Paramyosin, a Model Î±-Helical Protein

13 Paramyosin, a Model α-Helical Protein

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on December 15, 2015 | http://pubs.acs.org Publication Date: January 1, 1967 | doi: 10.1021/ba-1967-0063.ch013

LYNN M. RIDDIFORD

Biological Laboratories, Harvard University, Cambridge, Mass. 02138

Paramyosin

is proposed as a model protein

base optical rotatory dispersion tent.

helix upon which to

estimates of protein

It is suggested that this rigid two-chain residues comprising

helical

con

coiled coil is one

chain

with the proline

ORD

studies from 600 to 190 mµ for the native helix and to 205

mμ for the 7M guanidine-denatured analyzed.

the hairpin

molecule were performed and

In both the 600- to 300-mμ and the 315- to 240-mμ

region,

the Moffitt

parameters

are shown to be colinear

[m']

but not with λ , over the entire three-step helix-coil

tion.

An estimate of helical content for myoglobin

232

turn.

c

paramyosin

parameters

(using

b

0

based on the

as the most reliable

agrees well with the x-ray crystallographic

with transi index)

analysis.

/ ^ p t i c a l r o t a t o r y d i s p e r s i o n ( O R D ) is one of t h e c o m m o n m e t h o d s of i n vestigating protein conformation.

M o s t n a t i v e g l o b u l a r proteins show

s i m p l e d i s p e r s i o n i n t h e v i s i b l e s p e c t r u m (63),

a n d t h u s , t h e d a t a c a n be

described b y t h e o n e - t e r m D r u d e e q u a t i o n , (1) where [m'] is t h e r e d u c e d m e a n residue r o t a t i o n . x

B y contrast, the soluble

fibrous proteins a n d h e l i c a l s y n t h e t i c p o l y p e p t i d e s show c o m p l e x d i s p e r s i o n (12, 63).

I n 1956, M o f f i t t (39) proposed a t h e o r y t o e x p l a i n t h e c o m p l e x

d i s p e r s i o n of t h e α-helical p o l y p e p t i d e s , f r o m w h i c h arose t h e f o l l o w i n g p h e n o m e n o l o g i c a l e q u a t i o n (40) :

(λ

2

-

λ. ) 2

2

where bo a n d λ are m a i n l y f u n c t i o n s of t h e h e l i c a l b a c k b o n e a n d are r e l a 0

t i v e l y i n s e n s i t i v e t o e n v i r o n m e n t a l factors, a n d a

0

is a f u n c t i o n of b o t h

i n t r i n s i c residue r o t a t i o n s a n d i n t e r a c t i o n s w i t h i n t h e h e l i x a n d t h u s m a y v a r y w i t h the environment.

A l t h o u g h t h e t h e o r e t i c a l basis of t h i s e q u a 167

In Ordered Fluids and Liquid Crystals; Porter, R., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1967.

168

ORDERED FLUIDS AND LIQUID CRYSTALS

t i o n is i n c o m p l e t e , i t s e m p i r i c a l use for t h e e s t i m a t i o n of h e l i c a l c o n t e n t r e m a i n s v a l i d (54, 63,

66).

O R D measurements are c o m m o n l y extended i n t o t h e f a r - u l t r a v i o l e t region where t h e C o t t o n effects c h a r a c t e r i s t i c of the α - h e l i x (a t r o u g h a t 233 ιημ a n d a m a x i m u m near 200 ιημ) (8, 56) are f o u n d .

B y analyzing the

c i r c u l a r d i c h r o i c spectra of α - h e l i c a l p o l y p e p t i d e s , H o l z w a r t h a n d D o t y (24) h a v e s h o w n t h a t three r o t a t o r y b a n d s (the Πχ — π~ t r a n s i t i o n at 222 ιημ, t h e p a r a l l e l - p o l a r i z e d π° — ir

e x c i t o n t r a n s i t i o n a t 206 ιημ, a n d t h e p e r


p e n d i c u l a r l y p o l a r i z e d π° — ττ e x c i t o n t r a n s i t i o n at 190 πΐμ) c a n a c c o u n t for these C o t t o n effects.

W h e n t h e p o l y p e p t i d e c h a i n becomes d i s o r d e r e d ,

o n l y a single C o t t o n effect w i t h a t r o u g h a t 204 ιημ is seen

(8).

P r e v i o u s l y , a l l t h e estimates of h e l i c a l content of p r o t e i n s h a v e been based o n the p o l y p e p t i d e models.

I n d e p e n d e n t evidence is a v a i l a b l e for

the existence of these p o l y p e p t i d e s as α - h e l i c a l r i g i d rods or as r a n d o m coils i n aqueous s o l u t i o n , d e p e n d i n g u p o n p H [for reviews, see 27, 63].

Re

c e n t l y , Y a n g a n d M c C a b e (68) a n d M c D i a r m i d (37) h a v e s h o w n t h a t t h e m a g n i t u d e s of t h e h e l i c a l C o t t o n effects for t h e f u l l y h e l i c a l p o l y g l u t a m i c a c i d ( P G A ) v a r y w i t h p H a n d i o n i c s t r e n g t h ; t h i s p h e n o m e n o n m a y be cor r e l a t e d w i t h t h e aggregation of h e l i c a l chains w h i c h a p p a r e n t l y affects o n l y t h e specific r o t a t i o n a n d a b u t n o t fr (55). 0

an inadequate model helix.

0

I n a n y case, P G A seems t o be

N o t a l l h e l i c a l p o l y p e p t i d e s , especially those

of t h e a r o m a t i c a m i n o acids, e x h i b i t n o r m a l O R D b e h a v i o r , even i n t h e v i s i b l e range (27).

I n t h e u l t r a v i o l e t range h e l i c a l a r o m a t i c p o l y p e p t i d e s

s h o w C o t t o n effects i n t h e 260- t o 300 ιημ region as w e l l as a n o m a l o u s C o t t o n effects i n t h e f a r - u l t r a v i o l e t region (6, 7,17,18).

H e l i c a l polypeptides

c o m p o s e d of t w o or three a m i n o acids, s u c h as c o p o l y - L - t y r o s i n e - L - g l u t a m i c acid ( 5 %

t y r o s i n e ) ( P T G A ) (17, 61)

tamic acid ( P A L G A )

(19,

62)

and copoly-L-alanine-L-lysine-L-glu-

give somewhat

different v a l u e s for

M o f f i t t parameters t h a n t h e single p o l y a m i n o acids.

the

Presumably the dif

ferent values arise f r o m t h e effects of t h e increasing n u m b e r of s i d e - c h a i n i n t e r a c t i o n s possible i n these copolymers.

Y e t , w i t h o n l y t w o or three

a m i n o acids, t h e m a n y v a r i e d t y p e s of s i d e - c h a i n i n t e r a c t i o n s f o u n d i n p r o teins are s t i l l n o t i m p l i c i t l y a c c o u n t e d for i n t h e O R D of either t h e h e l i c a l or t h e r a n d o m c o n f o r m a t i o n or of a n y m i x t u r e s of t h e t w o

conformations.

C o n s e q u e n t l y , a b e t t e r m o d e l is a p r o t e i n w h i c h is n a t i v e l y a n α - h e l i c a l r i g i d r o d f r o m evidence independent of O R D a n d w h i c h c a n be t r a n s f o r m e d readily and reversibly into a random coil.

A l s o , it should contain a l l the

c o m m o n a m i n o acids except p r o l i n e , w h i c h d i s r u p t s t h e α - h e l i c a l s t r u c t u r e , a n d p o s s i b l y cystine. A s o r i g i n a l l y p o i n t e d o u t b y C o h e n a n d S z e n t - G y o r g y i (12), t h e soluble fibrous

proteins w h i c h possess t h e α - t y p e wide-angle x - r a y d i f f r a c t i o n d i a

g r a m , w i t h the exception of

fibrinogen,

show complex r o t a t o r y dispersion

s i m i l a r t o t h a t of t h e s y n t h e t i c p o l y p e p t i d e s .

Light meromyosin Fraction

I ( L M M F r . I ) , t r o p o m y o s i n , a n d p a r a m y o s i n a l l h a v e o v e r 9 0 % h e l i x (12)


13.

RiDDiFORD

169

Paramyosin

w h i c h is correlated w i t h t h e i r l o w p r o l i n e content (58).

T h e s e proteins

show a 5.1-A. m e r i d i o n a l reflection i n s t e a d of the 5.4-A. p i t c h reflection, characteristic of the α - h e l i c a l p o l y p e p t i d e s , w h i c h b o t h C r i c k (15)

and

P a u l i n g a n d C o r e y (44) h a v e a t t r i b u t e d to t h e presence of a coiled c o i l . T h e t w o - c h a i n coiled c o i l m o d e l is t h e best fit for the wide-angle x - r a y p a t t e r n of the p a r a m y o s i n - r i c h (over 5 0 % of the s t r u c t u r a l protein) anterior byssus r e t r a c t o r muscle of the m u s s e l Mytilus

edulis

(10).

T h e physico-

c h e m i c a l d a t a for p a r a m y o s i n , L M M F r . I , a n d t r o p o m y o s i n agree w i t h the


proposed t w o - c h a i n s t r u c t u r e (34~36, 65).

T h e i r c r y s t a l l i n e p a t t e r n s as

seen b y t h e electron microscope are different b u t h a v e some inherent s i m i l a r i t i e s — n a m e l y , a repeated occurrence of the 7 0 - A . a n d 140-A. spacings T h e o n l y n a t i v e p r o t e i n w h i c h appears to be a single α - h e l i c a l

(11, 23, 60). c h a i n is Pinna

nobilis t r o p o m y o s i n A ( s i m i l a r i n a m i n o a c i d c o m p o s i t i o n to

Venus p a r a m y o s i n ) (28, 29) ;

some c o n t r o v e r s y has arisen over existence

of t h i s p r o t e i n as a single c h a i n [for the c r i t i c i s m a n d its r e b u t t a l , see (36 and

30)].

C e r t a i n l y , Pinna

t r o p o m y o s i n is more u n s t a b l e t h a n

Venus

p a r a m y o s i n since i t is c o m p l e t e l y (and n o t e n t i r e l y reversibly) d e n a t u r e d i n SM 9.5M

u r e a (29).

Venus

p a r a m y o s i n is a b o u t t w o - t h i r d s d e n a t u r e d i n

u r e a (12) or i n 5M g u a n i d i n e - 1 . 2 M u r e a (48), a n d c o m p l e t e l y (and

r e v e r s i b l y ) d e n a t u r e d o n l y i n 7M g u a n i d i n e - H C l ( G - H C 1 ) (42, 43, 46)T h e coiled c o i l c o n f i g u r a t i o n of these proteins a p p a r e n t l y does not alter t h e i r r o t a t o r y b e h a v i o r , at least i n the v i s i b l e w a v e l e n g t h range as i n d i c a t e d b y bo (12).

T h i s r e l a t i v e i n s e n s i t i v i t y is n o t s u r p r i s i n g since the m a j o r

h e l i x of the coiled c o i l requires a t i l t angle r e l a t i v e to the m i n o r h e l i x of o n l y 10° a n d a t w i s t per residue of 2.86° as c o m p a r e d w i t h t h e t w i s t per residue i n the m i n o r h e l i x of 100° (14)·

Therefore, one of these

three

fibrous muscle proteins w o u l d be a good p r o t e i n m o d e l since they are a l l readily available. Paramyosin

as a Model

Helix

O f these three α - h e l i c a l proteins, p a r a m y o s i n is a suitable m o d e l h e l i x for the f o l l o w i n g rea-sons: P a r a m y o s i n is t h e o n l y one for w h i c h there is e x p e r i m e n t a l evidence of t h e existence of t h e t w o - c h a i n coiled c o i l in situ (10) as w e l l as i n t h e d r i e d fiber (4, 5) a n d i n s o l u t i o n (36). P a r a m y o s i n behaves as a n e x t r e m e l y a s y m m e t r i c α - h e l i c a l r i g i d r o d i n s o l u t i o n , as s h o w n b y its h y d r o d y n a m i c a n d l i g h t - s c a t t e r i n g properties (22, 36), its d y n a m i c viscoelastic b e h a v i o r (1,2), the h y p o c h r o m i c i t y of its f a r u l t r a v i o l e t a b s o r p t i o n s p e c t r u m (50), a n d its o p t i c a l r o t a t o r y properties (12, 56). P a r a m y o s i n is r e v e r s i b l y d e n a t u r e d b y 7M G - H C 1 (42, 4$, 46) whereas L M M F r . I is i r r e v e r s i b l y dissociated i n t o t h e p r o t o m y o s i n s b y 5M u r e a (59), a n d t h e 3 . 5 M G - H C 1 d e n a t u r a t i o n of t r o p o m y o s i n is o n l y 8 0 % r e v e r s i b l e (42, 4$)> A l l three proteins are insensitive t o p H d e n a t u r a t i o n , except t h a t a b o v e p H 10 t r o p o m y o s i n begins to u n f o l d p a r t i a l l y (34)»


170

ORDERED FLUIDS A N D LIQUID CRYSTALS

P a r a m y o s i n h a s , a t m o s t , 2 t o 3 prolines p e r 220,000 m o l e c u l a r w e i g h t (32, 47), a w e i g h t % s i m i l a r to t h a t f o u n d for L M M F r . I . a n d l o w e r t h a n t h a t for t r o p o m y o s i n (58).


P a r a m y o s i n has no c y s t i n e residues a n d a l o w n u m b e r of cysteine residues (11, 46, 4-7), whereas L M M F r . I (35) has h a l f - c y s t i n e s a n d shows no t r a c e of free s u l f h y d r y l groups b y t h e p - c h l o r o m e r c u r i b e n z o a t e t i t r a t i o n . T r o p o m y o s i n also has at least one disulfide b o n d (11) w h i c h m a y l i n k t h e t w o chains of t h e coiled c o i l (65). P a r a m y o s i n has 3 7 % h i g h l y h y d r o p h o b i c residues a n d 3 6 % charged a m i n o acids (34, 46), a n d l a c k s o n l y t r y p t o p h a n of t h e c o m m o n a m i n o acids (47). O n e u n c e r t a i n t y as t o p a r a m y o s i n s t r u c t u r e r e m a i n s :

whether the

coiled c o i l consists of t w o separate α-helical chains (each a b o u t 1400 A . long) or m e r e l y t w o p a r t s of one 2 8 0 0 - A . α-helical c h a i n w i t h a h a i r p i n t u r n n e a r i t s center.

F r o m t h e i n t r i n s i c v i s c o s i t y of t h e c o m p l e t e l y d e n a t u r e d

m o l e c u l e ( 6 i l f G - H C 1 , 43°), N o e l k e n (42) c a l c u l a t e d a r a d i u s of g y r a t i o n w h i c h agrees f a i r l y w e l l w i t h t h a t expected for a s i n g l e - c h a i n r a n d o m c o i l w i t h a m o l e c u l a r w e i g h t of 225,000.

H o w e v e r , he also s h o w e d t h a t t w o

r a n d o m l y coiled chains of m o l e c u l a r w e i g h t n e a r 110,000 c a n give e q u a l l y g o o d agreement.

H y d r o d y n a m i c a n d l i g h t - s c a t t e r i n g studies i n 7M

G-

H C 1 , i n w h i c h t h e molecule exists i n i t s r a n d o m c o n f o r m a t i o n at r o o m t e m p e r a t u r e (42, 46), are needed t o resolve t h e q u e s t i o n . S i n c e t h e molecule a l w a y s refolds t o i t s n a t i v e c o n f o r m a t i o n u p o n r e m o v a l of t h e g u a n i d i n e (see T a b l e I ) , e v e n after p r o l o n g e d h e a t i n g a t 5 0 ° C . i n t h i s solvent (46) a n d since t h e r e are no disulfide b o n d s or o t h e r k n o w n c o v a l e n t c r o s s - l i n k s (11, 47), I f a v o r t h e o n e - c h a i n h y p o t h e s i s . Table I.

Optical Rotatory Dispersion

Solvent

a

0

0.6MKC1, pH7.2

I t seems

-17°

b Wavelength Range 600-240 0

± 5°

- 4 4 0 ° ± 3°

Wavelength Range 600-300 0.SM KC1, p H 7 . 4 0 . 6 M KC1, p H 7 . 2 7 M guanidine + 0 . 6 M KC1, p H 7.2* c

d

- 1 ° ± 5.4° + 14° ± 1.4° - 5 4 3 ° ± 5°

- 5 7 4 ° ± 10° - 6 0 0 ° ± 3° + 2 0 ° ± 7°

Wavelength Range 315-240 0.6MKC1, pH7.2or 0 . 6 M N a F or K F , p H 7.2 7M guanidine + 0 . 6 M KC1, p H 7.2 0 . 6 M KC1, p H 7.2 after 7M guanidine

- 1 0 9 ° ± 9° - 4 7 6 ° ± 9° - 1 0 6 ° ± 10°

A l l values are averages for at least 6 different preparations. a b kurude, and \ are standard deviations for individual values. [ra'J values are limits of values averaged. Data from only two experiments calculated in this manner. α

0f

0}

c

- 3 4 3 ° ± 4° + 2 0 ° ± 4° - 3 4 3 ° ± 4° Errors indicated for Errors indicated for

6


13.

RIDDIFORD

171

Paramyosin

u n l i k e l y t h a t p a i r s of c o m p l e t e l y separated c h a i n s c o u l d reassemble i n t h e f a i r l y d i l u t e solutions (about 20 μΜ).

Also, although Ramakrishnan a n d

R a m a c h a n d r a n (45) h a v e r e c e n t l y s h o w n t h a t a n α - h e l i c a l c h a i n c a n i n c o r p o r a t e a n L - p r o l i n e residue t o w a r d s a n e n d w i t h a m i n i m u m 35° angle b e t w e e n t h e t w o h e l i c a l p o r t i o n s i f there is a s l i g h t d i s t o r t i o n of t h e p l a n a r i t y of t h e p e p t i d e g r o u p , t h e l o c a t i o n of t h e t w o prolines i n p a r a m y o s i n a t a h a i r p i n t u r n w o u l d b o t h a c c o u n t f o r t h e t u r n a n d least d i s r u p t t h e ah e l i c a l s t r u c t u r e of t h e t w o single chains of t h e coiled c o i l .


I n t h e f u l l y d e n a t u r e d m o l e c u l e (7M G - H C 1 a t 5 0 ° ) , w h e r e t h e r e is a b o u t 1 0 % r e s i d u a l h e l i x as c o m p a r e d w i t h p o l y p e p t i d e s (46), these p r o l i n e residues m i g h t r e s t r i c t complete r a n d o m n e s s b y a l l o w i n g i n t e r a c t i o n s b e t w e e n a m i n o acids t o r e m a i n i n a l o c a l i z e d r e g i o n ( a m i n i m u m of eight residues i n v o l v e d ) o n e i t h e r side of t h e m .

B r a n t a n d F l o r y (9) h a v e s h o w n

t h a t o n l y electrostatic i n t e r a c t i o n s b e t w e e n a m i d e groups a n d n o t specific s i d e - c h a i n o r s o l v e n t i n t e r a c t i o n s influence t h e c o n f i g u r a t i o n of r a n d o m p o l y p e p t i d e chains i f t h e p o l y p e p t i d e c h a i n is e n t i r e l y i n t h e t r a n s confor mation.

Y e t , t h e p r o l i n e p e p t i d e l i n k a g e c a n be either t h e t r a n s o r t h e cis

c o n f o r m a t i o n , a n d t h e existence of t h e cis c o n f o r m a t i o n i n d i c a t e s t h e p r e s ence of f a v o r a b l e i n t r a m o l e c u l a r i n t e r a c t i o n s w h i c h c o u n t e r a c t t h e h i g h e r energy of t h i s c o n f o r m a t i o n (54).

I n p a r a m y o s i n t h e existence of s u c h

i n t e r a c t i o n s i n t h e r a n d o m c o n f o r m a t i o n c a n n o t be r u l e d o u t . H y d r o phobic interactions certainly are i m p o r t a n t i n stabilizing t h e native coiled c o i l s t r u c t u r e (10, 34, 47, 48) a n d are i m p l i c a t e d i n t h e d e n a t u r e d s t a t e (46, T h e s e h y d r o p h o b i c i n t e r a c t i o n s m a y exist b e t w e e n side c h a i n s i n

48).

o t h e r p o r t i o n s of t h e u n f o l d e d m o l e c u l e as w e l l as i n t h e r e g i o n of t h e h a i r Parameters for Paramyosin

a

terude Χ IP

λ (τημ) β

τημ (λ = 218 πΐμψ 0

Nonlinear

Nonlinear

Nonlinear Nonlinear - 2 4 . 6 ± 0.2

Nonlinear Nonlinear 208.2 ± 0.8

( - 2 3 . 0 + 1.5)· - 2 3 . 2 ± 0.2 ( - 2 2 . 5 ± 1.4)'

(236.7 ± 0 . 7 ) · 214.2 ± 0.4 (236.7 + 0 . 7 ) ·

ιημ (λ = 212 ιημ) 0

τημ (λ = 220 ηΐμΥ 0

Ν'] 32 2

- 1 5 , 4 0 0 ° ± 150° -2090° ± 2 0 ° - 1 5 , 0 0 0 ° ± 200°

Μίθδ.δ + 7 0 , 2 0 0 ° + 500° ....

Recalculated from Riddiford and Scheraga (45), using Lorentz correction for dis persion of refractive index of water. From Table III (46). Parentheses indicate that these values based on assumption of a linear Drude plot are not strictly valid since larger standard deviations indicate nonlinearity. c

d

6


172

ORDERED FLUIDS A N D LIQUID CRYSTALS

pin turn.

A possible m e t h o d f o r t e s t i n g t h i s h y p o t h e s i s is n o w a v a i l a b l e .

I n a p r e l i m i n a r y note, W i l c h e k et al. (64) r e p o r t t h e use of s o d i u m i n l i q u i d a m m o n i a for t h e specific cleavage of i V - p r o l i n e peptides i n c l u d i n g p o l y - L p r o l i n e (molecular w e i g h t 1500).

I f t h i s m e t h o d is specific for p r o l i n e

l i n k a g e s i n proteins, t h e n t h i s t y p e of r e d u c t i v e cleavage

of

denatured

p a r a m y o s i n s h o u l d give t w o chains of a p p r o x i m a t e l y e q u a l l e n g t h a n d m o l e c u l a r w e i g h t 110,000, o n t h e a s s u m p t i o n t h a t t h e p r o l i n e residues are


involved i n the hairpin turn. ORD

Studies

of

Paramyosin

Experimental. A l l t h e O R D studies were m a d e w i t h p a r a m y o s i n p r e p a r e d f r o m t h e w h i t e p o r t i o n of t h e a d d u c t o r m u s c l e of t h e c l a m Venus mercenaria, as o u t l i n e d b y R i d d i f o r d a n d S c h e r a g a (47). T h e experimental p r o c e d u r e has been g i v e n i n d e t a i l (46). A l l O R D measurements were m a d e o n t h e C a r y m o d e l 60 r e c o r d i n g s p e c t r o p o l a r i m e t e r a t 23° ± 2 ° C . , except as otherwise n o t e d . T h e c o m p u t a t i o n s of t h e M o f f i t t p a r a m e t e r s (see E q u a t i o n 2 ) , u t i l i z i n g t h e s t a t i s t i c a l procedures w i t h error analyses developed b y S o g a m i , L e o n a r d , a n d F o s t e r (57) t o d e t e r m i n e t h e best λ v a l u e s , a n d of t h e D r u d e p a r a m eters {see E q u a t i o n 1; a m o d i f i e d D r u d e p l o t of [m'\ vs. [m']\ (67) was used} w i t h error analyses were p e r f o r m e d w i t h t h e I B M 7094 c o m p u t e r . C o r r e c t i o n for t h e dispersion of r e f r a c t i v e i n d e x of t h e solvent was m a d e as o u t l i n e d (46). 0

2

A s s h o w n i n F i g u r e 1, t h e M o f f i t t p l o t of t h e O R D d a t a f r o m 600 to 300 ιημ does n o t differ s i g n i f i c a n t l y w i t h K C 1 c o n c e n t r a t i o n .

T h e data in

λ -λ| 2

Figure 1.

Moffitt plot of optical rotatory dispersion of paramyosin from 600 to 300 ra/z at 24° and 20°C., respectively Ο 0.6M KCl, 0.01M phosphate buffer, pH 7.2 A 0.3M KCl, 0.01M phosphate buffer, pH 7.4 λ = 212 τημ 0



13.

RIDDIFORD

173

Paramyosin

λ -λ| 2

Figure 2. MoffiU plot of optical rotatory dispersion of paramyosin in 0.6M KCl, Ό.01Μ phosphate buffer, pH 7.2, from 600 to 240 my.

at 24°C λο

=

218 mμ

0 . 3 M K C l were o b t a i n e d o n a R u d o l p h m o d e l 200 p o l a r i m e t e r as described b y R i d d i f o r d a n d S c h e r a g a (48) a n d are t h e same as s h o w n i n F i g u r e 2 of that paper;

t h e y h a v e been r e c a l c u l a t e d t o i n c o r p o r a t e t h e c o r r e c t i o n for

t h e d i s p e r s i o n of r e f r a c t i v e i n d e x of w a t e r (16).

T h e best λ for these d a t a β

i n 0 . 3 M K C l is 215 ιημ, b u t t h e p o i n t s o n t h e figure are those c a l c u l a t e d with λ

0

as 212 ηΐμ since t h a t is t h e best v a l u e for t h e m o r e a c c u r a t e C a r y

d a t a i n 0 . 6 M K C l (the circles) for w h i c h t h e l i n e s h o w n is c o m p u t e d . W h e n t h e d a t a f r o m 300 to 240 ηΐμ are a d d e d , t h e t y p i c a l M o f f i t t p l o t for t h e n a t i v e p r o t e i n i n 0 . 6 M K C l is seen i n F i g u r e 2.

T h e best λ is n o w 0

f o u n d t o be 218 ηΐμ, b u t t h e d e v i a t i o n s f r o m t h e p l o t t e d l i n e (negative a b o v e 275 ηΐμ a n d p o s i t i v e below) are greater t h a n seen i n p l o t s for t h e t w o s e p a r a t e d regions ( F i g u r e s 1 a n d 3). F i g u r e 3 shows t y p i c a l M o f f i t t p l o t s for t h e n a t i v e a n d t h e 7M G - H C 1 d e n a t u r e d p r o t e i n i n t h e 240- to 315-ηΐμ region where λ is 220 ιημ. 0

I n this

w a v e l e n g t h range t h e D r u d e p l o t for t h e n a t i v e p r o t e i n appears l i n e a r a l t h o u g h the statistical analysis indicates m u c h larger standard deviations for t h e slope a n d t h e i n t e r c e p t , especially t h e l a t t e r [nearly t e n t i m e s as great as for t h e r a n d o m f o r m of t h e p r o t e i n (46)].

A l s o , X and the D r u d e c


174

O R D E R E D FLUIDS A N D LIQUID CRYSTALS

c o n s t a n t o b t a i n e d f r o m a L o w r y p l o t of t h e same d a t a for t h e n a t i v e p r o t e i n do n o t agree. a b o u t 2 5 % less.

X is o n l y a b o u t 8 ιημ l a r g e r , b u t t h e D r u d e c o n s t a n t is c

T a b l e I gives t h e p e r t i n e n t M o f f i t t a n d D r u d e p a r a m e t e r s

a n d t h e i r s t a n d a r d d e v i a t i o n s for these t h r e e ranges as w e l l as t h e v a l u e s of [m'] 32 a n d [m']i9 .5 (the t r o u g h a n d t h e m a x i m u m of t h e h e l i c a l C o t t o n effect, 2

8


respectively).

Figure 3.

Moffitt plots of optical rotatory dispersion of paramyosin from 315 to 240 mμ at at 22°C. (46)

Ο 0.6M KCl, 0.01M phosphate buffer, pH 7.3 • 7M guanidine-HCl (prepared with 0.6M KCl, 0.01M phosphate buffer, pH λ = 220 mμ

7.3)

0

T h e h e l i x - c o i l t r a n s i t i o n of p a r a m y o s i n as a f u n c t i o n of G - H C 1 c o n c e n t r a t i o n occurs i n three d i s t i n c t steps as m e a s u r e d b y [m'] 32 or b or a 0

2

(for either w a v e l e n g t h region) (46).

0

F i g u r e 4 shows these t r a n s i t i o n s of

ΝΊ232 (open s y m b o l s , s o l i d curve) as a f u n c t i o n of G - H C 1 c o n c e n t r a t i o n a n d also shows t h a t t h e D r u d e p a r a m e t e r X (for t h e 240- t o 315-ηΐμ range) c

(closed s y m b o l s , dashed curve) is n o t as sensitive t o these changes.

No

d i s t i n c t t r a n s i t i o n i n X coincides w i t h t h e first t r a n s i t i o n i n [m'] 32, a n d o n l y c

2

a s m a l l t r a n s i t i o n i n X is e v i d e n t i n t h e second step. c

T h e sharper t r a n s i

t i o n i n X coincides w i t h t h e t h i r d a n d final t r a n s i t i o n i n d i c a t e d b y [ra'] 3 c

2

2

a n d occurs as t h e molecule becomes less t h a n 3 0 % h e l i c a l a n d t h e rigorous s t a t i s t i c a l d e f i n i t i o n of t h e best X (67) ceases to h o l d . 0



13.

RIDDIFORD

175

Paramyosin

MOLARITY

Figure 4-

GUANIDINE-HCI

Reduced mean residue rotation and \ as a function of guanidine-HCl concentration at 25°C. c

Open symbols and left-hand ordinate. 232^μ minimum of helical Cotton effect (46) Solid symbols and right-hand ordinate. \ of one-term Drude equation for 315- to 240^μ range Triangles and circles indicate two different stock solutions c

Discussion M i z u k a m i (38) has s h o w n t h a t p a r a m y o s i n is a m i x t u r e of m o n o m e r s a n d d i m e r s (monomeric m o l e c u l a r w e i g h t 206,000) a t Γ / 2 = 0.25, p H 7.8, a n d of m o n o m e r s , d i m e r s , a n d t r i m e r s a t Γ / 2 = 0.4, p H 7.2, b u t is solely i n i t s m o n o m e r i c c o n d i t i o n a t Γ / 2 = 0.6, p H 7.2, as also f o u n d b y L o w e y , K u c e r a , a n d H o l t z e r (36).

T h e r e f o r e , t h e m o l e c u l a r w e i g h t of 330,000 o b

t a i n e d b y R i d d i f o r d a n d S c h e r a g a (47) a t Γ / 2 = 0.3, p H 7.4, is p r e s u m a b l y i n d i c a t i v e of a m i x t u r e of m o n o m e r s a n d d i m e r s .

Y e t , there is n o signifi

cant difference i n t h e v a l u e s of t h e M o f f i t t p a r a m e t e r s i n t h e v i s i b l e w a v e l e n g t h range (see F i g u r e 1 a n d T a b l e I) for these t w o states of aggregation. S c h u s t e r (55) h a s r e c e n t l y f o u n d for h e l i c a l P G A t h a t b is r e l a t i v e l y i n 0

sensitive t o t h e state of aggregation whereas a changes. 0

Perhaps the ag

g r e g a t i o n is m o r e extended i n his case. A l t h o u g h aggregation is n o t t h e same as s u p e r c o i l i n g , t h e r e l a t i v e i n s e n s i t i v i t y of t h e M o f f i t t p a r a m e t e r , b , t o aggregation of helices leads one Q

to suspect t h a t s u p e r c o i l i n g of t w o helices as occurs i n n a t i v e p a r a m y o s i n also w i l l h a v e l i t t l e effect o n this p a r a m e t e r .

Supercoiling m a y have a n

effect u p o n t h e specific r o t a t i o n a t one w a v e l e n g t h , p a r t i c u l a r l y t h e r o t a t i o n a t t h e t r o u g h of t h e h e l i c a l C o t t o n effect a t 233 ιημ. t h e difference i n

[ra'] 32 2

F o r paramyosin,

between t h e h e l i c a l a n d r a n d o m c o n f o r m a t i o n s is


176

ORDERED

FLUIDS AND

LIQUID

CRYSTALS

- 1 3 , 3 0 0 ° (46), w h i c h is s l i g h t l y less t h a n t h e v a l u e s for P G A of - 1 4 , 0 0 0 ° t o - 1 4 , 5 0 0 ° f o u n d b y v a r i o u s w o r k e r s (25, 26, 51, 68). Whether this de crease is caused b y s u p e r c o i l i n g or b y specific side chains a n d / o r t h e i r i n t e r actions w i t h i n t h e molecule is n o t k n o w n . O p t i c a l l y a c t i v e t r a n s i t i o n s of t h e a r o m a t i c chromophores of p h e n y l a l a n i n e , t y r o s i n e , t r y p t o p h a n , a n d h i s t i d i n e occur i n t h e 210- t o 230-ηΐμ r e g i o n as w e l l as i n t h e 250- t o 300-ηΐμ region (6, 7, 17, 18, 49). T h e r a n d o m i n c o r p o r a t i o n of as l i t t l e as 5 % t y r o s i n e or p h e n y l a l a n i n e i n t o P G A decreases t h e absolute m a g n i t u d e of N'] 33 (17, 51), a p p a r e n t l y a result of s u c h a r o m a t i c t r a n s i t i o n s . R o s e n b e r g (49) suggests t h a t these a r o m a t i c effects w i l l generally be a c c o m m o d a t e d i n t h e M o f f i t t p a r a m e t e r a unless v e r y large, i n w h i c h case t h e y w i l l d r a s t i c a l l y change λ , as is observed i n n a t i v e carbonic a n h y d r a s e (3, 49), where a r o m a t i c C o t t o n effects w h i c h are observable i n t h e 260- t o 300-ηΐμ region (3, 13, 41, 49) a p p a r e n t l y d o m i n a t e t h e f a r - u l t r a v i o l e t O R D (49).


2

0

0

bo

Figure 5. Reduced mean rotation and a as a function of the Moffitt parameter, b , for the entire Mix-coil transition of paramyosin in guanidine-HCl at 25°C. (46) Open symboh and left-hand ordinate. 232-mp minimum of helical Cotton effect Solid symbols and right-hand ordinate. Moffitt parameter a Moffitt parameters calculated from optical rotatory dispersion data from 315 to 240 mp using \ = 220 mμ 0

0

Q

0

F o r p a r a m y o s i n [m'] 32 a n d t h e M o f f i t t p a r a m e t e r s a a n d b are colinear for the entire h e l i x - c o i l t r a n s i t i o n (see F i g u r e 5), b u t since b represents a n a v e r a g i n g of r o t a t o r y c o n t r i b u t i o n s at m a n y w a v e l e n g t h s a n d is r e l a t i v e l y insensitive t o t h e e n v i r o n m e n t , i t is t h e preferable p a r a m e t e r for t h e es t i m a t i o n of h e l i x content. A s seen for t h e h e l i c a l p o l y p e p t i d e s (33, 61, 62), t h e extension of t h e M o f f i t t p l o t t o 240 ιημ for p a r a m y o s i n changes t h e best v a l u e of λ t o 218 2

0

0

0

0


13.

177

Paramyosin

RIDDIFORD

ηΐμ [(20, Ifi), see also T a b l e I]. Y e t t h i s v a l u e of λ does n o t g i v e p e r f e c t l y l i n e a r plots [Figure 2, (61, 62)] w h i c h are f o u n d if t h e w a v e l e n g t h range is s p l i t i n t o t w o separate regions (600 t o 300 ηΐμ a n d 315 t o 240 ηΐμ) (62) (see F i g u r e s 1 a n d 3). S i n c e t h e h e l i c a l p e p t i d e t r a n s i t i o n s d o m i n a t e t h e d i s p e r s i o n b e l o w 300 ιημ [native p a r a m y o s i n becomes m o r e l e v o r o t a t o r y t h a n t h e u n f o l d e d p r o t e i n a b o u t 310 ιημ (see F i g u r e 3)], a q u e s t i o n has been raised a b o u t t h e v a l i d i t y of u s i n g t h e M o f f i t t e q u a t i o n i n t h i s region (53, 57). Schecter a n d B l o u t (52) proposed t h e t w o - t e r m D r u d e e q u a t i o n as a b e t t e r m e t h o d for a n a l y z i n g O R D d a t a f r o m 700 t o 275 ιημ. T h i s t w o t e r m D r u d e a p p r o a c h gives n e a r l y t h e same estimates of h e l i x content for t h e v a r i o u s p o l y p e p t i d e s a n d for p a r a m y o s i n (52, 53) as does t h e M o f f i t t t r e a t m e n t . T h e s m a l l m a g n i t u d e of F at t h e m a x i m u m of F for t h e 315t o 240-ιημ range (46) shows t h a t the M o f f i t t e q u a t i o n is adequate to fit t h e O R D d a t a for a f u l l y h e l i c a l p r o t e i n molecule f r o m 300 t o 240 ιημ ( i n t h e absence of a n y observable C o t t o n effects i n t h i s region), as U r n e s (61, 62) has p r e v i o u s l y f o u n d for t h e p o l y p e p t i d e s P G A , P T G A , a n d P A L G A .


0

Q

T h e s t a t i s t i c a l c r i t e r i a for t h e best λ (a m a x i m u m i n F a n d a m i n i m u m i n F at t h e same w a v e l e n g t h ) f a i l w h e n p a r a m y o s i n becomes less t h a n 3 0 % h e l i c a l (46)—i.e., a b o v e 5 . 5 M G - H C 1 — w h i c h as expected (57) is precisely t h e p o i n t at w h i c h the o n e - t e r m D r u d e e q u a t i o n becomes sufficient t o describe t h e d a t a . T h e v a l u e of X begins t o decrease r a p i d l y ( F i g u r e 4 ) , a n d t h e s t a n d a r d d e v i a t i o n s i n t h e slope a n d i n t e r c e p t of t h e D r u d e p l o t decrease t o those c h a r a c t e r i s t i c of a r i g o r o u s l y l i n e a r p l o t ( e q u a l t o those f o u n d for the r a n d o m coil). T h e a p p a r e n t l i n e a r i t y of t h e D r u d e p l o t for the n a t i v e h e l i c a l p r o t e i n i n t h e 315- t o 240-ηΐμ range c a n be m i s l e a d i n g . A s seen i n F i g u r e 4, \ shows o n l y t w o steps of t h e three-step h e l i x - c o i l t r a n s i t i o n i n d i c a t e d b y [ra'] (or b y a or b , F i g u r e 5). F u r t h e r m o r e , if \ is u s e d t o e s t i m a t e h e l i x c o n t e n t (67), there is a great d i s c r e p a n c y w i t h estimates based o n either [m'] or b . F o r example, at 4 M G - H C 1 , t h e X e s t i m a t e gives 8 0 % h e l i x whereas either [m'] or b estimates g i v e a b o u t 3 5 % h e l i x . T h i s d i s c r e p a n c y is consistent w i t h t h e s t a t i s t i c a l i n d i c a t i o n of n o n l i n e a r i t y of t h e D r u d e p l o t since \ has no m e a n i n g if t h e p l o t is n o t linear. 0

Q

c

c

0

232

0

c

0

232

c

232

0

c

T h e M o f f i t t t r e a t m e n t r e m a i n s v a l i d where the o n e - t e r m D r u d e e q u a t i o n is sufficient if λ is k e p t c o n s t a n t (63). 0

T h e r e f o r e , i n t h e case of t h e

d e n a t u r e d h e l i c a l p r o t e i n o r of t h e n a t i v e g l o b u l a r p r o t e i n w i t h l o w h e l i x c o n t e n t , λ s h o u l d be m a i n t a i n e d at t h e v a l u e f o u n d for t h e h e l i c a l p o l y 0

peptides a n d for p a r a m y o s i n — λ (46, 61, 62) a n d λ

0

= 220 ιημ i n t h e 240- t o 315- ιημ range

= 212 ηΐμ i n t h e 300- to 600-ηΐμ range (46, 63).

0

Hence,

b c a n r e a d i l y be u s e d as a measure of h e l i c a l content a c c o r d i n g t o E q u a t i o n 0

3. f

_ bp ( n a t i v e protein) — b (unfolded protein) b (α-helix) — b ( r a n d o m coil) 0

Q

0


(3)

178

ORDERED FLUIDS AND LIQUID CRYSTALS

As seen in Table I, b in the lower wavelength range is —343° for the native protein and + 2 0 ° for the denatured protein. Therefore, using myo globin as an example, the value of —250° obtained by Harrison and Blout (21) gives 74% helix, and the value of —266°, corrected for the Soret Cotton effect, obtained by Urnes (61, 62) gives 79% helix. These two values agree well with the 77% amide bonds in helical array from the x-ray analysis of the myoglobin crystal (31) and also with the values of 73% and 77% helix, respectively, based on the polypeptide models (an average of the values for P G A , P T G A , and P A L G A ) (61, 62). Thus, in spite of the apparent lack of complete randomness and the possible effects of supercoiling on optical activity, paramyosin seems to be a satisfactory model upon which to base estimates of helical contents of proteins.


Q

Acknowledgment

t

I thank Judy Campbell for her technical assistance, Peter Urnes for many enlightening discussions and his criticisms of this manuscript, and John Edsall for his critical appraisal of the manuscript. For use of the Cary 60 spectropolarimeter I thank Paul Doty and the Harvard Chemistry Department. Literature

Cited

(1) Allis, J., Ferry, J., J. Am. Chem. Soc. 87, 4681 (1965). (2) Allis, J., Ferry, J., Proc. Natl. Acad. Sci. U. S. 54, 369 (1965). (3) Armstrong, J., Myers, D. V., Verpoorte, J. Α., Edsall, J. T., J. Biol. Chem. 241, 5137 (1966). (4) Bear, R. S., J. Am. Chem. Soc. 66, 2043 (1944). (5) Bear, R. S., Selby, C., J. Biophys. Biochem. Cytol. 2, 55 (1956). (6) Beychok, S., Fasman, G., Biochemistry 3, 1675 (1964). (7) Beychok, S., Pflumm, M . N . , Lehman, J. E., J. Am. Chem. Soc. 87, 3990 (1965). (8) Blout, E. R., Schmier, I., Simmons, N. S., J. Am. Chem. Soc. 84, 3193 (1962). (9) Brant, D. Α., Flory, P. J., J. Am. Chem. Soc. 87, 2788, 2791 (1965). (10) Cohen, C., Holmes, K . C., J. Mol. Biol. 6, 423 (1963). (11) Cohen,C.,Szent-Györgyi, A. G., "Fourth International Congress of Biochemistry," Vol. 8, "Proteins," p. 108, Pergamon Press, London, 1960. (12) Cohen, C., Szent-Györgyi, A. G., J. Am. Chem. Soc. 79, 248 (1957). (13) Coleman, J. E., Biochemistry 4, 2644 (1965). (14) Crick, F. C., Acta Cryst. 6, 639 (1953). (15) Crick, F. C., Nature 170, 882 (1952). (16) Fasman, G., "Methods in Enzymology," Vol. 6, p. 928, Academic Press, New York, 1963. (17) Fasman, G., Bodenheimer, E., Lindblow, C., Biochemistry 3, 1665 (1964). (18) Fasman, G., Landsberg, M . , Buchwald, M., Can. J. Chem. 43, 1588 (1965). (19) Friedman, E., Gill, T. J., Doty, P., J. Am. Chem. Soc. 84, 3485 (1962). (20) Harrap, B. S., private communication, 1960. (21) Harrison, S. C., Blout, E . R., J. Biol. Chem. 240, 299 (1965). (22) Hodge, Α., Proc. Natl. Acad. Sci. U. S. 38, 850 (1952). (23) Hodge, A. J., Rev. Mod. Phys. 34, 409 (1959). (24) Holzwarth, G., Doty, P., J. Am. Chem. Soc. 87, 218 (1965). (25) Iizuka, E., Yang, J. T., Biochemistry 4, 1249 (1965). (26) Jirgensons, B., J. Biol. Chem. 240, 1064 (1965).



13.

RIDDIFORD

Paramyosin

179

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Paramyosin, a Model Î±-Helical Protein

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