BIOCHIMIE, 1975, 57, 417-428.
Applications of Raman spectroscopy to biological macromolecules. W a r n e r L. PETIGOLAS ( * ) ~ . Institute Max yon Laue -- Paul Langevin
--
B P 156, 3 8 0 4 2 G r e n o b l e .
S u m m a r y . - - The Raman spectra of biological maeromolecules arise from the moleenlar vibrations of either the backbone chains or the side chains. The frequencies of the Raman bands lie in a region between 200 c m q and 3000 cm-1. From certain frequencies of the vibrations of the backbone chains one can determine the conformation or secondary structure of a maeromolecule. Thus for polypeptides and proteins the frequencies of the Amide I and Amide III vibrations allow one to determine the average conformation of their backbone chain. In polynueleotides and nucleic acids, the frequency of the phosphate diester stretch of the phosphate furanose chain varies between 814 em-a for A conformation and 790 era-1 for B conformation. Haman spectra of the bases in nucleic acids can be used to determine base stacking and hydrogen bonding interactions. Thus Raruan spectroscopy is an important tool for determining the conformation structure of proteins and nucleic acids.
I. - - INTRODUCTION. H a m a n S p e c t r o s c o p y has b e c o m e an i n c r e a s i n gly i m p o r t a n t tool for the study of biological mac r o m o l e c u l e s . T h e g r o w t h of this n e w field is due to the d e v e l o p m e n t of n e w t e c h n i q u e s --- laser light sources, double and t r i p l e g r a t i n g m o n o c h r o m a t o r s and i m p r o v e d e l e c t r o n i c d e t e c t i o n of w e a k light signals. T h e details of these t e c h n i q u e s h a v e been r e v i e w e d v e r y t h o r o u g h l y in the previous p a p e r by Dr. Delhaye. C o n s e q u e n t l y in this p a p e r w e w i l l c o n c e n t r a t e on the a p p l i c a t i o n s of R a m a n s p e c t r o s c o p y to the c h a r a c t e r i z a t i o n of b i o l o g i c a l m a c r o m o l e c u l e s . E x a m p l e s will be taken f r o m the w o r k of the author. R e a d e r s w i s h i n g to read f u r t h e r in this area should consult one or m o r e of the r e c e n t r e v i e w s in this area [i-5] as w e l l as the e x t e n s i v e o r i g i n a l literature. In this b r i e f d e s c r i p t i o n , w e w i l l discuss t w o types of m a t e r i a l s : (1) p r o t e i n s and p o l y p e p t i d e s , and (2) p o l y n u c l e o t i d e s and n u c l e i c acids. Particular emphasis w i l l be given to those Rainan bands w h i c h s h o w changes in i n t e n s i t y or freq u e n c y w i t h changes in c o n f o r m a t i o n . II. - - THE RAMAN EFFECT FROM POLYPEPTIDES AND PROTEINS. Much of the e a r l y w o r k o.n the R a m a n spect r o s c o p y of p r o t e i n s was done by Lord and colia(*) Guggenheim Fellow, 1973-74. Permanent address: Department of Chemistry, University of Oregon, Eugene, Oregon 97403.
b o r a t o r s and L o r d [5J has r e v i e w e d this field. Most of the o b s e r v e d b a n d s of p r o t e i n s comes f r o m the a m i n o acid side chains, p a r t i c u l a r l y the a r o m a t i c side chains. Ho'a'ever, t h e r e are t w o a m i d e bands, the a m i d e I and a m i d e I I I ~ h i c h arise f r o m the b a c k b o n e p o l y p e p t i d e c h a i n s of the p r o t e i n . These bands are k n o w n to be sensitive to c o n f o r m a t i o n . L a t e r w e w i l l discuss these bands in p r o t e i n s , but first, let us c o n s i d e r a m u c h s i m p l e r p r o b l e m - - the c o n f o r m a t i o n a l changes w h i c h o c c u r w h e n a simple p o l y p e p t i d e is taken from one s i m p l e c o n f o r m a t i o n to another. A r e v i e w of the early w o r k on p o l y p e p t i d e s and o t h e r b i o p o l y m e r s has been given by K o e n i g [2]. T a k i n g the early w o r k t o g e t h e r w i t h the later w o r k of others \ r e can list the R a m a n f r e q u e n c i e s w h i c h are e o n f o r m a t i o n a l l y d e p e n d e n t in four tables. The first, tab~e I, s h o w s the value of the A m i d e I band for v a r i o u s s i m p l e p o l y p e p t i d e s in the v a r i o u s s i m p l e structures, a-helical, B-sheet, and i o n i z e d or d i s o r d e r e d f o r m . T h e A m i d e I b a n d is the most r e l i a b l e of the c o n f o r m a t i o n a l l y d e p e n d e n t bands in the sense that the f r e q u e n c y of the a-helix, ~-sheet and d i s o r d e r e d f o r m seems to be i n d e p e n d e n t of the nature of tile side c h a i n and to d e p e n d only on the g e o m e t r y of the polyp e p t i d e backbone. Table l I gives the f r e q u e n c y of the A m i d e II b a n d w h i c h is R a m a n active only in cis-amides. F u r t h e r i n v e s t i g a t i o n of this i m p o r t a n t b a n d in c y c l i c p o l y p e p t i d e s w i t h p e p t i d e g e o m e t r y betw e e n cis and trans p o l y p e p t i d e s is m u c h to be desired. 30
W
418
L. Peticolas. q u e n c y w i t h t h e t y p e of s i d e c h a i n f o r t h e 13-struct u r e . Also it s h o u l d be n o t e d t h a t t h i s v i b r a t i o n is v e r y w e a k i n t h e a - h e l i c a l s t r u c t u r e . T h u s f r o m
T a b l e II1 g i v e s t h e f r e q u e n c y of t h e R a m a n a c t i v e A m i d e III b a n d i n s e v e r a l p o l y p e p t i d e s . Of p a r t i c u l a r i n t e r e s t is t h e v a r i a t i o n of t h i s f r e -
TABLE I.
Amide I Frequencies (cm-O From Polypeptides and Proteins From Laser Raman Measurements. a-Helical
Substance Polyglycinea , . . . . . . . . . . . . . . . . . . . . . . . Poly-L-alanine . . . . . . . . . . . . . . . . . . . . . . P o l y - L - g l u t a m i c acid ,I. . . . . . . . . . . . . . . . GlucagonO . . . . . . . . . . . . . . . . . . . . . . . . . . . P o l y - L - L y s i n e f H~O . . . . . . . . . . . . . . . . . D~O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
%Structur,,
1654 1658 (W)~,
I674 (S) 1666c
1658 1645 1632
1672 1670 (S) 1658 (.%
Random c0il
1665 1660 (S)
(a) Ref. 13 ; (b) Ref. 6 ; (c) Ref. 14 ; (d) Ref. 15 ; (e) Ref. 16 ; (f) Ref. 6.
a k n o w l e d g e of t h e A m i d e I I I f r e q u e n c y a l o n e , o n e c a n n o t tell t h e c o n f o r m a t i o n on a n a b s o l u t e basis.
TABLE II.
Amide II Frequencies of Cis-Amides From Laser Raman Measurements. Substance
Amide It (cm-t)
Diketopiperazinea . . . . . Hypoxanthineb . . . . . . . 1-MethyluraciF . . . . . . .
1456 (powder), 1385 (aqueous) 1464 1417 (aqueous) . . . . . . . . . . . . .
(a) Ref. 17 ; (b) Ref. 18 ; (e) Ref. 20.
T a b l e IV s h o w s t h e v i b r a t i o n s o f t h e C a - C s t r e t c h f o r p o l y - L - l y s i n e i n v a r i o u s s t r u c t u r e s Eft]. T h i s a s s i g n m e n t m u s t b e r e g a r d e d as t e n t a t i v e , b e c a u s e it falls i n a r e g i o n d i f f i c u l t to a s s i g n [6]. I n o r d e r to s e e h o w t h e s e v i b r a t i o n s c a n b e u s e d to s t u d y c o n f o r m a t i o n a l c h a n g e s i n p o l y p e p -
TABLF III.
Amide IIl Frequencies (in cm-O in Polypeplides From Laser Raman Measurements. Substance
Polyglycine:, . . . . . . . . . . . . . . . . Poly-L-alanine . . . . . . . . . . . . . Poly-L-glutamic acidd . . . . . . Glucagone . . . . . . . . . . . . . . . . . . Poly-L-lysinef . . . . . . . . . . . . . .
J
a-belical
~-structure
I ] I ! ~
1261 1261 (W)l,
1234 1239 (S) ~:
1266 1311
1232 1240
i
Random coil or structure ionized
1248 1248 1243
(a) Ref. 13 ; (b) Ref. 6 ; (c) Ref. 14 ; (d) Ref. 15 ; (e) Ref. 16 ; (f) Ref. 6.
TARLE IV.
Conformationally Dependent Band in the 1000 em-~ Region in Polg-L-lysine (a). Con[ormatiou
Frequency (cm-~)
Ionized
.q45 958 1 002
(a) Taken f r o m ref. 6.
BIOCHIMIE, 1975, 57, n ° 4.
tides, w e begin w i t h figure 1 w h i c h s h o w s the R a m a n s p e c t r a of p o l y - L - l y s i n e in the a a n d form. Clearly the three bands described in t a b l e s I, III, a n d I V c a n b e s e e n to c h a n g e f r o m t h e v a l u e s t y p i c a l o f t h e a - h e l i x to t h a t o f t h e (~-structure. F i g u r e 2 s h o w s R a m a n s p e c t r u m of the i o n i z e d f o r m of poly-L-lysine. F i g u r e 3 s h o w s t h e R a m a n s p e c t r u m o f p o l y - L - l y s i n e as a f u n c t i o n o f t e m p e r a t u r e w h e n it is u n d e r g o i n g t h e ct - - 9 ~ t r a n s i t i o n [5].
Raman
spectroscopg
of
biological
the a p p e a r a n c e of the bands c h a r a c t e r i s t i c of the d i s o r d e r e d form. Consequently, it has been cone l u d e d that the ct - - > ~ change o c c u r s d i r e c t l y and does not i n v o l v e the i o n i z e d or d i s o r d e r e d f o r m as an i n t e r m e d i a t e state [6].
F i g u r e 4 shows h o w the i n t e n s i t y of a n y one of the c o n f o r m a t i o n a l l y d e p e n d e n t b a n d s in figure 3 can be used to p l o t a t r a n s i t i o n curve. Since R a m a n s p e c t r a v a r y in i n t e n s i t y w i t h alignment, r e f r a c t i v e i n d e x etc., it is m o r e accu-
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macromolecules.
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x FIa. 1. - - Laser R a m a n spectra of (top) ~-helieal poly-L-htsine 3 p. ceM H~O pit 11.8 T m ~°C and (bottom) [3-sheet poly-L-Iysine 3 I). cent II~O ptI 11.8 "1" ~= 52 °.
rate to use one of the c o n f o r m a t i o n a l l y i n d e p e n dent b a n d s in the s p e c t r u m as an i n t e r n a l stand a r d and to m e a s u r e the ratio of the c o n f o r m a tionally d e p e n d e n t band to the c o n f o r m a t i o n a l l y i n d e p e n d e n t band. In this w a y fluctuations in l a s e r intensity, changes in r e f r a c t i v e i n d e x w i t h t e m p e r a t u r e , and slight changes in a l i g m e n t that m a y o c c u r f r o m one s p e c t r u m to a n o t h e r are a u t o m a t i c a l l y c o r r e c t e d , This ratio is also indep e n d e n t of p o l y m e r c o n c e n t r a t i o n , so that one can c o m p a r e samples at d i f f e r e n t c o n c e n t r a t i o n s . Thus in figure 4 we have plotted the ratio of the A m i d e I I I b a n d at 1240 cm-1 to the b a n d at 1446 c m -1 as a f u n c t i o n of the t e m p e r a t u r e . In this w a y one can follow the ~ - - > ~ t r a n s i t i o n in a quantitative manner. R e f e r r i n g again to figure 3 one can see in the i n t e r m e d i a t e r e g i o n a splitting of each of the t h r e e c o n f o r m a t i o n a l l y sensitive bands w i t h o u t BIOCHIMIE, 1975, 57, n ° 4.
N o w w e m i g h t ask h o w useful these bands are in the study of p r o t e i n t r a n s i t i o n s ? To a n s w e r this question let us c o n s i d e r the well-knov,'n - + plot of R a m a c h a n d r a n [7]. T h e c o n f o r m a t i o n of any p o l y p e p t i d e or any p r o t e i n can be described by a series of angles 9z +1; ~2 +2; . . . opt +i . . . ~_~. +~- and these points can be p l a c e d on a t w o d i m e n s i o n a l g r a p h of ? vs ,~. NOw for a s i m p l e p o l y p e p t i d e in a definite c o n f o r m a t i o n , all of the ~'s a n d +'s are equal so that for the righth a n d e d alpha helix, for example, one has a single point, ~?~, +~. W h e n the p o l y p e p t i d e changes from a-->~, all of the p e p t i d e residues change f r o m ? ~ , + a - > " ~ , +;~" Thus w e h a v e in this case a s h a r p value for each confo.rmationally d e p e n d e n t R a m a n line at the ¢~ f r e q u e n c y w h i c h changes to a sharp value at the ~ f r e q u e n c y . This type of c h a n g e is v e r y casy to p i c k up in the R a m a n s p e c t r u m . On the other h a n d for a protein, w e have, in genreal, a r a t h e r b r o a d d i s t r i b u t i o n
420
W. L. Peticolas.
the synthesis o.f the cellular proteins. A great deal has been w r i t t e n about these materials a n d 'we w i l l review here only those features necessary for an i n t e r p r e t a t i o n of the R a m a n spectra. Nucleic acids m a y be d i v i d e d into two distinct classes d e p e n d i n g upon the structure of their constituent sugars. R i b o n u c l e i e acid (RNA) contains D-ribos'e w h i l e d e o x y r i b o n u c l e i c acid (DNA)
of .% ,b valaes. The c o n f o r m a t i o n a l l y d e p e n d e n t t t a m a n b a n d s m a y become r a t h e r broad since a w i d e variety of c o n f o r m a t i o n s are present. W h e n the p r o t e i n is gently d e n a t u r e d , there is a change from one b r o a d d i s t r i b u t i o n of ¢p, ,} values to a n o t h e r b r o a d d i s t r i b u t i o n of ¢, ~ values. (,onseq u e n t l y w h a t is to be expected is a r a t h e r small shift in the r a t h e r broad R a m a n bands. Hence
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(bottom) 3 p, cent in D~O pD 3.7 both at 22°C.
one can see the limitatio:ns of R a m a n spectroscopy to the study of p r o t e i n eonformatio.n u s i n g the Amide I and Amide I l I bands. It is i m p o r t a n t , w i t h a n y technique, to keep the l i m i t a t i o n s as well as the strong points i n m i n d . Recently, studies of the r e s o n a n t R a m a n effect from colored e h r o m o p h o r e s on p r o t e i n s have p r o v i d e d n e w a p p l i c a t i o n of R a m a n spectroscopy [8-10]. III. - - T H E RAMAN E F F E C T FROM NUCLEIC ACIDS. The n u c l e i c acids are the molecules w h i c h c a r r y a n d t r a n s m i t genetic i n f o r m a t i o n used i n BIOCHIMIE, 1975, 57, n ° 4.
c o n t a i n s 2'-deoxy-D-ribose. The g e n e r a l p r i m a r y b o n d i n g b y m e a n s of ester linkages at the 3' and the 5' h y d r o x y positions of the sugar. The b a c k b o n e of the n u c l e i c acid c h a i n is thus a copol y m e r of a ribose sugar a n d p h o s p h o r i c acid. Attached to each of the sugar groups i n the one position is a nitro.genous base. N o r m a l l y this base will consist of one of the following : a d e n i n e (A), g u a n i n e (G), cytosine (C), u r a c i l (U) or 5-methylu r a c i l called t h y m i d i n e (T). It is a general rule that U is f o u n d only in RNA and T is f o u n d in DNA. Sometimes i n s t e a d of guanine, it is more c o n v e n i e n t to study h y p o x a n t h i n e w h i c h is identical to g u a n i n e except that it lacks the p e n d a n t
Baman
spectroscopy
of
- - N H 2 group. Polymers c o n t a i n i n g h y p o x a n t h i n e are often m u c h easier to w o r k with t h a n those c o n t a i n i n g g u a n i n e because the g u a n i n e base into-
biological
421
macromolecules.
a r r a y in o r d e r to direct tile biological processes of the cells. However, model nucleic acids can be m a d e i n w h i c h only one type of base is attached to each sugar i n the chain. These materials are called polynueleotides. F o r a given base, the polynucleotide has the following n a m e s : a d e n i n e p o l y a d e n y l i c acid or poly A ; uracil, p o l y u r i d y l i c acid or poly U ; t h y m i d i n e , p o l y - d - t h y m i d y l i c acid or poly T ; guanine~ p o l y g u a n y l i c acid or poly G ; cytosine, p o l y c y t i d y l i c acid or poly C ;
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Fro. 4. - - Plot of the relative height of the 1240 era-1 Amide III band of polg-L-lgsine to the 1~16 cm-1 band at pH 10.96 vs temperature sho~wing the a - - : ~ ~J transition,
h y p o x a n t h i n e , p o l y i n o s i n i c acid or poly I. The small 'd' in p o l y - d - t h y m i d y l i c acid r e m i n d s us that w i t h t h y m i d i n e we n o r m a l l y have only the deoxyribose sugar. B T r a n s i t i o n in D N A [11]. We w o u l d n o w like to show the use of R a m a n s p e c t r o s c o p y to s t u d y c o n f o r m a t i o n a l changes i n n u c l e i c acids. F i r s t let us c o n s i d e r the A ~--~" B c o n f o r m a t i o n change w h i c h occurs for all DNA's i n the fiber form as a f u n c t i o n of relative h u m i dity. This is a reasonable place to start since the structure of the A-type helix a n d the B-type helix has b e e n o b t a i n e d from X-ray spectroscopy. Furthermore, i n this t r a n s f o r m a t i o n , n o t h i n g changes except the m a c r o m o l e c u l a r c o n f o r m a t i o n . Thus the base ratio, the n u m b e r and type of h y d r o g e n b o n d s a n d the base stacking r e m a i n essentially the same. F i g u r e 5 shows the R a m a n s p e c t r u m of calf t h y m u s DNA in the fiber form at 75 p. cent A. T h e A ~ - ~
FIG. 3. - - Detailed laser Raman spectra of c t - - 9 lransition o[ poly-L-lgsine 3 p. cent in HzO pH 10.96. In the intermediate temperatures the transition is slow. In the spectra at 27°C, the time interval between ta), (b) and (c) is about 20 minutes.
racts very strongly both 'with itself a n d the other bases. I n l i v i n g cells, the n u c l e i c acids c o n t a i n all four types of bases a r r a n g e d in an i n f o r m a t i o n a l BIOCHIMIE, 1975, 57, n ° 4.
422
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relative humidity where the polymer has the A - t y p e s t r u c t u r e a n d 98 p. c e n t r e l a t i v e h u m i d i t y
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FIG. 5. - - Ramail spectra of a fiber of the sodium suit of calf t h y m u s DNA at 98 p. cent rehttioe h u m i d i t y (upper curve) and 75 p. cent relatioe h u m i d i t y (lower curve). These spectra are completely reversible w i t h change in h u m i d i t y .
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Applications of Raman spectroscopy to biological macromolecules. W a r n e r L. PETIGOLAS ( * ) ~ . Institute Max yon Laue -- Paul Langevin
--
B P 156, 3 8 0 4 2 G r e n o b l e .
S u m m a r y . - - The Raman spectra of biological maeromolecules arise from the moleenlar vibrations of either the backbone chains or the side chains. The frequencies of the Raman bands lie in a region between 200 c m q and 3000 cm-1. From certain frequencies of the vibrations of the backbone chains one can determine the conformation or secondary structure of a maeromolecule. Thus for polypeptides and proteins the frequencies of the Amide I and Amide III vibrations allow one to determine the average conformation of their backbone chain. In polynueleotides and nucleic acids, the frequency of the phosphate diester stretch of the phosphate furanose chain varies between 814 em-a for A conformation and 790 era-1 for B conformation. Haman spectra of the bases in nucleic acids can be used to determine base stacking and hydrogen bonding interactions. Thus Raruan spectroscopy is an important tool for determining the conformation structure of proteins and nucleic acids.
I. - - INTRODUCTION. H a m a n S p e c t r o s c o p y has b e c o m e an i n c r e a s i n gly i m p o r t a n t tool for the study of biological mac r o m o l e c u l e s . T h e g r o w t h of this n e w field is due to the d e v e l o p m e n t of n e w t e c h n i q u e s --- laser light sources, double and t r i p l e g r a t i n g m o n o c h r o m a t o r s and i m p r o v e d e l e c t r o n i c d e t e c t i o n of w e a k light signals. T h e details of these t e c h n i q u e s h a v e been r e v i e w e d v e r y t h o r o u g h l y in the previous p a p e r by Dr. Delhaye. C o n s e q u e n t l y in this p a p e r w e w i l l c o n c e n t r a t e on the a p p l i c a t i o n s of R a m a n s p e c t r o s c o p y to the c h a r a c t e r i z a t i o n of b i o l o g i c a l m a c r o m o l e c u l e s . E x a m p l e s will be taken f r o m the w o r k of the author. R e a d e r s w i s h i n g to read f u r t h e r in this area should consult one or m o r e of the r e c e n t r e v i e w s in this area [i-5] as w e l l as the e x t e n s i v e o r i g i n a l literature. In this b r i e f d e s c r i p t i o n , w e w i l l discuss t w o types of m a t e r i a l s : (1) p r o t e i n s and p o l y p e p t i d e s , and (2) p o l y n u c l e o t i d e s and n u c l e i c acids. Particular emphasis w i l l be given to those Rainan bands w h i c h s h o w changes in i n t e n s i t y or freq u e n c y w i t h changes in c o n f o r m a t i o n . II. - - THE RAMAN EFFECT FROM POLYPEPTIDES AND PROTEINS. Much of the e a r l y w o r k o.n the R a m a n spect r o s c o p y of p r o t e i n s was done by Lord and colia(*) Guggenheim Fellow, 1973-74. Permanent address: Department of Chemistry, University of Oregon, Eugene, Oregon 97403.
b o r a t o r s and L o r d [5J has r e v i e w e d this field. Most of the o b s e r v e d b a n d s of p r o t e i n s comes f r o m the a m i n o acid side chains, p a r t i c u l a r l y the a r o m a t i c side chains. Ho'a'ever, t h e r e are t w o a m i d e bands, the a m i d e I and a m i d e I I I ~ h i c h arise f r o m the b a c k b o n e p o l y p e p t i d e c h a i n s of the p r o t e i n . These bands are k n o w n to be sensitive to c o n f o r m a t i o n . L a t e r w e w i l l discuss these bands in p r o t e i n s , but first, let us c o n s i d e r a m u c h s i m p l e r p r o b l e m - - the c o n f o r m a t i o n a l changes w h i c h o c c u r w h e n a simple p o l y p e p t i d e is taken from one s i m p l e c o n f o r m a t i o n to another. A r e v i e w of the early w o r k on p o l y p e p t i d e s and o t h e r b i o p o l y m e r s has been given by K o e n i g [2]. T a k i n g the early w o r k t o g e t h e r w i t h the later w o r k of others \ r e can list the R a m a n f r e q u e n c i e s w h i c h are e o n f o r m a t i o n a l l y d e p e n d e n t in four tables. The first, tab~e I, s h o w s the value of the A m i d e I band for v a r i o u s s i m p l e p o l y p e p t i d e s in the v a r i o u s s i m p l e structures, a-helical, B-sheet, and i o n i z e d or d i s o r d e r e d f o r m . T h e A m i d e I b a n d is the most r e l i a b l e of the c o n f o r m a t i o n a l l y d e p e n d e n t bands in the sense that the f r e q u e n c y of the a-helix, ~-sheet and d i s o r d e r e d f o r m seems to be i n d e p e n d e n t of the nature of tile side c h a i n and to d e p e n d only on the g e o m e t r y of the polyp e p t i d e backbone. Table l I gives the f r e q u e n c y of the A m i d e II b a n d w h i c h is R a m a n active only in cis-amides. F u r t h e r i n v e s t i g a t i o n of this i m p o r t a n t b a n d in c y c l i c p o l y p e p t i d e s w i t h p e p t i d e g e o m e t r y betw e e n cis and trans p o l y p e p t i d e s is m u c h to be desired. 30
W
418
L. Peticolas. q u e n c y w i t h t h e t y p e of s i d e c h a i n f o r t h e 13-struct u r e . Also it s h o u l d be n o t e d t h a t t h i s v i b r a t i o n is v e r y w e a k i n t h e a - h e l i c a l s t r u c t u r e . T h u s f r o m
T a b l e II1 g i v e s t h e f r e q u e n c y of t h e R a m a n a c t i v e A m i d e III b a n d i n s e v e r a l p o l y p e p t i d e s . Of p a r t i c u l a r i n t e r e s t is t h e v a r i a t i o n of t h i s f r e -
TABLE I.
Amide I Frequencies (cm-O From Polypeptides and Proteins From Laser Raman Measurements. a-Helical
Substance Polyglycinea , . . . . . . . . . . . . . . . . . . . . . . . Poly-L-alanine . . . . . . . . . . . . . . . . . . . . . . P o l y - L - g l u t a m i c acid ,I. . . . . . . . . . . . . . . . GlucagonO . . . . . . . . . . . . . . . . . . . . . . . . . . . P o l y - L - L y s i n e f H~O . . . . . . . . . . . . . . . . . D~O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
%Structur,,
1654 1658 (W)~,
I674 (S) 1666c
1658 1645 1632
1672 1670 (S) 1658 (.%
Random c0il
1665 1660 (S)
(a) Ref. 13 ; (b) Ref. 6 ; (c) Ref. 14 ; (d) Ref. 15 ; (e) Ref. 16 ; (f) Ref. 6.
a k n o w l e d g e of t h e A m i d e I I I f r e q u e n c y a l o n e , o n e c a n n o t tell t h e c o n f o r m a t i o n on a n a b s o l u t e basis.
TABLE II.
Amide II Frequencies of Cis-Amides From Laser Raman Measurements. Substance
Amide It (cm-t)
Diketopiperazinea . . . . . Hypoxanthineb . . . . . . . 1-MethyluraciF . . . . . . .
1456 (powder), 1385 (aqueous) 1464 1417 (aqueous) . . . . . . . . . . . . .
(a) Ref. 17 ; (b) Ref. 18 ; (e) Ref. 20.
T a b l e IV s h o w s t h e v i b r a t i o n s o f t h e C a - C s t r e t c h f o r p o l y - L - l y s i n e i n v a r i o u s s t r u c t u r e s Eft]. T h i s a s s i g n m e n t m u s t b e r e g a r d e d as t e n t a t i v e , b e c a u s e it falls i n a r e g i o n d i f f i c u l t to a s s i g n [6]. I n o r d e r to s e e h o w t h e s e v i b r a t i o n s c a n b e u s e d to s t u d y c o n f o r m a t i o n a l c h a n g e s i n p o l y p e p -
TABLF III.
Amide IIl Frequencies (in cm-O in Polypeplides From Laser Raman Measurements. Substance
Polyglycine:, . . . . . . . . . . . . . . . . Poly-L-alanine . . . . . . . . . . . . . Poly-L-glutamic acidd . . . . . . Glucagone . . . . . . . . . . . . . . . . . . Poly-L-lysinef . . . . . . . . . . . . . .
J
a-belical
~-structure
I ] I ! ~
1261 1261 (W)l,
1234 1239 (S) ~:
1266 1311
1232 1240
i
Random coil or structure ionized
1248 1248 1243
(a) Ref. 13 ; (b) Ref. 6 ; (c) Ref. 14 ; (d) Ref. 15 ; (e) Ref. 16 ; (f) Ref. 6.
TARLE IV.
Conformationally Dependent Band in the 1000 em-~ Region in Polg-L-lysine (a). Con[ormatiou
Frequency (cm-~)
Ionized
.q45 958 1 002
(a) Taken f r o m ref. 6.
BIOCHIMIE, 1975, 57, n ° 4.
tides, w e begin w i t h figure 1 w h i c h s h o w s the R a m a n s p e c t r a of p o l y - L - l y s i n e in the a a n d form. Clearly the three bands described in t a b l e s I, III, a n d I V c a n b e s e e n to c h a n g e f r o m t h e v a l u e s t y p i c a l o f t h e a - h e l i x to t h a t o f t h e (~-structure. F i g u r e 2 s h o w s R a m a n s p e c t r u m of the i o n i z e d f o r m of poly-L-lysine. F i g u r e 3 s h o w s t h e R a m a n s p e c t r u m o f p o l y - L - l y s i n e as a f u n c t i o n o f t e m p e r a t u r e w h e n it is u n d e r g o i n g t h e ct - - 9 ~ t r a n s i t i o n [5].
Raman
spectroscopg
of
biological
the a p p e a r a n c e of the bands c h a r a c t e r i s t i c of the d i s o r d e r e d form. Consequently, it has been cone l u d e d that the ct - - > ~ change o c c u r s d i r e c t l y and does not i n v o l v e the i o n i z e d or d i s o r d e r e d f o r m as an i n t e r m e d i a t e state [6].
F i g u r e 4 shows h o w the i n t e n s i t y of a n y one of the c o n f o r m a t i o n a l l y d e p e n d e n t b a n d s in figure 3 can be used to p l o t a t r a n s i t i o n curve. Since R a m a n s p e c t r a v a r y in i n t e n s i t y w i t h alignment, r e f r a c t i v e i n d e x etc., it is m o r e accu-
-
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macromolecules.
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x FIa. 1. - - Laser R a m a n spectra of (top) ~-helieal poly-L-htsine 3 p. ceM H~O pit 11.8 T m ~°C and (bottom) [3-sheet poly-L-Iysine 3 I). cent II~O ptI 11.8 "1" ~= 52 °.
rate to use one of the c o n f o r m a t i o n a l l y i n d e p e n dent b a n d s in the s p e c t r u m as an i n t e r n a l stand a r d and to m e a s u r e the ratio of the c o n f o r m a tionally d e p e n d e n t band to the c o n f o r m a t i o n a l l y i n d e p e n d e n t band. In this w a y fluctuations in l a s e r intensity, changes in r e f r a c t i v e i n d e x w i t h t e m p e r a t u r e , and slight changes in a l i g m e n t that m a y o c c u r f r o m one s p e c t r u m to a n o t h e r are a u t o m a t i c a l l y c o r r e c t e d , This ratio is also indep e n d e n t of p o l y m e r c o n c e n t r a t i o n , so that one can c o m p a r e samples at d i f f e r e n t c o n c e n t r a t i o n s . Thus in figure 4 we have plotted the ratio of the A m i d e I I I b a n d at 1240 cm-1 to the b a n d at 1446 c m -1 as a f u n c t i o n of the t e m p e r a t u r e . In this w a y one can follow the ~ - - > ~ t r a n s i t i o n in a quantitative manner. R e f e r r i n g again to figure 3 one can see in the i n t e r m e d i a t e r e g i o n a splitting of each of the t h r e e c o n f o r m a t i o n a l l y sensitive bands w i t h o u t BIOCHIMIE, 1975, 57, n ° 4.
N o w w e m i g h t ask h o w useful these bands are in the study of p r o t e i n t r a n s i t i o n s ? To a n s w e r this question let us c o n s i d e r the well-knov,'n - + plot of R a m a c h a n d r a n [7]. T h e c o n f o r m a t i o n of any p o l y p e p t i d e or any p r o t e i n can be described by a series of angles 9z +1; ~2 +2; . . . opt +i . . . ~_~. +~- and these points can be p l a c e d on a t w o d i m e n s i o n a l g r a p h of ? vs ,~. NOw for a s i m p l e p o l y p e p t i d e in a definite c o n f o r m a t i o n , all of the ~'s a n d +'s are equal so that for the righth a n d e d alpha helix, for example, one has a single point, ~?~, +~. W h e n the p o l y p e p t i d e changes from a-->~, all of the p e p t i d e residues change f r o m ? ~ , + a - > " ~ , +;~" Thus w e h a v e in this case a s h a r p value for each confo.rmationally d e p e n d e n t R a m a n line at the ¢~ f r e q u e n c y w h i c h changes to a sharp value at the ~ f r e q u e n c y . This type of c h a n g e is v e r y casy to p i c k up in the R a m a n s p e c t r u m . On the other h a n d for a protein, w e have, in genreal, a r a t h e r b r o a d d i s t r i b u t i o n
420
W. L. Peticolas.
the synthesis o.f the cellular proteins. A great deal has been w r i t t e n about these materials a n d 'we w i l l review here only those features necessary for an i n t e r p r e t a t i o n of the R a m a n spectra. Nucleic acids m a y be d i v i d e d into two distinct classes d e p e n d i n g upon the structure of their constituent sugars. R i b o n u c l e i e acid (RNA) contains D-ribos'e w h i l e d e o x y r i b o n u c l e i c acid (DNA)
of .% ,b valaes. The c o n f o r m a t i o n a l l y d e p e n d e n t t t a m a n b a n d s m a y become r a t h e r broad since a w i d e variety of c o n f o r m a t i o n s are present. W h e n the p r o t e i n is gently d e n a t u r e d , there is a change from one b r o a d d i s t r i b u t i o n of ¢p, ,} values to a n o t h e r b r o a d d i s t r i b u t i o n of ¢, ~ values. (,onseq u e n t l y w h a t is to be expected is a r a t h e r small shift in the r a t h e r broad R a m a n bands. Hence
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(bottom) 3 p, cent in D~O pD 3.7 both at 22°C.
one can see the limitatio:ns of R a m a n spectroscopy to the study of p r o t e i n eonformatio.n u s i n g the Amide I and Amide I l I bands. It is i m p o r t a n t , w i t h a n y technique, to keep the l i m i t a t i o n s as well as the strong points i n m i n d . Recently, studies of the r e s o n a n t R a m a n effect from colored e h r o m o p h o r e s on p r o t e i n s have p r o v i d e d n e w a p p l i c a t i o n of R a m a n spectroscopy [8-10]. III. - - T H E RAMAN E F F E C T FROM NUCLEIC ACIDS. The n u c l e i c acids are the molecules w h i c h c a r r y a n d t r a n s m i t genetic i n f o r m a t i o n used i n BIOCHIMIE, 1975, 57, n ° 4.
c o n t a i n s 2'-deoxy-D-ribose. The g e n e r a l p r i m a r y b o n d i n g b y m e a n s of ester linkages at the 3' and the 5' h y d r o x y positions of the sugar. The b a c k b o n e of the n u c l e i c acid c h a i n is thus a copol y m e r of a ribose sugar a n d p h o s p h o r i c acid. Attached to each of the sugar groups i n the one position is a nitro.genous base. N o r m a l l y this base will consist of one of the following : a d e n i n e (A), g u a n i n e (G), cytosine (C), u r a c i l (U) or 5-methylu r a c i l called t h y m i d i n e (T). It is a general rule that U is f o u n d only in RNA and T is f o u n d in DNA. Sometimes i n s t e a d of guanine, it is more c o n v e n i e n t to study h y p o x a n t h i n e w h i c h is identical to g u a n i n e except that it lacks the p e n d a n t
Baman
spectroscopy
of
- - N H 2 group. Polymers c o n t a i n i n g h y p o x a n t h i n e are often m u c h easier to w o r k with t h a n those c o n t a i n i n g g u a n i n e because the g u a n i n e base into-
biological
421
macromolecules.
a r r a y in o r d e r to direct tile biological processes of the cells. However, model nucleic acids can be m a d e i n w h i c h only one type of base is attached to each sugar i n the chain. These materials are called polynueleotides. F o r a given base, the polynucleotide has the following n a m e s : a d e n i n e p o l y a d e n y l i c acid or poly A ; uracil, p o l y u r i d y l i c acid or poly U ; t h y m i d i n e , p o l y - d - t h y m i d y l i c acid or poly T ; guanine~ p o l y g u a n y l i c acid or poly G ; cytosine, p o l y c y t i d y l i c acid or poly C ;
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Fro. 4. - - Plot of the relative height of the 1240 era-1 Amide III band of polg-L-lgsine to the 1~16 cm-1 band at pH 10.96 vs temperature sho~wing the a - - : ~ ~J transition,
h y p o x a n t h i n e , p o l y i n o s i n i c acid or poly I. The small 'd' in p o l y - d - t h y m i d y l i c acid r e m i n d s us that w i t h t h y m i d i n e we n o r m a l l y have only the deoxyribose sugar. B T r a n s i t i o n in D N A [11]. We w o u l d n o w like to show the use of R a m a n s p e c t r o s c o p y to s t u d y c o n f o r m a t i o n a l changes i n n u c l e i c acids. F i r s t let us c o n s i d e r the A ~--~" B c o n f o r m a t i o n change w h i c h occurs for all DNA's i n the fiber form as a f u n c t i o n of relative h u m i dity. This is a reasonable place to start since the structure of the A-type helix a n d the B-type helix has b e e n o b t a i n e d from X-ray spectroscopy. Furthermore, i n this t r a n s f o r m a t i o n , n o t h i n g changes except the m a c r o m o l e c u l a r c o n f o r m a t i o n . Thus the base ratio, the n u m b e r and type of h y d r o g e n b o n d s a n d the base stacking r e m a i n essentially the same. F i g u r e 5 shows the R a m a n s p e c t r u m of calf t h y m u s DNA in the fiber form at 75 p. cent A. T h e A ~ - ~
FIG. 3. - - Detailed laser Raman spectra of c t - - 9 lransition o[ poly-L-lgsine 3 p. cent in HzO pH 10.96. In the intermediate temperatures the transition is slow. In the spectra at 27°C, the time interval between ta), (b) and (c) is about 20 minutes.
racts very strongly both 'with itself a n d the other bases. I n l i v i n g cells, the n u c l e i c acids c o n t a i n all four types of bases a r r a n g e d in an i n f o r m a t i o n a l BIOCHIMIE, 1975, 57, n ° 4.
422
W, L. Pelicolas.
relative humidity where the polymer has the A - t y p e s t r u c t u r e a n d 98 p. c e n t r e l a t i v e h u m i d i t y
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FIG. 5. - - Ramail spectra of a fiber of the sodium suit of calf t h y m u s DNA at 98 p. cent rehttioe h u m i d i t y (upper curve) and 75 p. cent relatioe h u m i d i t y (lower curve). These spectra are completely reversible w i t h change in h u m i d i t y .
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