Xanthan GumMolecular Conformation and Interactions

7 Xanthan Gum—Molecular Conformation and Interactions R. MOORHOUSE, M. D. WALKINSHAW, and S. ARNOTT

Downloaded by NORTH CAROLINA STATE UNIV on May 7, 2013 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch007

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907

Xanthan Gum, the extracellular p o l y s a c c h a r i d e produced by the microorganism Xanthomonas campestris has found widespread industrial use (1,2,3) because o f its unique r h e o l o g i c a l properties. The p o l y s a c c h a r i d e forms homogeneous aqueous d i s p e r s i o n s and s o l u t i o n s e x h i b i t i n g high viscosity, as w e l l as having characteristics of both p s e u d o p l a s t i c and plastic polymer systems ( 4 , 5 ) . Of particular s i g n i f i c a n c e is the a t y p i c a l insensitivity of s o l u t i o n viscosity to s a l t e f f e c t s and to heat, e s p e c i a l l y at h i g h ionic s t r e n g t h . Molecular weight measurements (6) i n d i c a t e p o l y d i s p e r s e systems of h i g h molecular weight (>2x10 ). The primary s t r u c t u r e of xanthan has r e c e n t l y been r e i n v e s t i g a t e d (7,8) and found to c o n s i s t of pentasaccharide r e p e a t i n g u n i t s (I). 6

Pyruvate is attached on average to about o n e - h a l f of the t e r m i n a l mannose r e s i d u e s ; 0 - a c e t y l groups correspond to one residue f o r each pentassaccharide r e p e a t i n g u n i t . When p r e v i o u s l y detected in bacterial p o l y s a c c h a r i d e s , pyruvate has u s u a l l y been observed on every r e p e a t i n g u n i t ( 9 , 1 0 ) . However, the c l o s e l y r e l a t e d 90

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


7.

MOORHOUSE ET

AL.

Xanthan

Gum

Conformation

and

Interactions

91

polysaccharides from other Xanthomonas species (11,12) also show differing pyruvate contents (13). We have prepared f i b e r s of both xanthan and the r e l a t e d p o l y s a c c h a r i d e from Xanthomonas p h a s e o l i (14). Using e s t a b l i s h e d techniques f o r f i b e r d i f f r a c t i o n and computer aided model b u i l d i n g (15,16,17,18,19) we have been able to examine the p o s s i b l e molecular conformations of xanthan. The almost i n d e n t i c a l X-ray d i f f r a c t i o n patterns,from a l a r g e number of p o l y s a c c h a r i d e samples from both X. campestris and X. p h a s e o l i , i n d i c a t e s an o v e r a l l s i m i l a r i t y of molecular conformation and primary sequence. R e s u l t s and D i s c u s s i o n I t i s u s u a l l y p o s s i b l e to prepare specimens of long h e l i c a l polymers i n which the molecules are a l i g n e d with t h e i r long axes parallel. Often f u r t h e r o r g a n i z a t i o n occurs, but r a r e l y to the degree of a t h r e e - d i m e n s i o n a l l y ordered s i n g l e c r y s t a l . The xanthan X-ray d i f f r a c t i o n p a t t e r n (Figure 1) showing both c o n t i n uous i n t e n s i t y d i s t r i b u t i o n and Bragg maxima, i s c h a r a c t e r i s t i c of an ordered a r r a y of h e l i c e s which have t h e i r axes p a r a l l e l but are not f u r t h e r ordered (20). The presence of continuous d i f f r a c t i o n along the l a y e r l i n e s i n d i c a t e s that the i n d i v i d u a l molecules have random t r a n s l a t i o n s along and r o t a t i o n s about t h e i r axes and are not packed i n t o a w e l l developed c r y s t a l l a t t i c e . However, d e s t r u c t i v e i n t e r f e r e n c e has occurred near the center of the equator, l e a v i n g one broad Bragg r e f l e c t i o n of spacing 1.9 nm, the a r r a y of molecules t h e r e f o r e has some order when viewed down a molecular screw a x i s at s u f f i c i e n t l y low r e s o l u t i o n . The l a y e r l i n e spacing i s c o n s i s t e n t with a h e l i x of p i t c h 4.70 nm; the m e r i d i o n a l r e f l e c t i o n s (0,0,£) o c c u r r i n g only when Z=5n, suggests a 5-fold helix. T h i s gives a r i s e per backbone d i s a c c h a r i d e of 0.94 nm (Figure 2). The s t e r i c e f f e c t of the branching mannose r e s i d u e together with the consequent removal of the c e l l u l o s e 0(3)A—0(5) hydrogen bond across a l t e r n a t e 8-1,4 l i n k a g e s (u9ing the n o t a t i o n i n Figure 2) means that the backbone can no longer have the 2^ screw symmetry of c e l l u l o s e . Instead of the usual extended β-1,4 ribbon (21), a more sinuous h e l i x of the type shown i n F i g u r e 3 i s obtained. A p r i o r i we could have no preference f o r any of the four p o s s i b l e 5 - f o l d h e l i c a l models. The 5/1 and 5/4 conformations are r i g h t and left-handed r e s p e c t i v e l y and have a s i n g l e t u r n per h e l i x p i t c h while the two other (5/2 and 5/3) models a l s o d i f f e r by being r i g h t and left-handed and have two turns per h e l i x p i t c h . I n i t i a l l y molecular models f o r each of these f o u r s i n g l e h e l i c a l p o s s i b i l i t i e s , were examined assuming standard bondlengths, bond-angles and sugar r i n g conformation angles (15). The models were f u r t h e r c o n s t r a i n e d to e x h i b i t symmetry and p e r i o d i c i t y c o n s i s t e n t with the d i f f r a c t i o n p a t t e r n . On the b a s i s of a minimum s t e r i c compression comparison, the 5/1 (Figure 3) and 5/2 (Figure 4) right-handed h e l i c e s were most favored,



92

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POLYSACCHARIDES

Figure 1. Diffraction pattern typ ical for both Xanthomonas campestris and Xanthomonas phaseoli polysaccharides showing five-fold helical symmetry. The sharp Bragg reflection on the equator has a spacing of 1.9 nm.

Figure 2. The pentasaccharide repeat ing unit of xanthan showing atom label ing and aisaccharide backbone height. The unlettered residue and residue A are Ό-glucose, Β and Ε are O-mannose, and C is O-glucuronate.


MOORHOUSE

E TA L .

Xanthan

Gum Conformation

and

Interactions


7.

Figure 3.

The isolated 5/1 xanthan helix viewed perpendicular to the helix axis


93


Figure 4.

The isolated 5/2 xanthan helix viewed perpendicular to the helix axis



7.

MOORHOUSE E T A L .

Xanthan

Gum

Conformation

and

Interactions

95

having g l y c o s i d i c conformation angles w i t h i n the normal o l i g o saccharide ranges (Table 1) and no overshort non-bonded separat i o n s . With the left-handed h e l i c e s (5/3 and 5/4), minimization did not r e l i e v e a l l of the unacceptably short i n t e r a t o m i c cont r a c t s even a f t e r o p t i m i z a t i o n . In i s o l a t i o n there i s no d r i v i n g f o r c e to h o l d the s i d e chains c l o s e to the backbone and the i s o l a t e d chain models suggest a diameter of 3.8 nm as opposed to a value of 1.9 nm obtained from l a t e r a l p e r i o d i c i t i e s i n the d i f f r a c t i o n p a t t e r n . Studies on other branched polysaccharides favor the s i d e chains l y i n g roughly p a r a l l e l to the backbone (18), and we have t h e r e f o r e undertaken a second study i n which both packing and conformational v a r i a t i o n s were considered f o r each of the models. The most symmetrical and commonly observed c l o s e packing of polymeric molecules, having n e a r l y c i r c u l a r cross s e c t i o n , i s hexagonal packing i n which each molecule i s e q u i d i s t a n t from i t s 6 nearest neighbors but not n e c e s s a r i l y f u r t h e r r e l a t e d . We t h e r e f o r e placed one xanthan h e l i c a l chain i n a hexagonal u n i t c e l l of s i d e a. = 2.19nm, c_ = 4.70nm, that i s c o n s i s t a n t with the e q u a t o r i a l Bragg r e f l e c t i o n indexed as (100). Minimizing s t e r i c r e p u l s i o n i n t h i s environment causes the side chain to f o l d down against the backbone. Stereochemically both the 5/4 and 5/3 h e l i c e s are u n l i k e l y as an unacceptable number of i n t r a m o l e c u l a r overshort contacts p e r s i s t a f t e r refinement. This r e i n f o r c e s our previous c o n c l u s i o n of right-handedness f o r the i s o l a t e d chains. Although the 5/1 and 5/2 h e l i c e s are s t e r i c a l l y acceptable, the 5/1 e x h i b i t s the more f a v o r a b l e comparison with o l i g o s a c c h a r i d e conformation angles. I t i s of i n t e r e s t to note that the backbone conformation angles shown i n Table I have v a r i e d l i t t l e during the process of wrapping the s i d e chains around the backbone. Further, the 5/1 packed' h e l i x (Figure 5) shows a number of p o t e n t i a l intramolec u l a r hydrogen bonds (Table I I and Figure 6). Relaxing the a t t r a c t i v e i n t e r a c t i o n (hydrogen bond) terms i n the refinement did not a l t e r the molecular conformation. Only the a d d i t i o n a l i n f l u e n c e of small p e r t u r b a t i o n s to the conformation angles about the branching mannose l i n k a g e caused the s t a b i l i s i n g i n f l u e n c e of the hydrogen bonds to be l o s t . The 'packed 5/2 h e l i x presents a much t i g h t e r s t r u c t u r e than the 5/1 model while e x h i b i t i n g some overshort i n t r a m o l e c u l a r contacts and few p o t e n t i a l hydrogen bonds and was considered u n l i k e l y on the b a s i s of t h i s a n a l y s i s . Our reasoning so f a r has been based on the premise that the e q u a t o r i a l Bragg r e f l e c t i o n on the d i f f r a c t i o n p a t t e r n (Figure 1), a r i s e s from the packing of s i n g l e molecular e n t i t i e s , the p a t t e r n does not t e l l us what form these take. In our examination of i n t e r - c h a i n i n t e r a c t i o n s , we have thus considered those i n t e r a c t i o n s that can a r i s e from some side-by-side arrangement of the 5/1 h e l i c e s and a l s o the case of c o a x i a l m u l t i p l e h e l i c e s . 1

1


96

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

TABLE I


Comparison o f backbone c o n f o r m a t i o n a n g l e s i n t h e i s o l a t e d and ' p a c k e d ' 5/1 a n d 5/2 h e l i c a l m o d e l s

Angle

-148

-119

-98

-63

-64

-76

-30

-10O>- -161

-111

-99

-98

-97

-92

-22

-81

-61

-78+

(d)

-78+

(a) - e [ c

( 1 ) A

"

e t 0

-

Using

9 [ 0

,o

-98

( 4 )

,c

( 4 )

,c

C

( 5 )

]

, C

(5)A, (l)A'°(4) (4)

(c) - 6 [ C (d)

helices 5/2

-121

(b) (c)

'Packed' 5/1

-136

-100+ -161

(a)

(b)

Isolated helices 5/1 5/2

Range

0

{ 1 )

> (4 C

) A

>

C

]

C

( 4 ) A

> (5) ] A

C

(5)' (1),°(4)A, (4)

] A

atom n o t a t i o n i n F i g u r e 2.


MOORHOUSE E T A L .

Xanthan

Gum Conformation

and

Interactions

97


7.

(a)

(b)

Figure 5. The 'packed' 5/1 helix viewed (a) perpendicular to and (b) down the helix axis




Figure 6. Possible hydrogen bonds ( ) that may stabilize the molecule. Some adjoining residues are omitted for clarity, the backbone having solid bonds. See also Table II.


Xanthan

MOORHOUSE ET AL.

Gum Conformation

and

Interactions

TABLE I I P o s s i b l e a t t r a c t i v e i n t e r a c t i o n i n the X and 5/2 h e l i c e s

aampestris


3

Model

Overshort

5/1

Potential Hydrogen bonds

contacts (nm)

none

5/1

°(3) 0

^ °(5)A

( 2 ) ~ -

°(8a)

D

°(6)

°(5)C

°(2)A

** °(7)B

°(3)B—* °(6) [

o

r

°(3)B

°(2)D 5/2

Η

°(5)Α··· (4)Α (0.195 nm)

°(3)~

Υ

" °(5)C

°(6b)C

> 0

(5)A

°(6)A

* °(5)

°(3)B

' °(5)C

°(3)C

°(5)D


]


100


P l a c i n g a s i n g l e 5/1 h e l i x i n o u r h e x a g o n a l c e l l r e v e a l s few i n t e r a c t i o n s w i t h i t s nearest neighbors. This suggests that t h e h e l i c e s a r e s l o t t i n g i n t o some g r o o v e t h a t i s w i d e enough t o a c c o m o d a t e them w i t h o u t s t e r i c c l a s h e s . A l t e r n a t i v e l y t h e molec u l e c o u l d be c o n s i d e r e d a s a r i g i d r o d o f p o l y s a c c h a r i d e s u r rounded by a c y l i n d e r o f w a t e r , i n which case v e r y few p o l y s a c c h a r i d e - p o l y s a c c h a r i d e i n t e r a c t i o n s would be a p p a r e n t . Furthermore, a s s u c h a s i t u a t i o n c l o s e l y m i m i c s t h e s o l u t i o n s t a t e , t h e unusual s o l u t i o n p r o p e r t i e s would p r o b a b l y a r i s e from i n t e r a c t i o n s b e t w e e n r e g i o n s o f O r d e r e d ' w a t e r some o f w h i c h may b e t i g h t l y bound t o t h e p o l y s a c c h a r i d e . Current X-ray f i b e r d i f f r a c t i o n t e c h n o l o g y c a n n o t e n a b l e u s t o l o c a t e t h i s amount o f w a t e r ( 1 8 ) , p o s s i b l y NMR s t u d i e s o n s o l u t i o n s may be a b l e t o l o c a t e ' o r d e r e d w a t e r b u t w i t h o u t t h e d e t a i l t h a t i s sometimes a v a i l a b l e f r o m d i f f r a c t i o n s t u d i e s ( 1 6 , 1 7 ) . On d r y i n g t h e s p e c i ment f o r p r o l o n g e d p e r i o d s we n o t e a r e d u c t i o n o f o v e r 5 0 % i n t h e c e l l volume c o n s i s t e n t w i t h a s h r i n k a g e i n t h e Bragg s p a c i n g on the e q u a t o r w h i l e t h e f i b e r a x i s d i m e n s i o n r e m a i n s u n a l t e r e d . Apparently the molecular conformation o f the xanthan molecule s u r v i v e s d r y i n g w i t h l i t t l e change a n d a s u b s t a n t i a l q u a n t i t y o f w a t e r w h i c h f i l l s o u t t h e s t r u c t u r e i s n o t f i r m l y bound. W h i l e i t i s p o s s i b l e t o c o n s t r u c t a double h e l i c a l model, u s i n g t h e 5/1 s i n g l e h e l i x a s p r e c u r s o r , i n w h i c h t h e s e c o n d c o a x i a l s t r a n d i s p a r a l l e l t o , a n d r e l a t e d t o , t h e f i r s t b y 180 r o t a t i o n some a p p a r e n t l y u n r e s o l v a b l e o v e r s h o r t i n t e r - s t r a n d contacts exist. I t i s p o s s i b l e t h a t r e l a x i n g t h e summetry s o t h a t the p a r a l l e l c o a x i a l s t r a n d s a r e n o t r e l a t e d b y a 180 f i b e r a x i s r o t a t i o n o r a r e a n t i p a r a l l e l t o one a n o t h e r , c o u l d r e s u l t i n a c c e p t a b l e i n t e r a c t i o n s between c h a i n s . Should t h i s be t h e case i t w i l l s t i l l be n e c e s s a r y t o o b t a i n s u p p o r t i n g e v i d e n c e from other sources t o demonstrate t h e e x i s t e n c e o f double h e l i c e s . N o r m a l l y t h i s would t a k e t h e form a comparison o f t h e model w i t h the X - r a y i n t e n s i t y d a t a f r o m a c r y s t a l l i n e d i f f r a c t i o n p a t t e r n (e.g. 16,17,18) p l u s e v i d e n c e f r o m s o l u t i o n s t u d i e s o f b i - m o l e c u l a r i t y ( e . g . 2 2 ) . We w o u l d s t r e s s however t h a t t h e r e i s no e v i d e n c e o f d o u b l e h e l i c e s e i t h e r i n s o l u t i o n (27) o r t h e s o l i d s t a t e . R e c e n t l y , we h a v e b e e n a b l e t o o b t a i n a d i f f r a c t i o n p a t t e r n t h a t e x h i b i t s i n c r e a s e d c r y s t a l l i n i t y and which h a s been t e n t a t i v e l y i n d e x e d o n a t e t r a g o n a l c e l l i n w h i c h f o u r 5/1 s i n g l e h e l i c e s w i l l p a c k w i t h t h e minimum o f s t e r i c c o m p r e s s i o n . A r e finement u s i n g b o t h s t e r e o c h e m i c a l and X-ray i n t e n s i t y d a t a has not y e t been completed. 1

Conclusions T h i s p r e l i m i n a r y s t u d y shows t h a t t h e o r d e r e d c o n f o r m a t i o n of xanthan i n t h e condensed s t a t e , and p r o b a b l y i n s o l u t i o n , i s r e l a t e d t o t h e 5/1 h e l i x o u t l i n e d h e r e .


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Gum

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and Interactions

101

The Interactions t h a t o c c u r i n solution, giving rise t o viscosity effects showing t h e characteristic o f both flexible a n d stiff cross-linked r e g i o n s (4,5) must arise f r o m associations of t h e o r d e r e d 5/1 helical regions. The order/disorder transition s e e n with change o f t e m p e r a t u r e in solution ( 2 3 , 2 4 ) , w o u l d seem l i k e l y t o a r i s e f r o m c o n f o r m a t i o n a l changes p r i m a r i l y w i t h i n t h e s i d e c h a i n a s i t moves away f r o m i t s c l o s e a s s o c i a t i o n w i t h t h e o r d e r e d backbone e i t h e r accompanied b y , o r b e f o r e , c o n f o r m a t i o n a l changes i n t h e b a c k b o n e . T h i s s p r e a d i n g o f t h e 'arms o f t h e p o l y s a c c h a r i d e would cause a n i n c r e a s e d hydrodynamic volume and hence p r o v i d e t h e v i s c o s i t y s t a b i l i t y noted a t e l e v a t e d tempera tures (1,2,3,4). A s s o c i a t i o n , i n s o l u t i o n o f s i n g l e h e l i c e s does n o t r e q u i r e gel formation, a fact that points strongly i n favor o f single h e l i c a l x a n t h a n , w h i c h d o e s n o t show g e l a t i o n a t room t e m p e r a t u r e s . Weak g e l a t i o n o b s e r v e d a t t e m p e r a t u r e s c l o s e t o 0 C i s p r o b a b l y due t o a n a g g r e g a t i o n phenomenon. I t i s i n t e r e s t i n g t o n o t e t h a t t h e 5/1 h e l i x p r e s e n t s two d i s t i n c t f a c e s ; one h a v i n g t h e s i d e c h a i n s a n d c h a r g e d g r o u p s , the o t h e r e s s e n t i a l l y t h e c e l l u l o s e backbone. As xanthan i n t e r a c t s s y n e r g i s t i c a l l y w i t h t h e 3-1,4 l i n k e d g a l a c t o m a n n a n s l o c u s t b e a n a n d g u a r gums, i t i s p o s s i b l e t h a t t h i s t a k e s p l a c e a t t h e c e l l u l o s e ' g r o o v e ' i . e . b e t w e e n s i m i l a r 8-1,4 l i n k e d g l y c a n s . I t i s t h o u g h t t h a t 'smooth' u n s u b s t i t u t e d r e g i o n s o f t h e g a l a c t o mannan a r e i n v o l v e d i n t h e a s s o c i a t i o n ( 2 3 , 2 5 ) . More d e t a i l e d i n t e r p r e t a t i o n s o f t h i s c o n t i n u i n g w o r k w i l l be p u b l i s h e d e l s e w h e r e ( 2 6 ) .


1

Acknowledgement s We w i s h t o t h a n k D r s . A. J e a n e s a n d P.A. S a n f o r d , U.S.D.A,, P e o r i a a n d D r . I.W. C o t t r e l l , K e l c o , San D i e g o , f o r t h e i r g e n e r ous g i f t s o f s a m p l e s .

Literature Cited 1. Jeanes, A. (1973) In "proceedings of the ACS Conference on Water Soluble Polymers", ed. N.M. Bikales, Plenum Press, New York. pp. 227-242. 2. Jeanes, A. (1974) J . Polymer S c i . , Symp. No. 45, 209-227. 3. McNeely, W.H. and Kang, K.S. (1973) In "Industrial Gums" R.L. Whistler and J.N. BeMiller eds., pp. 473-497, Academic Press, New York. 4. Jeanes, Α., Pittsley, J.E. and Senti, F.R. (1961) J . Appl. Polymer S c i . , 5,519-526. 5. Jeanes, A. and Pittsley, J.E. (1973) J . Appl. Polymer S c i . , 17,1621-1624. 6. Dintzis, F.R., Babcock, G.E. and Tobin, R. (1970) Carbohyd. Res. 13,257-267.


102

7. 8. 9. 10.


11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

EXTRACELLULAR MICROBIAL

POLYSACCHARIDES

Jansson, P.E., Kenne, L. and Lindberg, B. (1975) Carbohyd. Res. 45,275-282. Melton, L.D., Mindt, L., Rees, D.A. and Sanderson, G.R. (1976) Carbohyd. Res., 46,245-257. Choy, Y.M. and Dutton, G.G.A. (1973) Can. J. Chem. 51,198-207. Choy, Y.M., Fehmel, F . , Frank, N. and Stirm, S. (1975) J . Virology 16,581-590. Gorin, P . A . J . , and Spencer, J.F.T. (1961) Can. J . Chem. 39, 2282-2289. Gorin, P.A.J. and Spencer, J.F.T. (1963) Can. J . Chem. 41, 2357-2361. Orentas, D.G., Sloneker, J.H. and Jeanes, A. (1963) Can. J . Microbiol., 9,427-430. Lesley, S.M. and Hochster, R.M. (1959) Can. J . Physiol. 37, 513-529. Arnott, S. and Scott, W.E. (1972) J . Chem. Soc. (Perkin II) 324-335. Guss, J.M., Hukins, D.W.L., Smith, P.J.C., Winter, W.T., Arnott, S., Moorhouse, R. and Rees, D.A. (1975) J . Mol. Biol. 95,359-384. Winter, W.T., Smith, P.J.C. and Arnott, S. (1975) J . Mol. Biol. 99,219-235. Moorhouse, R., Winter, W.T. and Arnott, S. (1976) J . Mol. Biol, in press. Smith, P.J.C. and Arnott, S. (1976) Acta Crystallogr., in press. Arnott, S. (1973) Trans. Amer. Crystallogr. Assoc., 9,31-56. Rees, D.A. (1973) In ΜΤΡ International Review of Science: Organic Chemistry Series 1, vol. 7, G.O. Aspinall, ed. 251283. Arnott, S., Fulmer, Α., Scott, W.E., Dea, I.C.M., Moorhouse, R, and Rees, D.A. (1974) J . Mol. Biol., 90,269-284. Morris, E.R. and Rees, D.A. (1976) J . Biol. Chem., in press. Holzworth, G. (1976) J . Biol. Chem., in press. Dea, I.C.M., McKinnon, A.A. and Rees, D.A. (1972) J . Mol. Biol. 68,153-172. Moorhouse, R. and Arnott, S., J . Mol. Biol., in preparation. Morris, E.R. personal communication.


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