Molecular Modeling - ACS Publications - American Chemical Society

Chapter 15

Inhibitor-Induced Structural Changes in Serine Proteases Monitored by Fourier Transform Infrared Spectroscopy

Downloaded by MONASH UNIV on October 26, 2012 | http://pubs.acs.org Publication Date: December 14, 1994 | doi: 10.1021/bk-1994-0576.ch015

Rina Κ. Dukor and Michael N. Liebman Bioinformatics Program, Amoco Technology Company, Mail Code F-2, 150 West Warrenville Road, Naperville, IL 60563-8460

Serine proteases are an important family of enzymes whose members participate in a variety of biological activities such as digestion and coagulation. The three-dimensional structure of some of the digestive serine proteases, i t s i n h i b i t o r s and t h e i r complexes have been determined to a very high atomic resolution. In this paper, conformational perturbations in enzymes (trypsin subfamily) caused by binding of synthetic and natural inhibitors and solvent changes are examined. The changes are monitored by comparison of second-derivative FTIR spectra of inhibited and uninhibited enzymes in solution (H O). The results are compared to those obtained from X-ray crystallography studies. 2

I n f r a r e d a b s o r p t i o n s p e c t r o s c o p y was f i r s t i n t r o d u c e d as a t o o l f o r t h e study o f p e p t i d e and p r o t e i n conformations more t h a n 40 y e a r s ago (1). I n t h e IR a b s o r p t i o n s p e c t r u m , amide g r o u p s a r e s t r o n g chromophores t h a t g i v e r i s e t o n i n e s t r o n g c h a r a c t e r i s t i c bands, named amide A, Β a n d I - V I I . Among t h e s e bands, amide I band (which i s due m o s t l y t o t h e C=0 s t r e t c h i n g v i b r a t i o n s o f t h e p e p t i d e backbone) h a s been p r i m a r i l y used f o rp r o t e i n secondary s t r u c t u r e d e t e r m i n a t i o n s t u d i e s due t o i t s h i g h s e n s i t i v i t y t o s m a l l changes i n m o l e c u l a r geometry a n d h y d r o g e n b o n d i n g o f t h e p e p t i d e group. I t was r e c o g n i z e d e a r l y o n t h a t f o r p r o t e i n s i n s o l u t i o n , amide I band i s b r o a d a n d f e a t u r e l e s s and f o r p r o t e i n s t h a t c o n t a i n s e g m e n t s w i t h d i f f e r e n t c o n f o r m a t i o n s , t h e r e s u l t s a r e not a l w a y s e a s y t o i n t e r p r e t

0097-6156/94/0576-0235$08.00/0 © 1994 American Chemical Society In Molecular Modeling; Kumosinski, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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(2) Therefore, the e a r l y e x p e r i m e n t s were u s e d t o e s t i m a t e t h e p r e d o m i n a n t c o n f o r m a t i o n and t o q u a l i t a t i v e l y follow c o n f o r m a t i o n a l changes due t o changes i n s o l u t i o n e n v i r o n m e n t (3-5). Some a t t e m p t s have been made t o d e r i v e quantitative information b a s e d on curve-fitting the o r i g i n a l s p e c t r u m (6). The f i e l d o f a p p l i c a t i o n o f IR s p e c t r o s c o p y to the p r o t e i n s t r u c t u r a l s t u d i e s has e r u p t e d i n t h e e a r l y 80's w i t h commercial a v a i l a b i l i t y of F o u r i e r t r a n s f o r m i n f r a r e d spectrometers. Due to inherent advantages of F o u r i e r transform i n f r a r e d spectroscopy (FTIR) s u c h as h i g h S/N r a t i o s and h i g h wavenumber p r e c i s i o n i t now became p o s s i b l e t o o b t a i n s p e c t r a t h a t can be e a s i l y m a n i p u l a t e d by c o m p u t e r . This l e d to access to such mathematical t r e a t m e n t s as s e c o n d d e r i v a t i v e a n a l y s i s (7), F o u r i e r s e l f deconvolution (FSD) (8) and interactive spectral s u b t r a c t i o n (9). Interactive spectral subtraction i s especially useful f o r c o r r e c t i n g a b s o r p t i o n s p e c t r a of p r o t e i n s i n water. Water has a s t r o n g a b s o r p t i o n band (HOH b e n d i n g mode) a t ~ 1650 cm- , e x a c t l y a t t h e f r e q u e n c y o f amide I b a n d . T h i s p r o b l e m i s overcome e x p e r i m e n t a l l y by u s i n g D 0 as a solvent, which i s non-absorbing i n t h i s region. But t h i s i n t u r n c r e a t e s a problem because of i n c o m p l e t e H-D e x c h a n g e t h a t c a n l e a d t o band d i s p l a c e m e n t s and small s t r u c t u r a l changes (10). Therefore, a l l studies i n t h i s l a b o r a t o r y are performed f o r p r o t e i n s i n H 0-based s o l v e n t s y s t e m s and d a t a a n a l y s i s has e m p h a s i z e d t h e c a r e f u l c o r r e c t i o n f o r w a t e r bands i n t h e o b s e r v e d s p e c t r a .


c

1

2

2

I n t h e r e c e n t y e a r s , FTIR s t u d i e s o f p r o t e i n s h a v e c o n c e n t r a t e d on e x t r a c t i n g q u a n t i t a t i v e s e c o n d a r y s t r u c t u r e i n f o r m a t i o n from the s p e c t r a . The methods c u r r e n t l y u s e d a r e e i t h e r b a s e d on band n a r r o w i n g and c u r v e f i t t i n g o f t h e amide I band ( ' f r e q u e n c y - b a s e d ' a p p r o a c h ) o r on the p r i n c i p l e o f ' p a t t e r n r e c o g n i t i o n ' (such as f a c t o r a n a l y s i s and p a r t i a l l e a s t s q u a r e s method) ( t h e r e i s a l a r g e v a r i e t y o f good work on t h e s e t o p i c s ; f o r r e v i e w s see r e f . 11-14). A b i l i t y to o b t a i n q u a n t i t a t i v e secondary s t r u c t u r e i n f o r m a t i o n f r o m FTIR s p e c t r a i s o f p a r t i c u l a r v a l u e when no o t h e r t e c h n i q u e i s a v a i l a b l e f o r s i m i l a r a n a l y s i s (e.g. membrane-bound p r o t e i n s ) o r when a f a s t a p p r o x i m a t e r e s u l t i s of i n t e r e s t . We b e l i e v e , however, t h a t t h e r e a l power o f a p p l y i n g FTIR s p e c t r o s c o p y to p r o t e i n s l i e s not i n a n a l y s i s of p r o t e i n conformation but t o f o l l o w d i s c r e t e conformational changes induced by a variety of perturbations (e.g solvent, inhibitor, s i t e - d i r e c t e d m u t a t i o n , etc.)» The g o a l o f t h e work t h a t i s p a r t i a l l y d e s c r i b e d h e r e i s t o f i n d a c o n s i s t e n t , r e l i a b l e method f o r c o r r e l a t i n g s p e c t r a l i n f o r m a t i o n t o s t r u c t u r a l knowledge. To d a t e , t h e h i g h e s t r e s o l u t i o n s t r u c t u r a l i n f o r m a t i o n on p r o t e i n s comes f r o m x - r a y d i f f r a c t i o n and t h e r e f o r e we d e c i d e d t o f i r s t examine s p e c t r o s c o p i c a l l y t h o s e p r o t e i n s f o r w h i c h h i g h u

In Molecular Modeling; Kumosinski, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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r e s o l u t i o n x-ray data e x i s t s . To o p t i m i z e t h e s y s t e m under analysis to evaluate the l i m i t of r e s o l u t i o n of s t r u c t u r a l d e t a i l w h i c h t h e method c a n d e t e c t we c h o s e t o examine a s e r i e s o f h i g h l y homologous p r o t e i n s , s e r i n e p r o t e a s e s . Two s u b s e t s a r e c h o s e n : one c o n s i s t i n g o f t r y p s i n a n d a w i d e r a n g e o f i t s forms a n d t h e o t h e r c o n t a i n i n g o t h e r t r y p s i n - l i k e serine proteases. In the f i r s t subset t r y p s i n a n d i t s zymogen, t r y p s i n o g e n , are subjected to d i f f e r e n t e n v i r o n m e n t a l p e r t u r b a t i o n s s u c h as c h a n g e o f s o l v e n t , pH a n d i n h i b i t i o n by n a t u r a l a n d s y n t h e t i c inhibitors. To d e m o n s t r a t e t h e methods d e v e l o p e d , we d i s c u s s t h e r e s u l t s o f some o f t h i s s u b s e t . The r e s u l t s f o r t h e r e s t o f t h e p r o t e i n s w i l l be p u b l i s h e d s e p a r a t e l y . Materials

And

Methods

Sample Preparation. Trypsinogen (TG) a n d trypsin i n h i b i t o r ( P T I ) , b o t h from b o v i n e p a n c r e a s , were p u r c h a s e d from W o r t h i n g t o n B i o c h e m i c a l Corp. Some t r y p s i n o g e n (T1143, b o v i n e p a n c r e a s ) was p u r c h a s e d f r o m Sigma C h e m i c a l Co. f o r c o m p a r i s o n . A l l samples were u s e d w i t h o u t f u r t h e r purification. O t h e r c h e m i c a l s u s e d were o f r e a g e n t g r a d e . P r o t e i n s were p r e p a r e d a t a p p r o x i m a t e c o n c e n t r a t i o n s of 20-60 mg/ml i n H 0 b a s e d s o l u t i o n s u n d e r t h e same e n v i r o n m e n t a l c o n d i t i o n s as u s e d i n t h e c r y s t a l s t r u c t u r e d e t e r m i n a t i o n s ( i . e . pH, s o l v e n t , i n h i b i t o r s ) . Table I l i s t s c r y s t a l l i z a t i o n c o n d i t i o n s f o r t r y p s i n o g e n subset a l o n g w i t h B r o o k h a v e n P r o t e i n D a t a Bank (pdb) (15) name and a t o m i c r e s o l u t i o n o f x - r a y d a t a . 2

Spectroscopy Measurements and Data Analysis. I n f r a r e d a b s o r p t i o n s p e c t r a were c o l l e c t e d w i t h a Bomem MB100 FTIR s p e c t r o m e t e r e q u i p p e d w i t h S i C s o u r c e a n d DTGS detector. A l l s p e c t r a were r e c o r d e d a t 2 cm- r e s o l u t i o n by c o - a d d i n g 1000 s c a n s . S o l u t i o n s were p l a c e d i n a c e l l w i t h C a F windows and 6 μ m y l a r s p a c e r ( G r a s e b y S p e c a c ) . For e a c h p r o t e i n measurement, a s i n g l e beam s p e c t r u m o f empty c e l l a n d o f b u f f e r was c o l l e c t e d . B u f f e r spectrum was s u b t r a c t e d from t h a t o f p r o t e i n f o l l o w i n g p r o c e d u r e o f Pézolet e t a l (16). The i n s t r u m e n t was c o n t i n u o u s l y p u r g e d w i t h d r y a i r (FTIR Purge Gas G e n e r a t o r from B a l s t o n , I n c . ) to reduce atmospheric a b s o r p t i o n , but n o n e t h e l e s s , b e f o r e any m a t h e m a t i c a l t r e a t m e n t s , a w a t e r v a p o r absorbance s p e c t r u m , c o l l e c t e d u n d e r t h e same c o n d i t i o n s a s t h e sample, was s u b t r a c t e d . T h i s was done i n s u c h a way as t o e l i m i n a t e sharp r e s i d u a l r o t a t i o n a l - v i b r a t i o n a l peaks of v a p o r phase w a t e r i n t h e 1800-1730 cm- r e g i o n , i . e . r e g i o n where w a t e r a b s o r b s b u t sample does n o t . For d e t e r m i n a t i o n o f p r o t e i n c o n c e n t r a t i o n s UV-VIS s p e c t r a were c o l l e c t e d on Unicam UV-2 s p e c t r o p h o t o m e t e r on the same p r o t e i n samples as u s e d i n FTIR s t u d y . Samples were p l a c e d i n a q u a r t z demountable r e c t a n g u l a r c e l l o f 10 μ path length (Starna C e l l s , Inc.). A b s o r p t i o n was 1

2

1



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Table I. Subset of C r y s t a l l i z a t i o n T r y p s i n o g e n and Complexes

Conditions

pdb name (res Â)

inhibitor (molar r a t i o )

solvent

pH

1. 2 t g a (1.8)

BPTI (1/4)

MgS0

4

6.9

2 . 2tgp (1.9)

MgS0

4

6.9

BPTI

3. 3 t p i (1.9)

MgS0

4

6.9

BPTI + i l e - v a l

with

a

4. l t g c (1.8)

BPTI (1/4)

50%CH OH

7.0

5. l t g n (1.65)

benzamidine

30%C H OH

7.5

6. l t g b (1.8)

BPTI (1/4)

30% PEG

7.6

a

complex

for

3

2

5

Molar r a t i o of i n h i b i t o r to trypsinogen (moles i n h i b i t o r / moles t r y p s i n o g e n )


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m e a s u r e d a t 280 nm a n d c o n c e n t r a t i o n c a l c u l a t e d u s i n g t h e l i t e r a t u r e e p s i l o n v a l u e s a t 280 nm (17). P r o t e i n FTIR a b s o r p t i o n d a t a i s c o n v e r t e d i n t o t h e u n i t s o f m o l a r e x t i n c t i o n c o e f f i c i e n t s so t h a t d i f f e r e n t samples c o u l d be compared. The e x a c t p a t h l e n g t h o f IR c e l l i s d e t e r m i n e d by an i n t e r f e r e n c e f r i n g e method o f empty c e l l (18). O t h e r methods o f n o r m a l i z a t i o n o f amide I band were a t t e m p t e d s u c h as n o r m a l i z i n g t o a r e a o r t o t y r o s i n e band b u t were f o u n d t o be i n a d e q u a t e . Second d e r i v a t i v e s p e c t r a a r e c a l c u l a t e d u s i n g Maximum L i k e l i h o o d d e r i v a t i v e a l g o r i t h m (Spectrum Square A s s o c i a t e s , I n c . ) w i t h a 7 cmhalf-width f o ra l l proteins.


1

Computational Methods. A l l c o m p u t a t i o n a l methods u s e d for a n a l y s i s of c r y s t a l l o g r a p h i c s t r u c t u r e data a r e based on a l g o r i t h m s o f Liebman and c o - w o r k e r s (19-22). In short, t h r e e - d i m e n s i o n a l p r o t e i n s t r u c t u r e s a r e r e p r e s e n t e d by two g e o m e t r i c p a r a m e t e r s : α-carbon d i s t a n c e s ( l i n e a r d i s t a n c e v a l u e s - LD) and α-carbon t o r s i o n a n g l e s (backbone d i h e d r a l a n g l e s - BDA). The LD v a l u e o f an amino a c i d i n a p r o t e i n sequence , i , i s computed by summing up t h e d i s t a n c e s from i t s α-carbon t o e a c h o f t h e f o u r s u c c e s s i v e r e s i d u e acarbons. Therefore, e a c h LD v a l u e describes the conformation o f f i v e r e s i d u e s , and f o r a p r o t e i n o f Ν r e s i d u e s t h e r e a r e (N-4) LD v a l u e s . A p l o t o f LD v a l u e s v e r s u s sequence p o s i t i o n g i v e s a d e t a i l e d p r o f i l e o f l o c a l folding. To compare two forms o f t h e same p r o t e i n , t h e i r r e s p e c t i v e LD's a r e s u b t r a c t e d p a i r w i s e a n d a p l o t w i l l h i g h l i g h t r e g i o n s t h a t undergo change (20). The BDA i s a d i h e d r a l a n g l e between two p l a n e s , one c o n t a i n i n g i , i+1, i+2 and t h e s e c o n d c o n t a i n i n g i+1, i+2, 1+3. One BDA value describes the o r i e n t a t i o n of four residues. This p a r a m e t e r g i v e s r e p r e s e n t a t i o n o f handedness o f p o l y p e p t i d e c h a i n and i s used t o d i s t i n g u i s h between a m b i g u i t y i n distance-based a n a l y s i s alone. To d e t e r m i n e i f f r a g m e n t s o f p o l y p e p t i d e backbone i n d i f f e r e n t p r o t e i n s t r u c t u r e s a r e e q u i v a l e n t , an a l g o r i t h m , b a s e d on s i m i l a r i t y i n t h e LD a n d BDA v a l u e s , h a s been d e v e l o p e d (21 ) and was u s e d h e r e w i t h some m o d i f i c a t i o n s . The b a s i s f o r t h e a l g o r i t h m i s d e s c r i b e d i n d e t a i l by P r e s t r e l s k i e t a l . (21) and a d d i t i o n a l m o d i f i c a t i o n s ( K l e i n , G. C , a n d L i e b m a n , Μ. Ν. , u n p u b l i s h e d ) a r e summarized here. F i r s t , we d e f i n e a s u b s t r u c t u r e a s any octapeptide conformation t h a t o c c u r s i n more t h a n one p r o t e i n i n the set of proteins studied. Substructures are e s t a b l i s h e d by s c r e e n i n g t h e LD a n d BDA v a l u e s o f a l l p o t e n t i a l substructures i n the proteins i n the set using cost function. To a c c o m p l i s h t h i s , the proteins are c o n s i d e r e d s e q u e n t i a l l y and f r a g m e n t s o f a d e f i n e d l e n g t h (N=8) a r e compared w i t h a l l o t h e r f r a g m e n t s o f t h e same l e n g t h . The 'screened' f i l e c o n t a i n s a l l t h e s u b s t r u c t u r e s and their matches. One r e s u l t a n t t a b l e contains s u b s t r u c t u r e number, f r e q u e n c y o f i t s o c c u r r e n c e s i n t h e s e t a n d c o r r e s p o n d i n g LD and BDA v a l u e s . A second l i s t s


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t h e o r d e r o f s u b s t r u c t u r e s a s s i g n e d t o e a c h r e s i d u e a s one p r o g r e s s e s a l o n g t h e amino a c i d s e q u e n c e . A hierarchical o r d e r i n g of substructures i s then generated, c o n t a i n i n g 5 LD v a l u e s , t o f i n d e q u i v a l e n t r e g i o n s i n t h e p r o t e i n s i n the s e t . A l l c a l c u l a t i o n s d e s c r i b e d h e r e were p e r f o r m e d on a Sun computer.


Results

and

Discussion

The t r y p s i n - l i k e s u b f a m i l y o f s e r i n e p r o t e a s e s i s i d e a l f o r t h e s t u d y o f c o n f o r m a t i o n a l changes t h a t t a k e p l a c e upon e n v i r o n m e n t a l p e r t u r b a t i o n s due t o a l a r g e number o f h i g h r e s o l u t i o n c r y s t a l l o g r a p h i c data a v a i l a b l e f o r under a variety of conditions . F i g u r e 1 shows a f l o w - c h a r t ( s i m i l a r t o a minimum s p a n n i n g t r e e ) o f t h e a v a i l a b l e x - r a y d a t a (pdb name) a n d t h e c o r r e s p o n d i n g p e r t u r b a t i o n s . As a demonstration o f t h e a n a l y s i s method, FTIR d a t a i s presented f o r trypsinogen (2tga), trypsinogen-BPTI complex (2tgp) a n d t r y p s i n o g e n i n d i f f e r e n t s o l v e n t s ( l t g b , l t g c ) . The s u b s t r u c t u r e l i b r a r y c a l c u l a t i o n was c a r r i e d o u t u s i n g a l l s t r u c t u r e s shown i n F i g u r e 1. F i g u r e 2 shows d i f f e r e n c e l i n e a r d i s t a n c e p l o t s f o r (a) l t g b , (b) l t g c a n d (c) 2 t g p a s e a c h i s compared t o 2tga. The c r y s t a l s t r u c t u r e l t g b i s o f t r y p s i n o g e n i n 30% p o l y e t h y l e n e g l y c o l (24) . Polyethylene g l y c o l i s quite d i f f e r e n t f r o m MgS04 ( t h e s o l v e n t u s e d f o r 2 t g a {25) s t r u c t u r e ) i n s a l t c o n c e n t r a t i o n and d i e l e c t r i c c o n s t a n t . B a s e d o n t h e d i f f e r e n c e LD p l o t , t h e m a i n d i f f e r e n c e b e t w e e n t h e two s t r u c t u r e s i s i n t h e r e g i o n b e t w e e n r e s i d u e s 168-174 ( p o s i t i o n s a r e r e f e r r e d t o b a s e d o n t h e o r d e r e d r e s i d u e l i s t i n β-trypsin, i . e . 1-223 f o r 223 residues i n β-trypsin (20). The s e v e n t h residue i n t r y p s i n o g e n , i s o l e u c i n e , assumes p o s i t i o n number 1 . ) . T h i s r e g i o n i s p a r t o f t h e " a c t i v a t i o n domain" (26), i . e . regions which are conformationally d i f f e r e n t i n trypsinogen as compared t o t r y p s i n . In trypsinogen, the a c t i v a t i o n d o m a i n i s d i s o r d e r e d (26) b u t i t i s d i s o r d e r e d i n b o t h e n v i r o n m e n t s (24). Based on t h e m a g n i t u d e o f t h e change i n l i n e a r distance values (2.2 Â) a n d c o m p a r i s o n w i t h c h a n g e s upon b i n d i n g o f p a n c r e a t i c t r y p s i n inhibitor ( F i g u r e 2 c , s e e below) i t i s r e a s o n a b l e t o c o n c l u d e t h a t t h e changes s e e n upon a s o l v e n t change a r e c o n f o r m a t i o n a l i n n a t u r e a n d most l i k e l y a r e due t o r i g i d i f i c a t i o n o r ordering of a 'disordered' chain. The c r y s t a l s t r u c t u r e t r y p s i n o g e n l t g c (27) i s i n 50% methanol-water mixture. B a s e d on t h e d i f f e r e n c e LD p l o t , t h e ' l a r g e s t ' d i f f e r e n c e o b s e r v e d i s i n t h e same r e g i o n a s t h a t d e s c r i b e d above, r e s i d u e s 168-174, b u t t h e change i s about 4 times s m a l l e r . Therefore, i ti s concluded that t h e r e i s a v e r y s m a l l e f f e c t ( i f any a t a l l ) o f a l c o h o l on the conformation o f trypsinogen. B o v i n e p a n c r e a t i c t r y p s i n i n h i b i t o r (PTI) i s a s m a l l p r o t e i n o f 58 amino a c i d s t h a t b i n d s t r y p s i n o g e n w i t h t h e


15. DUKOR & LIEBMAN

30% PEG \

Structural Changes in Senne Proteases

J50%CH3OH DIP ,50%H2O

241

30% C2H50H (benzamidine as inhibitor)

Trypsinogen


2tga (MgS04) • N-terminal hexapeptide

BPTI

(%tT)«

2 t g a

purified

GtgjT)

β-Trypsin Itpo

lle-Val

V

APPA

BPTI

GtpT)

QtjD S t D \ ^

t

p

o

benzamidine

Gptb) commercial prep, (α + β)

Gptn )

DIP

XiptpJ F i g u r e 1. F l o w - c h a r t o f c r y s t a l l o g r a p h i c d a t a f o r t r y p s i n and i t s zymogen and t h e c o r r e s p o n d i n g perturbations. Each s t r u c t u r e i s i d e n t i f i e d by i t s P r o t e i n Data Bank (pdb) name.


MOLECULAR MODELING

Q

(Delta LDP = LDP OF 2TGA - LDP OF 1TGBJ

|o

6.06.04.02.0delta LDP-I 0.0Anatrpmg -2.0

N^^J^^ ]

-4.0


-6.0. 1 20 40 60 80 100 120 140 160 180 200 223| (Residue Number] I IALDP ΙΙΙΜ.ΟΙΡΓ^ΒΡ [Delta LDP = LDP OF 2TGA - LDP OF 1TGCJ lO.Ot 8.06.04.02.0delta LDP-

0.0-2.0 -4.0 -6.0 1 20 40 60 80 100 120 140 160 180 200 223| (Residue Number! IALDP

iiigLgbLig^UJiia|

[Delta LDP r LDP OF 2TGA - LDP OF 2TGPZJ

I»· U

1

ΙΟΟ .τ 8.06.04.02.0delta LDP-I 0.0Απ gstroms

Ά

-6.0r1 20 40 60 80 100 120 140 160 ISO 200 223| (Residue Number] I ULDP

p»i.tll-*-^IB3

F i g u r e 2. D i f f e r e n c e l i n e a r d i s t a n c e p l o t s f o r (a) l t g b ( t r y p s i n o g e n i n 3 0% p o l y e t h y l e n e g l y c o l ) , (b) l t g c ( t r y p s i n o g e n i n 50% m e t h a n o l - w a t e r m i x t u r e ) a n d (c) 2 t g p ( t r y p s i n o g e n - b o v i n e p a n c r e a t i c i n h i b i t o r complex) as compared t o 2 t g a ( t r y p s i n o g e n i n MgS0 ) . 4


15. DUKOR & LIEBMAN

243



1

b i n d i n g c o n s t a n t o f 106 M - (28) . Upon b i n d i n g o f P T I , t h e a c t i v a t i o n domain becomes s t r u c t u r e d (26) and o v e r a l l t h e trypsinogen component adopts a more trypsin-like conformation (28). The d i f f e r e n c e LD p l o t ( F i g u r e 2c) shows t h e d i f f e r e n c e between LD v a l u e s f o r f r e e t r y p s i n o g e n and a t r y p s i n o g e n - P T I complex. A g a i n t h e change i s o b s e r v e d i n t h e same a c t i v a t i o n domain r e g i o n , r e s i d u e s 168 t o 175, b u t t h e s i g n o f t h e d i f f e r e n c e p l o t f o r r e s i d u e s 170 t o 175 i s o p p o s i t e t o t h a t s e e n i n 2 t g a - l t g b p l o t ( F i g u r e 2a) .. T h i s d i f f e r e n c e i n s i g n f o r t h e two p l o t s probably implies that i f there i s ordering of the a c t i v a t i o n r e g i o n upon a c o m p l e x f o r m a t i o n o r s o l v e n t change, then, t h e s e c o n f o r m a t i o n a l changes t h a t r e s u l t f r o m t h e two p e r t u r b a t i o n s a r e d i f f e r e n t . The d i f f e r e n c e FTIR d e r i v a t i v e s p e c t r a f o r t r y p s i n o g e n i n 12 - 1 3 % p o l y e t h y l e n e g l y c o l (PEG) , 2 0 % CH OH a n d a complex with. PTI as compared t o f r e e t r y p s i n o g e n a r e shown i n F i g u r e s 3, 4 and 5 r e s p e c t i v e l y . A l l t h r e e s p e c t r a show a d i f f e r e n c e i n t h e amide I r e g i o n (1600 -1690 cm- ) and no d i f f e r e n c e s i n a m i d e I I (1480-1575 cm- ) r e g i o n a r e observed. "The amide I I r e g i o n r e s u l t s p r i m a r i l y f r o m NH b e n d i n g a n d CN s t r e t c h i n g . I t i s n o t as s e n s i t i v e t o s e c o n d a r y s t r u c t u r a l c h a n g e s a s amide I a n d when t h e changes are small, i t i s not expected to reveal differences. The l a r g e s t d i f f e r e n c e s s e e n i n amide I f o r a l l t h r e e s p e c t r a a r e i n t h e same ' r e g i o n ' , i . e. 16331643 cm- ( o t h e r s a s s i g n t h i s r e g i o n as β-sheet (11-14, 29, 30), p o s s i b l y i n d i c a t i n g t h a t t h e change i s w i t h i n t h e same s u b s t r u c t u r e and p o t e n t i a l l y even i n t h e same p o s i t i o n o f protein. The LD r e s u l t s , as d e s c r i b e d above, s u g g e s t t h a t i t i s s a f e zo assume t h a t t h e c o n f o r m a t i o n a l changes t h a t t a k e p l a c e i n s o l u t i o n a r e i n f a c t i n t h e same p a r t o f t h e polypeptide chain i n a l l three cases. Other small d i f f e r e n c e p e a k s o b s e r v e d a t h i g h e r f r e q u e n c i e s (1670-1690 cm- ) a r e due t o s m a l l changes i n l o o p s a n d t u r n s . It is important t o p o i n t out that the r e s u l t s presented here are p r e l i m i n a r y and s e v e r a l sample p r e p a r a t i o n techniques r e q u i r e f u r t h e r r e f i n e m e n t b e f o r e f u r t h e r a n a l y s i s c a n be made. A l s o , t h e spectrum o f t h e complex o f t r y p s i n o g e n w i t h PTI c o n t a i n s c o n t r i b u t i o n s from amides o f b o t h p o l y p e p t i d e s a n d s i n c e i t i s n o t d e t e r m i n e d e x a c t l y how much PTI i s bound t o t r y p s i n o g e n i t i s n o t c l e a r how much o f i t s s p e c t r u m t o s u b t r a c t from t h a t o f t h e complex. The p r o t e i n s u b s t r u c t u r e l i b r a r y u s e d i n t h i s a n a l y s i s was c r e a t e d f o r t h e p r o t e i n s e t shown i n F i g u r e 1 . A n a l y s i s of the l i b r a r y i n d i c a t e s that f o r trypsinogen c r y s t a l s t r u c t u r e s 2 t g a , l t g c and 2 t g p s t a r t i n g w i t h r e s i d u e number 166 t h e o c t a p e p t i d e s u b s t r u c t u r e i s o f t h e same t y p e t h r o u g h r e s i d u e number 173. The l i b r a r y a v e r a g e LD v a l u e s f o r t h i s s u b s t r u c t u r e a r e : 27.4, 29.7, 27.8, and 31.8 Â w h i c h a r e c h a r a c t e r i s t i c o f LD v a l u e s f o r more 'extended' structures, similar t o 2.2 helix (LD=29.08) (21). For ltgb, the octapeptide substructure 3

1

1

1

1

7


244

MOLECULAR MODELING

2TGA


1TGB

2TGA - 1TGB

1800

1400

1600 Frequency ( 1 / c m )

F i g u r e 3. D i f f e r e n c e FTIR d e r i v a t i v e s p e c t r a f o r t r y p s i n o g e n i n 12-13% p o l y e t h y l e n e g l y c o l as compared t o t r y p s i n o g e n i n MgS0 . 4

s t a r t s a t r e s i d u e 165 and has an a v e r a g e LD v a l u e s o f 22.2, 27.4, 2 9 . 8 , a n d 27.9 À w h i c h i n d i c a t e t h a t t h e f i r s t 5 r e s i d u e s a r e i n v o l v e d i n a t u r n ( p r o b a b l y deformed) and t h e n f o l d s i n t o a n ' e x t e n d e d ' s t r u c t u r e (I) . A t r e s i d u e 173, t h e f o l d i n g p a t t e r n becomes t h e same f o r 2 t g a , l t g b and l t g c b u t n o t f o r 2 t g p . A t t h i s p o i n t t h e c o n f o r m a t i o n changes f o r 2 t g p and t h e n changes a g a i n a t r e s i d u e s 174 and 175. A t r e s i d u e 176 a l l f o u r s t r u c t u r e s a d o p t t h e same conformation. These r e s u l t s a r e c o n s i s t e n t w i t h t h e d i f f e r e n c e LD p l o t s b u t g i v e more d e t a i l a n d i n s i g h t on t h e n a t u r e o f t h e e x a c t changes. The h i e r a r c h i c a l o r d e r i n g o f substructures indicates that f o r a l l other crystal s t r u c t u r e s i n F i g u r e 1 except l t g n , 2tgd and 4 p t i t h e r e g i o n s t a r t i n g w i t h r e s i d u e 166 c a n be d e f i n e d by t h e same s u b s t r u c t u r e (as d e s c r i b e d above t h i s r e g i o n i s p a r t o f t h e ' a c t i v a t i o n domain'). Conclusion

I n t h i s p a p e r , we d e s c r i b e a m e t h o d f o r a n a l y z i n g s t r u c t u r a l changes i n p r o t e i n s u s i n g a c o m b i n a t i o n o f experimental, i . e . FTIR s p e c t r o s c o p y and computational techniques. The method i n v o l v e s t h e c o m p a r i s o n o f p r o t e i n s i n d i f f e r e n t s t a t e s u s i n g second d e r i v a t i v e a n a l y s i s o f


DUKOR & LIEBMAN


2TGA


1TGC

2TGA -

1800

1TGC

1400

1600 F r e q u e n c y (1 / c m )

F i g u r e 4. D i f f e r e n c e FTIR d e r i v a t i v e s p e c t r a f o r t r y p s i n o g e n i n 20% m e t h a n o l as compared t o t r y p s i n o g e n i n MgS0 . 4

2TGA

2TGP

2TGA -

2TGP

I 1800

1600

1400

F r e q u e n c y (1 / c m )

F i g u r e 5. D i f f e r e n c e FTIR d e r i v a t i v e s p e c t r a f o r trypsincgen-bovine pancreatic trypsin i n h i b i t o r complex as compared t o t r y p s i n o g e n i n MgS0 . 4



246

MOLECULAR MODELING

F T I R s p e c t r a a n d d i f f e r e n c e LD v a l u e s c o m p u t e d from s t r u c t u r e s observed by x - r a y c r y s t a l l o g r a p h y . C o l l e c t i o n and a n a l y s i s o f d a t a f o r a f a m i l y o f p r o t e i n s , e.g. s e r i n e p r o t e a s e s , enables t h e assignment o f observed spectral bands t o p r o t e i n s u b s t r u c t u r e - b a s e d f e a t u r e s a n d f u r t h e r e n a b l e s a n a l y s i s o f s p e c t r a where no x - r a y s t r u c t u r e i s available. I t i s i m p o r t a n t t o n o t e t h a t a l t h o u g h we r e p o r t the i n i t i a l r e s u l t s o f t h i s study i t i s apparent t h a t t h e method we d e s c r i b e does n o t r e l y on b a n d f i t t i n g t h e s p e c t r a or on d e v e l o p i n g frequency-based band assignments. However, a s t h e methods f o r d a t a a n a l y s i s from FTIR s p e c t r a c o n t i n u e t o e v o l v e (see o t h e r c h a p t e r s i n t h i s volume) FTIR spectroscopy promises t o be a p o w e r f u l method f o r e v a l u a t i n g p r o t e i n s t r u c t u r e and monitoring protein c o n f o r m a t i o n a l response i n s o l u t i o n .

Acknowledgments We w i s h t o t h a n k Mr. Gary K l e i n f o r h i s h e l p i n d e v e l o p i n g programs t o c r e a t e p r o t e i n s u b s t r u c t u r e l i b r a r y .

Literature

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247

16. Dousseau, F . , Therrien, Μ., and Pézolet, M. Appl. Spec. 1989, 43, 538 17. Practical Handbook of Biochemistry and Molecular Biology ; Fasman, G. D., Ed.; CRC Press, Inc.: Boca Raton, FL., 1989 18. Ingle, Jr., J . D., and Crouch, S. R. Spectrochemical Analysis; Prentice Hall, Inc.: Englewood Cliffs, NJ, 1988; p.418 19. Liebman, M. Ν., Venanzi, C. Α., and Weinstein, H. Biopolymers 1985, 24, 1724 20. Liebman, Μ. Ν. Enzyme 1986, 36, 115 21. Prestrelski, S. J., Williams, Jr., A. L . , and Liebman, Μ. Ν. Proteins : Structure, Function, and Genetics. 1992, 430 22. Prestrelski, S. J., Byler, D. Μ., and Liebman, M. N. Proteins: Structure, Function, and Genetics. 1992, 440 23. Prestrelski, S. J . Ph.D. Thesis 1990, The City University of New York 24. Bode, W., and Huber, R. FEBS Letters 1978, 90, 265 25. Bode, W., Fehlhammer, H., and Huber, R. J. Mol. Biol. 1976, 106, 325 26. Huber, R., and Bode, W. Accts. Chem. Res. 1978, 11, 114 27. Singh, T. P., Bode W., and Huber, R. Acta Cryst. 1980, B36, 621 28. Bode, W., Schwager, P., and Huber, R. J. MOl. Biol. 1978, 118, 99 29. Kumosinski, T. F . , and Unruh, J . J . present publication 30. Dong, Α., Huang, P., and Caughey, W. S. Biochemistry 1990, 29, 3303 RECEIVED

June 9, 1994


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