HYDRATION OF LYSOZYME IN AQUEOUS SOLUTION AS STUDIED BY SELF-DIFFUSION NMR MEASUREMENTS AND BY KERR-DIELECTRIC RELAXATION

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HYDRATION OF LYSOZYME IN AQUEOUS SOLUTION AS STUDIED BY SELF-DIFFUSION

NMR MEASUREMENTS AND BY KERR-DIELECTRIC RELAXATION

D. Bourret, J. Parello

To cite this version:

D. Bourret, J. Parello. HYDRATION OF LYSOZYME IN AQUEOUS SOLUTION AS STUDIED BY SELF-DIFFUSION NMR MEASUREMENTS AND BY KERR-DIELECTRIC RELAXATION.

Journal de Physique Colloques, 1984, 45 (C7), pp.C7-255-C7-258. �10.1051/jphyscol:1984729�. �jpa-

00224294�

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HYDRATION O F LYSOZYME IN AQUEOUS SOLUTION A S S T U D I E D BY SELF-DIFFUSION NMR M E A S U R E M E N T S A N D B Y KERR-DIELECTRIC RELAXATION

D. Bourret and J. parello*

Laboratoire de Chimie StructuraZe, U. S . 2'. L. and * ~ ~ u i ~ e de Biophysique e t Biochimie StructuraZe, C . N.R.S., Place E. BataiZZon, 34060 MontpeZZier, France

Resume

-

L ' h y d r a t a t i o n du lysozyme (blanc d ' o e u f de poule) en s o l u t i o n (pH 4.7)

a t e

etudiee grdce a l a mesure du c o e f f i c i e n t de s e l f d i f f u s i o n de

l ' e a u par l a technique RMN des echos de s p i n e t grace aux r e l a x a t i o n s Kerr- d i e l e c t r i q u e par des methodes impulsionnelles. Par RMN on denombre 110

+

15 molecules d'eau q u i correspondent ti l ' e a u fortement s t r u c t u r e e

a

l a s u r f a c e de l a p r o t e i n e p a r l i a i s o n s hydrogene eau-proteine. Les mesures de r e l a x a - t i o n s K e r r - d i e l e c t r i q u e conduisent aux dimensions de l ' e l l i p s o i d e hydrodyna- miquement e q u i v a l e n t : 57 x 31 x 31 A avec une monocouche d ' h y d r a t a t i o n de

0

3,2 A d'epaisseur.

A b s t r a c t

-

An i n v e s t i g a t i o n o f t h e h y d r a t i o n p r o p e r t i e s o f HEW (hen-egg whi- m o z y m e i n aqueous s o l u t i o n s (pH 4.7) has been c a r r i e d o u t by measuring t h e s e l f - d i f f u s i o n c o e f f i c i e n t o f H20 u s i n g IH NMR ( s p i n echo technique) and by K e r r - d i e l e c t r i c r e l a x a t i o n u s i n g pulsed techniques. The 110

+

15 water molecules i n f e r r e d from our NMR measurements correspond t o t h e ordered water molecules i n t e r a c t i n g through hydrogen bonds w i t h p o l a r groups a t t h e pro- t e i n surface. From K e r r - d i e l e c t r i c r e l a x a t i o n measurements we comoute t h e

0

dimensions o f t h e hydrodynamically e q u i v a l e n t e l l i p s o i d i e . 57 x 31 x 31 A w i t h a continous water monolayer 3.2

A

i n thickness.

INTRODUCTION

The way i n w i c h p r o t e i n s i n t e r a c t w i t h water molecules i s o f fundamental importance t o t h e i r s t r u c t u r a l s t a b i l i t y and t h e i r b i o l o g i c a l a c t i v i t y / I / . Hydration o f lysozyme has been p r e v i o u s l y s t u d i e d by a v a r i e t y o f physico-chemical methods, i n c l u - d i n g r e c e n t h i g h r e s o l u t i o n X-ray c r y s t a l l o g r a p h y s t u d i e s o f t h e p r o t e i n i n t h e c r y s t a l l i n e s t a t e and d i e l e c t r i c measurements on powders o f low h y d r a t i o n / 2 , 3 / , The water molecules i n t h e lysozyme c r y s t a l can be d i v i d e d i n t o ordered and d i s o r - dered classes on t h e bases o f t h e i r X-ray and d i e l e c t r i c behavior ; th e p r o p o r t i o n of p r o t e i n surface covered by t h e ordered water l y i n g between 45% and 85%, probably 75% f o r best estimate.

We r e p o r t here t h e r e s u l t s o f h y d r a t i o n measurements o f HEW (hen-egg w h i t e ) lysozyme i n aqueous s o l u t i o n by 'H NMR ( s p i n echo technique) and by K e r r d i e l e c t r i c r e - l a x a t i o n u s i n g pulsed techniques.

EXPERIMENTAL NMR measurements

The h y d r a t i o n o f aqueous p r o t e i n s o l u t i o n s can be evaluated by measuring t h e r a t i o of t h e water s e l f - d i f f u s i o n c o e f f i c i e n t s D and

D,

( w i t h and w i t h o u t p r o t e i n

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:1984729

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C7-256 JOURNAL DE PHYSIQUE

r e s p e c t i v e l y ) as a f u n c t i o n o f t h e p r o t e i n c o n c e n t r a t i o n W ( % by w e i g h t o f d r y p r o - t e i n ) / 4 / .

S e l f - d i f f u s i o n c o e f f i c i e n t s have been measured by H NMR spin-echo t e c h n i q u e ( s t a - 1 t i c g r a d i e n t ) under c o n d i t i o n s o f v e r y r a p i d exchange / 5 , 6 / . Ovalbumin was f i r s t i n v e s t i g a t e d ( f i g . 1) i n o r d e r t o compare t h e r e s u l t s w i t h t h o s e p r e v i o u s l y r e p o r - t e d by WANG u s i n g t h e ''0 s e l f - d i f f u s i o n c a p i l l a r y t e c h n i q u e /4/. F o r HEW, l y s o z y - me under t h e c o n d i t i o n s p r e s e n t l y used

(W

l e s s t h a n 10%) t h e r a t i o D/D, r e p o r t e d f i g . 1 i s l i n e a r l y r e l a t e d t o W i n agreement w i t h WANG's t h e o r i c a l p r e d i c t i o n /4/.

The s l o p e o f t h e l i n e a r r e p r e s e n t a t i o n i s equal t o

-

n(VDdo + H)

-

H where a i s a shape f a c t o r , VD t h e a p p a r e n t s p e c i f i c volume o f t h e anhydrous p r o t e i n , do t h e den- s i t y o f w a t e r and H t h e mass h y d r a t i o n en g o f w a t e r p e r g o f d r y p r o t e i n . I t f o l - lows t h a t H = 0.14 k 0.02 g o f w a t e r / g o f d r y p r o t e i n . Such an amount o f w a t e r i s o n l y a b l e t o c o v e r a r e s t r i c t e d p a r t o f t h e s u r f a c e o f t h e p r o t e i n . I t corresponds t o a b o u t 110

+

15 w a t e r m o l e c u l e s p e r p r o t e i n , a v a l u e w h i c h i s t o be compared t o v a l u e s r e c e n t l y i n f e r r e d f r o m an e x t e n s i v e X-ray c r y s t a l l o g r a p h i c a n a l y s i s o f hu- man lysozyme (111 w a t e r m o l e c u l e s ) and o f t o r t o i s e egg-white lysozyme (143 molecu- l e s ) f o r o r d e r e d w a t e r m o l e c u l e s a t t h e s u r f a c e o f t h e p r o t e i n / 2 / .

Lysozyme

F i g u r e 1 : S e l f - d i f f u s i o n c o e f f i c i e n t s r a t i o D/Do a g a i n s t p r o t e i n c o n c e n t r a - t i o n W ( % by w e i g h t o f d r y p r o t e i n ) . Upper c u r v e f o r ovalbumin pH 4.7 :

o u r r e s u l t s , 0 Wang's r e s u l t s . Low c u r v e f o r HEW lysozyme pH 4.7, A : 20°C and : 50°C.

K e r r - d i e l e c t r i c r e 1 a x a t i o n measurements

D i e l e c t r i c r e l a x a t i o n measurements were c a r r i e d by Time Domain Spectroscopy, but, due t o s l i g h t p u l s e overshoot, o n l y c o n c e n t r a t e d s o l u t i o n s g i v e good a R ( t ) t i m e domain c u r v e s . Measurements were c a r r i e d o u t w i t h a 7% (w/w) HEW lysozyme s o l u t i o n

( f i g .

2)

and t h e y y i e l d two d i s t i n c t t i m e s T and ^T i n agreement w i t h t h e p r o l a t e

€7

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T

Figure 2 : a R ( t ) time domain curves versus time f o r a 7.06% by weight s o l u t i o n a t 26OC and pH 4.7.

experimental r e s u l t s ;

-

t h e o r i c a l with T =

1 0.148 us and r = 0.114 us.

>

. * A

0

1 2 3 4 :

t us

Figure 3 : A = Decay Log (an/ano) a g a i n s t time f o r a 0.6% by weight s o l u t i o n a t 27.5"C and pH 4.7. experimental r e s u l t s ;

-

t h e o r i c a l with -rbl = 0.009 us

and -rb2 = 0.125 us. B = Pulse voltage from photomultiplier with horizontal speed =

0.02 us/div.. T~~ i s probably due t o

a

small amount of dimeric lysozyme o r agregate.

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C7-258 JOURNAL

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PHYSIQUE

e l l i p s o i d shape o f lysozyme and from which t h e r a t i o P = b/a o f t h e hydrodynamical- l y e q u i v a l e n t e l l i p s o C d i s c a l c u l a t e d using PERRIN's equations /7/.

Owing t o i t s b e t t e r s e n s i t i t i v i t y , pulsed K e r r e f f e c t was used t o c a r r y o u t r e l a x a - t i o n measurements w i t h a 0.6%

(w/w)

p r o t e i n s o l u t i o n thus a f f o r d i n g T~ = T /3 (de-

I

cay r e l a x a t i o n ) f o r a r e l a t i v e l y d i 1 u t e s o l u t i o n ( f i g . 3). Combined ~ e r ~ - d i e l e c t r i c r e l a x a t i o n measurements thus a l l o w t h e dimensions o f t h e hydrodynamically equiva- - .

0 0

l e n t e l l i p s o y d t o be determined, i.e. 2a = 57

+

1 A and 2b = 31.5

+

1 A, assuming t h e r a t i o P t o be independent o f p r o t e i n c o n c e n t r a t i o n ( * ) . T h i s corresponds t o a

"

3

volume o f 29,800

i3

which exceeds by about 12,200 A t h e volume o f t h e p r o t e i n i t - self. The l a t t e r value i s assumed t o be t h e h y d r a t i o n volume o f t h e molecule and i t

0

i s e q u i v a l e n t t o a continuous water monolayer 3.2 A i n thickness.

DISCUSSION

The r e p o r t e d measurements w i t h HEW lysozyme by s e l f - d i f f u s i o n NMR and by Kerr-die- l e c t r i c r e l a x a t i o n do n o t p r o v i d e evidence f o r a p e r t u r b a t i o n o f t h e water surroun- d i n g t h e p r o t e i n which would extend beyong a water monolayer. The paradox between both types o f measurements i n r e l a t i o n t o t h e h y d r a t i o n number o f lysozyme i n s o l u - t i o n c o u l d be o n l y apparent i n t h e l i g h t o f an h y d r a t i o n model which d i s t i n g u i s h e s between two classes o f p r o t e i n - i n t e r a c t i n g water molecules.

The f i r s t c l a s s corresponds t o water molecules t i g h t l y bound t o t h e p r o t e i n which markedly d i f f e r from b u l k water by t h e i r s t r u c t u r a l and dynamic p r o p e r t i e s . Since t h e number o f water molecules i n f e r r e d from o u r NMR measurements i s c l o s e l y r e l a t e d t o t h a t given by t h e above-mentionned X-ray c r y s t a l l o g r a p h i c studies, i t i s t e n t a t i - v e l y concluded t h a t t h e I' NMR water molecules ^"correspond t o t h e ordered water molecules i n t e r a c t i n g through hydrogen bonds w i t h p o l a r groups a t t h e s u r f a c e o f t h e c r y s t a l l i n e p r o t e i n .

The second c l a s s o f water molecules, which a l s o c o n t r i b u t e t o lysozyme h y d r a t i o n ac- c o r d i n g t o o u r K e r r - d i e l e c t r i c r e l a x a t i o n measurements, would correspond t o l e s s o r - dered molecules i n c o n t a c t w i t h t h e remaining p a r t o f t h e p r o t e i n surface.

REFERENCES

/I/ I.D. KUNTZ, W. KANZMAN, Advan. P r o t e i n Chem., 1974, 28, 239.

/2/ C.C.F. BLAKE, W.C.A. PULFORD, P.J. ARTYMIUK, J. Mol . T i 0 1

.,

^1983,

^167,

^693.

/3/ S. BONE, R. PETHIG, 3 . Mol. Biol., 1982, 157, 571.

/4/ J.H. WANG, J. Am. Chem. Soc., 1954, 76, 47%.

/5/ A. SAINT-YVES, R. SEMPERE, J. REGNIEK J. PARELLO, 5th Eur. Exp. NMR Conf

.,

Ktinigstein-Frankfurt, 1981, 68.

/6/ J. ANDRASKO, Bio. Bioph. Acta, 1976, 428, 304.

/ 7 / F. PERRIN, J. de Phys. Fr., 1934,

VIITZ,

33.

(*) F o r 7% W/W s o l u t i o n 3Tb = 0,150 ps from K e r r - r e l a x a t i o n and r = 0,148 ys I

from d i e l e c t r i c r e l a x a t i o n so t h i s assumption i s r e a l i s t i c .