Chapter 14
Applications of Optical Spectroscopy to Protein Conformational Transitions
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Henry A. Havel Control Research and Development, Upjohn Company, Kalamazoo, MI 49001
Solution state protein structure investigations using optical spectroscopy (UV absorption, circular dichroism and fluorescence) are reviewed and are applied to studies of the forces which stabilize the native conformations of proteins. Structural rearrangements which occur as a protein folds or unfolds can be identified by using chemical denaturants (guanidine HCl or urea), heat or pH adjustment to unfold the protein and employing optical spectroscopic probes to monitor the unfolding transition. Recent results of unfolding studies with bovine growth hormone, a small M ( W = 22,000 daltons) polypeptide hormone, are summarized. In particular, optical spectroscopy of the single tryptophan residue in bGH has contributed to the determination that a self-associated form of a partially unfolded intermediate is populated during equilibrium unfolding; in addition, it is shown that the tryptophan residues of self-associated bGH molecules are likely to be held rigidly in a polar environment which is near the interface between selfassociating molecules. The study o f p r o t e i n s t r u c t u r e i n t h e s o l u t i o n s t a t e has b e e n , and c o n t i n u e s t o b e , an i m p o r t a n t a r e a f o r chemical r e s e a r c h . In r e c e n t y e a r s t h e r e has been i n c r e a s e d a c t i v i t y i n t h i s area as t h e r e v o l u t i o n i n b i o t e c h n o l o g y has a l l o w e d t h e p r o d u c t i o n o f l a r g e q u a n t i t i e s o f r e l a t i v e l y pure p r o t e i n s a t modest c o s t v i a recombinant DNA t e c h n i q u e s . T h i s paper w i l l d i s c u s s t h e u t i l i t y o f o p t i c a l s p e c t r o s c o p i c t e c h n i q u e s , d e f i n e d here as UV a b s o r p t i o n , f l u o r e s c e n c e and c i r c u l a r d i c h r o i s m s p e c t r o s c o p y , as important t o o l s i n p r o t e i n s t r u c t u r e i n v e s t i g a t i o n s . The i n f o r m a t i o n d e r i v e d from t h e s e t e c h n i q u e s i s unique and complements t h a t o b t a i n e d by h i g h e r r e s o l u t i o n t e c h n i q u e s such
0097-6156/88/0362-0177$06.00/0 © 1988 American Chemical Society
Phillips et al.; The Impact of Chemistry on Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
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178
as NMR and v i b r a t i o n a l s p e c t r o s c o p y i n t h e s o l u t i o n s t a t e (as w e l l as s o l i d s t a t e X - r a y s t r u c t u r e s t u d i e s ) w i t h o u t t h e added c o m p l e x i t i e s which t h e s e o t h e r methods possess i n i n s t r u m e n t a t i o n , data a n a l y s i s a n d / o r d a t a i n t e r p r e t a t i o n . This review w i l l not p r o v i d e an i n - d e p t h t r e a t m e n t o f any o f t h e o p t i c a l s p e c t r o s c o p i c t e c h n i q u e s , but i n s t e a d w i l l h i g h l i g h t t h e i r more i m p o r t a n t f e a t u r e s and i l l u s t r a t e how o p t i c a l s p e c t r o s c o p i c t e c h n i q u e s can s e r v e as important t o o l s i n t h e study o f p r o t e i n c o n f o r m a t i o n a l t r a n s i t i o n s . The a p p l i c a t i o n s p r e s e n t e d w i l l be from e q u i l i b r i u m f o l d i n g s t u d i e s done at The Upjohn Company on b o v i n e growth hormone (bGH, b o v i n e s o m a t o t r o p i n , b S t ) , a 22,000 d a l t o n p r o t e i n which i s o f i n t e r e s t because of i t s l a c t o g e n i c and growth-promotant a c t i v i t i e s . It i s an a p p r o p r i a t e example f o r t h e s e s t u d i e s because i t has been shown t o undergo e q u i l i b r i u m u n f o l d i n g through a p r o c e s s i n v o l v i n g at l e a s t one s t a b l e i n t e r m e d i a t e . Protein
Structure
P r o t e i n s a r e fundamental m o l e c u l e s f o r a l l l i v i n g organisms (1_) as they c a t a l y z e r e a c t i o n s , c a r r y m e s s a g e s , defend a g a i n s t f o r e i g n agents and s u p p o r t t h e o r g a n i s m ' s s t r u c t u r e . It i s widely held t h a t the molecular s t r u c t u r e of a g l o b u l a r p r o t e i n i s d e t e r m i n e d by i t s amino a c i d sequence (2_) and t h a t t h e molecular s t r u c t u r e determines i t s b i o l o g i c a l f u n c t i o n . Hence, i f s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s can be e s t a b l i s h e d i t w i l l be p o s s i b l e t o e n g i n e e r new b i o l o g i c a l a c t i v i t i e s i n t o p r o t e i n s t h r o u g h m o d i f i c a t i o n o f amino a c i d s e q u e n c e s . For p r o t e i n p h a r m a c e u t i c a l p r o d u c t s , d e s i r a b l e new a c t i v i t i e s i n c l u d e improved b i o l o g i c a l h a l f - l i f e , e l i m i n a t i o n o f d e l e t e r i o u s s i d e e f f e c t s , reduction in aggregation p r o p e r t i e s , e t c . P r o t e i n s t r u c t u r e i s d e s c r i b e d c o n v e n t i o n a l l y (3_) u s i n g a h i e r a r c h i c a l scheme. The f i r s t t y p e o f s t r u c t u r e i s termed p r i mary s t r u c t u r e and c o n s i s t s o f t h e c o v a l e n t bond s t r u c t u r e o f a p r o t e i n ; i . e . , i t s amino a c i d sequence and the l o c a t i o n s o f any d i s u l f i d e bonds. The next l e v e l i n s t r u c t u r e d e s c r i b e s how l o c a l regions i n a p r o t e i n are arranged i n t o organized a s s e m b l i e s and i s termed secondary s t r u c t u r e . Common secondary s t r u c t u r e elements o f g l o b u l a r p r o t e i n s a r e t h e α - h e l i x , βp l e a t e d s h e e t , β - t u r n and d i s o r d e r e d s t r u c t u r e s ; a r e c e n t a d d i t i o n to t h i s l i s t i s the ordered "Ω-loop" s t r u c t u r e (4). T h i r d l y , t h e r e i s t h e t e r t i a r y s t r u c t u r e , which i n v o l v e s T h e o r i e n t a t i o n o f , and non-bonded c o n t a c t s between, t h e v a r i o u s secondary s t r u c t u r e elements. The l a s t l e v e l o f s t r u c t u r e i s t h e q u a t e r n a r y s t r u c t u r e which r e l a t e s t h e t e r t i a r y s t r u c t u r e s o f s e v e r a l p r o t e i n c h a i n s t o one a n o t h e r ; e . g . , i t g i v e s the r e l a t i v e p o s i t i o n s of subunits in a m u l t i - s u b u n i t p r o t e i n . The complete t h r e e - d i m e n s i o n a l s t r u c t u r e o f a p r o t e i n i s the r e s u l t o f a myriad o f chemical i n t e r a c t i o n s ( h y d r o g e n b o n d i n g , e l e c t r o s t a t i c , h y d r o p h o b i c , e t c . ) between t h e twenty d i f f e r e n t amino a c i d s which compose a p r o t e i n c h a i n and i n t e r a c t i o n s o f t h e amino a c i d r e s i d u e s w i t h s o l v e n t ( w a t e r ) . Given t h e number o f i n t e r a c t i o n s i n v o l v e d and t h e l a r g e number
Phillips et al.; The Impact of Chemistry on Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
14.
HAVEL
Applications of Optical Spectroscopy
o f c o n f o r m a t i o n a l degrees o f f r e e d o m , i t i s not s u r p r i s i n g t h a t p r e d i c t i o n s o f p r o t e i n s t r u c t u r e from an amino a c i d sequence a r e d i f f i c u l t at b e s t . The i m p o r t a n c e o f t h i s p r o b l e m , however, has prompted numerous i n v e s t i g a t i o n s i n t o t h e comparison o f t h e o r e t i c a l and e x p e r i m e n t a l r e s u l t s , w i t h t h e most p r o g r e s s o c c u r r i n g i n the p r e d i c t i o n o f s e c o n d a r y , r a t h e r than t e r t i a r y , structure. The most common p r e d i c t i o n methods f o r p r o t e i n secondary s t r u c t u r e a r e t h o s e o f Lim ( 5 ) , Chou and Fasman [6) and Robson and c o - w o r k e r s (_7_). Although t h e r e has been c r i t i c i s m o f t h e a c c u r a c y o f t h e s e methods ( 8 , 9 ) , they a r e u s e f u l when X - r a y s t r u c t u r e s a r e not a v a i l a b T e and i f t h e r e s u l t s a r e not e x t r a p o l a t e d t o uses f o r which they were not i n t e n d e d ; c o m b i n a t i o n s o f p r e d i c t i o n methods seem t o work r e l i a b l y (10,11). Recent work by Kuntz and c o - w o r k e r s ( 1 2 , 1 3 ) has shown some promise o f improvement i n p r e d i c t a b i l i t y "by" a p p l y i n g an a c c u r a t e p r e d i c t i o n o f t h e l o c a t i o n o f t u r n s u s i n g a pattern-matching approach. Conformational
Transitions
I n f o r m a t i o n d e r i v e d from t h e study o f c o n f o r m a t i o n a l t r a n s i t i o n s o f p r o t e i n s , i . e . , s t u d i e s o f t h e p r o t e i n f o l d i n g p r o c e s s , can provide c o n t r i b u t i o n s to the understanding of s t a b i l i t y of protein s t r u c t u r e (14). By examining t h e s t r u c t u r a l rearrangements w h i c " i ï o c c u r as a p r o t e i n f o l d s o r u n f o l d s , i t s h o u l d be p o s s i b l e t o d e t e r m i n e t h e c r i t i c a l a t t r i b u t e s which confer s t a b i l i t y . In p a r t i c u l a r , t h e m o l e c u l a r s t r u c t u r e s o f i n t e r m e d i a t e s which a r e p o p u l a t e d d u r i n g t h e f o l d i n g p r o c e s s s h o u l d p r o v i d e c l u e s t o t h e f o r c e s which s t a b i l i z e t h e n a t i v e p r o t e i n s t r u c t u r e and t h e d e t e r m i n a t i o n o f t h e r e l a t i o n s h i p between s t r u c t u r e and s t a b i l i t y . Equilibrium denaturation c u r v e s can be used t o q u a n t i f y s t a b i l i t y and t e s t p r e d i c t i o n s o f t h e e f f e c t s o f amino a c i d s u b s t i t u t i o n s on p r o t e i n s t a b i l i t y . A p r a c t i c a l goal o f r e s e a r c h i n t o p r o t e i n f o l d i n g mechanisms i s t o p r o v i d e a fundamental framework which can be used t o d e v e l o p and o p t i m i z e m a n u f a c t u r i n g p r o c e s s e s f o r proteins in t h e i r b i o l o g i c a l l y a c t i v e , folded state using a s u i t a b l e host organism i n l a r g e s c a l e f e r m e n t a t i o n . Often t h e f o r e i g n p r o t e i n i s not e x c r e t e d from t h e h o s t but i s s e q u e s t e r e d i n t o " i n c l u s i o n b o d i e s " where t h e p r o t e i n i s u s u a l l y u n f o l d e d and has i t s d i s u l f i d e bonds r e d u c e d . E f f i c i e n t methods f o r i s o l a t i o n , s o l u b i l i z a t i o n , p u r i f i c a t i o n and f o l d i n g o f t h e p r o t e i n - c o n t a i n i n g g r a n u l e s a r e i m p o r t a n t f o r commercial p r o d u c t i o n o f recombinant p r o t e i n s i n l a r g e q u a n t i t i e s . S e v e r a l methods a r e a v a i l a b l e t o a l t e r p r o t e i n c o n f o r m a t i o n under e q u i l i b r i u m c o n d i t i o n s i n t h e l a b o r a t o r y ( 1 5 - 1 7 ) ; they may a l s o be used i n t h e p r e p a r a t i o n o f f o l d e d p r o t e i n s from inclusion bodies. The most c o n v e n i e n t o f t h e s e i n v o l v e t h e a d d i t i o n o f c h e m i c a l agents such as urea o r g u a n i d i n e h y d r o c h l o r i d e (Gdn HC1) which have been shown t o u n f o l d p r o t e i n s r e v e r s i b l y a n d , a t h i g h enough c o n c e n t r a t i o n (12 M urea o r 6 M Gdn HC1), y i e l d p r o t e i n c h a i n s t h a t a r e random c o i l s . In c o n t r a s t , most s u r f a c t a n t s b i n d i r r e v e r s i b l y t o p r o t e i n
Phillips et al.; The Impact of Chemistry on Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
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T H E IMPACT OF CHEMISTRY ON BIOTECHNOLOGY
molecules. Adjustment o f s o l u t i o n pH and h e a t i n g can a l s o be used but o f t e n a r e accompanied by p r o t e i n d e g r a d a t i o n which i s not r e v e r s i b l e and t h e s e methods a r e not a s s u r e d o f p r o d u c i n g a completely unfolded protein c h a i n . The comparison o f r e s u l t s from t h e use o f d i f f e r e n t means o f u n f o l d i n g a p r o t e i n can p r o v i d e f u r t h e r i n f o r m a t i o n about t h e n a t u r e o f t h e u n f o l d i n g pathway, f o r example, t h e a d d i t i o n o f Gdn HC1 i n c r e a s e s s o l u t i o n i o n i c s t r e n g t h whereas urea does n o t . Optical
Spectroscopy
The use o f o p t i c a l s p e c t r o s c o p y t o study p r o t e i n s t r u c t u r e i s dependent on t h e s e n s i t i v i t y o f e l e c t r o n i c energy l e v e l s t o protein s t r u c t u r e changes. S t r u c t u r a l i n f o r m a t i o n can be o b t a i n e d by o b s e r v i n g changes i n s p e c t r a l p r o p e r t i e s ( i n t e n s i t i e s , w a v e l e n g t h s , band s h a p e s , e t c . ) and c o r r e l a t i n g t h e s e d a t a w i t h r e s u l t s from model compounds. The p r i n c i p a l a b s o r b i n g components i n p r o t e i n s a r e p e p t i d e bonds and a r o m a t i c amino a c i d s ( t r y p t o p h a n , t y r o s i n e and p h e n y l a l a n i n e ) which a l l have a b s o r p t i o n maxima i n t h e UV (λ < 300 nm) (18). (The a d d i t i o n o f p r o s t h e t i c g r o u p s ; e . g . , hemes, f l a v i n s , o r p y r i d o x a l p h o s p h a t e , can d r a m a t i c a l l y change t h e UV a b s o r p t i o n s p e c t r u m , but p r o t e i n s c o n t a i n i n g t h e s e groups w i l l not be considered here.) Due t o t h e l a r g e number o f p e p t i d e bonds p r e s e n t i n a p r o t e i n , s p e c t r o s c o p i c s t u d i e s which probe t h e e l e c t r o n i c energy l e v e l s o f p e p t i d e bonds w i l l n e c e s s a r i l y p r o v i d e "average" s t r u c t u r a l i n f o r m a t i o n , w h i l e s p e c t r o s c o p y o f a r o m a t i c amino a c i d s , which a r e r e p r e s e n t e d l e s s f r e q u e n t l y i n t h e p r o t e i n c h a i n , can o f t e n p r o v i d e d e t a i l e d s t r u c t u r a l information. UV A b s o r p t i o n S p e c t r o s c o p y . The a b s o r p t i o n o f r a d i a t i o n by pept i d e bonds o c c u r s i n t h e f a r - U V p a r t o f t h e spectrum due t o a weak η -* π* t r a n s i t i o n ( a t about 215 nm, ε ~ 100) and a s t r o n g π + π* t r a n s i t i o n ( a t about 190 nm, ε - 7000). Aromatic amino a c i d s ( t r y p t o p h a n , t y r o s i n e and p h e n y l a l a n i n e ) a b s o r b energy i n both t h e n e a r - and f a r - U V due t o s t r o n g % %* t r a n s i t i o n s ( ε ~ 5000). The t y p i c a l UV a b s o r p t i o n s p e c t r a f o r p e p t i d e bonds (19) and a r o m a t i c amino a c i d s (20) i n aqueous s o l u t i o n have been recorded. The d e t e r m i n a t i o n o f p r o t e i n c o n c e n t r a t i o n can be done c o n v e n i e n t l y u s i n g t h e near-UV a b s o r p t i o n maximum o f p r o t e i n s due t o t h e a b s o r p t i o n o f t y r o s i n e and t r y p t o p h a n residues. A c o m p i l a t i o n o f molar a b s o r p t i v i t y v a l u e s f o r s e v e r a l hundred p r o t e i n s and p r o t e i n d e r i v a t i v e s has been undertaken i n a s e r i e s o f papers by Kirschenbaum (21). The s e c o n d a r y s t r u c t u r e o f t h e p r o t e i n c h a i n can a l t e r t h e a b s o r p t i o n maximum and i n t e n s i t y o f p e p t i d e bond t r a n s i t i o n s (19), but t h e o v e r l a p o f t r a n s i t i o n s makes t h e d e t e r m i n a t i o n o f secondary s t r u c t u r e d i f f i c u l t from t h e s e d a t a . It w i l l be shown l a t e r t h a t t h e c i r c u l a r d i c h r o i s m spectrum i n t h i s same wavelength range p r o v i d e s a b e t t e r d e t e r m i n a t i o n o f s e c o n d a r y structure.
Phillips et al.; The Impact of Chemistry on Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
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The a b s o r p t i o n s p e c t r a o f a r o m a t i c amino a c i d s a r e s e n s i t i v e t o the p o l a r i t y ( d i e l e c t r i c c o n s t a n t ) o f t h e i r environment due t o t h e e f f e c t o f s o l v e n t on e l e c t r o n i c energy levels. S i n c e t h e a r o m a t i c amino a c i d s a r e n o n - p o l a r , they tend t o r e s i d e i n n o n - p o l a r environments i n n a t i v e p r o t e i n s t r u c t u r e s but become exposed t o p o l a r s o l v e n t (water) upon u n f o l d i n g . T h i s change i n environment i s r e f l e c t e d i n t h e UV a b s o r p t i o n spectrum as shown i n F i g u r e 1 f o r bGH; t h e r e i s a b l u e - s h i f t o f t h e a b s o r p t i o n maximum o f about 5 nm upon u n f o l d i n g due t o a change i n s o l v a t i o n w i t h t h e maximum change o c c u r r i n g near 290 nm. The u n f o l d e d bGH spectrum i n F i g u r e 1 i s v i r t u a l l y t h e same as t h a t f o r a m i x t u r e o f t h e a r o m a t i c amino a c i d s c o n t a i n e d i n bGH (1 t r y p t o p h a n , 6 t y r o s i n e s and 13 p h e n y l a l a n i n e s ) . These data i l l u s t r a t e how t h e UV a b s o r p t i o n spectrum can be used t o monitor t e r t i a r y s t r u c t u r e changes. F i g u r e 1 i l l u s t r a t e s t h e poor r e s o l u t i o n which i s found i n a t y p i c a l p r o t e i n UV a b s o r p t i o n spectrum due t o the l a r g e number of overlapping e l e c t r o n i c t r a n s i t i o n s . T h i s s i t u a t i o n can be improved and peak p o s i t i o n s l o c a t e d more a c c u r a t e l y i f t h e d e r i v a t i v e o f a b s o r p t i o n w i t h r e s p e c t t o wavelength i s c a l c u l a t e d (22). U s u a l l y t h e s e c o n d - d e r i v a t i v e spectrum i s used (23-26) and maxima (peaks) i n t h e z e r o - o r d e r spectrum become minima ( t r o u g h s ) i n t h e s e c o n d - d e r i v a t i v e s p e c t r u m , but f o u r t h d e r i v a t i v e s p e c t r a have been shown t o have c e r t a i n advantages (27,28) among which i s t h a t z e r o - o r d e r peaks a r e a l s o peaks i n the f o u r t h - d e r i v a t i v e spectrum. Some o f t h e uses o f t h e s e data i n c l u d e the d e t e r m i n a t i o n o f t h e t y r o s i n e / t r y p t o p h a n r a t i o i n unknown p r o t e i n s ( 2 3 ) , t h e d e t e r m i n a t i o n o f t h e number o f t y r o s i n e r e s i d u e s exposed t o s o l v e n t i n a p r o t e i n (24) and c o n f o r m a t i o n a l comparisons between n a t i v e and unfoVcïëci p r o t e i n s (25) o r between p r o t e i n s w i t h s i m i l a r amino a c i d c o m p o s i t i o n (26) . Using model compounds f o r t y r o s i n e (N-acetyl-tyrosine eTFfyl e s t e r ) and t r y p t o p h a n ( N - a c e t y l - t r y p t o p h a n e t h y l ester), i t has been demonstrated (25) t h a t a change i n s o l v e n t d i e l e c t r i c c o n s t a n t from n o n - p o l a r t o p o l a r has t h e e f f e c t o f s h i f t i n g t h e s e c o n d - d e r i v a t i v e bands o f t y r o s i n e w i t h o u t changes i n band i n t e n s i t i e s , w h i l e f o r t r y p t o p h a n t h e e f f e c t i s one o f c h a n g i n g band i n t e n s i t i e s w i t h o u t s h i f t i n g i n t h e s e c o n d d e r i v a t i v e bands. It w i l l be shown l a t e r how t h e s e d a t a can be used t o i n t e r p r e t s e c o n d - d e r i v a t i v e s p e c t r a o f t h e t r y p t o p h a n i n bGH. F1uorescence S p e c t r o s c o p y . The i n t r i n s i c f l u o r e s c e n c e p r o p e r t i e s o f p r o t e i n s p r o v i d e unique s p e c t r o s c o p i c t o o l s f o r p r o t e i n s t r u c t u r e i n v e s t i g a t i o n s (29) u s i n g both s t e a d y - s t a t e and t i m e - r e s o l v e d t e c h n i q u e s ; by l a b e l i n g w i t h e x t r i n s i c probes t h e a p p l i c a t i o n s can be expanded even f u r t h e r . The u t i l i t y o f i n t r i n s i c f l u o r e s c e n c e d a t a i s due t o s e v e r a l f a c t o r s , some o f which a r e : (1) e m i s s i o n s p e c t r a a r e s e n s i t i v e t o f l u o r o p h o r e environment d i e l e c t r i c c o n s t a n t and t h e p r e s e n c e o f s u b s t r a t e s ; (2) f l u o r e s c e n c e p r o v i d e s a method t o study p r o t e i n dynamics as many p r o t e i n motions can o c c u r d u r i n g an e x c i t e d s t a t e l i f e t i m e ( t y p i c a l l y i n t h e nanosecond r e g i m e ) ; (3) t h e p o l a r i z a t i o n
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p r o p e r t i e s o f f l u o r e s c e n c e e m i s s i o n can be e x p l o i t e d ; (4) i t i s p o s s i b l e t o use e x t r i n s i c quenching agents t o probe f l u o r o p h o r e a c c e s s i b i l i t y ; (5) f l u o r e s c e n c e energy t r a n s f e r s t u d i e s can be performed t o d e t e r m i n e d i s t a n c e s between r e s i d u e s i n p r o t e i n s ; (6) f l u o r e s c e n c e e m i s s i o n i s u s u a l l y r e s t r i c t e d t o a small number o f amino a c i d s i n a p r o t e i n , p r o v i d i n g a good degree o f specificity. S t e a d y - S t a t e F l u o r e s c e n c e . The f l u o r e s c e n c e e m i s s i o n s p e c t r a o f t h e t h r e e a r o m a t i c amino a c i d s i n water y i e l d an e m i s s i o n maximum f o r t r y p t o p h a n a t 348 nm, f o r t y r o s i n e a t 303 nm and f o r p h e n y l a l a n i n e at 282 nm ( 3 0 ) . The e m i s s i o n maximum f o r both t r y p t o p h a n and t y r o s i n e i s a f f e c t e d by t h e d i e l e c t r i c c o n s t a n t o f t h e environment w i t h n o n - p o l a r s u r r o u n d i n g s p r o d u c i n g b l u e s h i f t e d e m i s s i o n p e a k s . The most i n t e n s e f l u o r e s c e n c e e m i s s i o n i n a p r o t e i n i s due t o t r y p t o p h a n r e s i d u e s w i t h l e s s due t o t y r o s i n e and p h e n y l a l a n i n e r e s i d u e s . If t y r o s i n e and t r y p t o p h a n r e s i d u e s a r e both p r e s e n t (as i n most p r o t e i n s ) , i t i s not p o s s i b l e to observe s e p a r a t e l y the emission of t y r o s i n e residues due t o e f f i c i e n t energy t r a n s f e r from t y r o s i n e t o t r y p t o p h a n and q u e n c h i n g o f t y r o s i n e f l u o r e s c e n c e by o t h e r f u n c t i o n s groups o f the p r o t e i n ( 2 9 ) . It i s p o s s i b l e t o i s o l a t e t h e f l u o r e s c e n c e o f t r y p t o p h a n r e s i d u e s by e x c i t i n g a t t h e red edge o f t h e t r y p t o p h a n a b s o r p t i o n s p e c t r u m , t y p i c a l l y a t 295 t o 300 nm, and m o n i t o r i n g f l u o r e s c e n c e e m i s s i o n a t l o n g wavelengths ( λ > 350 nm). Under t h e s e c o n d i t i o n s t h e e m i s s i o n from t y r o s i n e i s v i r t u a l l y z e r o . F l u o r e s c e n c e e m i s s i o n from p h e n y l a l a n i n e r e s i d u e s i s e x t r e m e l y weak. The e f f e c t o f p r o t e i n u n f o l d i n g on t h e f l u o r e s c e n c e e m i s s i o n spectrum o f bGH i s s u b s t a n t i a l f o r e x c i t a t i o n o f t h e t r y p t o p h a n r e s i d u e a l o n e ( F i g u r e 2) and e x c i t a t i o n o f both t r y p t o p h a n and t y r o s i n e r e s i d u e s ( e x c i t a t i o n at 280 nm, data not shown). In both c a s e s a s h i f t i n t h e e m i s s i o n maximum i s o b s e r v e d , r e f l e c t i n g a change i n environment from n o n - p o l a r t o p o l a r , and an i n c r e a s e d i n t e n s i t y i s seen i n t h e u n f o l d e d s t a t e , p r o b a b l y due t o i n t r a m o l e c u l a r f l u o r e s c e n c e quenching i n t h e native state. There a r e two maxima observed when u n f o l d e d bGH i s e x c i t e d at 280 nm which a r e the r e s o l v e d e m i s s i o n maxima o f t y r o s i n e a t 305 nm and t r y p t o p h a n at 350 nm; energy t r a n s f e r from t y r o s i n e t o t r y p t o p h a n i n t h e n a t i v e s t r u c t u r e p r e v e n t s t h e o b s e r v a t i o n o f most t y r o s i n e f l u o r e s c e n c e e m i s s i o n and o n l y one e m i s s i o n peak i s o b s e r v e d . T i m e - R e s o l v e d F l u o r e s c e n c e . The study o f p r o t e i n s t r u c t u r e w i t h t i m e - r e s o l v e d f l u o r e s c e n c e t e c h n i q u e s has been reviewed r e c e n t l y ( 3 1 , 3 2 ) as have methods f o r t h e measurement o f f l u o r e s c e n c e i n t e n s i t y and a n i s o t r o p y decay u s i n g t i m e - c o r r e l a t e d s i n g l e photon c o u n t i n g ( 3 3 ) ; measurements u s i n g m u l t i f r e q u e n c y phase techniques are d i s c u s s e d i n d e s c r i p t i o n s of s t a t e - o f - t h e - a r t instrumentation (34,35). F l u o r e s c e n c e l i f e t i m e measurements o f p r o t e i n s a r e compTTcated by t h e f a c t t h a t even s i n g l e t r y p t o p h a n p r o t e i n s such as p h o s p h o l i p a s e A2 ( 3 6 ) , p a r v a l b u m i n (37) and r i b o n u c l e a s e T l (38) o r s i n g l e t y r o s i n e p r o t e i n s such as h i s t o n e
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Wavelength (nm)
F i g u r e 1. U l t r a v i o l e t absorption spectra of native ( ) and u n f o l d e d ( ) bGH. S o l v e n t f o r t h e n a t i v e spectrum was 0.05 M ammonium b i c a r b o n a t e (pH 8.5) and f o r t h e u n f o l d e d spectrum was t h e same b u f f e r p l u s 6 M Gdn HC1.
Wavelength (nm)
F i g u r e 2. Fluorescence emission spectra of native ( ) and u n f o l d e d ( ) bGH w i t h e x c i t a t i o n a t 295 nm (to e x c i t e only the tryptophan r e s i d u e ) . Solvent f o r the n a t i v e spectrum was 0.05 M ammonium b i c a r b o n a t e (pH 8.5) and f o r t h e u n f o l d e d spectrum was t h e same b u f f e r p l u s 6 M Gdn HC1.
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HI (39) a r e o b s e r v e d t o have n o n - e x p o n e n t i a l decay k i n e t i c s . The f l u o r e s c e n c e decay f o r f r e e t r y p t o p h a n i n s o l u t i o n i s a l s o n o n - e x p o n e n t i a l (40,41) as i s t h e decay f o r f r e e t y r o s i n e (42) w i t h t h e o r i g i n i n both c a s e s a s c r i b e d t o d i f f e r e n t rotamers o f t h e s i d e c h a i n i n t h e ground s t a t e ; f o r p r o t e i n s t h e causes o f n o n - e x p o n e n t i a l decay a r e not u n d e r s t o o d e n t i r e l y , but i t i s known t h a t t h e r e i s a v a r i a b i l i t y i n t r y p t o p h a n decay k i n e t i c s among p r o t e i n s w i t h d i f f e r e n t amino a c i d sequences when i n t h e i r native state (43). T h i s v a r i a b i l i t y d i s a p p e a r s when p r o t e i n s are d e n a t u r e d , as a l l p r o t e i n s c o n t a i n i n g t r y p t o p h a n e x h i b i t dual e x p o n e n t i a l decay o f e m i s s i o n w i t h l i f e t i m e s o f 1.5 and 4 nanoseconds. These r e s u l t s suggest t h a t i f t h e complex p h o t o p h y s i c s o f a p r o t e i n can be u n r a v e l e d t h e p o t e n t i a l exists t o o b t a i n d e t a i l e d m o l e c u l a r i n f o r m a t i o n from f l u o r e s c e n c e l i f e t i m e determinations. F l u o r e s c e n c e a n i s o t r o p y decay s t u d i e s can be used t o d e t e r mine t h e r o t a t i o n a l c o r r e l a t i o n time o f a p r o t e i n as well as p r o v i d i n g a measurement o f o t h e r dynamic p r o c e s s e s which depolarize fluorescence emission (44,45). Recent work by Brand and c o - w o r k e r s (46) has demonstratecT"Row i t i s p o s s i b l e t o resolve t h e c o n t r i b u t i o n s o f d i f f e r e n t components t o total a n i s o t r o p y decay i n a r o t a t i o n a l l y heterogeneous system u s i n g a n i s o t r o p y decay a s s o c i a t e d f l u o r e s c e n c e s p e c t r a . The f l u o r e s c e n c e e m i s s i o n s p e c t r a o f t h e d i f f e r e n t components may then be used t o make s t r u c t u r a l i n t e r p r e t a t i o n s . The a p p l i c a t i o n o f t h e s e t e c h n i q u e s t o t h e study o f p r o t e i n u n f o l d i n g p r o c e s s e s s h o u l d a l l o w t h e motions o f d i f f e r e n t t r y p t o p h a n r e s i d u e s t o be s e p a r a t e d and q u a n t i f i e d , p r o v i d e d t h e i r emission spectra are d i f f e r e n t . In t h i s manner, i n t e r m e d i a t e s i n t h e u n f o l d i n g p r o c e s s may be c h a r a c t e r i z e d a c c o r d i n g t o t h e range o f motions a v a i l a b l e t o i t s t r y p t o p h a n residues. Fluorescence Quenching. F l u o r e s c e n c e quenching s t u d i e s o f p r o t e i n s p r o v i d e i n f o r m a t i o n about t h e p e n e t r a t i o n o f a quencher molecule i n t o the protein matrix (47-49). With such s t u d i e s i t
i s possible t o probe t h e accessibility o f f l u o r o p h o r e s ; e . g . , t y r o s i n e o r t r y p t o p h a n r e s i d u e s , t o t h e quencher as modulated by s t e r i c and d i e l e c t r i c c o n s t a n t e f f e c t s . The use o f d i f f e r e n t chemical s p e c i e s , such as i o d i d e ( 5 0 ) , oxygen ( 5 1 ) , cesium i o n , acrylamide (52) and trichloroethanoT ( 5 3 ) , can s e r v e as a way o f c h a r a c t e r i z i n g t h e environment around a f l u o r o p h o r e depending on t h e quenching e f f i c i e n c y o f t h e d i f f e r e n t q u e n c h e r s . The i n t e r p r e t a t i o n o f t h e s e d a t a must t a k e i n t o account t h e p o s s i b i l i t y t h a t t h e quenching mechanism i s dynamic ( c o m p l e x a t i o n o f quencher w i t h an e x c i t e d s t a t e f l u o r o p h o r e ) o r s t a t i c ( c o m p l e x a t i o n o f quencher w i t h a ground s t a t e f l u o r o p h o r e ) o r b o t h . F u r t h e r c o m p l i c a t i o n s can a r i s e i f t h e quencher e x h i b i t s p a r t i t i o n i n g a n d / o r b i n d i n g t o t h e p r o t e i n "phase" ( 5 4 ) . C i r c u l a r D i c h r o i s m S p e c t r o s c o p y . C i r c u l a r d i c h r o i s m (CD) s p e c t r o s c o p y p r o v i d e s a measurement o f m o l e c u l a r o p t i c a l a c t i v i t y t h r o u g h measurement o f t h e d i f f e r e n c e i n e x t i n c t i o n
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c o e f f i c i e n t between l e f t - and r i g h t - c i r c u l a r l y p o l a r i z e d l i g h t ( 5 5 , 5 6 ) . CD s t u d i e s o f p r o t e i n s a r e i n f o r m a t i v e because t h e amino a c i d s o f which t h e y a r e composed a r e o p t i c a l l y a c t i v e individually. When t h e amino a c i d s are combined i n a p r o t e i n t h e y can produce l a r g e o p t i c a l l y a c t i v e s t r u c t u r e s (such as a h e l i c e s , β - s h e e t s , e t c . ) whose s t r u c t u r e can be probed w i t h CD spectroscopy. T h i s d i s c u s s i o n w i l l be l i m i t e d t o t h e study o f p r o t e i n e l e c t r o n i c t r a n s i t i o n s w i t h CD a l t h o u g h r e c e n t i n v e s t i g a t i o n s o f v i b r a t i o n a l CD o f amino a c i d s ( 5 7 ) , p e p t i d e s and p o l y p e p t i d e s (58) have demonstrated t h a t c o n s i d e r a b l e s t r u c t u r a l i n f o r m a t i o n can a l s o be g l e a n e d from t h e VCD spectrum. Near-UV CD. The e l e c t r o n i c t r a n s i t i o n s o f p r o t e i n s i n t h e n e a r UV a r e due t o a b s o r p t i o n s o f t h e a r o m a t i c amino a c i d s and were discussed previously. The s i g n i f i c a n t o v e r l a p o f t r a n s i t i o n s i n t h e a b s o r p t i o n spectrum o f t e n l e a d s t o poor r e s o l u t i o n o f s p e c t r a l components, a problem which i s l e s s pronounced i n a CD spectrum as t r a n s i t i o n s can have d i f f e r e n t s i g n s ( 5 9 ) . A CD s p e c t r u m , t h e r e f o r e , has h i g h e r i n h e r e n t r e s o l u t i o n than an absorption spectrum. T h i s p r o p e r t y has been e x p l o i t e d t o a s s i g n t h e v i b r a t i o n a l f i n e s t r u c t u r e i n a b s o r p t i o n bands o f p h e n y l a l a n i n e ( 6 0 ) , t r y p t o p h a n (61) and t y r o s i n e (62) i n model compounds and p r o t e i n s a t low temperature (77 K ) . " m i s work has been extended by Puett and c o - w o r k e r s (63) t o bGH and o t h e r growth hormones i n an assignment o f t h e i r near-UV e l e c t r o n i c t r a n s i t i o n s from CD s p e c t r a . These s t u d i e s f o l l o w e d e a r l i e r work (64,65) which demonstrated t h e s e n s i t i v i t y o f t h e near-UV CD spectrum of bGH ( F i g u r e 3) t o s o l u t i o n c o n d i t i o n s and p r o v i d e d e v i d e n c e t h a t t h e t h e near-UV CD spectrum i s a r e l i a b l e i n d i c a t o r of protein t e r t i a r y s t r u c t u r e . F a r - U V CD. P r o t e i n s absorb energy i n t h e f a r - U V due t o e l e c t r o n i c t r a n s i t i o n s of peptide bonds. The a p p l i c a t i o n o f t h e CD spectrum f o r t h e d e t e r m i n a t i o n o f secondary s t r u c t u r e has been s t a n d a r d p r a c t i c e f o r s e v e r a l y e a r s ever s i n c e t h e p i o n e e r i n g work by Fasman and c o - w o r k e r s ( 6 6 ) . There has been c o n s i d e r a b l e e f f o r t expended t o improve t h e r e l i a b i l i t y o f t h e s e d e t e r m i n a t i o n s (67-70) and t o go beyond t h e d e t e r m i n a t i o n o f a h e l i x , β - s h e e t and remainder p e r c e n t a g e s t o s e p a r a t e p a r a l l e l and a n t i - p a r a l l e l β - s h e e t c o n t r i b u t i o n s and t o determine β - t u r n percentages (71-75). Most o f t h e methods r e l y on c o r r e l a t i n g t h e observed CD spectrum f o r r e f e r e n c e p r o t e i n s i n t h e s o l u t i o n s t a t e w i t h the secondary s t r u c t u r e d e t e r m i n a t i o n from X - r a y c r y s t a l s t r u c t u r e s o f t h e same p r o t e i n s . The a c c u r a c y o f the methods v a r i e s but t h e most r e l i a b l e parameter t o d e t e r m i n e w i t h a l l o f them i s t h e p e r c e n t α - h e l i x , due l a r g e l y t o i t s spectrum b e i n g i s o l a t e d from t h e s p e c t r a o f t h e o t h e r secondary s t r u c t u r e elements ( 7 3 ) . The e f f e c t s o f s e v e r a l e x p e r i m e n t a l e r r o r s on secondary s t r u c t u r e d e t e r m i n a t i o n s from CD s p e c t r a can be s i g n i f i c a n t and a r e t o be a v o i d e d i n o r d e r t o a c h i e v e good agreement between experiment and t h e o r y ( 7 6 ) . It s h o u l d a l s o be
Phillips et al.; The Impact of Chemistry on Biotechnology ACS Symposium Series; American Chemical Society: Washington, DC, 1988.
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T H E IMPACT OF CHEMISTRY ON BIOTECHNOLOGY
emphasized t h a t s p e c t r a w i t h h i g h s i g n a l - t o - n o i s e r a t i o s a r e required for accurate r e s u l t s . The f a r - U V CD spectrum o f bGH i s c h a r a c t e r i s t i c f o r a p r o t e i n c o n t a i n i n g l a r g e amounts o f α - h e l i x s t r u c t u r e (minima a t 222 and 208 nm, maximum a t 193 nm) and has been used t o e s t i m a t e t h a t bGH has about 50% α - h e l i x and 10% β - s h e e t s t r u c t u r e ( 6 3 , 6 5 ) . The spectrum o f u n f o l d e d bGH ( i n 6 M Gdn HC1 ) i s c o n s i s t e n t w i t h a random c o i l s p e c t r u m ; i n p a r t i c u l a r , t h e r e i s v i r t u a l l y no CD s i g n a l a t 222 nm, a wavelength t h a t can be used t o m o n i t o r secondary s t r u c t u r e d u r i n g u n f o l d i n g s t u d i e s . Optical
S p e c t r o s c o p y o f t h e S i n g l e Tryptophan i n bGH
The u n f o l d i n g o f bGH has been s t u d i e d by s e v e r a l workers ( 6 3 , 7 7 , 7 8 ) and found t o be a m u l t i s t a t e p r o c e s s w i t h a t l e a s t
one s t a T J T e e q u i l i b r i u m
intermediate: I
£ U
where Ν r e p r e s e n t s t h e n a t i v e s t a t e , I an i n t e r m e d i a t e and U t h e u n f o l d e d s t a t e o f bGH. It has a l s o been shown (79) t h a t a s e l f a s s o c i a t e d form ( o r forms) o f bGH i s (are) p o p u l a t e d under p a r t i a l l y d e n a t u r i n g c o n d i t i o n s (3.7 M Gdn HC1 o r 8.5 M u r e a ) so t h a t t h e complete u n f o l d i n g p r o c e s s under e q u i l i b r i u m c o n d i t i o n s can be r e p r e s e n t e d a s : Ν £
I £ U
4r where I represents the s e l f - a s s o c i a t e d intermediate s p e c i e ( s ) and η i s between 3 and 5. The e q u i l i b r i u m u n f o l d i n g o f bGH i n Gdn HC1 and urea a r e summarized i n T a b l e I.
Table
I.
Summary o f E q u i l i b r i u m U n f o l d i n g R e s u l t s f o r bGH
Transition Mid-point Method A b s o r p t i o n (290 nm) Second-derivative absorption (tyr) Second-derivative absorption (trp) Fluorescence (trp) S i z e - e x c l u s i o n HPLC C i r c u l a r D i c h r o i s m (222 nm)
Gdn HC1
urea 7.Ô
3.1 3.2 3.5 3.6 3.8 3.9
(M)
8.1 9.2
In t h e d i s c u s s i o n b e l o w , o p t i c a l s p e c t r o s c o p i c methods w i l l be d e s c r i b e d which c h a r a c t e r i z e t h e m o l e c u l a r s t r u c t u r e o f each o f t h e s e s p e c i e s as d e t e r m i n e d by p r o b i n g o f t h e l o n e t r y p t o p h a n r e s i d u e i n bGH.
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S e c o n d - D e r i v a t i v e A b s o r p t i o n S p e c t r o s c o p y . It was demonstrated above t h a t the s e c o n d - d e r i v a t i v e a b s o r p t i o n spectrum o f t r y p t o p h a n can be used t o probe t h e d i e l e c t r i c c o n s t a n t o f the t r y p t o p h a n environment i n a p r o t e i n . The s p e c t r a f o r bGH (79) i n d i f f e r e n t c o n f o r m a t i o n a l s t a t e s show d i f f e r e n c e s i n p o l a r i t y t h a t a r e i n d i c a t e d by t h e d i f f e r e n t i n t e n s i t i e s o f t h e peak at 295 nm and t h e t r o u g h a t 291 nm. When the i n t e n s i t i e s are measured f o r t h e u n f o l d i n g t r a n s i t i o n a t h i g h (0.3 mg/mL) and low (0.05 mg/mL) p r o t e i n c o n c e n t r a t i o n s ( 7 9 ) , i t i s c o n c l u d e d t h a t the d i e l e c t r i c c o n s t a n t (ε) o f t h e t r y p t o p h a n environment f o r the s e l f - a s s o c i a t e d intermediate s t a t e i s the l a r g e s t of a l l the c o n f o r m a t i o n a l s t a t e s w i t h the o t h e r s i n t h e f o l l o w i n g order:
%
e
>
U
>
ε
Ι
>
These data have been a n a l y z e d i n more d e t a i l
ε
Ν elsewhere
(79).
F l u o r e s c e n c e S p e c t r o s c o p y . When t r y p t o p h a n f l u o r e s c e n c e i s used t o m o n i t o r t h e u n f o l d i n g o f bGH w i t h Gdn HC1 a t low p r o t e i n c o n c e n t r a t i o n a t r a n s i t i o n c u r v e i s o b s e r v e d t h a t has a peak near 4 M Gdn HC1 ( F i g u r e 4); t h i s i s c l e a r e v i d e n c e t h a t a s i m p l e twos t a t e p r o c e s s cannot e x p l a i n t h e u n f o l d i n g o f bGH: at l e a s t one e q u i l i b r i u m i n t e r m e d i a t e (I) i s p o p u l a t e d . Figure 4 a l s o demonstrates t h a t t h e f l u o r e s c e n c e e m i s s i o n o f t h e t r y p t o p h a n i n the n a t i v e s t a t e i s quenched i n t r a m o l e c u l a r l y when compared t o t h e I and t h e U s t a t e s . When t h e p r o t e i n c o n c e n t r a t i o n i s i n c r e a s e d at 3.8 M Gdn HC1, t h e f l u o r e s c e n c e i n t e n s i t y i s o b s e r v e d t o d e c r e a s e m o n o t o n i c a l l y and l e v e l o f f , i n d i c a t i n g t h a t the f o r m a t i o n o f I i s accompanied by i n c r e a s e d fluorescence quenching. The f l u o r e s c e n c e quantum y i e l d s (φ) o f t r y p t o p h a n i n the v a r i o u s forms o f bSt a r e : n
Ψ
Ι
>
Φΐ
> η
>
>
*N
The e f f e c t s o f t h e f l u o r e s c e n c e quenching agents a c r y l a m i d e , i o d i d e and t r i c h l o r o e t h a n o l (TCE) on t h e f l u o r e s c e n c e e m i s s i o n o f t h e s i n g l e t r y p t o p h a n o f bGH i n i t s v a r i o u s c o n f o r m a t i o n a l s t a t e s have been determined (Kauffman, E . W . , The Upjohn Company, u n p u b l i s h e d d a t a ) w i t h t h e f o l l o w i n g results: (1) as e x p e c t e d , f o r a l l s t a t e s TCE i s t h e most e f f e c t i v e quencher f o l l o w e d by a c r y l a m i d e and i o d i d e , t h e u n f o l d e d s t a t e i s t h e most a c c e s s i b l e o f a l l t h e s t a t e s and t h e n a t i v e s t a t e i s the most p r o t e c t e d from TCE and a c r y l a m i d e ; (2) t h e s e l f - a s s o c i a t e d i n t e r m e d i a t e s t a t e o f bGH i s t h e most p r o t e c t e d from i o d i d e o f a l l the s t a t e s . The l a t t e r r e s u l t can be i n t e r p r e t e d as i n d i c a t i n g t h a t t h e r e a r e n e g a t i v e charges near the t r y p t o p h a n i n t h e I s t a t e which prevent i o d i d e from penetrating the p r o t e i n matrix. Q
C i r c u l a r P i c h r o i s m S p e c t r o s c o p y . The near-UV CD spectrum o f bGH ( F i g u r e 3) p r o v i d e s a c o n v e n i e n t probe f o r t h e s e l f - a s s o c i a t e d
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T H E IMPACT O F CHEMISTRY ON BIOTECHNOLOGY
F i g u r e 3. Near-UV CD s p e c t r a o f n a t i v e ( ) and s e l f associated intermediate ( ) bGH. S o l v e n t f o r the n a t i v e spectrum was 0.05M ammonium b i c a r b o n a t e (pH 8.5) and f o r the s e l f - a s s o c i a t e d i n t e r m e d i a t e was t h e same b u f f e r p l u s 3.7 M Gdn HC1. (Reproduced from R e f . 79. C o p y r i g h t 1986 American Chemical S o c i e t y ) .
Gdn HCI (M)
F i g u r e 4. U n f o l d i n g t r a n s i t i o n f o r 0.01 mg/mL bGH i n Gdn HCI as m o n i t o r e d by t r y p t o p h a n f l u o r e s c e n c e i n t e n s i t y .
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i n t e r m e d i a t e form o f bGH (79,80). A band i s o b s e r v e d a t 300 nm, due t o t h e t r y p t o p h a n r e s i d u e , t h a t grows i n i n t e n s i t y i n t h e I form. The s i t u a t i o n i s s i m i l a r t o t h a t o b s e r v e d p r e v i o u s l y f o r i n s u l i n (81) where an i n c r e a s e i n t h e CD o f t y r o s i n e r e s i d u e s upon s e l f - a s s o c i a t i o n was a s c r i b e d t o d i p o l e - d i p o l e c o u p l i n g o f r e s i d u e s on d i f f e r e n t i n s u l i n m o l e c u l e s . These data can be i n t e r p r e t e d as an i n d i c a t i o n t h a t t h e t r y p t o p h a n r e s i d u e s o f I a r e h e l d r i g i d l y near t h e i n t e r f a c e between s e l f - a s s o c i a t i n g molecules. n
n
Acknowledgments I am g r a t e f u l f o r t h e i m p o r t a n t c o n t r i b u t i o n s t o t h i s work by my c o l l a b o r a t o r s a t The Upjohn Company: D r . D . N . Brems has been a g r e a t h e l p i n many a r e a s o f p r o t e i n c h e m i s t r y , E.W. Kauffman and S . M . P l a i s t e d have c o n t r i b u t e d v a l u a b l e t e c h n i c a l a s s i s t a n c e and D r . R . D . White has been a c o n t i n u a l s o u r c e o f s u p p o r t and advice. I a l s o acknowledge B . S . Hanna f o r c o n d u c t i n g s e v e r a l f l u o r e s c e n c e s t u d i e s o f bGH w h i l e c o m p l e t i n g a Kalamazoo C o l l e g e SIP i n t e r n s h i p a t Upjohn and K. H e n d r i c k s f o r t y p i n g t h e manuscript.
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