Significance of Macromolecular Interaction and Stability in Functional

Chapter 2

Significance of Macromolecular Interaction and Stability in Functional Properties of Food Proteins

Downloaded by COLUMBIA UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: February 19, 1991 | doi: 10.1021/bk-1991-0454.ch002

Akio Kato Department of Agricultural Chemistry, Yamaguchi University, Yamaguchi 753, Japan

Beside protein hydrophobicity, the role of macromolecular interaction and conformational stability in the functional properties of proteins are described here. The importance of protein association in the foaming properties was confirmed using various polymeric forms of ovomucin or heat-induced ovalbumin aggregates, while electrostatic repulsion enhancing protein dissociation was indicated to be an important factor in the emulsifying properties of proteins using the polyanionic proteins, ovomucin and phosvitin. The role of conformational stability of proteins in the surface properties was proved by a protein engineering approach using several mutant tryptophan synthases with different stabilities which were obtained by a single amino acid substitution at position 49. Good correlations were observed between the conformational stability and the surface properties of mutant tryptophan synthases. R e c e n t l y many attempts t o d e v e l o p new f u n c t i o n a l f o o d p r o t e i n s have been done by f o o d r e s e a r c h e r s t o meet t h e w o r l d ' s p r o t e i n needs. A t the same t i m e , s i n c e i t i s o f importance t o e l u c i d a t e t h e m o l e c u l a r b a s i s o f p r o t e i n f u n c t i o n a l i t y f o r d e v e l o p i n g new f u n c t i o n a l p r o t e i n s , many f o o d c h e m i s t s have f o c u s e d t h e i r e f f o r t s on e l u c i d a t i n g t h e r e l a t i o n s between t h e s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s . Of t h e f u n c t i o n a l p r o p e r t i e s o f f o o d p r o t e i n s , much emphasis has been p l a c e d on s u r f a c e p r o p e r t i e s such as foaming and e m u l s i f y i n g p r o p e r t i e s because they a r e c r i t i c a l elements i n t h e development o f new f o o d p r o t e i n s . I f we c a n know t h e r e l a t i o n s h i p s between t h e s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s , i t would be p o s s i b l e t o make an e d u c a t e d d e s i g n o f new f u n c t i o n a l p r o t e i n s by means o f v a r i o u s p r o t e i n m o d i f i c a t i o n i n c l u d i n g p r o t e i n e n g i n e e r i n g . However, o u r u n d e r s t a n d i n g o f t h e d e t e r m i n a n t s o f p r o t e i n f u n c t i o n a l p r o p e r t i e s i s s t i l l l i m i t e d . We have p r o p o s e d (1) t h a t t h e s u r f a c e hydrophobicity o f p r o t e i n s i s the important s t r u c t u r a l f a c t o r

0097-6156/91/0454-0013$06.00/0 © 1991 American Chemical Society In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


14

INTERACTIONS OF FOOD PROTEINS

g o v e r n i n g f u n c t i o n a l p r o p e r t i e s . Many s t u d i e s (2-5) have s u p p o r t e d our h y p o t h e s i s . I n a d d i t i o n t o s u r f a c e h y d r o p h o b i c i t y o f p r o t e i n s , the r e l a t i o n o f p r o t e i n a s s o c i a t i o n / d i s s o c i a t i o n and p r o t e i n s t a b i l i t y t o t h e f u n c t i o n a l p r o p e r t i e s o f f o o d p r o t e i n s s h o u l d be s t u d i e d . However, t h e s e s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s a r e d i f f i c u l t t o d e f i n e because t h e s t r u c t u r a l p r o p e r t i e s o f p r o t e i n s compared i n many e x p e r i m e n t s , namely, m o l e c u l a r s i z e , n e t c h a r g e , s t a b i l i t y and s u r f a c e h y d r o p h o b i c i t y , were markedly d i f f e r e n t from one a n o t h e r . I n t h i s paper we use model p r o t e i n s t o g e t c l e a r - c u t s t r u c t u r a l and f u n c t i o n a l r e l a t i o n s h i p . F i r s t , e f f e c t s o f p r o t e i n a s s o c i a t i o n and d i s s o c i a t i o n on t h e e m u l s i f y i n g and foaming p r o p e r t i e s were i n v e s t i g a t e d u s i n g v a r i o u s p o l y m e r i c forms o f ovomucin and h e a t - i n d u c e d s o l u b l e a g g r e g a t e s o f o v a l b u m i n . Second, r o l e s o f e l e c t r o s t a t i c r e p u l s i o n enhancing p r o t e i n d i s s o c i a t i o n i n the f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s were s t u d i e d u s i n g p o l y a n i o n i c p r o t e i n s , ovomucin and p h o s v i t i n . F i n a l l y , s i g n i f i c a n c e o f p r o t e i n s t a b i l i t y on t h e f u n c t i o n a l p r o p e r t i e s was i n v e s t i g a t e d u s i n g mutant t r y p t o p h a n s y n t h a s e s p r e p a r e d by p r o t e i n e n g i n e e r i n g p r o c e d u r e . I t i s s t r o n g l y emphasized h e r e t h a t p r o t e i n e n g i n e e r i n g i s a v e r y p o w e r f u l approach t o i n t e r p r e t a t i o n o f t h e s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s o f food p r o t e i n s . M a t e r i a l s - and Methods Ovomucin was p r e p a r e d by t h e method d e s c r i b e d i n a p r e v i o u s p a p e r ( 6 ) . Ovomucin g e l was s o l u b i l i z e d i n 1/20 M T r i s - H C l b u f f e r a t pH 8. The s o l u b l e ovomucin was t r e a t e d w i t h a s o n i c a t o r a t 5 A f o r 5 min a t 20 °C. The s o n i c a t e d ovomucin was f u r t h e r r e d u c e d w i t h 0.01 M mercaptoethanol. Ovalbumin was c r y s t a l l i z e d w i t h sodium s u l f a t e from f r e s h egg w h i t e and r e c r y s t a l l i z e d f i v e t i m e s . The h e a t - i n d u c e d s o l u b l e a g g r e g a t e s o f ovalbumin were formed by h e a t i n g t o 80 °C i n 67 mM phosphate b u f f e r a t pH7 c o n t a i n i n g 0.1 M NaCl w i t h an i n c u b a t o r a t a r a t e o f 3°C/min and t h e n i m m e d i a t e l y c o o l e d a t room temperature f o r 0, 3 and 6 h r s t o g e t d i f f e r e n t m o l e c u l a r w e i g h t s o f a g g r e g a t e s . The m o l e c u l a r w e i g h t s o f s o l u b l e a g g r e g a t e s were d e t e r m i n e d by t h e lowa n g l e l a s e r l i g h t s c a t t e r i n g t e c h n i q u e combined w i t h h i g h performance g e l chromatography ( 7 ) . P h o s v i t i n was p r e p a r e d by t h e method o f Mecham and O l c o t t (8) and f u r t h e r p u r i f i e d by i o n exchange chromatography on a DEAESephadex A-50. The w i l d - t y p e t r y p t o p h a n s y n t h a s e α-subunits were o b t a i n e d by the method o f Y u t a n i e t a l . ( 9 ) . S i x mutant p r o t e i n s s u b s t i t u t e d by a l a n i n e , g l y c i n e , i s o l e u c i n e , l y s i n e , p h e n y l a l a n i n e and t h r e o n i n e i n p l a c e o f g l u t a m i c a c i d a t p o s i t i o n 49 were o b t a i n e d by s i t e - d i r e c t e d mutagenesis u s i n g s y n t h e t i c o l i g o n u c l e o t i d e s ( 1 0 ) . The s i n g l e - s t r a n d e d DNA t e m p l a t e used f o r t h e mutagenesis was an Ml3mpll d e r i v a t i v e c o n t a i n i n g t h e α-subunit gene i s o l a t e d from an H i n d i fragment(56326925) o f t r y p t o p h a n o p e r o n . A p l a s m i d d e r i v e d from p l a s m i d pUC8 (Pharmacia) c o n t a i n i n g t h e t r y p t o p h a n promoter i n t r o d u c e d from pDR720 (Pharmacia) was used as t h e e x p r e s s i o n v e c t o r . P u r i f i e d mutants (20-100 mg) were o b t a i n e d from 5 l i t e r s o f b r o t h . A quantitative method i s r e q u i r e d t o s t u d y t h e r e l a t i o n s h i p between t h e s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s . P e a r c e and K i n s e l l a ( 1 1 ) have d e v e l o p e d t h e t u r b i d i t y method t o measure

In Interactions of Food Proteins; Parris, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

2. KATO

Macromolecular Interaction and Stability

15

the e m u l s i f y i n g p r o p e r t i e s . We have d e v e l o p e d a c o n d u c t i v i t y method t o e s t i m a t e the foaming p r o p e r t i e s (12). These methods were used t o d e t e r m i n e the e m u l s i f y i n g and foaming p r o p e r t i e s , because o f t h e i r r e l i a b l e and q u a n t i t a t i v e y i e l d s .


Results

and

Discussion

E f f e c t s o f P r o t e i n - P r o t e i n I n t e r a c t i o n on Foaming P r o p e r t i e s . The r o l e of p r o t e i n - p r o t e i n i n t e r a c t i o n i n surface f u n c t i o n a l properties i s f i r s t s t u d i e d u s i n g v a r i o u s p o l y m e r i c forms o f ovomucin. Ovomucin i s a g l y c o p r o t e i n r e s p o n s i b l e f o r g e l s t r u c t u r e o f t h i c k egg w h i t e . And i t i s w e l l known as the b e s t foaming p r o t e i n among n a t u r a l p r o t e i n s . S o l u b l e ovomucin i s a p o l y m e r i c form (8300 KDa) of h e t e r o g e n e o u s s u b u n i t s c o n s i s t i n g o f a - and (3-ovomucin, and i s d i s s o c i a t e d with sonication or reduction i n t o smaller units of m o l e c u l a r weight o f 1100 KDa and 540 KDa, r e s p e c t i v e l y , w i t h o u t changes i n c h e m i c a l c o m p o s i t i o n (13). As shown i n F i g u r e 1, good foaming p r o p e r t i e s a r e o b s e r v e d i n t h e h i g h l y p o l y m e r i z e d form o f ovomucin, w h i l e i t d e c r e a s e s i n the d i s s o c i a t e d forms, depending on the m o l e c u l a r w e i g h t . On the o t h e r hand, the e m u l s i f y i n g p r o p e r t i e s o f ovomucins showed a good c o r r e l a t i o n w i t h t h e s u r f a c e h y d r o p h o b i c i t y , not w i t h the polymerization(14). S i m i l a r l y , v a r i o u s p o l y m e r i c forms o f ovalbumin a g g r e g a t e s were used t o s t u d y the e f f e c t o f p r o t e i n a s s o c i a t i o n on the s u r f a c e p r o p e r t i e s . When ovalbumin i s h e a t e d a t c o n t r o l l e d c o n d i t i o n s (at 80 ° C , pH 7.4), i t forms s o l u b l e a g g r e g a t e s . The average m o l e c u l a r w e i g h t s are i n c r e a s e d w i t h p r o l o n g e d s t a n d i n g a t room t e m p e r a t u r e a f t e r h e a t - t r e a t m e n t ( 7 ) . F i g u r e 2 shows the foaming p r o p e r t i e s o f s o l u b l e ovalbumin a g g r e g a t e s h e a t e d a t 80°C and then c o o l e d a t room temperature f o r 0, 3, 6 h r whose m o l e c u l a r w e i g h t s are 6,000,000, 7,800,000 and 9,500,000, r e s p e c t i v e l y . The foaming p r o p e r t i e s i n c r e a s e d w i t h i n c r e a s i n g m o l e c u l a r w e i g h t o f the s o l u b l e a g g r e g a t e s . In c o n t r a s t , the e m u l s i f y i n g p r o p e r t i e s were found t o be u n a f f e c t e d (data a r e n o t shown h e r e ) . From t h e s e r e s u l t s , i t i s s u g g e s t e d t h a t p r o t e i n a s s o c i a t i o n i s an i m p o r t a n t s t r u c t u r a l f a c t o r g o v e r n i n g the foaming p r o p e r t y , w h i l e the e m u l s i f y i n g p r o p e r t i e s are h a r d l y r e l a t e d t o such e v e n t s . Role o f e l e c t r o s t a t i c r e p u l s i o n i n the e m u l s i f y i n g p r o p e r t i e s . ! 1 l u s t r a t i o n o f the r o l e o f p r o t e i n - p r o t e i n i n t e r a c t i o n i n the s u r f a c e p r o p e r t i e s was a l s o a c h i e v e d when p o l y a n i o n - t y p e p r o t e i n s were used which i n h i b i t the i n t e r a c t i o n a t the i n t e r f a c e . Ovomucin and p h o s v i t i n were used as p o l y a n i o n - t y p e p r o t e i n s . Ovomucin has about 10 % s i a l i c a c i d s i n the t e r m i n a l c a r b o h y d r a t e c h a i n s , and p h o s v i t i n have about 30 % phosphate bound t o s e r i n e r e s i d u e s . These t e r m i n a l p o l y a n i o n s can e a s i l y be removed by e n z y m a t i c a c t i o n w i t h o u t a f f e c t i n g p e p t i d e bonds. F i g u r e 3 shows the e f f e c t o f t e r m i n a l s i a l i c a c i d r e s i d u e s on the e m u l s i f y i n g p r o p e r t y o f ovomucin (15). The t e r m i n a l s i a l i c a c i d was removed by s i a l i d a s e . Removal o f s i a l i c a c i d s g r e a t l y a f f e c t e d the f u n c t i o n a l p r o p e r t i e s . The e m u l s i f y i n g p r o p e r t i e s o f ovomucin were g r e a t l y d e c r e a s e d by the removal o f s i a l i c a c i d , s u g g e s t i n g t h a t s i a l i c a c i d p l a y s an i m p o r t a n t r o l e f o r the e m u l s i f y i n g p r o p e r t i e s . On the o t h e r hand, the foaming p r o p e r t i e s o f ovomucin were i n c r e a s e d by the removal o f s i a l i c a c i d , as shown i n a p r e v i o u s p a p e r ( 1 5 ) . T h i s may be because the d e c r e a s e i n n e g a t i v e l y




T I M E , min F i g u r e 1. Foaming p r o p e r t i e s o f native(·) , s o n i c a t e d ( • ) and r e d u c e d (O) ovomucins

TIME,min F i g u r e 2. Foaming p r o p e r t i e s o f the s o l u b l e a g g r e g a t e s o f ovalbumin h e a t e d t o 80 C and then c o o l e d a t room temperature f o r 0 (·) , 3 ( H ) and 6 (O) hours



2. KATO


17

c h a r g e d s i a l i c a c i d s enhances the i n t e r a c t i o n o f ovomucin m o l e c u l e s a t a i r - w a t e r i n t e r f a c e , t h u s l e a d i n g t o s t r e n g t h e n i n g o f the foam film. F i g u r e 4 shows the e f f e c t o f phosphate r e s i d u e s on the e m u l s i f y i n g p r o p e r t i e s o f p h o s v i t i n (16).The e m u l s i f y i n g p r o p e r t i e s were g r e a t l y d e c r e a s e d by removal o f phosphate by p h o s p h a t a s e . The foaming p r o p e r t i e s o f p h o s v i t i n were too p o o r t o d e t e c t any changes. Thus, removal o f s i a l i c a c i d and phosphate from ovomucin and phosvitin, respectively, greatly affected their functional properties. The foaming p r o p e r t i e s o f t h e s e p r o t e i n s were promoted by the removal o f p o l y a n i o n s , w h i l e the e m u l s i f y i n g p r o p e r t i e s were d e c r e a s e d . A c c o r d i n g l y , t h e s e r e s u l t s i l l u s t r a t e the importance o f e l e c t r o s t a t i c r e p u l s i v e f o r c e s i n the s t a b i l i z a t i o n o f e m u l s i o n d r o p l e t s . These o b s e r v a t i o n s run p a r a l l e l w i t h our p r o p o s a l t h a t surface h y d r o p h o b i c i t y i s i m p o r t a n t i n the e m u l s i f y i n g p r o p e r t i e s o f p r o t e i n s . The s u r f a c e h y d r o p h o b i c i t y may a c t as the major d r i v i n g f o r c e f o r the e m u l s i o n f o r m a t i o n and the e l e c t r o s t a t i c r e p u l s i o n may s e r v e t o s t a b i l i z e the formed e m u l s i o n by p r e v e n t i n g the c o a l e s c e n c e o f emulsion o i l - d r o p l e t s . A d d i t i o n a l e v i d e n c e r e g a r d i n g the importance o f e l e c t r o s t a t i c r e p u l s i o n i n the e m u l s i f y i n g p r o p e r t i e s o f p r o t e i n s has come from the f a c t t h a t the e m u l s i f y i n g p r o p e r t i e s o f p r o t e i n s were improved by d e a m i d a t i o n (17-19). D e a m i d a t i o n o f p r o t e i n s r e s u l t s i n an i n c r e a s e i n n e g a t i v e c h a r g e s which r e s u l t from the h y d r o l y s i s o f amide groups i n g l u t a m i n e and a s p a r a g i n e . The e f f e c t s o f d e a m i d a t i o n on the e m u l s i f y i n g p r o p e r t i e s a r e e s p e c i a l l y remarkable f o r wheat g l u t e n c o n t a i n i n g a h i g h amount o f g l u t a m i n e and a s p a r a g i n e ( 1 9 ) . Thus, the e f f e c t s o f e l e c t r o s t a t i c r e p u l s i o n on the e m u l s i f y i n g p r o p e r t i e s o f p r o t e i n s seems t o be f a i r l y d r a s t i c . S i g n i f i c a n c e o f p r o t e i n s t a b i l i t y i n the f u n c t i o n a l p r o p e r t i e s . P r o t e i n s t a b i l i t y seems t o be an i m p o r t a n t f a c t o r i n f l u e n c i n g s u r f a c e p r o p e r t i e s because i t i s g e n e r a l l y a c c e p t e d t h a t u n s t a b l e p r o t e i n m o l e c u l e s u n f o l d w i t h h y d r o p h i l i c segments o r i e n t e d toward the aqueous phase and h y d r o p h o b i c segments o r i e n t e d toward the a i r o r o i l phase t o cause the pronounced r e d u c t i o n o f s u r f a c e o r i n t e r f a c i a l t e n s i o n t h a t f a c i l i t a t e s foaming and e m u l s i f i c a t i o n . To p r o v e t h i s h y p o t h e s i s , we have employed a p r o t e i n e n g i n e e r i n g a p p r o a c h . The e f f e c t s o f s i n g l e amino a c i d s u b s t i t u t i o n s on the s t a b i l i t y o f p r o t e i n s have been s t u d i e d u s i n g s i t e - d i r e c t e d m u t a g e n e s i s . In t h i s r e s p e c t , a s e r i e s o f v a r i a n t α-subunits o f t r y p t o p h a n s y n t h a s e s u b s t i t u t e d by each o f 20 amino a c i d s a t p o s i t i o n 49 have been c o n s t r u c t e d by Y u t a n i e t a l . ( 1 0 ) . In the case o f the t r y p t o p h a n s y n t h a s e α-subunit s u b s t i t u t e d a t p o s i t i o n 49, the Gibbs f r e e energy o f u n f o l d i n g i n water o f v a r i o u s mutants, which i s a q u a n t i t a t i v e measure o f p r o t e i n s t a b i l i t y , has v a r i e d between 0.72 and 1.92 times t h a t o f w i l d - t y p e p r o t e i n s . T h e r e f o r e , t h e y formed an i d e a l s e t o f mutants w i t h which i t would be f r u i t f u l t o e s t i m a t e the r e l a t i o n between the s u r f a c e p r o p e r t i e s and c o n f o r m a t i o n a l stability. F o r t u n a t e l y , a l a r g e amount o f mutants (20-100 mg p e r 5 l i t e r s o f b r o t h ) are o b t a i n e d (10). F o r t h i s r e a s o n , we have s t u d i e d (20) the r e l a t i o n s h i p between the c o n f o r m a t i o n a l s t a b i l i t y and f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s u s i n g t y p i c a l mutant t r y p t o p h a n s y n t h a s e o f varying s t a b i l i t i e s . T a b l e 1 shows the Gibbs energy o f u n f o l d i n g o f w i l d - t y p e and s i x mutant p r o t e i n s . The f r e e energy o f u n f o l d i n g i s a f u n c t i o n o f the e q u i l i b r i u m c o n s t a n t (K) t h a t r e l a t e s the


18


1.0


ε

0

L_J 0

•

L_J 2

4

T I M E , min F i g u r e 3. E f f e c t s o f s i a l i d a s e t r e a t m e n t on t h e e m u l s i f y i n g p r o p e r t i e s o f ovomucin. ·,untreated; Ο / t r e a t e d w i t h s i a l i d a s e

(Reprinted with permission from ref. 15. Copyright 1987 Japan Society for Bioscience, Biotechnology, and Agrochemistry.) 1.5 ι

0

1

L_l

0

.

5

L_

10

TIME , m i n F i g u r e 4. E f f e c t s o f phosphatase t r e a t m e n t on t h e e m u l s i f y i n g properties o f p h o s v i t i n . # , untreated; Of t r e a t e d w i t h phosphatase

(Reprinted with permission from ref. 16. Copyright 1987 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)



2. KATO


19

c o n c e n t r a t i o n o f f o l d e d and u n f o l d e d forms ( A G = - R T l n K ) , and r e f l e c t s the s t a b i l i t y o f p r o t e i n s . The s t a b i l i t y o f t h e mutant p r o t e i n s s u b s t i t u t e d by i s o l e u c i n e i n p l a c e o f g l u t a m i c a c i d a t p o s i t i o n 49 i s about t w i c e t h a t o f the w i l d - t y p e a t pH 7 and 9. F i g u r e 5 shows t h e foaming p r o p e r t i e s o f w i l d - t y p e and mutant ots u b u n i t s o f t r y p t o p h a n s y n t h a s e s u b s t i t u t i o n a t t h e same p o s i t i o n 49 by s i t e - d i r e c t e d m u t a g e n e s i s . S i n c e t h e α-subunit o f t r y p t o p h a n s y n t h a s e c o n s i s t s o f 268 amino a c i d r e s i d u e s w i t h o u t a d i s u l f i d e bond, i t i s a v e r y f l e x i b l e p r o t e i n and shows good foaming and e m u l s i f y i n g p r o p e r t i e s . D e s p i t e o n l y a s i n g l e amino a c i d s u b s t i t u t i o n a t t h e same p o s i t i o n , a b i g change i n t h e foaming p r o p e r t i e s a s s o c i a t e d w i t h a c o n c o m i t a n t s h i f t i n t h e c o n f o r m a t i o n a l s t a b i l i t y was o b s e r v e d between w i l d - t y p e and mutant p r o t e i n s . Thus, the r e l a t i o n s h i p s between the s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s o f the α-subunit o f t r y p t o p h a n s y n t h a s e were i n v e s t i g a t e d u s i n g w i l d - t y p e and 6 mutant p r o t e i n s s u b s t i t u t e d a t p o s i t i o n 49. As shown i n F i g u r e 6, good c o r r e l a t i o n s were o b s e r v e d between t h e s u r f a c e t e n s i o n and t h e Δ G o f u n f o l d i n g . T h i s s u g g e s t s t h a t the s u r f a c e t e n s i o n o f p r o t e i n s c l o s e l y depends on t h e c o n f o r m a t i o n a l s t a b i l i t y . F i g u r e s 7 and 8 show t h e r e l a t i o n s h i p s between the Δ G o f u n f o l d i n g , the foaming power, and the foam s t a b i l i t y o f mutant p r o t e i n s , r e s p e c t i v e l y . As c l e a r l y shown i n f i g u r e s , good c o r r e l a t i o n s were o b t a i n e d between t h e Δ G f o r b o t h foaming power and foam s t a b i l i t y . These d a t a i n d i c a t e t h a t t h e foaming p r o p e r t i e s o f p r o t e i n s a r e r e l a t e d t o the c o n f o r m a t i o n a l stability. F i g u r e 9 shows the r e l a t i o n s h i p between t h e Δ G and e m u l s i f y i n g a c t i v i t y o f mutant p r o t e i n s . A good c o r r e l a t i o n i s o b s e r v e d between the A G and e m u l s i f y i n g a c t i v i t y . The c o r r e l a t i o n c o e f f i c i e n t s o f t h e s u r f a c e f u n c t i o n a l p r o p e r t i e s w i t h the s t a b i l i t y ( A G ) o f mutants o f t r y p t o p h a n s y n t h a s e a t pH 9 a r e summarized i n T a b l e 2. The c o r r e l a t i o n c o e f f i c i e n t s o f A G w i t h foaming p r o p e r t i e s were h i g h e r than t h o s e w i t h e m u l s i f y i n g p r o p e r t i e s . Thus, t h e c o n f o r m a t i o n a l s t a b i l i t y ( A G ) i s c o n f i r m e d t o be t h e i m p o r t a n t d e t e r m i n a n t f o r t h e f u n c t i o n a l p r o p e r t i e s , e s p e c i a l l y f o r foaming. A l t h o u g h the r e l a t i o n s h i p s between t h e s u r f a c e h y d r o p h o b i c i t y and the s u r f a c e p r o p e r t i e s o f mutant t r y p t o p h a n s y n t h a s e s were a l s o s t u d i e d , t h e i r c o r r e l a t i o n s were p o o r . A number o f r e s e a r c h e r s (1-5) have r e p o r t e d good c o r r e l a t i o n s between t h e s u r f a c e h y d r o p h o b i c i t y and the s u r f a c e p r o p e r t i e s o f p r o t e i n s . I t i s r e a s o n a b l e t o assume t h a t t h e s u r f a c e h y d r o p h o b i c i t y o f p r o t e i n s p l a y s an i m p o r t a n t r o l e i n e m u l s i f i c a t i o n and foam f o r m a t i o n , because a m p h i p h i l i c p r o t e i n s p o s s e s s i n g h i g h s u r f a c e h y d r o p h o b i c i t y a r e s t r o n g l y adsorbed a t t h e i n t e r f a c e between o i l o r a i r and water c a u s i n g a pronounced r e d u c t i o n of i n t e r f a c i a l or surface tension that r e a d i l y f a c i l i t a t e s e m u l s i f i c a t i o n and f o a m i n g . The r e s u l t s p r e s e n t e d h e r e d e l i n e a t e u n e q u i v o c a l l y t h a t t h e c o n f o r m a t i o n a l s t a b i l i t y o f p r o t e i n s s h o u l d be a l s o c o n s i d e r e d as an i m p o r t a n t f a c t o r g o v e r n i n g t h e s u r f a c e p r o p e r t i e s o f unstable g l o b u l a r p r o t e i n s . Since the values f o r the s u r f a c e h y d r o p h o b i c i t y o f p r o t e i n s i n s o l u t i o n may be d i f f e r e n t from t h o s e a t t h e o i l / w a t e r i n t e r f a c e , i t i s n o t enough t o e l u c i d a t e t h e r e l a t i o n s h i p s between the s t r u c t u r a l and e m u l s i f y i n g p r o p e r t i e s u s i n g o n l y s u r f a c e h y d r o p h o b i c i t y as a s t r u c t u r a l f a c t o r . T h e r e f o r e , i t s h o u l d be more r a t i o n a l t o c o n s i d e r the c o n f o r m a t i o n a l s t a b i l i t y as a n o t h e r s t r u c t u r a l f a c t o r g o v e r n i n g the e m u l s i f y i n g p r o p e r t i e s o f proteins.


20

INTERACTIONS OF

FOOD

PROTEINS

TABLE 1.


V a l u e s o f U n f o l d i n g Gibbs Energy (AG) f o r Seven α-Subunits o f T r y p t o p h a n Synthase S u b s t i t u t e d a t P o s i t i o n 49 by S i t e - D i r e c t e d M u t a g e n e s i s

Residue at p o s i t i o n 49 G l u (wild) Gly Ala He Phe Lys Thr

A G pH 8.8 7.1 8.5 16.8 11.2 7.9 8.8

7.0 ± 0.1 ± 0.1 + 0.2 ± 0.5 ± 0.2 t 0.5 ± 0.2

pH 4.9 6.4 6.8 10.0 8.3 7.5 7.0

9.0 ± 0.3 + 0.1 + 0.2 ± 0.3 + 0.2 ± 0.9 + 0.8

S O U R C E : Reprinted with permission from ref. 20. Copyright 1990 Oxford University Press.

3

TIME ,

min

F i g u r e 5. Foaming p r o p e r t i e s o f t y p i c a l mutants o f t r y p t o p h a n s y n t h a s e s u b s t i t u t e d a t p o s i t i o n 49. Ο / s u b s t i t u t e d with i s o l e u c i n e ; • , s u b s t i t u t e d w i t h g l y c i n e ; #, w i l d - t y p e (glutamic acid)


KATO

Macromolecular Interaction and Stability 52 / ο

°

ο

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F

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τ

8

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48

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1

8

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12

16

20

A G , kcal/mol F i g u r e 6. R e l a t i o n s h i p between A G and s u r f a c e t e n s i o n o f mutant t r y p t o p h a n s y n t h a s e s s u b s t i t u t e d a t p o s i t i o n 49 a t pH 7(0) and pH 9(·) . A, E , F, G, I , Κ and Τ i n d i c a t e the d a t a o f p r o t e i n s whose amino a c i d s a t 49 are a l a n i n e , g l u t a m i c a c i d , p h e n y l a l a n i n e , g l y c i n e , i s o l e u c i n e , l y s i n e and t h r e o n i n e , respectively. (Reprinted with permission from ref. 20. Copyright 1988 Oxford University Press.)

A G , kcal/mol F i g u r e 7. R e l a t i o n s h i p between AG and foaming power o f mutant tryptophan synthases s u b s t i t u t e d a t p o s i t i o n 49 a t pH 7 (O) and pH 9 ( · ) . A-T, a s i n F i g u r e 6. (Reprinted with permission from ref. 20. Copyright 1988 Oxford University Press.)


INTERACTIONS OF FOOD PROTEINS 1 2

-

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Κ

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A G , kcal/mol F i g u r e 8. R e l a t i o n s h i p between A G and foam s t a b i l i t y o f mutant t r y p t o p h a n s y n t h a s e s s u b s t i t u t e d a t 49 a t pH 7 (O) and pH9(#). A-T, as i n F i g u r e 6.

(Reprinted with permission from ref. 20. Copyright 1988 Oxford University Press.)

kcal/mol F i g u r e 9. R e l a t i o n s h i p between A G and e m u l s i f y i n g a c t i v i t y o f mutant t r y p t o p h a n s y n t h a s e s s u b s t i t u t e d a t 49 a t pH 9. A-T, as i n F i g u r e 6.

(Reprinted with permission from ref. 20. Copyright 1988 Oxford University Press.)



2. KATO


23

R e l a t i o n s h i p between s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s . Table3 summarizes the r e l a t i o n s h i p s between s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s which have been e l u c i d a t e d i n t h i s p a p e r . The e m u l s i f y i n g p r o p e r t i e s are d i s t i n g u i s h e d between e m u l s i f y i n g a c t i v i t y and e m u l s i o n s t a b i l i t y . Both the s u r f a c e h y d r o p h o b i c i t y and the c o n f o r m a t i o n a l s t a b i l i t y o f p r o t e i n s a r e i n v o l v e d i n the e m u l s i f y i n g a c t i v i t y . B e s i d e t h e s e s t r u c t u r a l f a c t o r s , the e l e c t r o s t a t i c repulsive force a c t i n g as an i n h i b i t o r o f the c o a l e s c e n c e o f o i l d r o p l e t s s h o u l d be added t o g e t s t a b l e e m u l s i o n s . S i m i l a r l y , foaming p r o p e r t i e s a r e d i s t i n g u i s h e d between foaming power and foam s t a b i l i t y . The main s t r u c t u r a l f a c t o r o f foaming power i s the conformational s t a b i l i t y o f p r o t e i n s . There i s no c o r r e l a t i o n between the foaming power and the s u r f a c e h y d r o p h o b i c i t y o f p r o t e i n s used h e r e . To g e t a s t a b l e foam, p r o t e i n s have t o i n t e r a c t w i t h t h e m s e l v e s t o s t r e n g t h e n foam f i l m . Thus, p r o t e i n - p r o t e i n i n t e r a c t i o n i s an i m p o r t a n t d e t e r m i n a n t f o r foam s t a b i l i t y . Conclusions The s t r u c t u r a l d e t e r m i n a n t s o f e m u l s i f y i n g and foaming p r o p e r t i e s o f p r o t e i n s have been e l u c i d a t e d . As the r e l a t i o n s h i p s between s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s become c l e a r l y e s t a b l i s h e d , we w i l l be a b l e t o d e s i g n more r e a d i l y f u n c t i o n a l p r o t e i n s u s i n g p r o t e i n e n g i n e e r i n g t e c h n i q u e s . C u r r e n t and f u t u r e advances i n the g e n e t i c e n g i n e e r i n g o f f o o d p r o t e i n s s h o u l d make p o s s i b l e the p r o d u c t i o n o f new f u n c t i o n a l p r o t e i n s , i m p r o v i n g t h e i r n u t r i t i o n a l o r o t h e r f u n c t i o n a l p r o p e r t i e s . As shown i n t h i s p a p e r , the g e n e t i c a l t e r a t i o n o f amino a c i d r e s i d u e s can d r a m a t i c a l l y a l t e r the f u n c t i o n a l p r o p e r t i e s o f p r o t e i n s , even a t the l e v e l o f a s i n g l e amino a c i d s u b s t i t u t i o n . T h i s s u g g e s t s the p o s s i b i l i t y o f the development o f new f u n c t i o n a l p r o t e i n s by p r o t e i n e n g i n e e r i n g approach.

TABLE 2. pH

C o r r e l a t i o n C o e f f i c i e n t s of Surface P r o p e r t i e s a t 9 w i t h the AG o f T r y p t o p h a n Synthase a-Subunits

Surface p r o p e r t i e s Surface tension Foaming power Foam s t a b i l i t y Emulsifying activity

TABLE 3.

(P (P (P (P

Significance of Macromolecular Interaction and Stability in Functional

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