raman intensities of a-helical, g-sheet and random coil components. The reference intensity profiles were computed by a stepby-step fit of the amide ! spectra of a number of proteins of known secondary structure (from x-ray). The method is applicable both to solid state and solution spectra. For solution spectra, this method solves, simultaneously, for the secondary structural components and the subtraction coefficient, which determines the solvent spectral contribution. Although the method does not incorporate the contributions of E-turns, which are known to give rise to well-defined frequencies [9,40,42,44,48], it is somewhat surprising that the correlation coefficient between x-ray data and the Raman results was as high as 0.97.

133

IV-A.6. Method of Bussian and Sander 162] This method makes use of amide ! bands due to a-helical and B-sheet structures, it is claimed to be very simple for solvent subtraction, aided by use of a divided spinning cell technique, numerically stable data-handling algorithm and a clear et'ah~ation of expected accuracy. The purported claim of accuracy is 6% for helix content and 5% for B-sheet content. They applied this method to check the structural purity of

structures, when one is dealing with extremely small quantities of samples, the baselines arc found to shift from sample to sample. However, small deviations in base-line position were found to have only insignificant effects on the computed percentages. It was also found that an appropriate single base line could not be drawn for spectra of proteins with extremely high a-helix contents.

genetically engineered proteins. IV-A.Z Resonance Raman methods of Spiro et al. [63,64] Copeland and Spiro [63] proposed a far UV (192-29,23 nm) UV Raman spectroscopic method for determining protein secondary structures. A linear correlation with a-helix content was reported for 192 nm excitation. Characteristic amide II Raman cross sections were derived for a-helical, B-sheet and random coil components, and was applied to a I- and B-purothionin. The authors proposed another method [64] which makes use of amide ll, Ill and amide !1' modes in UVRR spectra at 200-nm excitation. A linear relationship was reported between the a-helical content and amide II and amide Ill modes for myoglobin, BSA and cytochrome c. However, amide ! band #ttemity was fotmd to be nearly independent of a-helical content. This method has the advantage that only micromolar concentrations of samples are needed, whereas conventional Raman experiments require far higher concentrations, which may lead to aggregation. Proteins that absorb or flouresce in the visible cannot be studied by dispersive Raman technique. However, the limitation of this method is its exclusive applicability to a-helical conformation. IV-B. FT-IR spectroscopic methods IV-RI. Method of Susi et al. /65,66/ In this most commonly used IR method, second derivative FI'-IR spectra are recorded in DzO in the spectral region, 135(I-1800 c m - t . The resolved peaks are then assigned to the a-helix, B-sheet and B-turns and used to compute the percentages of different secondary structural components. IV-B.Z Method of Dong et aL /67] This method employs water subtraction and second derivative techniques to estimate the amounts of secondary structures present in the protein spectra in H,.O. High H zO absorbances are handled by using a cell with CaF, windows and 6 /zm path length, as compared with the typical 60 tLm path lengths used by Susi et al. [68] in DzO solutions. The procedure uses a-helix, B-sheet, B-turn and random coil as component

IV-B.3. Method of Let, et al. [69] This makes use of factor analysis followed by multiple linear regression. The standard errors of prediction were given to be 3.9%, 8.3% and 6.6% for a-helix, B-sheet and turns, respectively. The purported advantages of the method were, no curve-fitting is necessary, and the several amide I band components need not be assigned. IV-B.4. Methods of Pdzolet et aL [70, 711 A method initially developed by these authors [70] is limited to determining only the/]-structure in proteins. it uses amide Ii!' and amide I', and was applied to human immunoglobulin G. The second method [71] makes use of amide I and !I bands, least squares fitting and an algorithm developed previously [72] for spectral subtraction of water. Best results were obtained with the partial least squares method. However, attempts to include B-turns led to a loss of accuracy.

IV-B.5. Method of Eckert et at. [73,74] Eckert et al. [73,74] used amide I' bands in the IR spectra. Their initial attempts to distinguish bctween parallel and anti-parallel B-sheet structures failed because of overlap of bands due to undefined and parallel B-sheet conformations. However, the method was shown to be especially useful in checking independently the results obtained by CD. IV-B.6. Method of Venyanlinoc et aL [75-77/ In this method, solvent-subtracted IR spectra are used in the 1800-1000 c m - ' region. Five to eight spectra of each of the 13 proteins u ~ d in the study are obtained by using different cells, concentrations and sample preparations and the spectra averaged. The novelty of this procedure is that thc amino acid side chain absorption curves [75] are subtracted to obtain the pure peptide absorptions. Their previous work [75] showed that side chains of arginine, asparagine, glutamine, aspartic and glutamic acids, lysine, tyrosine, histidine and phenylalanine have absorption bands in the 1800-1400 cm- J region. The average experimental errors of the spectra at the maxima of amide ! and II bands were reported to be 3.0 and 1.5%, respectively.

134

V. Applications

creases in random coil contents in both BR and Ca -'~ ATPase.

V-A. Bacteriorhodopsbz I/-B. Structure and conformation of toxins For reviews o n this topic sec Rothschild [78] and Braiman and Rothschild [79]. Earnest et al. [8(11 investigated the secondary structure of bacteriorhodopsin (BR) by polarized FT-IR spectroscopy and H-D exchange, using resolution enhancement of observed bands and isotope labelling of the Schiff base to assign the peaks in the amide ! region. The a-helical component exhibited strong IR dichroism and was little affected by H-D exchange. A band at 16411 cm ~, previously a~signed to ,8-sheet structure by Downer et al. [811 and by Lee ct al. [82,83], was reassigned in part to the C=N stretch of protonatcd schiff base of the retinylidene chromophore. Earnest et al. [84] showed that a peak at 1529 cm-J. assigned previously to the amide I1 band of /3-structure [82,83], arises in part from the C---C cthylenic s mode of the chromophorc. Thus the 1640 and 1529 cm ~ bands might have contributions from non-peptide parts of the molecule. It was concluded that mostly a-helical regions span the membrane and that most of the /3-structure is located in surface regions. Earnest ct al. [85] applied lx~larized FT-IR difference spectroscopy to probe the orientation of the transition dipole moments of chromophore vibrations in the K¢,.~ and M4~_, intermediates and to measure the dipole moment direction of the C=O bond of an aspartic acid that is protonated in the BR57~D M4~_, transition. The 1762 c m - t positive peak arising from the (7=0 stretching mode of an aspartic group in M4t,_ was found to be oriented about 45 ° from the membrane plane. The peaks at 1669 (negative) and 1661 (positive) cm- t in the BR.~7o--* K¢,3~ ~ spectrum were found only in the parallel polarization. These peaks were assigned to the amidc I mode of a-helical conformation of the protein backbone. Rothschild and Clark [86] assigned the 1669- and 1661 - c m - t peaks to the op and ip components of the amide i vibration in oriented BR films. These frequencies arc close to the op and ip amide 1 modes computed for the a u helix [84]. Earnest et al. [85] used the low intensity of these peaks to conclude that they were inconsis~.ent with a large change in the tilt angle of the a-helices during the BR.~7c~-'*M4~,. transition, as suggested by Draheim and Cassim [88]. Lee ct al. [82] reported second derivative FT-IR spectroscopic studies of the secondary, structures of bacteriorhodopsin and Ca 2+ATPase. The spectra indicated that both BR and Ca-" '-ATPasc contain helical,/3-sheet and random coil conformations. Purification in SR lipids (so as not to include contributions from other SR membrane proteins) or reconstitution into dimyristoylphosphatidylcholine bilayers had little effect on Ca-' ~-ATPase. Solubilization by Triton X-I()0 was found to lead to in-

Harada ct al. [89] studied the Raman spectra of a number of native and denatured neurotoxins. The loss of toxicity was correlated with the conversion of the ,8-sheet to the random coil structure and the breakage of the strong H-bond of Tyr-25. Replacement of Asp-31 in the component Lslll by Asparagine led to a reduction in toxicity by 88%. The loss of /3-sheet structure was also concluded to be partly responsible for this. Nabedryk-Viala et al. [90] used temperature-dependent H-D exchange technique and IR spectra to study the molecular dynamics of two homologous neurotoxins. They reported differences in solvent accessibility and temperature-dependent changes in flexibility and secondary structure. Existence of two conformer families which might be related to the unusual biphasic kinetics of dissociation of the toxin-receptor complex [91] was postulated. In a related study [92], it was found that the presence of a (nitrophenyl) sulfcnyl txmnd to the Trp does not disturb the secondary strucure of the toxin. FT-IR was applied by Surewicz et al. [93] to study the secondary structures of three snake venom cardiotoxins. The aqueous solution spectra of the three cardiotoxins were similar and high in ,8-structure. Lipid-free protein contained 34-43% /3-structure and 3 2 - 3 9 ~ unordered structure, the rest percentage being duc to/3-turns. Upon binding to lipid bilayers, the unordered structure d c c r c a ~ d by 8-1(1% and ,8-structure increased by about this amount. Surewicz et ai. [94] carried out structural studies of isolated B and A subunits of cholera toxin and its receptor by FI'-IR. The B unit was found to contain mainly/3-structure and was found to be hghly folded. The A subunit was less ordered than B. I="F-IR spectra showed no significant changes when the B subunit was bound to its GM~ receptor. They, therefore, concluded that the main role of the B subunit was to provide a water-soluble carrier to the A subunit and to place it in close proximity of the membrane surface [95]. Thomas ctal. [96] employed Raman spectroscopy to investigate the structures of a-bungarotoxin and cobratoxin. Both exhibited similar secondary structures with about 47~ and 53% for ,8-structure and coil, respectively.

I/-C. Stn~cture and conformation of gramicidin A Gramicidin A (GA) is a pcntadecapeptide with alternating i. and D residues. It facilitates the passive transport of ions across membranes and lipid bilayers [97l. A number of IR [99-106] and Raman [9,106-108]

135 studies have been carried out on GA and its analogs. Nebedryk et al. [103] used polarized IR to study GA in vesicles and concluded that the GA transmembrane channel was oriented normally to the bilayer with the helix axis at less than 15° with respect to the normal. Naik and Krimm [1(}9] have pointed out that effects of deuteration (experiments in DzO) on peptide group frequencies were neglected in some studies [ 102,1114,1(}5]. By comparing the Raman spectra of crystalline native, crystalline Cs+-bound and vesicle-bound GA and carrying out NCA on the/]4.4, /],.3/].~.f, and 1' J,/J 7.-" structures of GA, 1' ,L/]~~', 1' ,I,/]7.2 and /]"~ structures were assigned to the above, respectively [.see Table Xlll]. These are in good agreement with recent x-ray findings [i 10,11 I].

V-D. Stnwture of cap.sid proteins of viruse.~"and pha.qe.~ Thomas [112] has reviewed Raman spectroscopic applications to structure determinations of viru~s and phages. Li et al. [! 13] carried out structural studies of bean pod mottle virus (BPMV), capsid and RNA in solid and solution states by means of Raman spectroscopy. The protein subunits of the empty capsid contained 45-55%/]-sheet and /,C-turn structure; with packaging of RNA, this increased by a small amount (5%). The subunits contained less than 25% a-helix. The molecular environments of tour Trp residues per subunit were altered upon packaging RNA; however, Tyr, Cys S-H, Asp and Glu did not show any changes. Although significant rearrangement of aliphatic side chains within the viral capsid was found to occur with RNA packaging, BPMV middle and bottom components exhibited no differences in subunit secondary structure. Turano et al. [114] studied by Raman spectroscopy the structure of turnip yellow mosaic virus (TYMV). The amide I and Ill bands indicated the absence of helical and /]-structural components and the prepon-

dercnce of disordered conformation. Using the prediction scheme of Chou and Fasman [! 15], they predicted the secondary structure of the TYMV coat-protein molecule. TYMV was found to contain 16% a-helix, 41% /]-structure and 43% irregular structure. They thought it likely that the Raman spectra missed the 16% a-helix, they could not explain the discrepancy concerning the/]-structure. Prevelige et al. [116] used Raman spectroscopy to study eonformational states of the bacteriophage P22 capsid subunit in relation to self-assembly. Coat protein subunits which polymerized into closed procapsid shells (PS) in the presence of scaffolding protein contained mostly /]-structure and some a-helix. A clear conversion was reported from a-helical structure in subunits of PS particles to the/3-structure in subunits of polydisperse multimers, called 'associated subunits'. In a subsequent communication [I 17], they studied the N-terminal domain of the bacteriophage l. repressor which binds specifically to the n. operators. This was found to be 7(1% helical, in agreement with x-ray findings. Dunker et al. [118] reported UV and Raman spectra of filamentous phage fd. Raman spectra of the intact phage, and following removal of the coat protein, showed that the original a-helical conformation changed to that of/i-sheet. Sargent et ai. [119] used Raman spectroscopy for studying the secondary structure of the wild-type and a temperature-sensitive folding mutant of tailspike bacteriophage P22. Analysis of the Raman amide ! and I!! bands led t o 52-61% /]-structure, 24-27% a-helix, 15-21%/]-turn, and 0-10% other structures. The secondary structure of the wild-type tailspike, as monitored by the conformationally-sensitive amide I and !!! bands, was stable to 80°C, denatured reversibly between 80 and 90°C, and irreversibly above 90°C. The structure of the temperature-sensitive folding mutant (tsU38) was identical. This supported the previous re-

TABLE XIII

Ob.~erved and cah'ulated amble I frequencies (m cm Structure

~44 /j,.t T ,~ ,B5~' T J,/~7.2

/) fiJr smgh,- aful double-~tranded gmmk'idin A .~mwmre.v

Calculated "

Obse~'ed

v ( I R ) t,

Jv c

v (R) ~

v (IR)

Jv

v (R)

State

1 631 1 643 1 636 i 632

17 9 32-39 43-54

1 663 I 650 1666 1669

~ I 6411 " 1 638 ~ 1630 ~

42 ~ 55 g

1 654 I 1666 ~ 1668 ~

GA-lipid G A (tryst) GA-Cs '(cryst)

F r o m Refs. 221 a n d 222. u Strong parallel-polarized IR band [1116]. Difference between s t r o n g parallel and strongest ( t h r o u g h weaker) perpendicular IR mode [Iq16]. a Mid-point of IR-weak frequencies, expected to be strong in R a m a n . " Estimated from observations on vesicles [102.105]: see also Ref. 109. f From Ref. 109. From Re[. 223.

136 suits that the mutational defect at 40°C affects intermediates in the folding pathway rather than thc native structure. Bamford et al. [120] used Raman spectroscopy to study lipid-containing v i r u s n t h c icosahedral bacteriophage PRDI and its capsid protein. The major coat protein P3 was found to be predominantly in the g-sheet conformation which is maintained in the fully assembled PRDI virion, as well as in the empty capsid.

V-E. Miscellaneous applications Alvarez et al. [121] used second derivative FT-IR spectra to determine the frequencies of amide 1 and !1 components for concanavalin A. both in the presence "9+ and absence of Mn- , Ca ~'+ and a-methylmannose. Mn 2+ and Ca -'+ were found to shift amide I and !! bands by ! and 4 - 6 cm-m taken to imply changes in the g-sheet regions of concanavalin A. H-D exchange showed that the 1532 cm -~ component exchanged faster than the 1563 cm- ~ component, suggesting that the g-structure is more accessible to solvent than the beta turns. Wantyghem et al. [122] reported the secondary structural characteristics of a lectin, RPA3. The g-structure comprised 60% of the overall structure. RPA3 was shown to interact with phospholipid bilayers through polar interactions between the polar heads of the phospholipids and specific peptide units. Second and fourth derivative FT-IR spectra were used to study the secondary structure of the human erythrocyte glucose transporter protein [123]. The amide 1 and !1 band components at 1658 and 1548 c m - ' were assigned to an a-helical component while the amide ! components at 1691, 1639 and 1630 erawere assigned to g-structure. The weak component at 1681 cm -° was assigned, following Bandekar and Krimm [39,40,42,48], to g-turns. It was pointed out that the amide I components at 1691 and 1658 cm-n may also be due partly to g-turns [39,40,42,48]. Anderle et ai. [124] studied the FF-IR spectra of native and thermally denatured proteins by using the amide 111 region of the spectra. Although it was claimed that the amide !11 region does not require either subtraction of water or use of DzO as solvent, it must be noted that the problem with this approach is that amide ill modes are not localized in NH ib but contain contributions from side-chain modes. This is the reason Williams et al. [57] chose to work with the amide !!1' region of the spectra. Caughey et al. [125] applied their FT-IR method [67] to determine the secondary structure of the proteinase K resistant core of scrapie-associated protein PrP 27-31) as it exists in highly infectious fibril preparations. The analysis indicated that the protein is rich in g-structure (47%), g-turns and a-helix contributing the

remaining 31% and 17%, respectively. This will be discussed later (section VII). Chen and Lord [126] studied the Raman spectra of a-chymotrypsin and trypsin. The presence of an inten~ amide ! band at 1668 cm -a in both led them to conclude that there was a large amount of g-structure present. The contour of the amide !11 band, as proposed by Lord [3], was used to conclude that trypsin 'ontains more a-helical conformation than a-chymotrypsin. They also reported slight differences in solid and solution state conformations. Kaiden et al. [127] used the amide Ill bands of albumin, myoglobin and y-globulin to study heat denaturation. Bands in the regions 1300, 1260 and 1235 c m - a were respectively assigned to a-helix, disordered, and g-structures. This technique might be useful in characterizing the species of secondary structures of proteins adsorbed on material surfaces. Arrondo et al. [128] carried out FT-IR studies of sarcoplasmic rcticulum proteins, in H : O and D,O. By monitoring the a-helical and g-sheet amide 1 and il bands, they concluded that ATP induces conformationai changes in the above proteins, increasing the a-helical and g-sheet contents. On the other hand, Ca -'+ was found to disrupt the a-helical content. Bellissimo and Cooper [129] used the FT-IR-ATR technique to study plasma protein adsorption. Differcnt amide I11 peak positions for fibrinogen adsorption on germanium and Biomer '~' were taken to be indicative of fibrinogen conformational changes. T h e ~ findings are useful in studying catheter-blood protein interactions. Breton and Nabcdryk [130] used UV CD to determine secondary structure and IR dichroism for transmembrane orientation, of a-helices of chlorophylls in photosynthetic pigment-protein complexes. The a-helical segments of a large variety of intrinsic membrane chlorophyll-protein complexes were found to be tilted by an average angle of 30-35 ° away from the membrane normal. Raman spectroscopy and the method of Williams et al. [59,60] were employed to study the conformation of the extracellular enzyme levansucrase [131]. Fifty-one pcrcent g-sheets, 18% a-helix (7% ordered and 11% disordered), 17% turns and 13% undefined structures were reported. The presence of iron ( l i d ions was found to increase the ordered a-helical content from 7% to 16% and to modify the environment of the Trp and Phe residues. Byler and Purcell [132] used second derivative FT-IR spectra to study thermal denaturation of the three proteins from bovine whey, namely, g-lactoglobulin (gLG), serum albumin (BSA) and a-lactaibumin (aLA). in g L G and BSA, it was found that intermolecularly H-bonded g-strands are formed before the onset of denaturation.

137 Kato et al. [133] studied heat denaturation of albumin by using the intensity ratio of amide ii to amide ! bands. They reported a correlation between the ratio of amide !! to amide ! band intensities and the a-helix content. They applied this method to analyze the ATR spectra of albumin adsorbed on polyethylene oxide surface. Kirsch and Koenig [134] collected the F r - I R spectra of y-globulin, chymotrypsin, serum albumin and /j-lactoglobulin as a function of temperature, in order to minimize intermolecular interactions, low concentrations (1%) of proteins were used. The intensity of amide !I! band was found to be very weak. With /j-lactoglobulin, a band at 1672 cm-~ was assigned to /j-sheet structures, usually observed only in the Raman spectra. Cozar et al. [135] carried out FT-IR studies of proteolipid protein (PLP) from brain. They found that the a-helical content of PLP diminished when it was delipidated. In the reconstituted PLP, the a-helical content was found to be restored; however, there was an increase in the/j-sheet content. Following previous assignments [39,40,42,48] the bands at 1661) and 1675 era-n where assigned to/j-turns. Vincent et al. [136] carried out an IR study of the pH-dependent secondary structure of brain clathrin. At pH 8.5 clathrin dissociates from the bilayer membrane to form trimers and at pH 6.5, the trimers form cagelike structures. As the trimers formed cages, the a-helical content decrea~d. Fermandjian et al. [137] reported the vibrational spectra of angiotensin I! and an analog. Based on the positions of amide 1, !1 and I11 bands, angiotensin !! was shown to adopt an antiparallel /j-conformation both in the solid state and in aqueous solution. Fry et al. [138] used 2-D NMR, FT-IR and CD techniques to study the solution conformation of the 45-residue MgATP-binding peptide of adenylate kinase. Although the /j-sheet content of the peptide alone determined by the above methods was in reasonable agreement, a discrepancy was reported between NMR, X-ray and FI'-IR results foe the a-helical content: the a-helical content is 47%, 24%, < 10%, and 5-13%, by x-ray, NMR, FT-IR, and CD methods, repectively; the /j-structure was 22%, 38%, 27-42%, and 22-26%; and the unordered structure percentages were 31%, 38%, 48-73%, and 62-70%. FT-IR thus predicted far less a-helix than x-ray and NMR. This was attributed to the ability of x-ray and N M R to detect short helical lengths which FT-IR and CD cannot. Gorga et ai. [139] examined the secondary structures of human class 1 and class !I histocompatibility antigens by CD and FT-IR. Second derivative FT-IR spectra gave helix contents of 17%, 8% and 10% and /j-sheet contents of 41%, 48% and 53%, respectively, for HLA-A2, HLA-B7 and DRI. BY CD the corre-

sponding values wcrc 8%, 2(1%, and 17~, for a-helix, and 74%, 29% and 42% for fl-structure. The anomaly in HLA-A2 results was interpreted as arising from the Trp-107 that is missing in thc other two antigens. The discrepancies found in the other two antigens were considered insignificant because of the uncertainties inherent in both the quantitation procedures. Hall and Jagodzinski [140] used the method of Lippert et al. [53] to determine secondary structure of porcine mitochondrial malate dehydrogenase. The 35% fl-sheet computed was in agreement with the x-ray value of 34%. Yue et al. [55] used Raman spectroscopy to study the secondary structure of liver alcohol dehydrogenase (LADH). By the method of Lippert ct al. [53], LADH contained 21% a-helix, 35% /3-structure, and 37% random coil. X-ray reports [141] give 29% a-helix, 34% /j-structure, and 37% random coil. Heremans and Hcrcmans [142], using Raman spectroscopy, studied pH- and pressure-induced conformational changes in chymotrypsin. Pressure changes of up to 3 kbar had ,lo effect while pH changes did affect the secondary strt'.cturc. Holloway and Mantsch [143] studied the structure of cytochrome b 5 in solution by FT-IR and Fourier selfdeconvolution techniques. Cytochrome b s is an amphipathic integral membrane protein found in the endoplasmic reticulum [144]. it was found that the non-polar membrane-binding domain was more a-helical than the polar domain. Cutting the protein into two fragments resulted in a partial loss of a-helix content. Kitagawa et al. [145] studied the Raman spectra of Bcnce-Jones proteins (BJP's) by means of Raman spectroscopy. They made use of amide i and !!1 bands to study protein denaturation. All of the native BJP'S studied gave amide I bands in the 1670-1675 cm -n region and amide !II at 1242-1246 cm-n. Amide I shifted to 1667 cm-n upon LiBr, acid, and thermal denaturation but amide I!I remained unaltered; however, the amide 111 band was broadened. Morre et al. [146] provided IR spectroscopic evidence for conformational changes of plant plasma membranes upon exposure to the growth hormone analog, 2,4-dichlorophenoxyacetic acid (2,4-D). Amide I band broadening and changes in the absorbance ratios of amide I and 11 modes were observed at 2,4-D concentrations of as low as 10 -~ M. Nakamura et al. [147] used IR spectroscopy to study outer and cytoplasmic membranes of E. coli. They found that a large proportion of outer membrane proteins were rich in /J-sheet structure while the cytoplasmic membrane proteins hardly contained /j-structure. IR spectroscopy was shown to be useful for rapid distinction between outer and cytoplasmic membranes. Surewicz et al. [148] used FT-IR spectroscopy to study interactions between myelin basic protein and

138 lipid bilayers. In aqueous solution, the protein was found to contain mainly//-turns. Upon binding to lipid bilayers, it was found to contain 53%//-structure, 15% a-helix and 15%/3-turns. They extended these studies to a hydrophobic myelin protein, lipophilin [149]. in a phospholipid e~wironment, lipophilin was found to contain 55% a-helix, and 36%//-structure. Nabedryk et al. [150] used CD and polarized FT-IR techniques to respectively determine the secondary structure and orientation of a-helix in the thylakoid membrane and in the light-harvesting complex. FT-IR data demonstrated that transmembrane a-helices were present in both thylakoid and light-harvesting complex (LHC) with the a-helix axes tilted at less than 3(I° in LHC and 40° in thyalkoid with respect to the membrane normal. In thyalkoids, an orientation of the polar C---O ester groups of the lipids parallel to the membrane was detected. A similar approach was applied to characterize protein secondary, structure in purple membrane [151]. A large amount (74 + 5%) of transmembrane a-helix was detected (with no significant contribution of //-strands) running perpendicular to the membrane plane. These data were found to contradict the recent model of Jap et al. [152]. Similar study on porin [153] showed that the transition moments of the amide ! and II vibrations are on the average tilted at 47 + 3° from the membrane normal. Pande et al. [154] carried out Raman, IR and CD studies on metailothionein. The protein was found to be unusually rich in /]-turns. Prescott et al. [155] have reported Raman study of hen egg white yolk phosvitin. In lyophilized solid and in acidic solutions (pH 1.5) /3-structure was predominant; but at physiological pH, intense amide I band, observed at 1685 cm --t, was 10-40 cm- J higher than proteins in aqueous solutioas studied so far. This peak position does not correspond to any of the conventional secondary structural components. Renugopalakrishnan et al. [156] reported FT-IR and FT-IR photoacoustic spectra of phosvitin. The spectra indicated the presence of//-structure and/3-turns and no a-helix. The unusual thing was the appearance of the 1630 cm -~ band as a shoulder, which is usually very intense in the normal FT-IR spectra. What is unclear is if this is unique to the FT-IR photoacoustic spectra. Prestrelski et al. [157] determined, using FT-IR spectroscopy, the secondary structure of two recombinant human growth factors, platelet-derived growth factor and basic fibroblast growth factor. Both the proteins were found to be rich in //-structure with some a-helix; the basic fibroblast growth factor was found to be richer in //-turns. They also studied [158] amide I bands in a-lactalbumin and identified bands due to 3~- and a-helices. If true, the FT-IR technique might have a clear advantage over the UV-CD teeh-

nique, which is unable to distinguish between them [159]. By comparing FT-IR spectra with the x-ray resuits on bovine trypsin, they subsequently attempted to delineate bands due to loop and disordered structures [160]. They concluded that amide !' bands in the region of 1655 cm-~ may be assigned to loop structures; bands near 1645 c m - t were assigned to disordered structure. Renugopalakrishnan et al. [161] studied the secondary structure of a core protein from pig skin proteodermatan sulfate. The spectra showed the presence of a-helix, //-turns and disordered structure, carbohydrate portions of the molecule had no effect on the structure. This was later on confirmed by Raman findings [ 162]. Sucharda-Sobczyk [163] studied tuftsin and 21 different analogs by FT-IR spectroscopy. The peptides were found to bc intramolecularly H-bonded. IR evidence was given for the existence of//-turns for peptides containing proline at position 3; however, substitution of proline led to the formation of more than one folded form of the corresponding peptide. Vogel and Jahnig [164] used Raman spectroscopic results to propose models for the structure of outermembrane proteins of E. coil, namely, porin, maltporin and OmpA protein. The three proteins had similar structures consisting of 50-60% //-sheet, 20% /3-turn and about 15% a-helix. Employing a method that accounts for amphipathie//-sheets, folding models were developed for porin and OmpA protein. In a similar study, Raman results were used, in conjunction with the prediction method of Williams [59,60] to propose amphipathic helical models for lactose permease of E. coli [165]. VI. Critical assessment of amide modes

Results on VGG and IGG, two peptides adopting identical conformations but with different side-chains [166], have shown that the amide I and II bands were identical but not the amide ill [58]. Similar results were obtained for cyclo (D-Phe-t.-Pro-Giy-D-Ala-i.-Pro) and cyclo (Gly-L-Pro-Gly-D-Ala-Pro), two peptides with almost identical conformations but different amino acid residues at the first position [52, J. Bandekar, unpublished results]. Tables IV and V, respectively of/3- and a- PLA, show that the amide Ill bands are spread over a rather broad range. Amide Ill modes are thus affected by the nature of the side-chains. Recent VCD results [167] have also found the existence of coupling between C-H and N-H coordinates for amide Ill modes. The ab initio results on two peptides with identical conformation but different side-chains [58], have also led to different amide Ill modes. These results thus do not support the proposed correlations between amide Ill bands and the dihedral angle, ~b [3,168]. In the case

139 of BJP's, it was lound [145] that, upon denaturation, only the amidc ! band shifted and the amide !11 band was unshifted but only broadened [see section V-D). Also in need of better theoretical understanding are the amide V bands [27,169,170]. Vii. Perspectives for the future Considerable progress has been made in treating IR relative intensities (see, for example, Refs. 6, 171 and 172). Some work has been done on Raman intensities [173-175] but more needs to be done. Escribano el al. [176,177] have di~ussed the use of IR and Raman absolute band intensities in force constant calculations. As concerns methods for secondary structure determination, they differ from each other significantly [178]. It is very important to have one acceptable way of defining secondary structure. The side-chains contribute to the amide ! band intensities [3,179]. Ironically enough, although it was first in the Raman reports of Lord et al. [3,180] that the side-chain contributions were subtracted from the amide I bands, this has not been done so in the Raman methods of estimating secondary structure. However, the FT-IR method of Venyaminov et al. [75-77] incorporates side-chain contributions in the amide ! region. The advantage of FT-IR methods over Raman methods is that the former can make use of (in addition to amide I bands) amide 11 bands (absent in the Raman). On the other hand, amide !!! bands are usually weak in the IR and Raman methods provide a means for analyzing the amide I!! bands. Since the quantitative methods of secondary structure determination express the amidc ! (or other amide bands) band intensity as a linear combination of component bands, the success of these methods will depend on how well they incorporate all components contributing to intensity. There is, therefore, a need to characterize other conformations like T-helix [181], 1r-helix [182,183], to-helix [184], collagentype helix [185] and ~-helix [186] as well as non-repeating conformations. The intriguing findings of the linear relationships between the a-helical content and amide I! and I11 bands {and not amide I bands) in proteins [63,64] await theoretical interpretation. The currently available normal mode analysis treatments for studying protein conformations could be considerably improved upon. For example, although the current normal mode treatment of biopolymers represents significant progress [6], one should be cautious in reading too much into potential energy distributions (PED's), since the PED's in the low frequency regions of model polypeptides [36,38] seem to have little meaning. The cross-beta conformation [187, J. Bandekar, unpublished work] deserves further study. Although not yet identified in proteins, it is found in (i) proteins from Alzheimer's patients [188], (ii) the distal half of the

bacteriophage T4 [189], (iii) denatured proteins [1911,191]. It may also be important in protein-nucleic acid interactions [192] and in understanding the origin of life [193,194]. Recent Pq'-IR results of Caughey et al. [125] on the structure of the protcinasc K resistant core of the scrapie-associated protein PrP 27-3[} have tbued 47% H-structure, 31%/3-turns, and 17% or-helix in this protein {see section V-E). This is a protein with amyloid-like properties. If one notes that there is no random coil structure present and that a-helix could also be a type I!1 /j-turn, one comes up with about equal percentages of B-structure and fl-turns, it is, therefore, tempting to speculate if what one is looking at is a cross-/3 structure in this protein. Attempts have been made [195,196] to characterize the handedness of the helix by IR. No theoretical explanation exists for this. Once understood, it will lead to a better understanding of the left-handed helix like the to-helix [184] and enable one to study reversal in helix sense [197]. So far amide bands of only type I and type 1I /.J-turns have been well understood [39-48]. This is because model peptides of known/j-turn conformation are available [41,43,198]. It is important to study/j-turns of other types. X-ray and NMR studies can provide the lead in this case. Two-dimensional NMR techniques have greatly advanced the determination of protein structure in solution [199,200]. NMR is proving to bc an important complement to X-ray crystallography [201]. Recently, Oschkinat et al. [2{12] reported threedimensioanl NM R spectroscopy of proteins in solution. NMR and computer-simulation approaches have been applied to peptides [203,2114]. l_x~ops [21)5], /j-bulges [2{}6] and /3-barrels [2117,2{18] await spectroscopic characterization. Although deviations in amide ! peak positions from ideal /j-structure values have been observed, it has not been possible to interpret these deviations. For example, the band due to fl-structure in /J-lactoglobulin and concanavalin A appears at 1623 c m - ~, while in ribonuclease A it is found at 1636 cm t [209]. One of the reasons for such significant deviations might bc the twist observed in /3-sheets [210,211]. This deserves further investigation. There is controversy in the literature on the amide modes due to parallel /3-structure. Since there is no model polypeptide of known x-ray structure for this conformation, Bandekar and Krimm [212,213] computed the normal mode spectrum of a Pauling-Corey [214] parallel/3-sheet. The characteristic amide ! modes were predicted at 1663 (Raman) and 1642 (IR) for a single sheet. Susi and Byler [66] disagreed with this. They assigned instead IR bands at 1626-1639 (strong) and 1675 (weak) cm- J to the parallel/J-chains. Clearly more work is needed in this direction. Baron el al. [215,216] have reported IR spectra of oligopeptides of i.-valine, L-isoleucine and t.-phcnylalanine; these pep-

140 tides are thought to assume parallel g-structure conformation [215,216, M.H. Baron, personal communication] and their spectra are in agreement with the results of Bandekar and Krimm [212,213]. X-ray work on these oligomers, as well as Raman spectra, will help resolve this controversy. In the studies of model polymers for a-helix and g-sheet, the respective polymers are treated as being infinite. In proteins, however, what one observes are finite segments of the above. So far no attempts have been made to study the effects due to this chain length. In studies of small peptides, space group symmetries are taken into account and the observed fine structure is interpreted as being due to intermolecular TDC interactions [6,213,217]. In proteins the fine structure in a-helical and g-structural segments arises from local order. The forces responsible for fine structure from non-repeating segments like g-turns are not yet identified. Fry et al. [138] found that FT-IR predicted the correct percentage of 3-structure, but not ~-hclix, in MgATP-binding peptide of adenylate kinase. This needs further investigation. Pcptides containing a,gdehydro residues will have C=C contributions to further complicate the amide ! bands [218]. Such nonamide contributions should be considered in determinations of secondary structure, in the Raman spectra of anti freeze glycoprotins [219], very unusual amide i bands have been reported: glycoprotein 4 has three bands, at 1657, 1677 and 1690 cm- ~. while component 8 shows only one band at 1668 cm- ~. The likelihood of glycoprotein 4 containing g-turn structures should be considered. VIII. Acknowledgements ! am indebted to the following persons who helped me during the course of writing: Prof. Spiro (Princeton), Prof. Rothschild (Boston), Prof. Pezolet (Quebec), Prof. Williams (USUHS, Bethesda), Prof. Thomas (UM, Kansas City), Dr. Naik (Michigan-Dearborn), Mr. Purcell (USDA, Philadelphia), Dr. R~.nugopalakrishnan (Harvard Medical School), and Dr. Byler (College of Textiles, Philadelphia). l would like to thank the BOC Group for supporting this work, Dr. Robert Lieberman (of AT&T, Murray Hill) for many helpful discussions and Allan EIlgren for constructive criticism of the manuscript, encouragement and support. The author is indebted to one of the referees for many constructive comments that have considerably improved this article. it is my wish to dedicate this article to the memory of my late father who passed away in March 1989. References I Sutherland. G.B.B.M. 119521 Adv. Protein Chem, 7. 291-318. 2 Tobin. M.C. 119721 in Methods in En~moh~y. Parl C. Vol.

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