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SOLVENT EFFECTS IN BIOMOLECULAR SYSTEMS AND PROCESSES
J. Finney
To cite this version:
J. Finney. SOLVENT EFFECTS IN BIOMOLECULAR SYSTEMS AND PROCESSES. Journal de
Physique Colloques, 1984, 45 (C7), pp.C7-197-C7-210. �10.1051/jphyscol:1984722�. �jpa-00224287�
JOURNAL DE PHYSIQUE
Colloque C7, supplkment a u n09, Tome 45, septembre 1984 page C7-197
SOLVENT E F F E C T S I N B I O M O L E C U L A R SYSTEMS AND PROCESSES
J . L . F i n n e y
Department o f CrystaZZography, Birkbeck CoZZege, MaZet S t r e e t , London WClE 7 H X , U.K.
Rdsumd
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L ' i n f l u e n c e d u s o l v a n t dans l e s p r o c e s s u s d ' i n t e r a c t i o n e t d e changement de c o n f o r m a t i o n des moldcules b i o l o g i q u e s e s t d i s c u t d e e n a y a n t e n vue l e s problsmes s o u l e v d s p a r l ' i n f l u e n c e apparemment s u b t i l e des d i f f d - r e n t s e f f e t s e n t r o p i q u e s e t d n e r g g t i q u e s . On n o t e l ' i m p o r t a n c e p o s s i b l e des c o n t r i b u t i o n s h y d r o p h i l e s e t d e t y p e v a n d e r Waals, t o u t e n remarquant que n o t r e c o n n a i s s a n c e a c t u e l l e du n i v e a u m o l d c u l a i r e n ' e s t p a s s u f f i s a n t e pour a f f i r m e r ou r e j e t e r l a domination g d n s r a l e m e n t admise des i n t e r a c t i o n s hydrophobes dans l e s p r o c e s s u s b i o m o l d c u l a i r e s t y p i q u e s . On montre l e b e s o i n d ' a m d l i o r e r n o t r e comprghension d e s s o l u t i o n s aqueuses s i m p l e s p a r d e s mgthodes e x p d r i m e n t a l e s d i r e c t e s , e t q u e l q u e s r d s u l t a t s r d c e n t s s u r d e s s o l u - t i o n s d e moldcules p o l a i r e s e t d e s c r i s t a u x de l a coenzyme B12 s o n t p r d s e n t d s . La d i f f u s i o n des n e u t r o n s e s t p a r t i c u l i s r e m e n t b i e n a d a p t d e pour r d p o n d r e B beaucoup d e c e s q u e s t i o n s e t d e s p r o g r s s r 6 c e n t s dans l ' i n s t r u m e n t a t i o n e tl'avbnement de s o u r c e s pulsCes c o n t r i b u e r o n t c e r t a i n e m e n t B a g r a n d i r d e s p o s s i b i l i t 6 s e x p d r i m e n t a l e s q u i Q t a i e n t a u p a r a v a n t m a r g i n a l e s .
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The s o l v e n t dependence of b i o m o l e c u l e f o l d i n g and i n t e r a c t i o n p r o c e s s e s i s d i s c u s s e d w i t h p a r t i c u l a r r e f e r e n c e t o t h e problems r a i s e d by t h e a p p a r e n t s u b t l e t y of t h e v a r i o u s e n t r o p i c and e n e r g y e f f e c t s i n v o l v e d . The p o s s i b l e s i g n i f i c a n c e of h y d r o p h i l i c and van d e r Waals c o n t r i b u t i o n s a r e s t r e s s e d , n o t i n g t h a t o u r c u r r e n t m o l e c u l a r - l e v e l knowledge i s i n a d e q u a t e t o a s s e r t o r deny t h e g e n e r a l l y assumed dominance of h y d r o p h o b i c i n t e r a c t i o n s i n t y p i c a l b i o m o l e c u l a r i n t e r a c t i o n s . A need t o improve o u r u n d e r s t a n d i n g of s i m p l e r aqueous s o l u t i o n s by d i r e c t e x p e r i m e n t a l t e c h n i q u e s i s a r g u e d and some r e c e n t r e s u l t s on b o t h p o l a r m o l e c u l e s o l u t i o n s and coenzyme B 1 2 c r y s t a l s a r e d i s c u s s e d . Neutrons a r e p a r t i c u l a r l y w e l l s u i t e d t o a d d r e s s i n g many o f t h e o u t s t a n d i n g q u e s t i o n s , and r e c e n t p r o g r e s s i n i n s t r u m e n t a t i o n ,and t h e a d v e n t of p u l s e d s o u r c e s , a r e l i k e l y t o open up p r e v i o u s l y m a r g i n a l e x p e r i m e n t a l p o s s i b i l i t i e s .
I
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INTRODUCTIONIt i s c o n v e n t i o n a l wisdom t h a t w a t e r p l a y s more t h a n a ~ a s s i v e r o l e i n many b i o - m o l e c u l a r i n t e r a c t i o n s and p r o c e s s e s . S i n c e Kauzmann's c e l e b r a t e d 1959 p a p e r / I / h y d r o p h o b i c i n t e r a c t i o n s have been c o n s i d e r e d i m p o r t a n t
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p e r h a p s dominant-
i n t h e f o l d i n g o f p r o t e i n s , and many w o r k e r s have s i n c e a r g u e d f o r a s i m i l a r dominance i n p r o t e i n - p r o t e i n , e n z y m e - s u b s t r a t e , and hormone-receptor i n t e r a c t i o n s . Although t h e r e i s d i r e c t e x p e r i m e n t a l e v i d e n c e of t h e i r p o s s i b l e r e l e v a n c e i n b i o m o l e c u l a r i n t e r a c - t i o n s , c h a r g e , hydrogen bonding, and v a n d e r Waals i n t e r a c t i o n s h a v e r e c e i v e d much l e s s a t t e n t i o n .D e s p i t e much s p e c u l a t i o n , o u r u n d e r s t a n d i n g o f r e l e v a n t s o l v e n t e f f e c t s a t t h e m o l e c u l a r l e v e l remains poor. T h i s i s p a r t l y due t o t h e c o m p l e x i t y of t h e m u l t i - component s y s t e m s i n v o l v e d , and a l s o p a r t l y t o t h e p r o b a b l e s u b t l e t y o f many o f t h e s e e f f e c t s . At t h e b a s i c l e v e l , we need an u n d e r s t a n d i n g of t h e consequences of p e r t u r b a t i o n s i n weak
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i n t e r a c t i o n s whose s t e r e o c h e m i s t r y and e n e r g e t i c s a r e d i f f i c u l t t o s p e c i f y . T h i s i s f a r from a s t r a i g h t f o r w a r d problem, and p r o g r e s s r e q u i r e s v e r y c a r e f u l c h o i c e s of s u i t a b l e e x p e r i m e n t a l s y s t e m s , and a p p r o p r i a t e( p r e f e r a b l y d i r e c t ) e x p e r i m e n t a l and t h e o r e t i c a l methods, t h e r e s u l t s o f which s h o u l d b e i n t e r p r e t a b l e w i t h a minimum ( i d e a l l y z e r o ) dependence on c o n c e p t u a l models.
Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1984722
C7-198 JOURNAL
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PHYSIQUEI n what f o l l o w s , t h e b a s i c s o f a p o s s i b l e m u l t i d i s c i p l i n a r y approach a r e d i s c u s s e d . F i r s t , a s an example o f a s o l v e n t - a f f e c t e d b i o l o g i c a l p r o c e s s , we summarize
b r i e f l y a s i m p l e model of h y d r a t i o n - r e l a t e d e f f e c t s t h a t a p p e a r t o b e e s s e n t i a l f o r t h e a c t i v a t i o n o f d r i e d lysozyme. Then, i n o r d e r t o u n d e r l i n e t h e s u b t l e t y of many of t h e s o l v e n t e f f e c t s we a r e i n t e r e s t e d i n , some thermodynamic c o n t r i b u t i o n s t o t h e f r e e energy change of a c l a s s o f b i o m o l e c u l a r p r o c e s s ( p r o t e i n f o l d i n g , enzyme-substrate b i n d i n g ) a r e d i s c u s s e d . T h i s d i s c u s s i o n s t r e s s e s a v i t a l need t o improve o u r u n d e r s t a n d i n g of t h e b a s i c w a t e r - i n v o l v e d i n t e r a c t i o n s such a s hydrogen bonding. F i n a l l y , p o s s i b l e r o u t e s t o improving o u r u n d e r s t a n d i n g i n b o t h r e l a t i v e l y s i m p l e (aqueous s o l u t i o n s ) and complex ( b i o m o l e c u l a r c r y s t a l s and s o l u t i o n s ) systems a r e s u g g e s t e d . P r e l i m i n a r y r e s u l t s a r e d i s c u s s e d of some r e c e n t u n p u b l i s h e d work o f t h e B i r k b e c k L i q u i d L a b o r a t o r y u s i n g n e u t r o n s , which a r e s e e n t o b e c r u c i a l i n improving o u r u n d e r s t a n d i n g of s o l v e n t i n t e r a c t i o n s i n b o t h s i m p l e and complex systems.
I1
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LYSOZYME ACTIVATIONDry lysozyme i s i n a c t i v e . However, a s d e m o n s t r a t e d by t h e p i o n e e r i n g work of Rupley / 3 / , f u l l s o l u t i o n c o n d i t i o n s a r e n o t n e c e s s a r y f o r a c t i v i t y t o recommence.
Making d i f f i c u l t measurements a t d i f f e r e n t v a l u e s of pH, t h e y showed t h a t a c t i v i t y r e t u r n e d a t a h y d r a t i o n of about 0.2 g w a c e r I g p r o t e i n . A t t h i s w a t e r c o n t e n t ,
which
i s l e s s t h a n monolayer coverage, t h e
m rote in
e x i s t s a s asolid
f i l m o r powder, depending upon t h e method of sample p r e p a r a t i o n . The i n t e r e s t i n g q u e s t i o n t h a t f o l l o w s i s how do t h e s e 160 o r s o w a t e r m o l e c u l e s p e r enzyme molecule f a c i l i t a t e a c t i v i t y ? A t t h e m o l e c u l a r l e v e l ,how
a r e t h e y i n t e r a c t i n g w i t h t h e enzyme, and what changes-
i f any-
o c c u r t o t h e enzyme a s a consequence which might be r e l a t e d t o t h e o n s e t of a c t i v i t y a t t h i s c r i t i c a l h y d r a t i o n v a l u e ?These q u e s t i o n s cannot be answered s i m p l y , b u t r a t h e r r e q u i r e a b a t t e r y o f hope- f u l l y complementary e x p e r i m e n t a l t e c h n i q u e s , t h e r e s u l t s from which can b e i n t e r - p r e t e d i n a way which i s c o n s i s t e n t w i t h e a c h o t h e r and o u r knowledge o f t h e m o l e c u l a r ( e s p e c i a l l y surface-exposed) s t r u c t u r e . Such m u l t i t e c h n i q u e work h a s been performed by Rupley / 4 / and o u r s e l v e s / 5 / .
On t h e b a s i s of Raman, d i r e c t d i f f e r e n c e i n f r a - r e d , NllR p r o t o n back-exchange, and uv s p e c t r o s c o p y , coupled w i t h a knowledge of t h e n a t u r e of t h e p r o t e i n s u r f a c e , t h e f o l l o w i n g model can be argued which we t h i n k g i v e s some i n s i g h t i n t o t h e m o l e c u l a r - l e v e l r o l e of w a t e r i n t h e o n s e t of enzyme a c t i v i t y . A t t h e l o w e s t h y d r a t i o n s , t h e a c i d i c and b a s i c groups i o n i s e t o r e s t o r e t h e i r normal pK o r d e r . F u r t h e r w a t e r a d d i t i o n h y d r a t e s b o t h t h e s e charged groups and surface-exposed p o l a r
r o u p s , and seems t o f a c i l i t a t e a f l e x i b i l i t y increase-the enzvme. between a b o r 3 . 0 7
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0.10 g i g . T h i s i n t e r p r e t a t i o n was o r i g i n a l l y based on NMR back-exchangemeasurements / 6 / , a n d was a d m i t t e d l y somewhat model-dependent. It i s , however, c o n s i s t e n t w i t h ESR / 4 / , p e p t i d e hydrogen exchange measurements / 4 / , and d i e l e c t r i c r e s u l t s / 7 / from o t h e r groups. P r e l i m i n a r y i n e l a s t i c n e u t r o n s c a t t e r i n g measurements performed e a r l i e r t h i s y e a r a t t h e ILL on I N 5 seem t o d i r e c t l y c o n f i r m t h i s i n t e r - p r e t a t i o n (J.C. Smith, S. Cusack, P.L.Poole and J.L. F i n n e y , u n p u b l i s h e d work. See a l s o t h e p a p e r by P.L.Poole i n t h i s volume).
F u r t h e r w a t e r a d d i t i o n c o n t i n u e s t h e p r o c e s s of p o l a r group h y d r a t i o n , a l t h o u g h amide group h y d r a t i o n a p p e a r s t o be s t i l l i n c o m p l e t e a t t h e c r i t i c a l h y d r a t i o n l e v e l of 0 . 2 g / g . C o n f o r m a t i o n a l l y s e n s i t i v e Raman b a n d s , do, however show s i g n i f i c a n t changes below t h i s h y d r a t i o n ; a t 0 . 2 g / g , t h e s e bands a r e i n d i s t i n a u i s a b l e from t h o s e observed from s o l u t i o n s . Thus, we-see e v i d e n c e f o r some ( p r o b a b l y s m a l l ) c o n f o r m a t i o n a l changes i n t h e p r o t e i n which a r e completed b e f o r e t h e o n s e t of enzyme a c t i v i t y . The n a t u r e o f t h e s e changes remains u n c e r t a i n , b u t may r e l a t e t o an i n c r e a s e d o r d e r i n g of t h e h e l i c a l r e g i o n s / 5 / .
Thus, we a r e l e f t w i t h t h e f o l l o w i n g model of lysozyme h y d r a t i o n . A t low water c o n t e n t , t h e molecule i s r e l a t i v e l y i n f l e x i b l e , w i t h an i n t e r n a l s t r u c t u r e which d e v i a t e s ( p r o b a b l y o n l y l o c a l l y ) from t h a t i n t h e c r y s t a l and i n s o l u t i o n . A small amount of w a t e r (50-100 molecules p e r p r o t e i n molecule) i s r e q u i r e d b e f o r e t h e
enzyme b e g i n s t o "loosen up" t o a l l o w s o l v e n t a c c e s s t o b u r i e d amide groups. T h i s h y d r a t i o n - i n d u c e d f l e x i b i l i t y t h e n f a c i l i t a t e s c o n f o r m a t i o n a l s h i f t s - p e r h a p s i n t h e h e l i c a l r e g i o n s
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which f u l l y r e s t o r e t h e s o l u t i o n s t r u c t u r e . These changes a r e complete b e f o r e t h e o n s e t of enzyme a c t i v i t y . These r e s u l t s a r e p r e s e n t e d i n more d e t a i l i n t h e accompanying p a p e r by P o o l e .T h i s seems t o us an i n t e r e s t i n g d i r e c t d e m o n s t r a t i o n o f t h e involvement of s o l v e n t e f f e c t s i n a b i o m b l e c u l a r p r o c e s s . Water i n t e r a c t i o n s seems t o b e r e q u i r e d t o
"prime'1 t h e p r o t e i n , i n b o t h a dynamic and s t r u c t u r a l way, f o r a c t i o n . The exper- i m e n t a l t e c h n i q u e s g i v e u s i n f o r m a t i o n on where t h e w a t e r m o l e c u l e s go, and what happens c o n s e q u e n t l y t o t h e p r o t e i n . The more fundamental q u e s t i o n of t h e w a t e r e f f e c t s t h e s e changes remains u n c l e a r .
111
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SOLVENT EFFECTS I N PROTEIN FOLDING AND INTERACTIONSWe d i s c u s s h e r e t h e k i n d of c o n t r i b u t i o n s t h a t a r e t h o u g h t t o b e i m p o r t a n t t o t h e f r e e energy change AG a s s o c i a t e d w i t h t y p i c a l b i o m o l e c u l a r p r o c e s s e s s u c h a s p r o t e i n f o l d i n g . P a r t i c u l a r a t t e n t i o n i s p a i d t o ( a ) t h e o r i g i n s of t h e v a r i o u s e f f e c t s , (b) t h e d e g r e e t o which t h e y i n v o l v e t h e s o l v e n t , and ( c ) o u r a b i l i t y t o e s t i m a t e t h e i r o r d e r s of magnitude from o u r c u r r e n t knowledge. I t s h o u l d b e s t r e s s e d a t t h e o u t s e t t h a t t h e n u m e r i c a l e s t i m a t e s g i v e n a r e o n l y p o s s i b l e o r d e r s of magnitude, p r e s e n t e d t o i l l u s t r a t e t h e d i s c u s s i o n . They must
not
b e t a k e n a s n e c e s s a r i l y r e a l i s t i c v a l u e s . S e v e r a l , though n o t a l l , c o n t r i b u t i o n s t o AG can c o n v e n i e n t l y be c o n s i d e r e d a s e i t h e r mainly e n t h a l p i c o r e n t r o p i c i n n a t u r e . B e a r i n i n mind t h a t o v e r a l l AG v a l u e s a p p e a r t o b e r e l a t i v e l y s m a l l (x 10-20 kcal.mol.-B t y p i c a l l y f o r t h e f o l d i n g of lysozyme o r RNAse-S) compared w i t h e x p e c t e d magnitudes o f i n d i v i d u a l c o n t r i b u t i o n s , a s s e r t i n g t h e dominance of a p a r t i c u l a r c o n t r i b u t i o n (e.g. hydrophobic e f f e c t s ) remains p r o b l e m a t i c a l . Moreover, t h e l a r g e number o f i n d i v i d u a l i n t e r - a c t i o n s (e.g. hydrogen bonds, n o n - s p e c i f i c w a t e r p e r t u r b a t i o n s a t t h e p r o t e i n - w a t e r i n t e r f a c e ) t h a t need b e c o n s i d e r e d when comparing t h e n a t i v e w i t h a h y p o t h e t i c a l u n f o l d e d s t a t e , s u g g e s t s t h a t r e l a t i v e l y s m a l l changes a t t h e l e v e l of a n i n d i v i d u a l i n t e r a c t i o n may a g g r e g a t e up t o q u i t e l a r g e f r e e e n e r g i e s f o r t h e t o t a l system. Thus q u i t e s u b t l e changes may be i n v o l v e d , which by d e f i n i t i o n w i l l n o t be e a s y t o quan- t i f y a t a s u f f i c i e n t l y p r e c i s e l e v e l . E a r l i e r d i s c u s s i o n s a l o n g s i m i l a r l i n e s have been g i v e n by P a i n 181 and Finney e t a l . / 9 / .For c l a r i t y t h i s d i s c u s s i o n i s f o r m u l a t e d i n terms of an a n n o t a t e d l i s t a s f o l l o w s . (1) AG c h a r g e
O r i g i n . A change i n charge-charge i n t e r a c t i o n on f o l d i n g . Assuming no dependence of t h e c h a r g e q; of i o n i s a b l e groups upon environment ( c l e a r l y i n c o r r e c t
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s e e( 2 ) b e l o w ) , t h e E n t e r c h a r g e s e p a r a t i o n s r i j w i l l b e d i f f e r e n t i n u n f o l d e d and n a t i v e c o n f o r m a t i o n s . Hence
1
' i q j w i l l change.i j
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S o l v e n t D e ~ e n d e n c e . Through what i s o f t e n c a l l e d t h e " e f f e c t i v e d i e l e c t r i c c o n s t a n t "
---
o f t h e i n t e r v e n i n g s ~ l v e n t / ~ r o t e i n medium, which "along t h e s u r f a c e " o f a p r o t e i n w i l l b e d i f f e r e n t from t h a t through u n p e r t u r b e d s o l v e n t . How t o t r e a t t h i s " e f f e c t i v e
El'
i s an unsolved problem; we t e n d t o t h e view t h a t a t t h e m o l e c u l a r l e v e l , t h e m o l e c u l a r and e l e c t r o n i c p o l a r i s a b i l i t y e f f e c t s t h a t g i v e r i s e t o t h e macroscopic p e r m i t t i v i t y need t o b e t a k e n s p e c i f i c account of t h r o u g h a s u i t a b l e model / 1 0 , 1 1 / . g ~ d e r of blagnitude i s e s s e n t i a l l y n o t known. There i s a s t r o n g tendency t o assume i t i s a b o u t z e r o n e a r t h e i s o e l e c t r i c p o i n t .( 2 ) A G t i t r .
g ~ & g j n . Changes i n t h e pKa of i o n i s a b l e groups r e s u l t i n g from changes i n t h e i r l o c a l environments ( e . g . from b u r i a l o r p a r t i a l b u r i a l i n t h e n a t i v e c o n f o r m a t i o n ) . S o l v e n t D e ~ e n d e n c e i s a g a i n t h r o u g h t h e e f f e c t on " l o c a l p e r m i t t i v i t y " o f p e r t u r - b a t i o n s i n t h e s t a t e of n e a r b y s o l v e n t .
C7-200 SOURNAL DE PHYSIQUE
Magnitude i s c l e a r l y unknown. A v a l u e o f a b o u t
-
5 K c a l mol-' was e s t i m a t e d by P a i n / 8 / f o r RNase f o l d i n g . Although t h i s i s a r e l a t i v e l y s m a l l v a l u e compared t o o t h e r c o n t r i b u t i o n s below, i t i s a s i g n i f i c a n t f r a c t i o n (25-50 %) o f t h e t o t a l AG.( 3 ) AG, : "Hydrophobic E f f e c t s "
C)zigjn i s much d i s c u s s e d , and i s n o t f u l l y u n d e r s t o o d . Thermodynamically t h e e f f e c t i s c h a r a c t e r i s e d by a l a r g e , p o s i t i v e e n t r o p y change AS, w i t h a s m a l l e n t h a l p i c t e r m AH < 0 ; t h e o v e r a l l AG i s t h e r e f o r e s t r o n g l y n e g a t i v e , d o m i n a t e d by t h e e n t r o p i c term. C o n v e n t i o n a l l y , t h e e f f e c t i s t h o u g h t of i n t e r m s o f a g e n e r a l l y a c c e p t e d model of a p o l a r h y d r a t i o n o f t e n c a l l e d
-
p e r h a p s u n f o r t u n a t e l y-
h y d r o p h o b i c h y d r a t i o n . Although t h e r e w i l l b e a weak a t t r a c t i o n b e t w e e n a w a t e r m o l e c u l e and an a p o l a r ( e . g . methyl) g r o u p , t h e s t r e n g t h o f t h i s i n t e r a c t i o n w i l l g e n e r a l l y b e p e r h a p s an o r d e r o f magnitude l e s s t h a n a t y p i c a l w a t e r - w a t e r hydrogen bond (% 4-5 k c a l mol-I). Thus, f o r a n ensemble o f one a p o l a r s r o u p and s e v e r a l w a t e r m o l e c u l e s , t h e o v e r a l l e n e r g y i s l i k e l y t o b e l o w e r i f t h e w a t e r s c a n a r r a n g e t o hydrogen bond w i t h e a c h o t h e r r a t h e r t h a n " w a s t i n g " a hydrogen-bond s i t e on t h e a p o l a r group. T h i s , however, w i l l have e n t r o p i c a l l y u n f a v o u r a b l e c o n s e q u e n c e s i n t h a t t h e c o n f i g u r a t i o n a l s p a c e volume a v a i l a b l e t o t h e w a t e r m o l e c u l e s i s reduced. The w a t e r m o l e c u l e s c l o s e t o t h e s o l u t e a r e t h u s i n some way r e s t r i c t e d . For a n ensemble o f two a p o l a r s o l u t e m o l e c u l e s i n a b a t h o f w a t e r , t h i s e n t r o p i c p e n a l t y c a n b e r e d u a i f t h e two s o l u t e m o l e c u l e s come i n t o c o n t a c t . I n d o i n g s o , some of t h e p r e v i o u s l y r e s t r i c t e d w a t e r s w i l l b e r e l e a s e d t o t h e b u l k s o l v e n t w i t h a c o n s e q u e n t e n t r o p i c g a i n . Although t h i s s i m p l e p i c t u r e of t h e h y d r o p h o b i c e f f e c t i s g e n e r a l l y c o n s i d e r e d t o be r e a s o n a b l e , i t i s p r o b a b l y more c o m p l i c a t e d i n r e a l i t y . For example, t h e r e i s e x p e r i m e n t a l , t h e o r e t i c a l , and c o m p u t e r
s i m u l a t i o n e v i d e n c e (12-14) f o r t h e e x i s t e n c e of a f a v o u r a b l e " s o l v e n t s e p a r a t e d "
minimum i n t h e p o t e n t i a l o f mean f o r c e f o r t h e h y d r o p h o b i c i n t e r a c t i o n . I n t h i s p a r t i c u l a r c o n f i g u r a t i o n , t h e two a p o l a r m o l e c u l e s do n o t make c o n t a c t , b u t remain s e p a r a t e d by an i n t e r v e n i n g (presumably v e r y r e s t r i c t e d , and t h e r e f o r e e n t r o p i c a l l y e x p e n s i v e ) p l a n e o f w a t e r s . I n t h i s c o n t e x t , "phobic" would seem a n a p p r o p r i a t e d e s c r i p t i o n , and t h e e x i s t e n c e of t h i s s o l v e n t - s e p a r a t e d s t a t e would seem t o c o m p l i c a t e t h e s i m p l e b i o m o l e c u l a r arguments o f t e n made f o r t h e dominance o f t h i s i n t e r a c t i o n i n p r o t e i n f o l d i n g and i n t e r a c t i o n s . Although n o t i n a n y way c l a i m i n g i t t o be a good model s y s t e m f o r s t u d y i n g t h e s t r u c t u r a l c o n s e q u e n c e s o f hydro- p h o b i c i n t e r a c t i o n s , h i g h r e s o l u t i o n n e u t r o n and x-ray r e f i n e m e n t o f t h e coenzyme B 1 2 c r y s t a l s t r u c t u r e ( s e e s e c t i o n 5 ( 2 ) below) does show s i m i l a r " s o l v e n t s e p a r a t e d 1 ' m e t h y l g r o u p s /15,16/. We s h o u l d n o t e a t t h i s p o i n t t h a t t h e a p p r o x i m a t e ( e a s i l y d i s t o r t a b l e ) t e t r a h e d r a l i t y o f w a t e r - w a t e r i n t e r a c t i o n i s s t r o n g l y c o n s i s t e n t w i t h
" c l a t h r a t e - t y p e q q c a g e f o r m a t i o n a r o u n d e f f e c t i v e l y i n e r t s o l u t e s .
S o l v e n t d e ~ e n d e n c e . AS t h e e f f e c t depends upon ( p r o b a b l y s u b t l e ) p e r t u r b a t i o n o f
--- ---
w a t e r s t r u c t u r e , and presumably dynamics, c l o s e t o a r e l a t i v e l y i n e r t s u r f a c e , i t s s o l v e n t dependence i s e f f e c t i v e l y c o m p l e t e .
Magnitude. T h i s r e m a i n s c o n t r o v e r s i a l f o r p r o t e i n i n t e r a c t i o n s , t h o u g h c o n v e n t i o n -
-- ---
a l l y i t i s t h o u g h t t o d o m i n a t e p r o t e i n f o l d i n g ( b u t s e e r e f s . 2 , 9 ) . S i n c e C h o t h i a f i r s t p r o p o s e d / 1 7 / a c o r r e l a t i o n between t h e b u r i a l o f a c c e s s i b l e s u r f a c e a r e a and h y d r o p h o b i c f r e e e n e r g y , t h i s h a s been a p o p u l a r r e c i p e f o r e s t i m a t i n g AG$
from a t o m i c c o o r d i n a t e s , a l t h o u g h t h e r e a r e n o t i n s i g n i f i c a n t p h y s i c a l l y - b a s e d o b j e c t i o n s t o t h e p r o c e d u r e / l a / . 'lsing t h i s method f o r t h e f o l d i n g o f RNase-S and lysozyme, we o b t a i n v a l u e s o f -130 and -160 Kcal mol-I r e s p e c t i v e l y . A s t h e o v e r a l l e x p e r i m e n t a l AG f o r t h e s e p r o c e s s e s i s o n l y a b o u t 10 % o f t h e s e v a l u e s , o n t h i s e s t i m a t e , t h e h y d r o p h o b i c c o n t r i b u t i o n i s c l e a r l y l a r g e and f a v o u r a b l e .
( 4 ) AH+ : Hydrogen bonding e n e r g i e s
O r i g i g . I n a f o l d e d p r o t e i n , t h e r e a r e many i n t e r n a l i n t r a m o l e c u l a r h y d r o g e n
---
bonds (?. 150 f o r lysozyme). I n a h y p o t h e t i c a l e x t e n d e d s t a t e , many o f t h e s e w i l l b e r e p l a c e d by hydrogen b o n d i n g i n t e r a c t i o n s o f t h e r e l e v a n t p o l a r g r o u p s ( p i ) t o
s o l v e n t w a t e r (w). I n t h e s i m p l e s t c a s e , a l a r g e number of t r a n s i t i o n s o f t h e t y p e
would o c c u r . D i f f e r e n c e s i n s t r e n g t h s between t h e d i f f e r e n t hydrogen bonds can t h e r e f o r e g i v e r i s e t o a non-zero e n e r g e t i c c o n t r i b u t i o n t o t h e t o t a l A G . The s i t u a t i o n i s made more complex when we a l s o c o n s i d e r p o s s i b l e n o n - c o n s e r v a t i o n o f number of hydrogen bonds, and hydrogen bond d i s t o r t i o n by s t e r e o c h e m i c a l r e s - t r i c t i o n s imposed by t h e p r o t e i n ' s t e r t i a r y s t r u c t u r e . C r y s t a l l o g r a p h i c d a t a c a n a s s i s t i n e s t i m a t i n g t h e p o s s i b l e i m p o r t a n c e of t h e s e two l a t t e r p o s s i b i l i t i e s 191.
S o l v e n t Dependence. The magnitude of t h i s c o n t r i b u t i o n c l e a r l y depends i n t i m a t e l y on w a t e r hydrogen bonding.
O r d e r of-m_ngn_.Lgu_d_e_, and even t h e s i g n , of t h i s p o s s i b l e c o n t r i b u t i o n i s d i f f i c u l t t o e s t i m a t e , depend,ing a s i t does upon p o s s i b l e
small
and u n c e r t a i n d i f f e r e n c e s i n hydrogen bonds s t r e n g t h s , which, b e c a u s e of t h e l a r g e number of s u c h i n t e r a c t i o n s(% 150 f o r lysozyme) c o u l d e a s i l y a g g r e g a t e t o a large t o t a l A$ c o n t r i b u t i o n . We c a n make two f i n a l a s s u m p t i o n s t o g e t some f e e l i n g f o r t h e ~ o s s i b l e i m p o r t a n c e of AH$. I n t h e t r i v i a l c a s e where a l l hydrogen bonds a r e of e q u a l s t r e n g t h and u n d i s t o r t e d ( c l e a r l y u n t r u e ! ) a n d x e r e a r e no u n m a d e l u n s a t u r a t e d hydrogen bonds
( a g a i n c o n t r a d i c t o r y t o e x p e r i m e n t a l e v i d e n c e / 191 ) , t h e n A H $ - 0 . Secondly, a g a i n assuming no d i s t o r t i o n and hydrogen bond c o n s e r v a t i o n , b u t t a k i n g
Upipj ( a l l i , j ) = ( 1 . 0
+
. l ) Upw,f o r t h e 150 o r s o hydrogen bonds i n RNase-S, AH$ a g g r e g a t e s up t o a b o u t +. 75 k c a l mol-l. ~ h even f o r o n l y a 10 ~ ~ , % d i f f e r e n c e i n hydrogen bond s t r e n g t h s ( e q u i v a l e n t t o a n energy of a p p r o x i m a t e l y kT), t h i s hydrogen bonding c o n t r i b u t i o n i s p o s s i b l y of a n o r d e r of magnitude s i m i l a r t o t h e above e s t i m a t e d hydrophobic c o n t r i b u t i o n . We s h o u l d u n d e r l i n e h e r e t h a t t h i s r e l a t i v e l y l a r g e a g g r e g a t e e n e r g y r e s u l t s from
o n l y a v e r y s m a l l d i f f e r e n c e i n t h e s t r e n g t h s of d i f f e r e n t i n d i v i d u a l hydrogen bonds.Thus, r e l a t i v e l y s m a l l , s u b t l e e f f e c t s a r e p o t e n t i a l l y c a p a b l e of d o m i n a t i n g a p a r t i c u l a r p r o c e s s .
The s i z e of t h i s c o n t r i b u t i o n i s d i f f i c u l t t o e s t i m a t e r e a l i s t i c a l l y . Although e s t i m a t e d e r r o r s a r e l a r g e , t h e r e i s quantum mechanical e v i d e n c e fo; p e p t i d e ' h y d r o g e n bonds b e i n g up t o 20 % s t r o n g e r t h a n t h e a v e r a g e i n t e r a c t i o n w i t h w a t e r ( 2 0 ) .
Assuming no d i f f e r e n c e i n s t r e n g t h between i d e a l p o l a r f r o u p - w a t e r and water-water hydrogen bonds, a c o n s e r v a t i v e f i g u r e of 0 . 5 K c a l mol- f o r t h e a d d i t i o n a l s t r e n g t h of a n i n t e r n a l hydrogen band o v e r a p o l a r group-water i n t e r a c t i o n y i e l d s a favou- r a b l e AH* of -75 k c a l mol-1 f o r t h e f o l d i n g of RNaSe-S. We s h o u l d n o t e , however, t h a t s t r e n g t h d i f f e r e n c e s between p o l a r group-water and w a t e r - w a t e r i n t e r a c t i o n s of t h e same o r d e r would a c t t o r e d u c e t h i s f i g u r e , a s would d i s t o r t i o n s of i n t e r n a l hydrogen bonds and u n s a t i s f i e d hydrogen bond a c c e p t o r s and d o n o r s i n s i d e t h e mole- c u l e . E s t i m a t e s b a s e d on c r y s t a l l o g r a p h i c d a t a 1 9 1 s u g g e s t t h e s e e f f e c t s may e a c h b e o f t h e o r d e r o f up t o 100 k c a l mol-I And d e s t a b i l i s i n g . The v a l i d i t y of u s i n g a v e r a g e c o o r d i n a t e s from c r y s t a l s t r u c t u r e r e f i n e m e n t s f o r t h e s e c a l c u l a t i o n s can, however b e q u e s t i o n e d .
I n summary on hydrogen bonding e f f e c t s , we can s a y o n l y t h a t , depending on d e t a i l s of t h e hydrogen bonding i n t e r a c t i o n s i n v o l v e d , p o s s i b l e c o n t r i b u t i o n s t o A G of f o l d i n g may b e s i g n i f i c a n t . With o u r p r e s e n t l a c k o f knowledge of r e l a t i v e hydrogen bond s t r e n g t h s o f t h e r e q u i r e d l e v e l of a c c u r a c y , we cannot d i s m i s s them from c o n s i d e r a t i o n a s p o s s i b l y i m p o r t a n t c o n t r i b u t i o n s . The energy b a l a n c e of hydrogen bonding i s complex, b u t t h a t i s n o t s u f f i c i e n t r e a s o n t o i g n o r e o r n e g l e c t i t . I n t h i s c o n t e x t , we might u s e f u l l y s t r e s s one p o s s i b l e r o l e of w a t e r a s a "hydrogen bond s i n k " i n macromolecular s t r u c t u r e s 1211. Although n a t i v e p r o t e i n s do seem t o b e v e r y e f f i c i e n t l y hydrogen bonded, w i t h o n l y a few i s o l a t e d p o l a r groups u n a b l e t o hydrogen bond 1191, each such u n s a t u r a t e d group may c o n t r i b u t e % 5 K c a l mol-l o f i n s t a b i l i t y
-
a s i g n i f i c a n t f r a c t i o n o f t h e t o t a l Ahm. The v e r s a f i l i t y o f w a t e rC7-202 JOURNAL DE PHYSIQUE
hydrogen b o n d i n g ( r e l a t i v e l y s o f t o r i e n t a t i o n a l s t e r e o c h e m i c a l c o n s t r a i n t s / 2 2 , 2 3 / ) and i t s s m a l l m o l e c u l a r s i z e a l l o w i t t o make o t h e r w i s e i m p o s s i b l e hydrogen bonds and t h u s s a v e a n o t h e r w i s e u n s t a b l e n a t i v e s t r u c t u r e . T h i s i s e s p e c i a l l y t r u e o f l a r g e r p r o t e i n s , where many i n t e r n a l w a t e r s a r e o f t e n found 1211. Without t h i s i n - t e r n a l w a t e r , t h e p r o t e i n would b e u n s t a b l e r e l a t i v e t o t h e u n f o l d e d s t a t e . (5) ASW,,,l : E n t r o p i c E f f e c t s from Water R e l e a s e
h i g i n .
I n a d d i t i o n t o t h e e n t h a l p i c consequences o f hydrogen bond s w i t c h i n g j u s t d i s c u s s e d , t h e r e w i l l b e i n a d d i t i o n an e n t r o p i c g a i n on r e l e a s i n g p r e v i o u s l y r e s t r i c t e d polar-group-bonded w a t e r m o l e c u l e s t o t h e b u l k l i q u i d . The a s s o c i a t e d c o n f i g u r a t i o n a l r e s t r i c t i o n of t h e p r o t e i n m o l e c u l e c o n s e q u e n t upon t h e t r a n s i t i o n d e s c r i b e d by e q u a t i o n (1) i s d i s c u s s e d s e p a r a t e l y below. T h i s e n t r o p i c g a i n on w a t e r r e l e a s e i s c o n c e p t u a l l y s i m i l a r t o t h e r e l e a s e o f a p o l a r r e s t r i c t e d w a t e r s t o t h e b u l k a s d i s c u s s e d w i t h r e s p e c t t o t h e h y d r o p h o b i c i n t e r a c t i o n ( / 4 1 a b o v e ) . I n t h e p r e s e n t c a s e , t h e n a t u r e o f t h e w a t e r r e s t r i c t i o n s may be d i f f e r e n t , and c l e a r l y t h e e n t h a l p i c t e r m a r i s i n g from t h e s o l u t e - s o l u t e i n t e r a c t i o n s a r e a n o r d e r of magnitude d i f f e r e n t .S o l v e n t dependence a g a i n i s c o m p l e t e . The magnitude o f t h e , e f f e c t c l e a r l y depends - - -
- - -
- -- -
-- -
-- - --
c r u c i a l l y on t h e s t a t e o f w a t e r c l o s e t o t h e v a r i o u s s u r f a c e g r o u p s .
Magnitude i s t h e r e f o r e v e r y d i f f i c u l t t o e s t i m a t e , a s o u r u n d e r s t a n d i n g o f t h e p h a s e s p a c e r e s t r i c t i o n T w a t e r n e a r p o l a r ( a s w i t h a p o l a r ) groups i s v e r y
i n c o m p l e t e . A maximum l i m i t , deduced from t h e m e l t i n g e n t r o p y of i c e , i s a b o u t -480 k c a l mol-1 f o r t h e 300 o r s o w a t e r m o l e c u l e s r e l e a s e d on f o l d i n g lysozyme from a f u l l y e x t e n d e d c o n f o r m a t i o n . Although NMR r e s u l t s s u g g e s t a few i n t e r n a l w a t e r s may b e r e s t r i c t e d t o t h i s e x t e n t / 2 4 / , most w i l l b e l e s s s o . A more r e a l i s -
t i c e s t i m a t e would p e r h a p s assume on t h e a v e r a g e a b o u t a q u a r t e r o f t h e i c e v a l u e ; even t h i s s c a l e s up t o 3 -120 K c a l m o l - l , a v a l u e comparable t o t h e h y d r o p h o b i c c o n t r i b u t i o n d i s c u s s e d e a r l i e r .
A s p e c u l a t i v e comparison of t h e thermodynamic consequences o f hydrogen-bond s w i t c h i n g d i s c u s s e d i n t h i s and t h e p r e v i o u s s e c t i o n , w i t h t h e h y d r o p h o b i c i n t e r - a c t i o n , i s p e r h a p s i n t e r e s t i n g . F o r t h e h y d r o p h o b i c c a s e , AH+
&
0 , t h e main c o n t r i b u t i o n b e i n g a n " e n t r o p y o f r e l e a s e " term._ For hydrogen b o n d i n g ,t h e d i s c u s s i o n i n s e c t i o n ( 4 ) above s u g g e s t s !AH$
1
i1
AH*/ .
Thus, u n l e s s o u r e s t i m a t e s o f t h e e n t r o p i c t e r m s i n t h e two c a s e s a r e w i d e l y i n c o r r e c t (which t h e y may b e ) , t h i s s u g g e s t s a AG+ ( d e f i n e d a s AH+-
TA S, rel) may be l a r g e r and more n e g a t i v e t h a n AG+ ! Prima f a c i e , t h e r e f o r e , t h e r e s e & s a n o n - n e g l i g i b l e c a s e f o r a " h y d r o p h i l i c e f f e c t " o f t h e same o r d e r o f m a g n i t u d e , i f n o t g r e a t e r t h a n , t h e h y d r o p h o b i c e f f e c t . C l e a r l y , o u r c u r r e n t knowledge i s i n a d e q u a t e t o a r g u e t h i s one way o r t h e o t h e r . I t d o e s , however, s u g g e s t we admit o u r i g n o r a n c e , r a t h e r t h a n i m p l i c i t l y assuming h y d r o p h o b i c dominance i n b i o m o l e c u l a r i n t e r a c t i o n s .( 6 ) A s c o n f i g .
23igip. From hydrogen bond s w i t c h i n g on f o l d i n g , a s d i s c u s s e d i n ( 4 ) and ( 5 ) above (and o t n e r d r i v i n g f o r c e s l e a d i n g t o t h e f o l d e d s t a t e ) . A c o n s i d e r a b l e l o s s of c o n f i g u r a t i o n a l freedom r e s u l t s from i n t e r n a l hydrogen bonding.
S o l v e n t dependence i s weak and i n d i r e c t .
--- ---
Magnitude a g a i n i s d i f f i c u l t t o e s t i m a t e . V a l u e s o f 2 t o 5 c a l K-' r e s i d u e mol-' h a v e b e e n q u o t e d 1251, which s c a l e up t o a n u n f a v o u r a b l e f r e e e n e r g y c o n t r i b u t i o n of 70-200 K c a l mol-l f o r lysozyme f o l d i n g . A g a i n , t h i s i s a r e l a t i v e l y l a r g e number, a b o u t a n o r d e r of m a g n i t u d e g r e a t e r t h a n t h e t o t a l f r e e e n e r g y change.
We m e n t i o n b r i e f l y h e r e some o t h e r p o s s i b l y i m p o r t a n t c o n t r i b u t i o n s f o r which o u r knowledge i s , i f a n y t h i n g , more i n c o m p l e t e t h a n t h e e f f e c t s d i s c u s s e d above.
( a ) Any change i n t h e dynamics of t h e p r o t e i n on f o l d i n g w i l l r e s u l t i n a v i b r a t i o n a l e n t r o p y c o n t r i b u t i o n . C o n v e n t i o n a l wisdom t a l k s o f a f i g h t e n - i n g up of t h e p r o t e i n on f o l d i n g , and a l s o on b i n d i n g . T h i s c o n t r i b u t i o n w i l l t h e r e f o r e be e x p e c t e d t o be u n f a v o u r a b l e . The s o l v e n t dependence i s u n c l e a r , b u t p r o b a b l y n o n - n e g l i g i b l e .
( b ) Van d e r Waals I n t e r a c t i o n s , AHvdw
Although e x p e r i m e n t a l work 121 h a s f o c u s s e d on t h e p o s s i b l y s i g n i f i c a n t magnitude of van d e r Waals e f f e c t s , l i t t l e s e r i o u s a t t e n t i o n h a s been p a i d t o t h i s p o s s i b l e s o u r c e o f s t a b i l i z i n g f r e e energy. T h i s i s p r o b a b l y b e c a u s e , i n comparison w i t h hydrogen bonding, van d e r Waals e n e r g i e s a r e r e l a t i v e l y s m a l l ( p e r h a p s an o r d e r of magnitude l o w e r ) . However, we know from c r y s t a l l o g r a p h y t h a t t h e i n t e r i o r of a p r o t e i n i s q u i t e d e n s e l y packed 126, 271, each atom group b e i n g i n r e l a t i v e l y c l o s e non-bonded c o n t a c t w i t h s e v e r a l n e i g h b o u r s . I n c o n t r a s t , i n a n extended c h a i n c o n f o r m a t i o n i n s o l v e n t , i n t e r m o l e c u l a r c l o s e c o n t a c t s would b e much l e s s e v i d e n t and i n t e r a c t i o n s w i t h s o l v e n t would be governed l a r g e l y by d i r e c t i o n a l hydrogen bonding i n t e r a c t i o n s ( f o r p o l a r groups) and t h e t r a n s i e n t ( p o s s i b l y c a g e - l i k e ) w a t e r s t r u c t u r e s around t h e a p o l a r g r o u p s . I n e i t h e r c a s e , t h e p a c k i n g d e n s i t i e s would be r e l a t i v e l y low, s a y o f t h e o r d e r of t h a t of w a t e r , which i s a p p r o x i m a t e l y o n l y 50 % of t h a t of a s i m p l e l i q u i d of atoms w i t h n o n - d i r e c t i o n a l i n t e r a t o m i c i n t e r a c t i o n s . Thus we can r e a s o n a b l y c o n c l u d e t h a t t h e p a c k i n g e f f i c i e n c y o f t h e f o l d e d p r o t e i n p l u s w a t e r system i s much g r e a t e r t h a n f o r an extended p r o t e i n p l u s w a t e r . There a r e a l s o a v e r y l a r g e number of a t o m i c c e n t r e s i n a p r o t e i n ; t h u s , even a s m a l l i n d i v i d u a l van d e r Waals e n e r g y c o u l d g r o s s up t o a l a r g e t o t a l ( f a v o u r a b l e ) energy c o n t r i b u t i o n . Assuming a n a v e r a g e van d e r Waals energy o f , s a y , 0.2 Kcal mol-l, a change i n a v e r a g e a t o m i c c o o r d i n a t i o n from 4 t o 8 on f o l d i n g , and t h a t about 50 % of t h e 1000 o r s o non-hydrogen atoms i n RNase-S o r lysozyme a r e b u r i e d , we o b t a i n a s u r p r i s i n g l y l a r g e AHvdw term of about
-
200 Kcal mol-1, l a r g e r t h a n o u r c r u d e e s t i m a t e of t h e hydrophobic c o n t r i b u t i o n .T h i s e s t i m a t e i s c o m p l i c a t e d by o t h e r e f f e c t s . For example, some of t h i s Hvdw w i l l a l r e a d y be accounted f o r i n A G ; how much i s d i f f i c u l t t o d e c i d e w i t h o u t a f u l l e r u n d e r s t a n d i n g o f t h e hydrophobic e f f e c t i t s e l f . Secondly, t h e r e
i s c r y s t a l l o g r a p h i c e v i d e n c e from s h o r t p o l a r group
-
a p o l a r group d i s t a n c e s i n p r o t e i n s f o r some enhanced d i p o l e-
induced d i p o l e i n t e r a c t i o n s which c o u l d double t h e i n d i v i d u a l van d e r Waals e n e r g i e s f o r c e r t a i n p a i r s / 9 , 1 9 , 2 1 / . Thus, o u r J A H ~ ~ ~ J e s t i m a t e may be t o o low by a s i g n i f i c a n t amount, and i t s s t a b i l i s i n g c o n t r i b u t i o n c o r r e s p o n d i n g l y g r e a t e r .Summary
From t h e above s p e c u l a t i v e d i s c u s s i o n , t h e f o l l o w i n g main p o i n t s seem t o emerge.
( a ) The o v e r a l l AG of f o l d i n g o r b i n d i n g i s s m a l l , of t h e o r d e r t y p i c a l l y of 10-20 Kcal mol-l, e q u i v a l e n t t o 2-5 h y d r o g e n b o n d s .
(b) P r o t e i n s a r e macromolecules, w i t h many i n t e r a t o m i c i n t e r a c t i o n s . Thus, even
-
s m a l l e f f e c t s a t t h e l e v e l of t h e i n d i v i d u a l i n t e r a t o m i c i n t e r a c t i o n c a n b e m a g n i f i e d t o g i v e l a r g e o v e r a l l c o n t r i b u t i o n s t o AG.
( c ) I n d i v i d u a l s o l v e n t - r e l a t e d e f f e c t s a r e p r o b a b l y s u b t l e
-
and t h e r e f o r e d i f f i c u l t t o u n d e r s t a n d a d e q u a t e l y - y e t a g g r e g a t e t o l a r ~ e c o n t r i b u t i o n s t o AG (a 100-
200 Kcal mol-1).To u n d e r s t a n d how s o l v e n t s a f f e c t s c o n t r o l o r a f f e c t b i o m o l e c u l a r p r o c e s s t h e r e f o r e p l a c e s e x t r e m e l y s e v e r e demands on t h ep r e c i s i o n o f o u r u n d e r s t a n d i n g of t h e s e e f f e c t s a t t h e m o l e c u l a r l e v e l . (d) Our c u r r e n t u n d e r s t a n d i n g i s s u c h t h a t q u a n t i t a t i v e e s t i m a t e s o f t h e v a r i o u s
s o l v e n t - r e l a t e d c o n t r i b u t i o n s t o AG+ a r e e x t r e m e l y poor.
( e ) T h e r e f o r e , i t seems dangerous t o a s s e r t which
-
i f any-
e f f e c t dominates.C7-204 JOURNAL DE PHYSIQUE
The generally-assumed dominance of hydrophobic d r i v i n g f o r c e s i s QI~ borne o u t c l e a r l y by t h i s d i s c u s s i o n . They- dominate, b u t o u r q u a n t i t a t i v e knowledge of m o l e c u l a r - l e v e l s o l v e n t e f f e c t s i s p r e s e n t l y i n a d e q u a t e t o confirm
-
o r deny-
t h i s c o n v e n t i o n a l wisdom. There a r e s t r o n g s u g g e s t i o n s - s u p p o r t e d by d i r e c t e x p e r i m e n t a l e v i d e n c e / 2 /-
t h a t hydrophobic and van d e r Waals i n t e r a c t i o n s may have been underemphasised.F i n a l l y , t h e above d i s c u s s i o n emphasises o u r need of a
much
b e t t e r u n d e r s t a n d i n g of t h e geometry and e n e r g e t i c s o f hydrogen bonding ( i ) o f w a t e r2
w a t e r ; ( i i )to
w a t e r
from
o t h e r groups ( p o l a r , a p o l a r , c h a r g e d ) . w i t h o u t a d e t a i l e d u n d e r s t a n d i n g of s i m p l e r aqueous s o l u t i o n s , it seems p e r h a p s a l i t t l e o v e r o p t i m i s t i c t o e x p e c t t o b e a b l e t o u n d e r s t a n d t h e c o m p l e x i t i e s of multicompoonent macromolecular aqueous systems.With t h i s a s background, we p r o c e e d t o d i s c u s s some p o s s i b l e approaches a t t h e l e v e l s of b o t h "simple" model s y s t e m s , and w e l l - c h a r a c t e r i s e d macromolecular a s s e m b l i e s , t o t h e s e problems o f b i o m o l e c u l a r s o l v e n t e f f e c t s .
I V . A WAY FORWARD ?
Two major a s p e c t s o f w a t e r i n b i o l o g i c a l s y s t e m s seem t o be p a r t i c u l a r l y r e l e v a n t t o t h e d e s i g n of e x p e r i m e n t a l and t h e o r e t i c a l approaches aimed t o improve o u r u n d e r s t a n d i n g of s o l v e n t e f f e c t s . F i r s t , t h e systems themselves a r e complex, c o n t a i n i n g many components. I t would t h u s be o v e r o p t i m i s t i c t o e x p e c t c o n c l u s i v e r e s u l t s from s i n g l e e x p e r i m e n t s . R a t h e r t h i s complexity i m p l i e s t h a t a v a r i e t y of h o p e f u l l y complementary e x p e r i m e n t a l and t h e o r e t i c a l t e c h n i q u e s a r e needed t o p r o b e
(a) p e r t u r b a t i o n s o f t h e b i o m o l e c u l a r system by t h e s o l v e n t , and (b) p e r t u r b a t i o n s of t h e s o l v e n t by t h e b i o m o l e c u l a r s o l u t e , and o t h e r components t h a t may b e i n t h e system.
G e n e r a l l y , t h e f i r s t approach i s more t r a c t a b l e , and l i k e l y t o y i e l d r e s u l t s l e a s t p a i n f u l l y , o n e example b e i n g t h e m u l t i t e c h n i q u e i n v e s t i g a t i o n of lysozyme h y d r a t i o n and a c t i v a t i o n d i s c u s s e d i n s e c t i o n 2 above. I t i s much more d i f f i c u l t t o e x t r a c t c l e a r c o n c l u s i o n s from t h e second approach - l o o k i n g f o r p e r t u r b a t i o n s o f s o l v e n t by t h e b i o m o l e c u l e . A s we have a r g u e d i n t h e p r e c e d i n g s e c t i o n , t h e s e
- a r e l i k e l y t o be s m a l l , and hence d i f f i c u l t b o t h t o d e t e c t and t o c h a r a c t e r i s e . Furthermore, t h e p e r t u r b e d s o l v e n t i n a r e a s o n a b l y t r a c t a b l e model system i s
l i k e l y t o be only a s m a l l f r a c t i o n o f t h e
total
s o l v e n t , and t h u s t h e e f f e c t s of t h e p e r t u r b a t i o n s w i l l b e , f o r most e x p e r i m e n t a l t e c h n i q u e s , d i l u t e d . Moreover, i n most c a s e s , a n i n t e r p r e t i v e model i s needed which i t s e l f may b e m i s l e a d i n g , and hence y i e l d e r r o n e o u s c o n c l u s i o n s . A p a r t i c u l a r example i s t h e u s e of NMR t o t r y t o measuret h e p e r t u r b a t i o n of w a t e r dynamics c l o s e t o a p r o t e i n , where v e r y d i f f e r e n t con- c l u s i o n s have been drawn by d i f f e r e n t w o r k e r s , p a r t l y b e c a u s e of t h e d i f f e r e n t i n t e r p r e t i v e models used / 2 8 , 2 9 / . A review of t h e t e c h n i q u e s used t o s t u d y w a t e r p e r t u r b a t i o n s , and t h e i r r e s u l t s , i s g i v e n e l s e w h e r e / 2 9 / .
The s u b t l e t y of t h e s o l v e n t e f f e c t s s t r o n g l y i m p l i e s we s h o u l d a l s o t r y t o g a i n a b e t t e r u n d e r s t a n d i n g o f s i m p l e r aqueous systems
-
i n p a r t i c u l a r aqueous s o l u t i o n s of model molecules c o n t a i n i n g t h e chemical groups o f p a r t i c u l a r i n t e r e s t i n our b i o m o l e c u l a r system. We e s s e n t i a l l y need a much d e e p e r u n d e r s t a n d i n g a t t h em o l e c u l a r l e v e l of hydrogen bonding, hydrophobic i n t e r a c t i o n s , and aqueous s o l u t i o n s of i o n s , p o l a r , and a p o l a r m o l e c u l e s .
We c a n d i s c u s s t h e i n f o r m a t i o n we need i n terms of t h e two t y p e s o f c o n t r i b u t i o n s t o f r e e e n e r g y o f f o l d i n g d i s c u s s e d i n t h e p r e v i o u s s e c t i o n , namely e n t h a l p i c (ener- g e t i c ) and e n t r o p i c c o n t r i b u t i o n s . The f i r s t l a r g e l y depends d i r e c t l y on t h e i n t e r - m o l e c u l a r f o r c e s
-
c o d s i d e r e d h e r e a s e s s e n t i a l l y two body e f f e c t s . I f we do n o t know t h e r e l a t i v e s t r e n g t h s of NH-water and water-water hydrogen bonds, we saw i n t h e p r e v i o u s s e c t i o n t h a t we cannot s a y much a b o u t even t h e s i g n of t h e r e s ~ l t a n t i n t e r n a l e n e r g y c o n t r i b u t i o n t o AG. The prime need h e r e , t h e r e f o r e , i s t o o b t a i n a b e t t e r b a s i c u n d e r s t a n d i n g of i n t e r m o l e c u l a r f o r c e s i n s i m p l e r systems. T h i s i n i t s e l f i s a major, somewhat t e d i o u s , problem, w i t h a l o n g h i s t o r y . Most " d a t a b a s e s "of i n t e r m o l e c u l a r f o r c e s a r e d e r i v e d from f i t t i n g e x p e r i m e n t a l d a t a t o assumed a n a l y t i c a l forms f o r t h e i n t e r a c t i o n s ( e . g . r e f . / 3 0 / ) . O t h e r approaches v i a
quantum mechanics appear t o b e i n p r i n c i p l e i n c a p a b l e of y i e l d i n g t h e r e q u i r e d a b s o l u t e e n e r g i e s t o b e t t e r t h a n a b o u t 1 K c a l mol-l ( / l o / , 1 2 3 1 ) . We know from o u r e a r l i e r work t h a t s o l v e n t o r g a n i s a t i o n i n r e l a t i v e l y s i m p l e s y s t e m s i s s t r o n g l y dependent on t h e c h o i c e of p o t e n t i a l f u n c t i o n from t h e l i t e r a t u r e 1321. I n a d d i t i o n t o p u r s u i n g new approaches ( / l o / , 1311) p e r h a p s we s h o u l d t r y t o a s s e s s r e a l i s t i c a l l y how a c c u r a t e p o t e n t i a l f u n c t i o n s must b e i n o r d e r t o b e u s e f u l i n a b i o m o l e c u l a r c o n t e x t .
Perhaps even more d i f f i c u l t t o u n d e r s t a n d a r e t h e e n t r o p i c e f f e c t s , which, a s i n t h e hydrophobic i n t e r a c t i o n d i s c u s s e d above, depend on changes i n t h e r e s t r i c t i o n of w a t e r m o l e c u l e s a s t h e y move from t h e neighbourhood of a n a p o l a r group t o t h e b u l k l i q u i d . These e f f e c t s a r e e s s e n t i a l l y many-body, and hence d i f f i c u l t t o h a n d l e t h e o r e t i c a l l y . As t h e y depend upon a s u b t l e i n t e r p l a y between t h e f o r c e s i n v o l v e d , t h e y a r e a g a i n l i k e l y t o b e v e r y s t r o n g l y dependent upon b o t h r e l a t i v e s t r e n g t h s and o r i e n t a t i o n a l dependencies of t h e assumed p o t e n t i a l f u n c t i o n s . Such problems can b e t a c k l e d i n p r i n c i p l e by computer s i m u l a t i o n t e c h n i q u e s . I n p r a c t i c e , however, t h e r e a r e problems 1331, 1341, and i t t h e r e f o r e seems e s s e n t i a l t o check we c a n reproduce e x p e r i m e n t a l d a t a i n r e a l , w e l l - c h a r a c t e r i s e d ( s i m p l e and complex) s y s t e m s b e f o r e moving on t o p r o b e more b i o l o g i c a l l y i n t e r e s t i n g systems ( s e e below).
Thus we would a r g u e f o r an approach t o b i o m o l e c u l a r s o l v a t i o n problems on two l e v e l s , which can b e f o l l o w e d i n p a r a l l e l . F i r s t , a t t h e more b a s i c l e v e l , p o t e n t i a l func- t i o n s f o r t h e v a r i o u s i n t e r a c t i o n s r e q u i r e d e v e l o p i n g and t e s t i n g w e l l - c h a r a c t e r i s e d systems. These t e s t systems a r e b o t h s u i t a b l y - c h o s e n h y d r a t e c r y s t a l s , and aqueous s o l u t i o n s f o r which d i r e c t i n f o r m a t i o n on w a t e r c o o r d i n a t i o n around r e l e v a n t groups can be o b t a i n e d by t h e n e u t r o n f i r s t o r d e r d i f f e r e n c e method developed f o r e l e c t r o - l y t e s o l u t i o n s by Enderby and N e i l s o n / 3 5 / . ( S e e a l s o t h e p a p e r by N e i l s o n i n t h i s volume).On t h e complex system l e v e l , r e c e n t developments i n h i g h r e s o l u t i o n
r e f i n e m e n t of macromolecule c r y s t a l s g i v e us w e l l - c h a r a c t e r i s e d ( p o s s i b l y ~ a r t i a l l y - o r d e r e d ) s o l v e n t i n f o r m a t i o n which can t h e n be used t o t e s t ( u s i n g computer s i m u l a t - i o n c a l c u l a t i o n s ) t h e p o t e n t i a l f u n c t i o n s developed from t h e s i m p l e r system work.
Once a d e q u a t e agreement (which i s n o t e a s y t o a s s e s s / 3 6 / , / 3 7 / ) i s o b t a i n e d i n such w e l l c h a r a c t e r i s e d s y s t e m s , we w i l l have some c o n f i d e n c e i n e x t e n d i n g t h e compu- t a t i o n a l work t o s o l u t i o n c o n d i t i o n s o f b i o l o g i c a l i n t e r e s t , s u c h a s enzyme-substrate i n t e r a c t i o n s (F. Sussman, J . M . Goodfellow, J.L. Finney and P . B a r n e s , u n p u b l i s h e d work)
.
V. SOME EXAMPLES
To i l l u s t r a t e t h e s e approaches a t t h e two l e v e l s , we summarise h e r e some r e s u l t s of r e c e n t ( s o f a r u n p u b l i s h e d ) work f r o m t h e B i r k b e c k l a b ~ r a t o r ~ . I n t h e examples chosen, t h e use of n e u t r o n s h a s been c r u c i a l i n o b t a i n i n g t h e e x p e r i m e n t a l d a t a , which i n some c a s e s h a s begun t o throw new l i g h t on w a t e r i t s e l f , w i t h i n t e r e s t i n g i m p l i c a - t i o n s f o r how we s h o u l d modify t h e way we model t h e w a t e r i n t e r a c t i o n s t h e m s e l v e s .
(1) H y d r a t i o n of aqueous s o l u t i o n s of b i o l o g i c a l l y r e l e v a n t p o l a r m o l e c u l e s . T h i s i s p a r t o f o u r w i d e r programme a t b o t h ILL and t h e R u t h e r f o r d Appleton Labo- r a t o r y d e s i g n e d t o s t u d y d i r e c t l y t h e h y d r a t i o n of v a r i o u s groups ( c h a r g e d , p o l a r , a p o l a r ) i n b i o l o g i c a l l y - r e l e v a n t model m o l e c u l e s . I t a p p l i e s t h e n e u t r o n i s o t o p e d i f f e r e n c e method a s developed by Enderby and N e i l s o n 1351 t o molecules i n aqueous s o l u t i o n . I n o u r i n i t i a l a p p l i c a t i o n t o p e p t i d e group h y d r a t i o n , we a r e u s i n g n i t r o g e n i s o t o p e s u b s t i t u t i o n i n a s e r i e s o f amides c u l m i n a t i n g w i t h t h e p e p t i d e model N-methyl a c e t a m i d e . Thus, we can e f f e c t i v e l y s i t on t h e n i t r o g e n atom, measure d i r e c t l y t h e w a t e r c o o r d i n a t i o n of t h e -ND and -ND2 g r o u p s , and t e s t t h e o r - e t i c a l p r e d i c t i o n s a g a i n s t t h e e x p e r i m e n t a l d a t a . I n a d d i t i o n , d a t a can be o b t a i n e d on any changes t h a t may o c c u r i n t h e s t r u c t u r e o f t h e molecule i t s e l f i n s o l u t i o n . So f a r , we have examined o u r p r e l i m i n a r y d a t a on u r e a 1381 (of i n t e r e s t i t s e l f a s a p r o t e i n d e n a t u r a n t p r o b a b l y o ~ e r a t i n g through s o l v e n t e f f e c t s ) , and have concluded t h a t t h e ND2 group i s n o t s t r o n g l y h y d r a t e d , t h u s e n a b l i n g us t o r e j e c t s t r o n g h y d r a t i o n models. The n i t r o g e n - w a t e r c o r r e l a t i o n f u n c t i o n s o b t a i n e d from two computer s i m u l a t i o n s u s i n g two d i f f e r e n t w a t e r models (ST2 and TlPS2) a r e both a t v a r i a n c e w i t h t h e e x p e r i m e n t a l r e s u l t s , i m p l y i n g n e i t h e r p o t e n t i a l i s a d e q u a t e
JOURNAL
DE
PHYSIQUEf o r b i o m o l e c u l e s i m u l a t i o n s t u d i e s (J. T u r n e r , u n p u b l i s h e d work). H y d r a t i o n models based on quantum mechanical c a l c u l a t i o n s u s i n g t h e s u p e r m o l e c u l e approach /39/ a r e a l s o a t v a r i a n c e w i t h t h e e x p e r i m e n t a l r e s u l t s . T h i s l a t t e r c o n c l u s i o n may have some s e r i o u s i m p l i c a t i o n s f o r t h e v a l i d i t y of s i m i l a r s m a l l b a s i s - s e t quantum mechanical h y d r a t i o n models of more complex systems ( e . g . DNA).
A more d e t a i l e d a n a l y s i s of d a t a on u r e a and o t h e r amides i s c u r r e n t l y i n p r o g r e s s . Other e x p e r i m e n t a l work on m o l e c u l a r i o n s and b i f u n c t i o n a l ( p o l a r and a p o l a r ) molecules i s e i t h e r under way, o r p l a n n e d . With t h e almost q u a l i t a t i v e i n s t r u m e n t a l r e c e n t improvements a t t h e ILL (D4B, and h o p e f u l l y D20), and t h e a d v e n t of t h e SERC s p a l l a t i o n s o u r c e a t t h e R u t h e r f o r d Appleton L a b o r a t o r y , t h e p r o s p e c t s f o r s o r t i n g
o u t d i r e c t l y t h e h y d r a t i o n of c h a r g e d , p o l a r , and even a p o l a r groups i n molecules of b i o l o g i c a l i n t e r e s t , a r e e x t r e m e l y b r i g h t .
(2) S o l v e n t O r g a n i s a t i o n i n coenzyme B12
A h i g h r e s o l u t i o n (% 0.9
i)
n e u t r o n and X-ray r e f i n e m e n t o f (H-D exchanged) v i t a m i n B12 coenzyme h a s r e s u l t e d i n a n e x t r e m e l y d e t a i l e d model of t h e w a t e r o r g a n i s a t i o n i n t h e c r y s t a l . T h i s molecule ( m o l e c u l a r weight 1580 D a l t o n s , about 56 w a t e r molecules i n t h e u n i t c e l l ) was chosen i n p r e f e r e n c e t o a s m a l l p r o t e i n a s i t was known t o d i f f r a c t t o v e r y h i g h r e s o l u t i o n , l a r g e c r y s t a l s s u i t a b l e f o r n e u t r o n d i f f r a c t i o n could be grown, and t h e molecule c o n t a i n e d chemical groups r e l e v a n t t o b o t h p r o t e i n s and n u c l e i c a c i d s . The aims of t h i s work were two f o l d . F i r s t , t o o b t a i n a r e l i a b l e model of t h e w a t e r o r g a n i s a t i o n i n a biomacromolecule c r y s t a l , which would g i v e us d i r e c t i n f o r m a t i o n on t h e h y d r a t i o n of r e l e v a n t s u r f a c e g r o u p s , t o g e t h e r w i t h i n f o r - mation on water-water i n t e r a c t i o n s i n t h e neighbourhood of t h e s u r f a c e . Secondly, g i v e n a w e l l - c h a r a c t e r i z e d s o l v e n t o r g a n i s a t i o n , t h i s c o u l d t h e n b e used t o t e s t computer s i m u l a t i o n p r e d i c t i o n s , i n o r d e r t o a s s e s s t h e v i a b i l i t y of o u r c u r r e n t models o f w a t e r i n t e r a c t i o n s . F o r b o t h p u r p o s e s , d a t a approaching a t o m i c r e s o l u t i o n i s e s s e n t i a l i f many of t h e u n c e r t a i n t i e s i n h e r e n t i n u s i n g s o l v e n t d a t a from macromolecule c r y s t a l s a r e t o be a v o i d e d / 2 1 / , / 4 0 / , / 4 1 / . Neutrons were e s s e n t i a l a s i n f o r m a t i o n on w a t e r o r i e n t a t i o n s r e q u i r e s t h e l o c a t i o n of t h e w a t e r hydrogens( d e u t e r o n s ) ; our p r e v i o u s knowledge of o r i e n t a t i o n s was e x t r e m e l y s p a r s e . The d a t a was t a k e n on D 8 a t t h e ILL w i t h P.A. Timmins, P.F. L i n d l e y , and J. A l l i b o n / 4 2 / , and t h e r e f i n e m e n t was performed by H.F.J. Savage ( / 1 5 / ; a l s o u n p u b l i s h e d work). Some of t h e main r e s u l t s of t h i s s o l v e n t r e f i n e m e n t c a n be summarised a s f o l l o w s .
(a) Two s o l v e n t r e g i o n s
-
t h e p o c k e t and t h e c h a n n e l - a r e s e p a r a t e d by a ( p a r t i a l l y - o r d e r e d ) a c e t o n e molecule ( c r y s t a l s were grown from acetone-waters o l u t i o n s ) . I n t h e p o c k e t r e g i o n when t h e a c e t o n e molecule i s p r e s e n t , t h e r e i s one major w a t e r network, though w i t h some l o c a l d i s o r d e r . The s o l v e n t i n t h e c h a n n e l i s l e s s w e l l - o r d e r e d , and s e v e r a l s e l f - c o n s i s t e n t d i f f e r e n t networks c a n b e proposed from t h e d a t a . Thus, t h e w a t e r i n t h i s c r y s t a l i s p a r t l y o r d e r e d and p a r t l y
d i s o r d e r e d , p r o v i d i n g a c r i t i c a l t e s t of models o f w a t e r i n t e r a c t i o n s .
(b) The w a t e r c o o r d i n a t i o n i s v a r i a b l e , w i t h a b o u t 7 0 % b e i n g 3-coordinated ( o f t e n t r i g o n a l p l a n a r ) and 30 % having f o u r - f o l d c o o r d i n a t i o n . T h i s v a r i a b l e , mainly t r i g o n a l , c o o r d i n a t i o n i s c o n s i s t e n t w i t h o t h e r t l ~ e o r e t i c a l and e x p e r i m e n t a l work which a r g u e s t h a t t h e generally-assumed t e t r a h e d r a l i t y of w a t e r molecule i n t e r a c t i o n s i s o f t e n o v e r - s t r e s s e d / 1 9 / , / 2 2 / , / 2 3 / . I t a l s o u n d e r l i n e s t h e "bonding" v e r s a - t i l i t y of t h e w a t e r molecule which e n a b l e s i t t o soak up hydrogen-bond donor/
a c c e p t o r imbalance though i t s a c t i o n a s a "hydrogen bond s i n k " ( s e c t i o n 3.4 above).
( c ) D i s t r i b u t i o n s of hydrogen bond d i s t a n c e s ( b o t h f o r water-water and w a t e r - coenzyme i n t e r a c t i o n s ) a r e c o n s i s t e s t w i t h g e n e r a l l y - a c c e p t e d v a l u e s , w i t h a few s h o r t d i s t a n c e s down t o 2.4 o r 2.5 A. These s h o r t d i s t a n c e s a r e p r o b a b l y r e a l , and a g a i n p r e s e n t a d i f f i c u l t t e s t f o r o u r models o f w a t e r i n t e r a c t i o n s , which g e n e r a l l y seem t o g i v e narrow hydrogen bond d i s t a n c e d i s t r i b u t i o n s w i t h a h i g h e r s h o r t d i s t a n c e c u t o f f ( s e e n e x t s e c t i o n ) . Hydrogen bond a n g l e s a r e a l s o c o n s i s t e n t w i t h e x p e c t a t i o n s from s m a l l molecule s t r u c t u r e s . The m a j o r i t y of X-D...Y a n g l e s
l i e between 150° and 170".
(d) Two p a r t i c u l a r l y i n t e r e s t i n g r e s u l t s emerged from l o c a t i n g t h e hydrogen p o s i t i o n s , which a p p e a r n o t t o have been n o t i c e d p r e v i o u s l y , and which may w e l l h a t e i m p l i c a t i o n s f o r t h e way we normally model w a t e r w i t h o u t e x p l i c i t hydrogen c e n t r e s . F i r s t , around t h e coenzyme c a r b o n y l g r o u p s , t h e w a t e r oxygens appear t o
